Jeffrey J. Wine

All Publications

• Calibrating sweat chloride levels to CFTR activity via ETI effects on CF subjects with one or two F508DEL mutations. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society Wine, J. J. 2024

  Abstract

  It is difficult to determine CFTR activity following highly effective CFTR modulator therapies (HEMT). The sweat gland provides two biomarkers of CFTR activity: a linear readout via the β-sweat rate and a logarithmic readout via sweat chloride concentration (SCC). In prior work, different logarithmic functions were generated to calibrate SCC with the percent of healthy control CFTR activity (HCCFTR). Two functions, A and B, were fit to SCC means from healthy controls set = 100 % and CF carriers measured as 50 % HCCFTR. A and B differ in the % HCCFTR activity assigned to SCC for minimal function mutations = 0.01 % for A and 1 % for B.Here, the functions are evaluated based on retrospective analysis of three multi-center studies of CF subjects with one or two F508del mutations treated with Elexacaftor/Tezacaftor/Ivacaftor (ETI). Predictions of the percent HCCFTR activity for one vs two mutations were compared for the two functions. The expectation is that after ETI treatment, subjects with two responsive mutations will have 2-fold higher HCCFTR activity than subjects with only one. The hypothesis is that the SCCHCCFTR function that most closely fits that expectation provides the more accurate prediction of CFTR activity.In two separate comparisons, function B most accurately predicted a 2-fold (1.9, 2.3-fold) higher level of HCCFTR activity in subjects on ETI with two vs. one responsive mutation. Function A predicted a 4, 5.5-fold higher level.Function B predicts that 60 mmol/L SCC, the cutoff for a CF diagnosis, is associated with 10 % HCCFTR activity. Comparing HEMT effects on subjects with one or two mutations provides an additional tool for calibrating SCC to CFTR activity.

  View details for DOI 10.1016/j.jcf.2024.09.004

  View details for PubMedID 39406575

• How the sweat gland reveals levels of CFTR activity. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society Wine, J. J. 2022

  Abstract

  CFTR is an anion channel that causes cystic fibrosis (CF) when its activity, equal to channel number x open probability x conductance (n·PO·gamma) is absent or nearly so. CFTR modulators increase CFTR activity, but estimates of in vivo efficacy vary. This review shows how values from the simple and widely used sweat chloride test can be calibrated to provide more accurate estimates of CFTR activity as a percent of the average for healthy control (HC) subjects (hereafter 'CFTR activity'). Sweating stimulated by beta-adrenergic agonists (beta-sweat) is rate-limited by CFTR, producing a near linear, ratio scale of CFTR activity with carriers=50% and CF=0% of HC values set=100%, but the beta-sweat assay is difficult to use. Here, sweat chloride is calibrated to CFTR activity by plotting mean sweat chloride values, taken from numerous studies and the CFTR2 database against mean beta-sweat rates for CF, carriers and HC. The resulting inverse logarithmic relations indicate that sweat chloride values ≥60mmol/L occur when CFTR activity is below 1.2% -10% of HC. These are lower than most previous estimates, which resulted from setting nasal potential difference (NPD) as linear rather than logarithmic measures of CFTR activity. Features of the sweat gland coil and duct are used to explain why readouts of CFTR activity are linear for beta-sweat and logarithmic for sweat chloride. Sweat chloride values fall steeply for small increments of CFTR activity above zero-the most clinically relevant region. Thus, large health benefits can be achieved by restoring low levels of CFTR activity, especially if this is done before irreversible lung damage. Truncated Abstract: CFTR is an anion channel that causes cystic fibrosis (CF) when its activity, equal to channel number x open probability x conductance (n·PO·gamma) is absent or nearly so. CFTR modulators increase CFTR activity, but estimates of in vivo efficacy vary. This review shows how values from the sweat chloride test can be calibrated to provide accurate estimates of CFTR activity as a percent of the average for healthy control (HC) subjects. Sweating stimulated by beta-adrenergic agonists is rate-limited by CFTR, producing a near linear, ratio scale of CFTR activity, but the assay is difficult to use. Here, sweat chloride is calibrated to CFTR activity by plotting it against mean beta-sweat rates for different groups. The resulting logarithmic relations indicate that CF sweat chloride values occur when CFTR activity is below 1.2% -10% of HC, and that large health benefits can be achieved by restoring low levels of CFTR activity if this is done early.

  View details for DOI 10.1016/j.jcf.2022.02.001

  View details for PubMedID 35184981

• Combined agonists act synergistically to increase mucociliary clearance in a cystic fibrosis airway model. Scientific reports Joo, N. S., Cho, H., Shinbashi, M., Choi, J. Y., Milla, C. E., Engelhardt, J. F., Wine, J. J. 2021; 11 (1): 18828

  Abstract

  Mucus clearance, a primary innate defense mechanism of airways, is defective in patients with cystic fibrosis (CF) and CF animals. In previous work, the combination of a low dose of the cholinergic agonist, carbachol with forskolin or a beta adrenergic agonist, isoproterenol synergistically increased mucociliary clearance velocity (MCCV) in ferret tracheas. Importantly, the present study shows that synergistic MCCV can also be produced in CF ferrets, with increases~55% of WT. Synergistic MCCV was also produced in pigs. The combined agonists increased MCCV by increasing surface fluid via multiple mechanisms: increased fluid secretion from submucosal glands, increased anion secretion across surface epithelia and decreased Na+ absorption. To avoid bronchoconstriction, the cAMP agonist was applied 30min before carbachol. This approach to increasing mucus clearance warrants testing for safety and efficacy in humans as a potential therapeutic for muco-obstructive diseases.

  View details for DOI 10.1038/s41598-021-98122-5

  View details for PubMedID 34552115

• EARLY CONTINUOUS ANTI-INFECTIVES: CONCEPTS AND CONCERNS Wine, J. J. WILEY. 2019: S75–S76

  View details for Web of Science ID 000484544000032

• High-Efficiency, Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia. Cell stem cell Vaidyanathan, S. n., Salahudeen, A. A., Sellers, Z. M., Bravo, D. T., Choi, S. S., Batish, A. n., Le, W. n., Baik, R. n., de la O, S. n., Kaushik, M. P., Galper, N. n., Lee, C. M., Teran, C. A., Yoo, J. H., Bao, G. n., Chang, E. H., Patel, Z. M., Hwang, P. H., Wine, J. J., Milla, C. E., Desai, T. J., Nayak, J. V., Kuo, C. J., Porteus, M. H. 2019

  Abstract

  Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Mortality in CF patients is mostly due to respiratory sequelae. Challenges with gene delivery have limited attempts to treat CF using in vivo gene therapy, and low correction levels have hindered ex vivo gene therapy efforts. We have used Cas9 and adeno-associated virus 6 to correct the ΔF508 mutation in readily accessible upper-airway basal stem cells (UABCs) obtained from CF patients. On average, we achieved 30%-50% allelic correction in UABCs and bronchial epithelial cells (HBECs) from 10 CF patients and observed 20%-50% CFTR function relative to non-CF controls in differentiated epithelia. Furthermore, we successfully embedded the corrected UABCs on an FDA-approved porcine small intestinal submucosal membrane (pSIS), and they retained differentiation capacity. This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF.

  View details for DOI 10.1016/j.stem.2019.11.002

  View details for PubMedID 31839569

• How to live a long and healthy life with cystic fibrosis: Lessons from the CF ferret. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society Wine, J. J. 2018

  View details for PubMedID 30361142

• Ivacaftor restores CFTR-dependent sweat gland fluid secretion in cystic fibrosis subjects with S945L alleles JOURNAL OF CYSTIC FIBROSIS Kim, J., Davies, Z., Dunn, C., Wine, J. J., Milla, C. 2018; 17 (2): 179–85

  Abstract

  To determine in vivo effects of CFTR modulators on mutation S945L.We measured effects of CFTR modulators on CFTR-dependent sweating ('C-sweat') in two pancreatic sufficient cystic fibrosis (CF) subjects. S1 (S945L/G542X) took ivacaftor and S2 (S945L/F508del) took ivacaftor+tezacaftor. Sweating was stimulated pharmacologically to produce sequentially both CFTR-independent (methacholine stimulated) M-sweat and C-sweat; and the ratio of these was compared. Sweat secretion was measured with two methods: real time secretory rate quantitative recording and by optically measuring the growth of sweat bubbles under oil from multiple identified glands.Using the quantitative recorder, we saw zero C-sweat secretion off-drug, but when on-drug the C-sweat responses for both subjects were comparable to those seen in carriers. The on-drug response was further quantified using the sweat bubble method. Each subject again showed robust C-sweat responses, with C-sweat/M-sweat ratios~half of the ratio determined for a cohort of 40 controls tested under identical conditions.These in vivo results, consistent with prior in vitro findings, indicate that the drug treatments restore near-normal function to S945L-CFTR, and support the use of ivacaftor as a treatment for CF patients who carry this allele.

  View details for PubMedID 29279204

• The magnitude of ivacaftor effects on fluid secretion via R117H-CFTR channels: Human in vivo measurements PLOS ONE Char, J. E., Dunn, C., Davies, Z., Milla, C., Moss, R. B., Wine, J. J. 2017; 12 (4)

  Abstract

  We optically measured effects of orally available ivacaftor (Kalydeco®) on sweat rates of identified glands in 3 R117H subjects, each having a unique set of additional mutations, and compared them with 5 healthy control subjects tested contemporaneously. We injected β-adrenergic agonists intradermally to stimulate CFTR-dependent 'C-sweat' and methacholine to stimulate 'M-sweat', which persists in CF subjects. We focused on an R117H-7T/F508del subject who produced quantifiable C-sweat off ivacaftor and was available for 1 blinded, 3 off ivacaftor, and 3 on ivacaftor tests, allowing us to estimate in vivo fold-increase in sweat rates produced by ivacaftor's effect on the open probability (PO) of R117H-CFTR. Measured sweat rates must be corrected for sweat losses. With estimated sweat losses of 0.023 to 0.08 nl·gland-1·min-1, ivacaftor increased the average C-sweat rates 3-7 fold, and estimated function as % of WT were 4.1-12% off ivacaftor and 21.9-32% on ivacaftor (larger values reflect increased loss estimates). Based on single tests, an R117H-7T/ R117H-7T subject showed 6-9% WT function off ivacaftor and 28-43% on ivacaftor. Repeat testing of an R117H-5T/F508del subject detected only trace responding to ivacaftor. We conclude that in vivo, R117H PO is strongly increased by ivacaftor, but channel number, mainly determined by variable deletion of exon 10, has a marked influence on outcomes.

  View details for DOI 10.1371/journal.pone.0175486

  View details for Web of Science ID 000399875200023

  View details for PubMedID 28419121

• Marked increases in mucociliary clearance produced by synergistic secretory agonists or inhibition of the epithelial sodium channel SCIENTIFIC REPORTS Joo, N. S., Jeong, J. H., Cho, H., Wine, J. J. 2016; 6

  Abstract

  Mucociliary clearance (MCC) is a critical host innate defense mechanism in airways, and it is impaired in cystic fibrosis (CF) and other obstructive lung diseases. Epithelial fluid secretion and absorption modify MCC velocity (MCCV). We tested the hypotheses that inhibiting fluid absorption accelerates MCCV, whereas inhibiting fluid secretion decelerates it. In airways, ENaC is mainly responsible for fluid absorption, while anion channels, including CFTR and Ca(2+)-activated chloride channels mediate anion/fluid secretion. MCCV was increased by the cAMP-elevating agonists, forskolin or isoproterenol (10 μM) and by the Ca(2+)-elevating agonist, carbachol (0.3 μM). The CFTR-selective inhibitor, CFTRinh-172, modestly reduced MCCV-increases induced by forskolin or isoproterenol but not increases induced by carbachol. The ENaC inhibitor benzamil increased basal MCCV as well as MCCV increases produced by forskolin or carbachol. MCC velocity was most dramatically accelerated by the synergistic combination of forskolin and carbachol, which produced near-maximal clearance rates regardless of prior treatment with CFTR or ENaC inhibitors. In CF airways, where CFTR-mediated secretion (and possibly synergistic MCC) is lost, ENaC inhibition via exogenous agents may provide therapeutic benefit, as has long been proposed.

  View details for DOI 10.1038/srep36806

  View details for Web of Science ID 000388090100001

  View details for PubMedID 27830759

  View details for PubMedCentralID PMC5103292

• Inhibition of airway surface fluid absorption by cholinergic stimulation. Scientific reports Joo, N. S., Krouse, M. E., Choi, J. Y., Cho, H., Wine, J. J. 2016; 6: 20735-?

  Abstract

  In upper airways airway surface liquid (ASL) depth and clearance rates are both increased by fluid secretion. Secretion is opposed by fluid absorption, mainly via the epithelial sodium channel, ENaC. In static systems, increased fluid depth activates ENaC and decreased depth inhibits it, suggesting that secretion indirectly activates ENaC to reduce ASL depth. We propose an alternate mechanism in which cholinergic input, which causes copious airway gland secretion, also inhibits ENaC-mediated absorption. The conjoint action accelerates clearance, and the increased transport of mucus out of the airways restores ASL depth while cleansing the airways. We were intrigued by early reports of cholinergic inhibition of absorption by airways in some species. To reinvestigate this phenomenon, we studied inward short-circuit currents (Isc) in tracheal mucosa from human, sheep, pig, ferret, and rabbit and in two types of cultured cells. Basal Isc was inhibited 20-70% by the ENaC inhibitor, benzamil. Long-lasting inhibition of ENaC-dependent Isc was also produced by basolateral carbachol in all preparations except rabbit and the H441 cell line. Atropine inhibition produced a slow recovery or prevented inhibition if added before carbachol. The mechanism for inhibition was not determined and is most likely multi-factorial. However, its physiological significance is expected to be increased mucus clearance rates in cholinergically stimulated airways.

  View details for DOI 10.1038/srep20735

  View details for PubMedID 26846701

  View details for PubMedCentralID PMC4742893

• Airway Gland Structure and Function. Physiological reviews Widdicombe, J. H., Wine, J. J. 2015; 95 (4): 1241-1319

  Abstract

  Submucosal glands contribute to airway surface liquid (ASL), a film that protects all airway surfaces. Glandular mucus comprises electrolytes, water, the gel-forming mucin MUC5B, and hundreds of different proteins with diverse protective functions. Gland volume per unit area of mucosal surface correlates positively with impaction rate of inhaled particles. In human main bronchi, the volume of the glands is ∼50 times that of surface goblet cells, but the glands diminish in size and frequency distally. ASL and its trapped particles are removed from the airways by mucociliary transport. Airway glands have a tubuloacinar structure, with a single terminal duct, a nonciliated collecting duct, then branching secretory tubules lined with mucous cells and ending in serous acini. They allow for a massive increase in numbers of mucus-producing cells without replacing surface ciliated cells. Active secretion of Cl(-) and HCO3 (-) by serous cells produces most of the fluid of gland secretions. Glands are densely innervated by tonically active, mutually excitatory airway intrinsic neurons. Most gland mucus is secreted constitutively in vivo, with large, transient increases produced by emergency reflex drive from the vagus. Elevations of [cAMP]i and [Ca(2+)]i coordinate electrolyte and macromolecular secretion and probably occur together for baseline activity in vivo, with cholinergic elevation of [Ca(2+)]i being mainly responsive for transient increases in secretion. Altered submucosal gland function contributes to the pathology of all obstructive diseases, but is an early stage of pathogenesis only in cystic fibrosis.

  View details for DOI 10.1152/physrev.00039.2014

  View details for PubMedID 26336032

• Mucociliary clearance and submucosal gland secretion in the ex vivo ferret trachea. American journal of physiology. Lung cellular and molecular physiology Jeong, J. H., Joo, N. S., Hwang, P. H., Wine, J. J. 2014; 307 (1): L83-93

  Abstract

  In many species submucosal glands are an important source of tracheal mucus, but the extent to which mucociliary clearance (MCC) depends on gland secretion is unknown. To explore this relationship, we measured basal and agonist-stimulated MCC velocities in ex vivo tracheas from adult ferrets and compared the velocities with previously measured rates of ferret glandular mucus secretion (Cho HJ, Joo NS, Wine JJ. Am J Physiol Lung Cell Mol Physiol 299: L124-L136, 2010). Stimulated MCC velocities (mm/min, means ± SE for 10- to 35-min period poststimulation) were as follows: 1 μM carbachol: 19.1 ± 3.3 > 10 μM phenylephrine: 15.3 ± 2.4 ≈ 10 μM isoproterenol: 15.0 ± 1.9 ≈ 10 μM forskolin: 14.6 ± 3.1 > 1 μM vasoactive intestinal peptide (VIP): 10.2 ± 2.2 > basal (t15): 1.8 ± 0.3; n = 5-10 for each condition. Synergistic stimulation of MCC was observed between low concentrations of carbachol (100 nM) and isoproterenol (300 nM). Bumetanide inhibited carbachol-stimulated MCC by ~70% and abolished the increase in MCC stimulated by forskolin + VIP, whereas HCO3 (-)-free solutions did not significantly inhibit MCC to either intracellular Ca(2+) concentration or intracellular cAMP concentration ([cAMP]i)-elevating agonists. Stimulation and inhibition of MCC and gland secretion differed in several respects: most importantly, elevating [cAMP]i increased MCC much more effectively than expected from its effects on gland secretion, and bumetanide almost completely inhibited [cAMP]i-stimulated MCC while it had a smaller effect on gland secretion. We conclude that changes in glandular fluid secretion are complexly related to MCC and discuss possible reasons for this.

  View details for DOI 10.1152/ajplung.00009.2014

  View details for PubMedID 24793168

• A Little CFTR Goes a Long Way: CFTR-Dependent Sweat Secretion from G551D and R117H-5T Cystic Fibrosis Subjects Taking Ivacaftor PLOS ONE Char, J. E., Wolfe, M. H., Cho, H., Park, I., Jeong, J. H., Frisbee, E., Dunn, C., Davies, Z., Milla, C., Moss, R. B., Thomas, E. A., Wine, J. J. 2014; 9 (2)

  Abstract

  To determine if oral dosing with the CFTR-potentiator ivacaftor (VX-770, Kalydeco) improves CFTR-dependent sweating in CF subjects carrying G551D or R117H-5T mutations, we optically measured sweat secretion from 32-143 individually identified glands in each of 8 CF subjects; 6 F508del/G551D, one G551D/R117H-5T, and one I507del/R117H-5T. Two subjects were tested only (-) ivacaftor, 3 only (+) ivacaftor and 3 (+/-) ivacaftor (1-5 tests per condition). The total number of gland measurements was 852 (-) ivacaftor and 906 (+) ivacaftor. A healthy control was tested 4 times (51 glands). For each gland we measured both CFTR-independent (M-sweat) and CFTR-dependent (C-sweat); C-sweat was stimulated with a β-adrenergic cocktail that elevated [cAMP]i while blocking muscarinic receptors. Absent ivacaftor, almost all CF glands produced M-sweat on all tests, but only 1/593 glands produced C-sweat (10 tests, 5 subjects). By contrast, 6/6 subjects (113/342 glands) produced C-sweat in the (+) ivacaftor condition, but with large inter-subject differences; 3-74% of glands responded with C/M sweat ratios 0.04%-2.57% of the average WT ratio of 0.265. Sweat volume losses cause proportionally larger underestimates of CFTR function at lower sweat rates. The losses were reduced by measuring C/M ratios in 12 glands from each subject that had the highest M-sweat rates. Remaining losses were estimated from single channel data and used to correct the C/M ratios, giving estimates of CFTR function (+) ivacaftor  = 1.6%-7.7% of the WT average. These estimates are in accord with single channel data and transcript analysis, and suggest that significant clinical benefit can be produced by low levels of CFTR function.

  View details for DOI 10.1371/journal.pone.0088564

  View details for Web of Science ID 000331254600090

  View details for PubMedID 24520399

  View details for PubMedCentralID PMC3919757

• In vivo readout of CFTR function: ratiometric measurement of CFTR-dependent secretion by individual, identifiable human sweat glands. PloS one Wine, J. J., Char, J. E., Chen, J., Cho, H., Dunn, C., Frisbee, E., Joo, N. S., Milla, C., Modlin, S. E., Park, I., Thomas, E. A., Tran, K. V., Verma, R., Wolfe, M. H. 2013; 8 (10)

  Abstract

  To assess CFTR function in vivo, we developed a bioassay that monitors and compares CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (∼50) individual, identified glands in each subject. Sweating was stimulated by intradermally injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-layer that contained dispersed, water soluble dye particles that partitioned into the sweat bubbles, making them highly visible. CFTR-independent secretion (M-sweat) was stimulated with methacholine, which binds to muscarinic receptors and elevates cytosolic calcium. CFTR-dependent secretion (C-sweat) was stimulated with a β-adrenergic cocktail that elevates cytosolic cAMP while blocking muscarinic receptors. A C-sweat/M-sweat ratio was determined on a gland-by-gland basis to compensate for differences unrelated to CFTR function, such as gland size. The average ratio provides an approximately linear readout of CFTR function: the heterozygote ratio is ∼0.5 the control ratio and for CF subjects the ratio is zero. During assay development, we measured C/M ratios in 6 healthy controls, 4 CF heterozygotes, 18 CF subjects and 4 subjects with 'CFTR-related' conditions. The assay discriminated all groups clearly. It also revealed consistent differences in the C/M ratio among subjects within groups. We hypothesize that these differences reflect, at least in part, levels of CFTR expression, which are known to vary widely. When C-sweat rates become very low the C/M ratio also tended to decrease; we hypothesize that this nonlinearity reflects ductal fluid absorption. We also discovered that M-sweating potentiates the subsequent C-sweat response. We then used potentiation as a surrogate for drugs that can increase CFTR-dependent secretion. This bioassay provides an additional method for assessing CFTR function in vivo, and is well suited for within-subject tests of systemic, CFTR-directed therapeutics.

  View details for DOI 10.1371/journal.pone.0077114

  View details for PubMedID 24204751

• Substance P stimulates human airway submucosal gland secretion mainly via a CFTR-dependent process JOURNAL OF CLINICAL INVESTIGATION Choi, J. Y., Khansaheb, M., Joo, N. S., Krouse, M. E., Robbins, R. C., Weill, D., Wine, J. J. 2009; 119 (5): 1189-1200

  Abstract

  Chronic bacterial airway infections are the major cause of mortality in cystic fibrosis (CF). Normal airway defenses include reflex stimulation of submucosal gland mucus secretion by sensory neurons that release substance P (SubP). CFTR is an anion channel involved in fluid secretion and mutated in CF; the role of CFTR in secretions stimulated by SubP is unknown. We used optical methods to measure SubP-mediated secretion from human submucosal glands in lung transplant tissue. Glands from control but not CF subjects responded to mucosal chili oil. Similarly, serosal SubP stimulated secretion in more than 60% of control glands but only 4% of CF glands. Secretion triggered by SubP was synergistic with vasoactive intestinal peptide and/or forskolin but not with carbachol; synergy was absent in CF glands. Pig glands demonstrated a nearly 10-fold greater response to SubP. In 10 of 11 control glands isolated by fine dissection, SubP caused cell volume loss, lumen expansion, and mucus flow, but in 3 of 4 CF glands, it induced lumen narrowing. Thus, in CF, the reduced ability of mucosal irritants to stimulate airway gland secretion via SubP may be another factor that predisposes the airways to infections.

  View details for DOI 10.1172/JCI37284

  View details for Web of Science ID 000265843400020

  View details for PubMedID 19381016

  View details for PubMedCentralID PMC2673871

• Synergistic airway gland mucus secretion in response to vasoactive intestinal peptide and carbachol is lost in cystic fibrosis JOURNAL OF CLINICAL INVESTIGATION Choi, J. Y., Joo, N. S., Krouse, M. E., Wu, J. V., Robbins, R. C., Ianowski, J. P., Hanrahan, J. W., Wine, J. J. 2007; 117 (10): 3118-3127

  Abstract

  Cystic fibrosis (CF) is caused by dysfunction of the CF transmembrane conductance regulator (CFTR), an anion channel whose dysfunction leads to chronic bacterial and fungal airway infections via a pathophysiological cascade that is incompletely understood. Airway glands, which produce most airway mucus, do so in response to both acetylcholine (ACh) and vasoactive intestinal peptide (VIP). CF glands fail to secrete mucus in response to VIP, but do so in response to ACh. Because vagal cholinergic pathways still elicit strong gland mucus secretion in CF subjects, it is unclear whether VIP-stimulated, CFTR-dependent gland secretion participates in innate defense. It was recently hypothesized that airway intrinsic neurons, which express abundant VIP and ACh, are normally active and stimulate low-level gland mucus secretion that is a component of innate mucosal defenses. Here we show that low levels of VIP and ACh produced significant mucus secretion in human glands via strong synergistic interactions; synergy was lost in glands of CF patients. VIP/ACh synergy also existed in pig glands, where it was CFTR dependent, mediated by both Cl(-) and HCO(3) (-), and clotrimazole sensitive. Loss of "housekeeping" gland mucus secretion in CF, in combination with demonstrated defects in surface epithelia, may play a role in the vulnerability of CF airways to bacterial infections.

  View details for DOI 10.1172/JCI31992

  View details for Web of Science ID 000249894400042

  View details for PubMedID 17853942

  View details for PubMedCentralID PMC1974867

• DURATIONS OF UNITARY SYNAPTIC POTENTIALS HELP TIME A BEHAVIORAL SEQUENCE SCIENCE Wine, J. J., Hagiwara, G. 1978; 199 (4328): 557-559

  Abstract

  Recordings in identified neurons and muscles that mediate crayfish tailflips reveal inhibitory postsynaptic potentials of two distinct durations. Those of long duration are recorded in five classes of cells in the flexion circuit, while those of short duration are recorded in three classes of cells in the extension circuit. The durations of the inhibitory postsynaptic potentials are matched to the durations of inhibition required by the different phases of the behavior.

  View details for Web of Science ID A1978EH73900025

  View details for PubMedID 622557

• ORGANIZATION OF ESCAPE BEHAVIOR IN CRAYFISH JOURNAL OF EXPERIMENTAL BIOLOGY Wine, J. J., Krasne, F. B. 1972; 56 (1): 1-?

  View details for Web of Science ID A1972L917600001

  View details for PubMedID 21046844

• The lysogenic filamentous Pseudomonas bacteriophage phage Pf slows mucociliary transport. PNAS nexus Burgener, E. B., Cai, P. C., Kratochvil, M. J., Rojas-Hernandez, L. S., Joo, N. S., Gupta, A., Secor, P. R., Heilshorn, S. C., Spakowitz, A. J., Wine, J. J., Bollyky, P. L., Milla, C. E. 2024; 3 (9): pgae390

  Abstract

  Pseudomonas aeruginosa is a major pulmonary pathogen causing chronic pulmonary infections in people with cystic fibrosis (CF). The P. aeruginosa filamentous and lysogenic bacteriophage, Pf phage, is abundant in the airways of many people with CF and has been associated with poor outcomes in a cross-sectional cohort study. Previous studies have identified roles for Pf phage in biofilm formation, specifically forming higher-order birefringent, liquid crystals when in contact with other biopolymers in biofilms. Liquid crystalline biofilms are more adherent and viscous than those without liquid crystals. A key feature of biofilms is to enhance bacterial adherence and resist physical clearance. The effect of Pf phage on mucociliary transport is unknown. We found that primary CF and non-CF nasal epithelial cells cultured at air-liquid interface treated with Pf phage exhibit liquid crystalline structures in the overlying mucus. On these cell cultures, Pf phage entangles cilia but does not affect ciliary beat frequency. In both these in vitro cell cultures and in an ex vivo porcine trachea model, introduction of Pf phage decreases mucociliary transport velocity. Pf phage also blocks the rescue of mucociliary transport by CF transmembrane conductance regulator modulators in CF cultures. Thus, Pf phage may contribute to the pathogenesis of P. aeruginosa-associated CF lung disease via induction of liquid crystalline characteristics to airway secretions, leading to impaired mucociliary transport. Targeting Pf phage may be useful in treatment CF as well as other settings of chronic P. aeruginosa infections.

  View details for DOI 10.1093/pnasnexus/pgae390

  View details for PubMedID 39301510

  View details for PubMedCentralID PMC11412248

• Transgenic ferret models define pulmonary ionocyte diversity and function. Nature Yuan, F., Gasser, G. N., Lemire, E., Montoro, D. T., Jagadeesh, K., Zhang, Y., Duan, Y., Ievlev, V., Wells, K. L., Rotti, P. G., Shahin, W., Winter, M., Rosen, B. H., Evans, I., Cai, Q., Yu, M., Walsh, S. A., Acevedo, M. R., Pandya, D. N., Akurathi, V., Dick, D. W., Wadas, T. J., Joo, N. S., Wine, J. J., Birket, S., Fernandez, C. M., Leung, H. M., Tearney, G. J., Verkman, A. S., Haggie, P. M., Scott, K., Bartels, D., Meyerholz, D. K., Rowe, S. M., Liu, X., Yan, Z., Haber, A. L., Sun, X., Engelhardt, J. F. 2023

  Abstract

  Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

  View details for DOI 10.1038/s41586-023-06549-9

  View details for PubMedID 37730992

  View details for PubMedCentralID 6295155

• Variation in CFTR-dependent 'beta-sweating' among healthy adults. PloS one DeRose, L., Kim, J., Farahmand, M., Shinbashi, M. Y., Joo, N. S., Wine, J. J. 2022; 17 (3): e0265432

  Abstract

  The genetic disease cystic fibrosis (CF) results when mutations in the gene for the anion channel CFTR reduce CFTR's activity below a critical level. CFTR activity = N·PO·gamma (number of channels x open probability x channel conductance). Small molecules are now available that partially restore CFTR function with dramatic improvements in health of CF subjects. Continued evaluation of these and other compounds in development will be aided by accurate assessments of CFTR function. However, measuring CFTR activity in vivo is challenging and estimates vary widely. The most accurate known measure of CFTR activity in vivo is the 'beta/M' ratio of sweat rates, which is produced by stimulation with a beta-adrenergic agonist cocktail referenced to the same individual's methacholine-stimulated sweat rate. The most meaningful metric of CFTR activity is to express it as a percent of normal function, so it is critical to establish beta/M carefully in a population of healthy control subjects. Here, we analyze beta/M from a sample of 50 healthy adults in which sweat rates to cholinergic and beta-adrenergic agonists were measured repeatedly (3 times) in multiple, (~50) identified sweat glands from each individual (giving ~20,000 measurements). The results show an approximately 7-fold range, 26-187% of the WT average set to 100%. These provide a benchmark against which other measures of CFTR activity can be compared. Factors contributing to beta/M variation in healthy controls are discussed.

  View details for DOI 10.1371/journal.pone.0265432

  View details for PubMedID 35312728

• Targeted replacement of full-length CFTR in human airway stem cells by CRISPR/Cas9 for pan-mutation correction in the endogenous locus. Molecular therapy : the journal of the American Society of Gene Therapy Vaidyanathan, S. n., Baik, R. n., Chen, L. n., Bravo, D. T., Suarez, C. J., Abazari, S. M., Salahudeen, A. A., Dudek, A. M., Teran, C. A., Davis, T. H., Lee, C. M., Bao, G. n., Randell, S. H., Artandi, S. E., Wine, J. J., Kuo, C. J., Desai, T. J., Nayak, J. V., Sellers, Z. M., Porteus, M. H. 2021

  Abstract

  Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR/Cas9 and two adeno-associated viruses (AAV) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial basal stem cells (HBECs). The modified cells were enriched to obtain 60-80% tCD19+ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.

  View details for DOI 10.1016/j.ymthe.2021.03.023

  View details for PubMedID 33794364

• Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers JOURNAL OF BIOLOGICAL CHEMISTRY Trillo-Muyo, S., Nilsson, H. E., Recktenwald, C. V., Ermund, A., Ridley, C., Meiss, L. N., Baehr, A., Klymiuk, N., Wine, J. J., Koeck, P. B., Thornton, D. J., Hebert, H., Hansson, G. C. 2018; 293 (15): 5746–54

  Abstract

  Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca2+ in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1-D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles.

  View details for PubMedID 29440393

  View details for PubMedCentralID PMC5900763

• Progress in understanding mucus abnormalities in cystic fibrosis airways. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society Wine, J. J., Hansson, G. C., Konig, P., Joo, N. S., Ermund, A., Pieper, M. 2018; 17 (2S): S35–S39

  Abstract

  Normal airways below the carina maintain an essentially sterile environment via a multi-pronged innate defence system that includes mucus clearance via mucociliary clearance and cough, multiple antimicrobials and cellular components including macrophages and neutrophils. In cystic fibrosis (CF), loss of CFTR function compromises these defences, and with present standard of care virtually all people with CF eventually develop mucus accumulation, plugging and chronic infections. This review focuses on how mucus is affected by CFTR loss.

  View details for PubMedID 28951068

• Sweat rate analysis of ivacaftor potentiation of CFTR in non-CF adults. Scientific reports Kim, J. n., Farahmand, M. n., Dunn, C. n., Milla, C. E., Horii, R. I., Thomas, E. A., Moss, R. B., Wine, J. J. 2018; 8 (1): 16233

  Abstract

  To determine if ivacaftor (Kalydeco) influences non-CF human CFTR function in vivo, we measured CFTR-dependent (C-sweat) and CFTR-independent (M-sweat) rates from multiple identified sweat glands in 8 non-CF adults. The two types of sweating were stimulated sequentially with intradermal injections of appropriate reagents; each gland served as its own control via alternating off-on drug tests on both arms, given at weekly intervals with 3 off and 3 on tests per subject. We compared drug effects on C-sweating stimulated by either high or low concentrations of β-adrenergic cocktail, and on methacholine-stimulated M-sweating. For each subject we measured ~700 sweat volumes from ~75 glands per arm (maximum 12 readings per gland), and sweat volumes were log-transformed for statistical analysis. T-tests derived from linear mixed models (LMMs) were more conservative than the familiar paired sample t-tests, and show that ivacaftor significantly increased C-sweating stimulated by both levels of agonist, with a larger effect in the low cocktail condition; ivacaftor did not increase M-sweat. Concurrent sweat chloride tests detected no effect of ivacaftor. We conclude that ivacaftor in vivo increases the open channel probability (PO) of WT CFTR, provided it is not already maximally stimulated.

  View details for PubMedID 30389955

• The normal trachea is cleaned by MUC5B mucin bundles from the submucosal glands coated with the MUC5AC mucin BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Ermund, A., Meiss, L. N., Rodriguez-Pineiro, A. M., Baehr, A., Nilsson, H. E., Trillo-Muyo, S., Ridley, C., Thornton, D. J., Wine, J. J., Hebert, H., Klymiuk, N., Hansson, G. C. 2017; 492 (3): 331–37

  Abstract

  To understand the mucociliary clearance system, mucins were visualized by light, confocal and electron microscopy, and mucus was stained by Alcian blue and tracked by video microscopy on tracheal explants of newborn piglets. We observed long linear mucus bundles that appeared at the submucosal gland openings and were transported cephalically. The mucus bundles were shown by mass spectrometry and immunostaining to have a core made of MUC5B mucin and were coated with MUC5AC mucin produced by surface goblet cells. The transport speed of the bundles was slower than the airway surface liquid flow. We suggest that the goblet cell MUC5AC mucin anchors the mucus bundles and thus controls their transport. Normal clearance of the respiratory tree of pigs and humans, both rich in submucosal glands, is performed by thick and long mucus bundles.

  View details for PubMedID 28859985

  View details for PubMedCentralID PMC5596833

• Impaired PGE2-stimulated Cl- and HCO3- secretion contributes to cystic fibrosis airway disease. PloS one Sellers, Z. M., Illek, B. n., Figueira, M. F., Hari, G. n., Joo, N. S., Sibley, E. n., Souza-Menezes, J. n., Morales, M. M., Fischer, H. n., Wine, J. J. 2017; 12 (12): e0189894

  Abstract

  Airway mucociliary clearance (MCC) is an important defense mechanism against pulmonary infections and is compromised in cystic fibrosis (CF). Cl- and HCO3- epithelial transport are integral to MCC. During pulmonary infections prostaglandin E2 (PGE2) production is abundant.To determine the effect of PGE2 on airway Cl- and HCO3- secretion and MCC in normal and CF airways.We examined PGE2 stimulated MCC, Cl- and HCO3- secretion using ferret trachea, human bronchial epithelial cell cultures (CFBE41o- with wildtype CFTR (CFBE41 WT) or homozygous F508del CFTR (CFBE41 CF) and human normal bronchial submucosal gland cell line (Calu-3) in Ussing chambers with or without pH-stat.PGE2 stimulated MCC in a dose-dependent manner and was partially impaired by CFTRinh-172. PGE2-stimulated Cl- current in ferret trachea was partially inhibited by CFTRinh-172, with niflumic acid eliminating the residual current. CFBE41 WT cell monolayers produced a robust Cl- and HCO3- secretory response to PGE2, both of which were completely inhibited by CFTRinh-172. CFBE41 CF cells exhibited no response to PGE2. In Calu-3 cells, PGE2 stimulated Cl- and HCO3- secretion. Cl- secretion was partially inhibited by CFTRinh-172, with additional inhibition by niflumic acid. HCO3- secretion was completely inhibited by CFTRinh-172.PGE2 stimulates bronchotracheal MCC and this response is decreased in CF. In CF airway, PGE2-stimulated Cl- and HCO3- conductance is impaired and may contribute to decreased MCC. There remains a CFTR-independent Cl- current in submucosal glands, which if exploited, could represent a means of improving airway Cl- secretion and MCC in CF.

  View details for PubMedID 29281691

  View details for PubMedCentralID PMC5744969

• Glandular Proteome Identifies Antiprotease Cystatin C as a Critical Modulator of Airway Hydration and Clearance AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY Evans, T. I., Joo, N. S., Keiser, N. W., Yan, Z., Tyler, S. R., Xie, W., Zhang, Y., Hsiao, J. J., Cho, H., Wright, M. E., Wine, J. J., Engelhardt, J. F. 2016; 54 (4): 469-481

  Abstract

  Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel lead to viscous secretions from submucosal glands that cannot be properly hydrated and cleared by beating cilia in cystic fibrosis (CF) airways. The mechanisms by which CFTR, and the predominant epithelial sodium channel (ENaC), control the hydration and clearance of glandular secretions remain unclear. We used a proteomics approach to characterize the proteins contained in CF and non-CF submucosal gland fluid droplets and found that differentially regulated proteases (cathepsin S and H) and their antiprotease (cystatin C) influenced the equilibration of fluid on the airway surface and tracheal mucociliary clearance (MCC). Contrary to prevailing models of airway hydration and clearance, cystatin C, or raising the airway surface liquid (ASL) pH, inhibited cathepsin-dependent ENaC-mediated fluid absorption and raised the height of ASL, and yet decreased MCC velocity. Importantly, coupling of both CFTR and ENaC activities were required for effective MCC and for effective ASL height equilibration after volume challenge. Cystatin C-inhibitable cathepsins controlled initial phases of ENaC-mediated fluid absorption, whereas CFTR activity was required to prevent ASL dehydration. Interestingly, CF airway epithelia absorbed fluid more slowly owing to reduced cysteine protease activity in the ASL but became abnormally dehydrated with time. Our findings demonstrate that, after volume challenge, pH-dependent protease-mediated coupling of CFTR and ENaC activities are required for rapid fluid equilibration at the airway surface and for effective MCC. These findings provide new insights into how glandular fluid secretions may be equilibrated at the airway surface and how this process may be impaired in CF.

  View details for DOI 10.1165/rcmb.2015-0090OC

  View details for Web of Science ID 000373707500003

  View details for PubMedID 26334941

  View details for PubMedCentralID PMC4821051

• Evaporimeter and Bubble-Imaging Measures of Sweat Gland Secretion Rates. PloS one Kim, J., Farahmand, M., Dunn, C., Davies, Z., Frisbee, E., Milla, C., Wine, J. J. 2016; 11 (10)

  Abstract

  Beta-adrenergically-stimulated sweat rates determined by evaporimetry or by sweat bubble imaging are useful for measuring CFTR function because they provide a near-linear readout across almost the full range of CFTR function. They differentiate cystic fibrosis (CF) subjects from CF carriers and carriers from controls. However, evaporimetry, unlike bubble imaging, appears to be unable to detect improved levels of CFTR function in G551D subjects taking the CFTR modulator ivacaftor. Here, we quantify the sensitivity of evaporimetry and bubble imaging methods for assessing low levels of CFTR-dependent sweat rates. To establish sensitivity, we did dose-ranging studies using intradermally injected [cAMP]i-elevating cocktails. We reduced isoproterenol/aminophylline levels while maintaining a high level of atropine to block muscarinic elevation of [Ca2+]i. We stimulated the same sets of glands for both assays and recorded responses for 20 min. In response to a 3-log dilution of the stimulating cocktail (0.1%), bubble responses were detected in 12/12 tests (100%), with 49% ± 3% of glands secreting to produce an aggregate volume of 598 nl across the 12, 20-min tests. This was ~5% of the response to full cocktail. Evaporimetry detected responses in 3/12 (25%) tests with an aggregate secretion volume of 175 nl. After stimulation with a still more dilute cocktail (0.03%), bubble imaging detected 15 ± 13% of glands secreting at a rate ~0.9% of the response to full cocktail, while zero responding was seen with evaporimetry. The bubble imaging method detected secretion down to aggregate rates of <0.2 nl/(cm2·min), or ~1/30th of the average basal transepithelial water loss (TEWL) in the test subject of 4 g/m2·hr or 6.7 nl/(cm2·min). The increased sensitivity of bubble imaging may be required to detect small but physiologically important increases in secretion rates produced by CFTR modulators.

  View details for DOI 10.1371/journal.pone.0165254

  View details for PubMedID 27768743

  View details for PubMedCentralID PMC5074501

• Secretion rates of human nasal submucosal glands from patients with chronic rhinosinusitis or cystic fibrosis. American journal of rhinology & allergy Jeong, J. H., Hwang, P. H., Cho, D., Joo, N. S., Wine, J. J. 2015; 29 (5): 334-338

  Abstract

  A majority of patients with cystic fibrosis (CF) have chronic rhinosinusitis (CRS) and/or nasal polyps, both of which may be secondary to reduced fluid secretion from nasal submucosal glands.To determine whether decreased fluid secretion from nasal submucosal glands also occurs in patients without CF and with CRS.Inferior turbinates of the nasal cavity were harvested from controls, subjects with CRS, and subjects with CF (n = 5-7 per group). The secretion rates of the nasal submucosal glands of the three groups in response to carbachol and forskolin were measured by using time lapse digital imaging of mucus bubbles from single glands as they formed on the mucosal surface under oil.Carbachol-stimulated secretion rates were the following: controls, 1670 ± 381 pl·min(-1)·gland(-1); CRS, 965 ± 440 pl·min(-1)·gland(-1); and CF, 933 ± 588 pl·min(-1)·gland(-1) (p = 0.23, Kruskal-Wallis test). Forskolin-stimulated secretion rates were the following: controls, 229 ± 14 pl·min(-1)·gland(-1); CRS, 154 ± 48 pl·min(-1)·gland(-1); and CF, 22 ± 15 pl·min(-1)·gland(-1) (p = 0.008, Kruskal-Wallis test). The ratio of the average secretion rate induced by forskolin to that induced by carbachol was 13.7% in the controls, and 15.9% in CRS and 2.3% in CF groups.The only significant difference in this small study was decreased forskolin-stimulated secretion in subjects with CF relative to the other subjects. However, there was a trend toward reduced carbachol-stimulated secretion rates in subjects with CRS and with and without CF relative to controls. Additional studies are needed to determine if nasal submucosal gland hyposecretion occurs in CRS either as a contributor to or as a consequence of CRS pathogenesis.

  View details for DOI 10.2500/ajra.2015.29.4213

  View details for PubMedID 26358343

• Secretion rates of human nasal submucosal glands from patients with chronic rhinosinusitis or cystic fibrosis AMERICAN JOURNAL OF RHINOLOGY & ALLERGY Jeong, J. H., Hwang, P. H., Cho, D., Joo, N. S., Wine, J. J. 2015; 29 (5): 334-338

  Abstract

  A majority of patients with cystic fibrosis (CF) have chronic rhinosinusitis (CRS) and/or nasal polyps, both of which may be secondary to reduced fluid secretion from nasal submucosal glands.To determine whether decreased fluid secretion from nasal submucosal glands also occurs in patients without CF and with CRS.Inferior turbinates of the nasal cavity were harvested from controls, subjects with CRS, and subjects with CF (n = 5-7 per group). The secretion rates of the nasal submucosal glands of the three groups in response to carbachol and forskolin were measured by using time lapse digital imaging of mucus bubbles from single glands as they formed on the mucosal surface under oil.Carbachol-stimulated secretion rates were the following: controls, 1670 ± 381 pl·min(-1)·gland(-1); CRS, 965 ± 440 pl·min(-1)·gland(-1); and CF, 933 ± 588 pl·min(-1)·gland(-1) (p = 0.23, Kruskal-Wallis test). Forskolin-stimulated secretion rates were the following: controls, 229 ± 14 pl·min(-1)·gland(-1); CRS, 154 ± 48 pl·min(-1)·gland(-1); and CF, 22 ± 15 pl·min(-1)·gland(-1) (p = 0.008, Kruskal-Wallis test). The ratio of the average secretion rate induced by forskolin to that induced by carbachol was 13.7% in the controls, and 15.9% in CRS and 2.3% in CF groups.The only significant difference in this small study was decreased forskolin-stimulated secretion in subjects with CF relative to the other subjects. However, there was a trend toward reduced carbachol-stimulated secretion rates in subjects with CRS and with and without CF relative to controls. Additional studies are needed to determine if nasal submucosal gland hyposecretion occurs in CRS either as a contributor to or as a consequence of CRS pathogenesis.

  View details for DOI 10.2500/ajra.2015.29.4213

  View details for Web of Science ID 000364337200011

  View details for PubMedID 26358343

• Proteomic Analysis of Pure Human Airway Gland Mucus Reveals a Large Component of Protective Proteins PLOS ONE Joo, N. S., Evans, I. A., Cho, H., Park, I., Engelhardt, J. F., Wine, J. J. 2015; 10 (2)

  Abstract

  Airway submucosal glands contribute to innate immunity and protect the lungs by secreting mucus, which is required for mucociliary clearance and which also contains antimicrobial, anti-inflammatory, anti-proteolytic and anti-oxidant proteins. We stimulated glands in tracheal trimmings from three lung donors and collected droplets of uncontaminated mucus as they formed at the gland orifices under an oil layer. We analyzed the mucus using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analysis identified 5486 peptides and 441 proteins from across the 3 samples (269-319 proteins per subject). We focused on 269 proteins common to at least 2 0f 3 subjects, of which 102 (38%) had protective or innate immunity functions. While many of these have long been known to play such roles, for many others their cellular protective functions have only recently been appreciated in addition to their well-studied biologic functions (e.g. annexins, apolipoproteins, gelsolin, hemoglobin, histones, keratins, and lumican). A minority of the identified proteins are known to be secreted via conventional exocytosis, suggesting that glandular secretion occurs via multiple mechanisms. Two of the observed protective proteins, major vault protein and prohibitin, have not been observed in fluid from human epithelial cultures or in fluid from nasal or bronchoalveolar lavage. Further proteomic analysis of pure gland mucus may help clarify how healthy airways maintain a sterile environment.

  View details for DOI 10.1371/journal.pone.0116756

  View details for Web of Science ID 000350662100049

  View details for PubMedID 25706550

  View details for PubMedCentralID PMC4338240

• Measuring mucociliary transport and mucus properties in multiple regions of airway epithelial surfaces helps clarify cystic fibrosis defects. American journal of respiratory and critical care medicine Wine, J. J. 2014; 190 (4): 364-5

  View details for DOI 10.1164/rccm.201407-1247ED

  View details for PubMedID 25127304

• Medicine. Letting go of mucus. Science (New York, N.Y.) Wine, J. J. 2014; 345 (6198): 730-1

  View details for DOI 10.1126/science.1258493

  View details for PubMedID 25124411

• Mucociliary clearance and submucosal gland secretion in the ex vivo ferret trachea AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Jeong, J. H., Joo, N. S., Hwang, P. H., Wine, J. J. 2014; 307 (1): L83-L93

  Abstract

  In many species submucosal glands are an important source of tracheal mucus, but the extent to which mucociliary clearance (MCC) depends on gland secretion is unknown. To explore this relationship, we measured basal and agonist-stimulated MCC velocities in ex vivo tracheas from adult ferrets and compared the velocities with previously measured rates of ferret glandular mucus secretion (Cho HJ, Joo NS, Wine JJ. Am J Physiol Lung Cell Mol Physiol 299: L124-L136, 2010). Stimulated MCC velocities (mm/min, means ± SE for 10- to 35-min period poststimulation) were as follows: 1 μM carbachol: 19.1 ± 3.3 > 10 μM phenylephrine: 15.3 ± 2.4 ≈ 10 μM isoproterenol: 15.0 ± 1.9 ≈ 10 μM forskolin: 14.6 ± 3.1 > 1 μM vasoactive intestinal peptide (VIP): 10.2 ± 2.2 > basal (t15): 1.8 ± 0.3; n = 5-10 for each condition. Synergistic stimulation of MCC was observed between low concentrations of carbachol (100 nM) and isoproterenol (300 nM). Bumetanide inhibited carbachol-stimulated MCC by ~70% and abolished the increase in MCC stimulated by forskolin + VIP, whereas HCO3 (-)-free solutions did not significantly inhibit MCC to either intracellular Ca(2+) concentration or intracellular cAMP concentration ([cAMP]i)-elevating agonists. Stimulation and inhibition of MCC and gland secretion differed in several respects: most importantly, elevating [cAMP]i increased MCC much more effectively than expected from its effects on gland secretion, and bumetanide almost completely inhibited [cAMP]i-stimulated MCC while it had a smaller effect on gland secretion. We conclude that changes in glandular fluid secretion are complexly related to MCC and discuss possible reasons for this.

  View details for DOI 10.1152/ajplung.00009.2014

  View details for Web of Science ID 000338923400008

• A little CFTR goes a long way: CFTR-dependent sweat secretion from G551D and R117H-5T cystic fibrosis subjects taking ivacaftor. PloS one Char, J. E., Wolfe, M. H., Cho, H., Park, I., Jeong, J. H., Frisbee, E., Dunn, C., Davies, Z., Milla, C., Moss, R. B., Thomas, E. A., Wine, J. J. 2014; 9 (2)

  Abstract

  To determine if oral dosing with the CFTR-potentiator ivacaftor (VX-770, Kalydeco) improves CFTR-dependent sweating in CF subjects carrying G551D or R117H-5T mutations, we optically measured sweat secretion from 32-143 individually identified glands in each of 8 CF subjects; 6 F508del/G551D, one G551D/R117H-5T, and one I507del/R117H-5T. Two subjects were tested only (-) ivacaftor, 3 only (+) ivacaftor and 3 (+/-) ivacaftor (1-5 tests per condition). The total number of gland measurements was 852 (-) ivacaftor and 906 (+) ivacaftor. A healthy control was tested 4 times (51 glands). For each gland we measured both CFTR-independent (M-sweat) and CFTR-dependent (C-sweat); C-sweat was stimulated with a β-adrenergic cocktail that elevated [cAMP]i while blocking muscarinic receptors. Absent ivacaftor, almost all CF glands produced M-sweat on all tests, but only 1/593 glands produced C-sweat (10 tests, 5 subjects). By contrast, 6/6 subjects (113/342 glands) produced C-sweat in the (+) ivacaftor condition, but with large inter-subject differences; 3-74% of glands responded with C/M sweat ratios 0.04%-2.57% of the average WT ratio of 0.265. Sweat volume losses cause proportionally larger underestimates of CFTR function at lower sweat rates. The losses were reduced by measuring C/M ratios in 12 glands from each subject that had the highest M-sweat rates. Remaining losses were estimated from single channel data and used to correct the C/M ratios, giving estimates of CFTR function (+) ivacaftor  = 1.6%-7.7% of the WT average. These estimates are in accord with single channel data and transcript analysis, and suggest that significant clinical benefit can be produced by low levels of CFTR function.

  View details for DOI 10.1371/journal.pone.0088564

  View details for PubMedID 24520399

• In Vivo Readout of CFTR Function: Ratiometric Measurement of CFTR-Dependent Secretion by Individual, Identifiable Human Sweat Glands PLOS ONE Wine, J. J., Char, J. E., Chen, J., Cho, H., Dunn, C., Frisbee, E., Joo, N. S., Milla, C., Modlin, S. E., Park, I., Thomas, E. A., Tran, K. V., Verma, R., Wolfe, M. H. 2013; 8 (10)

  Abstract

  To assess CFTR function in vivo, we developed a bioassay that monitors and compares CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (∼50) individual, identified glands in each subject. Sweating was stimulated by intradermally injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-layer that contained dispersed, water soluble dye particles that partitioned into the sweat bubbles, making them highly visible. CFTR-independent secretion (M-sweat) was stimulated with methacholine, which binds to muscarinic receptors and elevates cytosolic calcium. CFTR-dependent secretion (C-sweat) was stimulated with a β-adrenergic cocktail that elevates cytosolic cAMP while blocking muscarinic receptors. A C-sweat/M-sweat ratio was determined on a gland-by-gland basis to compensate for differences unrelated to CFTR function, such as gland size. The average ratio provides an approximately linear readout of CFTR function: the heterozygote ratio is ∼0.5 the control ratio and for CF subjects the ratio is zero. During assay development, we measured C/M ratios in 6 healthy controls, 4 CF heterozygotes, 18 CF subjects and 4 subjects with 'CFTR-related' conditions. The assay discriminated all groups clearly. It also revealed consistent differences in the C/M ratio among subjects within groups. We hypothesize that these differences reflect, at least in part, levels of CFTR expression, which are known to vary widely. When C-sweat rates become very low the C/M ratio also tended to decrease; we hypothesize that this nonlinearity reflects ductal fluid absorption. We also discovered that M-sweating potentiates the subsequent C-sweat response. We then used potentiation as a surrogate for drugs that can increase CFTR-dependent secretion. This bioassay provides an additional method for assessing CFTR function in vivo, and is well suited for within-subject tests of systemic, CFTR-directed therapeutics.

  View details for DOI 10.1371/journal.pone.0077114

  View details for Web of Science ID 000326152300015

  View details for PubMedID 24204751

  View details for PubMedCentralID PMC3811985

• CHOLINERGIC POTENTIATION OF CFTR-DEPENDENT SECRETION BY INDIVIDUAL, IDENTIFIABLE SWEAT GLANDS Wine, J., Chen, J., Modlin, S., Tran, K., Kennedy, M. H., Verma, R., Joo, N., Frisbee, E., THOMAS, E. A., Cho, H. WILEY-BLACKWELL. 2012: 299–299

  View details for Web of Science ID 000308882000304

• Defective Fluid Secretion from Submucosal Glands of Nasal Turbinates from CFTR-/- and CFTR Delta F508/Delta F508 Pigs PLOS ONE Cho, H., Joo, N. S., Wine, J. J. 2011; 6 (8)

  Abstract

  Cystic fibrosis (CF), caused by reduced CFTR function, includes severe sinonasal disease which may predispose to lung disease. Newly developed CF pigs provide models to study the onset of CF pathophysiology. We asked if glands from pig nasal turbinates have secretory responses similar to those of tracheal glands and if CF nasal glands show reduced fluid secretion.Unexpectedly, we found that nasal glands differed from tracheal glands in five ways, being smaller, more numerous (density per airway surface area), more sensitive to carbachol, more sensitive to forskolin, and nonresponsive to Substance P (a potent agonist for pig tracheal glands). Nasal gland fluid secretion from newborn piglets (12 CF and 12 controls) in response to agonists was measured using digital imaging of mucus bubbles formed under oil. Secretion rates were significantly reduced in all conditions tested. Fluid secretory rates (Controls vs. CF, in pl/min/gland) were as follows: 3 µM forskolin: 9.2±2.2 vs. 0.6±0.3; 1 µM carbachol: 143.5±35.5 vs. 52.2±10.3; 3 µM forskolin + 0.1 µM carbachol: 25.8±5.8 vs. CF 4.5±0.9. We also compared CF(ΔF508/ΔF508) with CFTR(-/-) piglets and found significantly greater forskolin-stimulated secretion rates in the ΔF508 vs. the null piglets (1.4±0.8, n = 4 vs. 0.2±0.1, n = 7). An unexpected age effect was also discovered: the ratio of secretion to 3 µM forskolin vs. 1 µM carbachol was ∼4 times greater in adult than in neonatal nasal glands.These findings reveal differences between nasal and tracheal glands, show defective fluid secretion in nasal glands of CF pigs, reveal some spared function in the ΔF508 vs. null piglets, and show unexpected age-dependent differences. Reduced nasal gland fluid secretion may predispose to sinonasal and lung infections.

  View details for DOI 10.1371/journal.pone.0024424

  View details for Web of Science ID 000294680800088

  View details for PubMedID 21935358

  View details for PubMedCentralID PMC3164206

• Properties of substance P-stimulated mucus secretion from porcine tracheal submucosal glands AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Khansaheb, M., Choi, J. Y., Joo, N. S., Yang, Y., Krouse, M., Wine, J. J. 2011; 300 (3): L370-L379

  Abstract

  Human and pig airway submucosal glands secrete mucus in response to substance P (SubP), but in pig tracheal glands the response to SubP is >10-fold greater than in humans and shares features with cholinergically produced secretion. CFTR-deficient pigs provide a model for human cystic fibrosis (CF), and in newborn CF pigs the response of tracheal glands to SubP is significantly reduced (Joo et al. J Clin Invest 120: 3161-3166, 2010). To further define features of SubP-mediated gland secretion, we optically measured secretion rates from individual adult porcine glands in isolated tracheal tissues in response to mucosal capsaicin and serosal SubP. Mucosal capsaicin (EC(50) = 19 μM) stimulated low rates of secretion that were partially inhibited by tetrodotoxin and by inhibitors for muscarinic, VIP, and SubP receptors, suggesting reflex stimulation of secretion by multiple transmitters. Secretion in response to mucosal capsaicin was inhibited by CFTR(inh)-172, but not by niflumic acid. Serosal SubP (EC(50) = 230 nM) stimulated 10-fold more secretion than mucosal capsaicin, with a V(max) similar to that of carbachol. Secretion rates peaked within 5 min and then declined to a lower sustained rate. SubP-stimulated secretion was inhibited 75% by bumetanide, 53% by removal of HCO(3)(-), and 85% by bumetanide + removal of HCO(3)(-); it was not inhibited by atropine but was inhibited by niflumic acid, clotrimazole, BAPTA-AM, nominally Ca(2+)-free bath solution, and the adenylate cyclase inhibitor MDL-12330A. Ratiometric measurements of fura 2 fluorescence in dissociated gland cells showed that SubP and carbachol increased intracellular Ca(2+) concentration by similar amounts. SubP produced rapid volume loss by serous and mucous cells, expansion of gland lumina, mucus flow, and exocytosis but little or no contraction of myoepithelial cells. These and prior results suggest that SubP stimulates pig gland secretion via CFTR- and Ca(2+)-activated Cl(-) channels.

  View details for DOI 10.1152/ajplung.00372.2010

  View details for Web of Science ID 000287915100007

  View details for PubMedID 21131402

• Measurement of fluid secretion from intact airway submucosal glands. Methods in molecular biology (Clifton, N.J.) Wine, J. J., Joo, N. S., Choi, J. Y., Cho, H., Krouse, M. E., Wu, J. V., Khansaheb, M., Irokawa, T., Ianowski, J., Hanrahan, J. W., Cuthbert, A. W., Tran, K. V. 2011; 742: 93-112

  Abstract

  Human airways are kept sterile by a mucosal innate defense system that includes mucus secretion. Mucus is secreted in healthy upper airways primarily by submucosal glands and consists of defense molecules mixed with mucins, electrolytes, and water and is also a major component of sputum. Mucus traps pathogens and mechanically removes them via mucociliary clearance while inhibiting their growth via molecular (e.g., lysozyme) and cellular (e.g., neutrophils, macrophages) defenses. Fluid secretion rates of single glands in response to various mediators can be measured by trapping the primary gland mucus secretions in an oil layer, where they form spherical bubbles that can be optically measured at any desired interval to provide detailed temporal analysis of secretion rates. The composition and properties of the mucus (e.g., solids, viscosity, pH) can also be determined. These methods have now been applied to mice, ferrets, cats, pigs, sheep, and humans, with a main goal of comparing gland secretion in control and CFTR-deficient humans and animals.

  View details for DOI 10.1007/978-1-61779-120-8_6

  View details for PubMedID 21547728

• Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis JOURNAL OF CLINICAL INVESTIGATION Sun, X., Sui, H., Fisher, J. T., Yan, Z., Liu, X., Cho, H., Joo, N. S., Zhang, Y., Zhou, W., Yi, Y., Kinyon, J. M., Lei-Butters, D. C., Griffin, M. A., Naumann, P., Luo, M., Ascher, J., Wang, K., Frana, T., Wine, J. J., Meyerholz, D. K., Engelhardt, J. F. 2010; 120 (9): 3149-3160

  Abstract

  Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments.

  View details for DOI 10.1172/JCI43052

  View details for Web of Science ID 000281458800021

  View details for PubMedID 20739752

  View details for PubMedCentralID PMC2929732

• Hyposecretion of fluid from tracheal submucosal glands of CFTR-deficient pigs JOURNAL OF CLINICAL INVESTIGATION Joo, N. S., Cho, H., Khansaheb, M., Wine, J. J. 2010; 120 (9): 3161-3166

  Abstract

  Cystic fibrosis (CF) results from mutations that disrupt CF transmembrane conductance regulator (CFTR), an anion channel found mainly in apical membranes of epithelial cells. CF leads to chronic infection of the airways with normally innocuous bacteria and fungi. Hypotheses to explain the pathophysiology of CF airways have been difficult to test because mouse models of CF do not develop human-like airway disease. The recent production of pigs lacking CFTR and pigs expressing the most common CF-causing CFTR mutant, DeltaF508, provide another model that might help clarify the pathophysiology of CF airway disease. Here, we studied individual submucosal glands from 1-day-old piglets in situ in explanted tracheas, using optical methods to monitor mucus secretion rates from multiple glands in parallel. Secretion rates from control piglets (WT and CFTR+/-) and piglets with CF-like disease (CFTR-/- and CFTR-/DeltaF508) were measured under 5 conditions: unstimulated (to determine basal secretion), stimulated with forskolin, stimulated with carbachol, stimulated with substance P, and, as a test for synergy, stimulated with forskolin and a low concentration of carbachol. Glands from piglets with CF-like disease responded qualitatively to all agonists like glands from human patients with CF, producing virtually no fluid in response to stimulation with forskolin and substantially less in response to all other agonists except carbachol. These data are a step toward determining whether gland secretory defects contribute to CF airway disease.

  View details for DOI 10.1172/JCI43466

  View details for Web of Science ID 000281458800022

  View details for PubMedID 20739758

  View details for PubMedCentralID PMC2929738

• Mucus secretion from individual submucosal glands of the ferret trachea AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Cho, H., Joo, N. S., Wine, J. J. 2010; 299 (1): L124-L136

  Abstract

  Mucus secretion from individual tracheal glands in adult ferrets was studied with time-lapse optical imaging of mucus droplets under an oil layer. Density of functional glands (determined by responses to 1 muM carbachol) was 1.5 +/- 0.3 per mm(2) (n = 6). Secretion rates (in pl.min(-1).gland(-1)) were as follows: 4.1 +/- 0.7 basal (unstimulated; n = 27, 669 glands), 338 +/- 70 to 10 microM forskolin (n = 8, 90 glands), 234 +/- 13 to 1 microM VIP (n = 6, 57 glands), 183 +/- 92 to 10 microM isoproterenol (n = 3, 33 glands), 978 +/- 145 to 1 microM carbachol (n = 11, 131 glands), and 1,348 +/- 325 to 10 muM phenylephrine (n = 7, 74 glands). The potency (EC(50), in microM) and efficacy (V(max), in pl x min(-1) x gland(-1)) were 7.6 (EC(50)) and 338 +/- 16 (V(max)) to forskolin, 1.0 (EC(50)) and 479 +/- 19 (V(max)) to VIP, 0.6 (EC(50)) and 1,817 +/- 268 (V(max)) to carbachol, and 3.7 (EC(50)) and 1,801 +/- 95 (V(max)) to phenylephrine. Although carbachol and phenylephrine were equally effective secretagogues, only carbachol caused contractions of the trachealis muscle. Synergy was demonstrated between 300 nM isoproterenol and 100 nM carbachol, which, when combined, produced a secretion rate almost fourfold greater than predicted from their additive effect. The dependence of fluid secretion on Cl(-) and HCO(3)(-) varied depending on the mode of stimulation. Secretion stimulated by VIP or forskolin was reduced by approximately 60% by blocking either anion, while carbachol-stimulated secretion was blocked 68% by bumetanide and only 32% by HEPES replacement of HCO(3)(-). These results provide parametric data for comparison with fluid secretion from glands in ferrets lacking CFTR.

  View details for DOI 10.1152/ajplung.00049.2010

  View details for Web of Science ID 000281388900014

  View details for PubMedID 20435689

  View details for PubMedCentralID PMC2904090

• The Development of Lung Disease in Cystic Fibrosis Pigs SCIENCE TRANSLATIONAL MEDICINE Wine, J. J. 2010; 2 (29)

  Abstract

  The leading cause of death in cystic fibrosis (CF) patients is lung disease; yet despite enormous research efforts, its pathogenesis is not well understood. Because CF mice do not develop human-like airway disease, CF pigs were produced on the premise that they would offer insights into the pathogenesis of CF lung disease. But CF pigs develop severe intestinal blockage and often die shortly after birth. Now, a team of scientists report results from five CF pigs that lived for ~2 to 6 months; during this time, they developed some of the key features of human CF lung disease and revealed that infection precedes inflammation.

  View details for DOI 10.1126/scitranslmed.3001130

  View details for Web of Science ID 000277304700004

  View details for PubMedID 20427819

• Lubiprostone stimulates secretion from tracheal submucosal glands of sheep, pigs, and humans AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Joo, N. S., Wine, J. J., Cuthbert, A. W. 2009; 296 (5): L811-L824

  Abstract

  Lubiprostone, a putative ClC-2 chloride channel opener, has been investigated for its effects on airway epithelia (tracheas). Lubiprostone is shown to increase submucosal gland secretion in pigs, sheep, and humans and to increase short-circuit current (SCC) in the surface epithelium of pigs and sheep. Use of appropriate blocking agents and ion-substitution experiments shows anion secretion is the driving force for fluid formation in both glands and surface epithelium. From SCC concentration-response relations, it is shown that for apical lubiprostone K(d) = 10.5 nM with a Hill slope of 1.08, suggesting a single type of binding site and, from the speed of the response, close to the apical surface, confirmed the rapid blockade by Cd ions. Responses to lubiprostone were reversible and repeatable, responses being significantly larger with ventral compared with dorsal epithelium. Submucosal gland secretion rates following basolateral lubiprostone were, respectively, 0.2, 0.5, and 0.8 nl gl(-1) min(-1) in humans, sheep, and pigs. These rates dwarf any contribution surface secretion adds to the accumulation of surface liquid under the influence of lubiprostone. Lubiprostone stimulated gland secretion in two out of four human cystic fibrosis (CF) tissues and in two of three disease controls, chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis (COPD/IPF), but in neither type of tissue was the increase significant. Lubiprostone was able to increase gland secretion rates in normal human tissue in the continuing presence of a high forskolin concentration. Lubiprostone had no spasmogenic activity on trachealis muscle, making it a potential agent for increasing airway secretion that may have therapeutic utility.

  View details for DOI 10.1152/ajplung.90636.2008

  View details for Web of Science ID 000265581300011

  View details for PubMedID 19233902

• MUCUS SECRETION FROM INDIVIDUAL FERRET SUBMUCOSAL GLANDS Cho, H., Joo, N., Wu, J., Krouse, M. E., Wine, J. J. WILEY-BLACKWELL. 2009: 241–241

  View details for Web of Science ID 000270703400167

• MUCUS HYPOSECRETION FROM SUBMUCOSAL GLANDS FROM NASAL TURBINATE OF CF PIGLETS Cho, H., Joo, N., Khansaheb, M., Karp, P. H., Stoltz, D. A., Meyerholz, D. K., Welsh, M. J., Wine, J. J. WILEY-BLACKWELL. 2009: 242–242

  View details for Web of Science ID 000270703400168

• HYPOSECRETION OF MUCUS FROM AIRWAY SUBMUCOSAL GLANDS OF CF PIGLETS Joo, N., Cho, H., Khansaheb, M., Karp, P. H., Stoltz, D. A., Meyerholz, D. K., Welsh, M. J., Wine, J. J. WILEY-BLACKWELL. 2009: 236–236

  View details for Web of Science ID 000270703400152

• Substance P stimulates CFTR-dependent fluid secretion by mouse tracheal submucosal glands PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY Ianowski, J. P., Choi, J. Y., Wine, J. J., Hanrahan, J. W. 2008; 457 (2): 529-537

  Abstract

  The mucosa of the proximal airways defends itself and the lower airways from inhaled irritants such as capsaicinoids, allergens, and infections by several mechanisms. Sensory nerves monitor the luminal microenvironment and release the tachykinin substance P (SP) to stimulate mucus secretion. Here, we have studied the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in SP stimulation by comparing mouse airway submucosal gland responses in wild-type (WT) and CFTR-/- mice. Capsaicinoids (chili pepper oil) increased fluid secretion by glands from WT mice five-fold, and this response was abolished by exposing the basolateral aspect of the tracheas to L-732,138 (10 micromol/l), a specific antagonist of the neurokinin-1 receptor. Secretion was also stimulated 25-fold by basolateral application of SP, and this response was strongly inhibited by the CFTR inhibitor CFTR(inh)172. In contrast, submucosal glands from CFTR knockout mice failed to secrete when stimulated by SP (1 micromol/l), although those from wild-type control littermates were responsive. SP stimulation of wild-type glands was also abolished by clotrimazole (25 micromol/l), a blocker of Ca(2+)-activated K(+) channels. These results indicate that SP mediates local responses to capsaicinoids through a mechanism involving coordinated activation of CFTR and K(+) channels. To our knowledge, this is the first study in which CFTR-dependent responses to substance P have been directly demonstrated. Since CFTR regulation is qualitatively similar in human and mouse glands, loss of this local regulation in CF may contribute to reduced innate defenses in CF airways.

  View details for DOI 10.1007/s00424-008-0527-0

  View details for Web of Science ID 000262414200024

  View details for PubMedID 18509672

• The inexhaustible mouse nose. Focus on "Olfactory epithelia exhibit progressive functional and morphological defects in CF mice" AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Wine, J. J. 2007; 293 (2): C537-C539

  View details for DOI 10.1152/ajpcell.00183.2007

  View details for Web of Science ID 000248581000003

  View details for PubMedID 17507433

• Parasympathetic control of airway submucosal glands: Central reflexes and the airway intrinsic nervous system AUTONOMIC NEUROSCIENCE-BASIC & CLINICAL Wine, J. J. 2007; 133 (1): 35-54

  Abstract

  Airway submucosal glands produce the mucus that lines the upper airways to protect them against insults. This review summarizes evidence for two forms of gland secretion, and hypothesizes that each is mediated by different but partially overlapping neural pathways. Airway innate defense comprises low level gland secretion, mucociliary clearance and surveillance by airway-resident phagocytes to keep the airways sterile in spite of nearly continuous inhalation of low levels of pathogens. Gland secretion serving innate defense is hypothesized to be under the control of intrinsic (peripheral) airway neurons and local reflexes, and these may depend disproportionately on non-cholinergic mechanisms, with most secretion being produced by VIP and tachykinins. In the genetic disease cystic fibrosis, airway glands no longer secrete in response to VIP alone and fail to show the synergy between VIP, tachykinins and ACh that is observed in normal glands. The consequent crippling of the submucosal gland contribution to innate defense may be one reason that cystic fibrosis airways are infected by mucus-resident bacteria and fungi that are routinely cleared from normal airways. By contrast, the acute (emergency) airway defense reflex is centrally mediated by vagal pathways, is primarily cholinergic, and stimulates copious volumes of gland mucus in response to acute, intense challenges to the airways, such as those produced by very vigorous exercise or aspiration of foreign material. In cystic fibrosis, the acute airway defense reflex can still stimulate the glands to secrete large amounts of mucus, although its properties are altered. Importantly, treatments that recruit components of the acute reflex, such as inhalation of hypertonic saline, are beneficial in treating cystic fibrosis airway disease. The situation for recipients of lung transplants is the reverse; transplanted airways retain the airway intrinsic nervous system but lose centrally mediated reflexes. The consequences of this for gland secretion and airway defense are poorly understood, but it is possible that interventions to modify submucosal gland secretion in transplanted lungs might have therapeutic consequences.

  View details for DOI 10.1016/j.autneu.2007.01.008

  View details for Web of Science ID 000246056400004

  View details for PubMedID 17350348

  View details for PubMedCentralID PMC1989147

• Mucus secretion by single tracheal submucosal glands from normal and cystic fibrosis transmembrane conductance regulator knockout mice JOURNAL OF PHYSIOLOGY-LONDON Ianowski, J. P., Choi, J. Y., Wine, J. J., Hanrahan, J. W. 2007; 580 (1): 301-314

  Abstract

  Submucosal glands line the cartilaginous airways and produce most of the antimicrobial mucus that keeps the airways sterile. The glands are defective in cystic fibrosis (CF), but how this impacts airway health remains uncertain. Although most CF mouse strains exhibit mild airway defects, those with the C57Bl/6 genetic background have increased airway pathology and susceptibility to Pseudomonas. Thus, they offer the possibility of studying whether, and if so how, abnormal submucosal gland function contributes to CF airway disease. We used optical methods to study fluid secretion by individual glands in tracheas from normal, wild-type (WT) mice and from cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice (Cftr(m1UNC)/Cftr(m1UNC); CF mice). Glands from WT mice qualitatively resembled those in humans by responding to carbachol and vasoactive intestinal peptide (VIP), although the relative rates of VIP- and forskolin-stimulated secretion were much lower in mice than in large mammals. The pharmacology of mouse gland secretion was also similar to that in humans; adding bumetanide or replacement of HCO(3)(-) by Hepes reduced the carbachol response by approximately 50%, and this inhibition increased to 80% when both manoeuvres were performed simultaneously. It is important to note that glands from CFTR knockout mice responded to carbachol but did not secrete when exposed to VIP or forskolin, as has been shown previously for glands from CF patients. Tracheal glands from WT and CF mice both had robust secretory responses to electrical field stimulation that were blocked by tetrodotoxin. It is interesting that local irritation of the mucosa using chili pepper oil elicited secretion from WT glands but did not stimulate glands from CF mice. These results clarify the mechanisms of murine submucosal gland secretion and reveal a novel defect in local regulation of glands lacking CFTR which may also compromise airway defence in CF patients.

  View details for DOI 10.1113/jphysiol.2006.123653

  View details for Web of Science ID 000245646800027

  View details for PubMedID 17204498

• Capsaicin induces CFTR-dependent mucus secretion via local reflexes in airway submucosal gland Choi, J., Joo, N., Krouse, M., Wine, J. J. WILEY-BLACKWELL. 2007: 239–239

  View details for Web of Science ID 000250105000188

• Flagellin activates P38, PI3 kinase, NF-kappa B and CFTR-dependent CL secretion in airway gland epithelial cells Machen, T., Illek, B., Fu, Z., Lerner, S., Choi, J., Wine, J. J. WILEY-BLACKWELL. 2007: 234–234

  View details for Web of Science ID 000250105000173

• Substance P induces mucus secretion via CFTR-dependent mechanism in pig airway gland Choi, J., Joo, N., Ianowski, J., Hanrahan, J. W., Wine, J. J. WILEY-BLACKWELL. 2007: 239–240

  View details for Web of Science ID 000250105000189

• Cholinergic stimulation: A novel pathway regulating airway surface ENaC activity Joo, N., Krouse, M. E., Choi, J., Wine, J. J. WILEY-BLACKWELL. 2007: 240–241

  View details for Web of Science ID 000250105000192

• Substance P stimulates CFTR-dependent fluid secretion by mouse tracheal submucosal glands Ianowski, J. P., Choi, J., Wine, J. J., Hanrahan, J. WILEY-BLACKWELL. 2007: 259–259

  View details for Web of Science ID 000250105000243

• Acinar origin of CFTR-dependent airway submucosal gland fluid secretion AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Wu, J. V., Krouse, M. E., Wine, J. J. 2007; 292 (1): L304-L311

  Abstract

  Cystic fibrosis (CF) airway disease arises from defective innate defenses, especially defective mucus clearance of microorganisms. Airway submucosal glands secrete most airway mucus, and CF airway glands do not secrete in response to VIP or forskolin. CFTR, the protein that is defective in CF, is expressed in glands, but immunocytochemistry finds the highest expression of CFTR in either the ciliated ducts or in the acini, depending on the antibodies used. CFTR is absolutely required for forskolin-mediated gland secretion; we used this finding to localize the origin of forskolin-stimulated, CFTR-dependent gland fluid secretion. We tested the hypothesis that secretion to forskolin might originate from the gland duct rather than or in addition to the acini. We ligated gland ducts at various points, stimulated the glands with forskolin, and monitored the regions of the glands that swelled. The results supported an acinar rather than ductal origin of secretion. We tracked particles in the mucus using Nomarski time-lapse imaging; particles originated in the acini and traveled toward the duct orifice. Estimated bulk flow accelerated in the acini and mucus tubules, consistent with fluid secretion in those regions, but was constant in the unbranched duct, consistent with a lack of fluid secretion or absorption by the ductal epithelium. We conclude that CFTR-dependent gland fluid secretion originates in the serous acini. The failure to observe either secretion or absorption from the CFTR and epithelial Na(+) channel (ENaC)-rich ciliated ducts is unexplained, but may indicate that this epithelium alters the composition rather than the volume of gland mucus.

  View details for DOI 10.1152/ajplung.00286.2006

  View details for Web of Science ID 000243399900037

  View details for PubMedID 16997881

• Hyposecretion, not hyperabsorption, is the basic defect of cystic fibrosis airway glands JOURNAL OF BIOLOGICAL CHEMISTRY Joo, N. S., Irokawa, T., Robbins, R. C., Wine, J. J. 2006; 281 (11): 7392-7398

  Abstract

  Human airways and glands express the anion channel cystic fibrosis transmembrane conductance regulator, CFTR, and the epithelial Na(+) channel, ENaC. Cystic fibrosis (CF) airway glands fail to secrete mucus in response to vasoactive intestinal peptide or forskolin; the failure was attributed to loss of CFTR-mediated anion and fluid secretion. Alternatively, CF glands might secrete acinar fluid via CFTR-independent pathways, but the exit of mucus from the glands could be blocked by hyperabsorption of fluid in the gland ducts. This could occur because CFTR loss can disinhibit ENaC, and ENaC activity can drive absorption. To test these two hypotheses, we measured single gland mucus secretion optically and applied ENaC inhibitors to determine whether they augmented secretion. Human CF glands were pretreated with benzamil and then stimulated with forskolin in the continued presence of benzamil. Benzamil did not rescue the lack of secretion to forskolin (50 glands, 6 CF subjects) nor did it increase the rate of cholinergically mediated mucus secretion from CF glands. Finally, neither benzamil nor amiloride increased forskolin-stimulated mucus secretion from porcine submucosal glands (75 glands, 7 pigs). One possible explanation for these results is that ENaC within the gland ducts was not active in our experiments. Consistent with that possibility, we discovered that human airway glands express Kunitz-type and non-Kunitz serine protease inhibitors, which might prevent proteolytic activation of ENaC. Our results suggest that CF glands do not display excessive, ENaC-mediated fluid absorption, leaving defective, anion-mediated fluid secretion as the most likely mechanism for defective mucus secretion from CF glands.

  View details for DOI 10.1074/jbc.M512766200

  View details for Web of Science ID 000236030900059

  View details for PubMedID 16410244

• Acid in the airways. Focus on "Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis" AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Wine, J. J. 2006; 290 (3): C669-C671

  View details for DOI 10.1152/ajpcell.00525.2005

  View details for Web of Science ID 000235232700002

  View details for PubMedID 16469862

• Acid and base secretion in the Calu-3 model of human serous cells AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Krouse, M. E., Talbott, J. F., Lee, M. M., Joo, N. S., Wine, J. J. 2004; 287 (6): L1274-L1283

  Abstract

  Submucosal glands are the primary source of airway mucus, a critical component of lung innate defenses. Airway glands are defective in cystic fibrosis (CF), showing a complete absence of secretion to vasoactive intestinal peptide or forskolin, which increase intracellular cAMP concentration. This defect is attributed to gland serous cells, which express the cystic fibrosis transmembrane conductance regulator. Calu-3 cells, which mimic many features of serous cells, secrete Cl(-) and HCO(3)(-), with HCO(3)(-) secretion predominating for forskolin stimulation and Cl(-) secretion predominating for stimuli that open basolateral K(+) channels to hyperpolarize the cells. We used pH stat and ion substitution experiments to clarify the mechanisms and consequences of these two modes of secretion. We confirm that Calu-3 cells secrete primarily HCO(3)(-) in response to forskolin. Unexpectedly, HCO(3)(-) secretion continued in response to K(+) channel openers, with Cl(-) secretion being added to it. Secretion of HCO(3)(-) from hyperpolarized cells occurs via the conversion of CO(2) to HCO(3)(-) and is reduced by approximately 50% with acetazolamide. A gap between the base equivalent current and short-circuit current was observed in all experiments and was traced to secretion of H(+) via a ouabain-sensitive, K(+)-dependent process (possibly H(+)-K(+)-ATPase), which partially neutralized the secreted HCO(3)(-). The conjoint secretion of HCO(3)(-) and H(+) may help explain the puzzling finding that mucus secreted from normal and CF glands has the same acidic pH as does mucus from glands stimulated with forskolin or ACh. It may also help explain how human airway glands produce mucus that is hypotonic.

  View details for DOI 10.1152/ajplung.00036.2004

  View details for Web of Science ID 000224956900027

  View details for PubMedID 15310554

• An inwardly rectifying potassium channel in apical membrane of Calu-3 cells JOURNAL OF BIOLOGICAL CHEMISTRY Wu, J. V., Krouse, M. E., Rustagi, A., Joo, N. S., Wine, J. J. 2004; 279 (45): 46558-46565

  Abstract

  Patch clamp methods and reverse transcription-polymerase chain reaction (RT-PCR) were used to characterize an apical K+ channel in Calu-3 cells, a widely used model of human airway gland serous cells. In cell-attached and excised apical membrane patches, we found an inwardly rectifying K+ channel (Kir). The permeability ratio was PNa/PK = 0.058. In 30 patches with both cystic fibrosis transmembrane conductance regulator and Kir present, we observed 79 cystic fibrosis transmembrane conductance regulator and 58 Kir channels. The average chord conductance was 24.4 +/- 0.5 pS (n = 11), between 0 and -200 mV, and was 9.6 +/- 0.7 pS (n = 8), between 0 and 50 mV; these magnitudes and their ratio of approximately 2.5 are most similar to values for rectifying K+ channels of the Kir4.x subfamilies. We attempted to amplify transcripts for Kir4.1, Kir4.2, and Kir5.1; of these only Kir4.2 was present in Calu-3 lysates. The channel was only weakly activated by ATP and was relatively insensitive to internal pH. External Cs+ and Ba2+ blocked the channel with Kd values in the millimolar range. Quantitative modeling of Cl- secreting epithelia suggests that secretion rates will be highest and luminal K+ will rise to 16-28 mm if 11-25% of the total cellular K+ conductance is placed in the apical membrane (Cook, D. I., and Young, J. A. (1989) J. Membr. Biol. 110, 139-146). Thus, we hypothesize that the K+ channel described here optimizes the rate of secretion and is involved in K+ recycling for the recently proposed apical H+ -K+ -ATPase in Calu-3 cells.

  View details for DOI 10.1074/jbc.M406058200

  View details for Web of Science ID 000224832400028

  View details for PubMedID 15328350

• A "virtual gland" method for quantifying epithelial fluid secretion AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Irokawa, T., Krouse, M. E., Joo, N. S., Wu, J. V., Wine, J. J. 2004; 287 (4): L784-L793

  Abstract

  We developed a new apparatus, the virtual gland (VG), for measuring the rate of fluid secretion (Jv), its composition, and the transepithelial potential (TEP) in cultured epithelial cells under open circuit. The VG creates a 10-microl chamber above the apical surface of epithelial cells on a Costar filter with a small hole leading to an oil-filled reservoir. After the chamber is primed with a fluid of choice, secreted fluid is forced through the hole into the oil, where it forms a bubble that is monitored optically to determine Jv and collected for analysis. Calu-3 cells were mounted in the VG with a basolateral bath consisting of Krebs-Ringer bicarbonate buffer at 37 degrees C. Basal Jv was 2.7 +/- 0.1 microl x cm(-2) x h(-1) (n = 42), and TEP was -9.2 +/- 0.6 mV (n = 33); both measures were reduced to zero by ouabain (n = 6) x Jv and TEP were stimulated 64 and 59%, respectively, by 5 microM forskolin (n = 10), 173 and 101% by 1 mM 1-ethyl-2-benzimidazolinone (n = 5), 213 and 122% by 333 nM thapsigargin (n = 5), and 520 and 240% by forskolin + thapsigargin (n = 6). Basal Jv and TEP were inhibited to 82 and 63%, respectively, with 10 microM bumetanide (n = 5), 71 and 82% with 100 microM acetazolamide (n = 5), and 47 and 56% with 600 microM glibenclamide (n = 4). Basal Jv and TEP were 52 and 89% of control values, respectively, after HCO3- replacement with HEPES (n = 16). The net HCO3- concentration of the secreted fluid was close to that of the bath (25 mM), except when stimulated with forskolin or VIP, when it increased (approximately 80 mM). These results validate the use of the VG apparatus and provide the first direct measures of Jv in Calu-3 cells.

  View details for DOI 10.1152/ajplung.00124.2004

  View details for Web of Science ID 000223762200019

  View details for PubMedID 15169677

• Regulation of antiprotease and antimicrobial protein secretion by airway submucosal gland serous cells JOURNAL OF BIOLOGICAL CHEMISTRY Joo, N. S., Lee, D. J., Winges, K. M., Rustagi, A., Wine, J. J. 2004; 279 (37): 38854-38860

  Abstract

  Airway submucosal gland serous cells express the cystic fibrosis transmembrane conductance regulator (CFTR) and secrete antimicrobial, anti-inflammatory, and antioxidant molecules. In cystic fibrosis, diminished gland secretion may impair innate airway host defenses. We used Calu-3 cells as a serous cell model to study the types of proteins released, the pathways that release them, and the possible involvement of CFTR activity in protein release. Many proteins were secreted constitutively into the apical fluid and showed increased release to agonists. We identified some of them by high pressure liquid chromatography-mass spectrometry and reverse transcriptase PCR, including lysozyme, siderocalin (the protein NGAL), which inhibits bacterial growth by binding iron-containing siderophores, HSC-71, which is thought to have anti-inflammatory properties, and the serine protease inhibitors alpha-1-antitrypsin and alpha-1-antichymotrypsin, which may function as antimicrobials as well as play a potential role in diminishing the activation of epithelial Na(+) channels by serine proteases. We used an enzyme-linked immunosorbent assay to quantify lysozyme secretion by Calu-3 cells in response to various agonists and inhibitors. Forskolin increased the lysozyme secretion rate (J(lyz)) from 32 to 77 ng/hr/cm(2) (n = 36, p < 0.005). Thapsigargin increased J(lyz) from 40 to 63 ng/h/cm(2) (n = 16, p < 0.005), and forskolin plus thapsigargin further increased the forskolin-stimulated J(lyz) by 48% (n = 9, p < 0.05). 1-Ethyl-benzimidazolinone and carbachol were less effective. Glibenclamide inhibited basal and stimulated J(lyz), but clotrimazole was without effect. CFTR(inh)172 caused a small (15%) but significant inhibition of forskolin-stimulated J(lyz) without affecting basal J(lyz). Thus, Calu-3 cells secrete diverse proteins that in aggregate would be expected to suppress microbial growth, protect the airways from damage, and limit the activation of epithelial Na(+) channels via serine proteases.

  View details for DOI 10.1074/jbc.M407077200

  View details for Web of Science ID 000223684100098

  View details for PubMedID 15234967

• Submucosal glands and airway defense. Proceedings of the American Thoracic Society Wine, J. J., Joo, N. S. 2004; 1 (1): 47-53

  Abstract

  Most airway mucus is produced by submucosal glands in response to neural signals. Gland mucus traps microbes, inhibits their replication, and clears them from the airways. In cystic fibrosis mucus clearance is compromised, allowing pathogens to persist in static mucus. These trigger an influx of inflammatory cells, but optimal effectiveness of inflammation, and especially its resolution, also requires effective mucus clearance. Our objective is to understand the basis for defective mucus clearance in cystic fibrosis. We discovered that in subjects with cystic fibrosis, submucosal gland secretion in response to agents that elevate intracellular cyclic AMP level is completely lost and mucus stimulated by elevating intracellular Ca2+ level is thicker. We hypothesize that loss of functional cystic fibrosis transmembrane conductance regulator from gland serous cells renders them unable to secrete anions and fluid in response to any stimulus, resulting in thickened gland mucus that can be tethered to the gland ducts. In primary ciliary dyskinesias, mucus is normal, but the dysfunctional cilia lining the gland ducts may also lead to inadequate clearance of mucus from glands. Thus, understanding of lung pathology in each disease may require that an improved understanding of gland structure and function be added to our rapidly growing understanding of surface epithelia.

  View details for PubMedID 16113412

• Absent secretion to vasoactive intestinal peptide in cystic fibrosis airway glands JOURNAL OF BIOLOGICAL CHEMISTRY Joo, N. S., Irokawa, T., Wu, J. V., Robbins, R. C., Whyte, R. I., Wine, J. J. 2002; 277 (52): 50710-50715

  Abstract

  We are testing the hypothesis that the malfunctioning of airway gland serous cells is a component of cystic fibrosis (CF) airway disease. CF is caused by mutations that disrupt CF transmembrane conductance regulator, an anion channel essential for proper fluid secretion in some epithelia. Submucosal glands supply most of the mucus in upper airways, and gland serous cells are the primary site of CF transmembrane conductance regulator expression in airways. We have discovered a major defect in CF glands by in situ optical monitoring of secretions from single human airway glands. CF glands did not secrete to agents that elevated [cAMP](i) (0 responses/450 glands, 8 subjects), whereas glands were responsive in all donor tracheas (605/827 glands, 15 subjects) and in bronchi from subjects who were transplanted because of other lung diseases (148/166 glands, n = 10). CF glands secreted to cholinergic stimulation, and serous cells were abundant in glands from all CF subjects. The complete absence of secretion to agents that elevate [cAMP](i) suggests that altered secretion of gland mucus could contribute to CF lung disease.

  View details for DOI 10.1074/jbc.M208826200

  View details for Web of Science ID 000180177700068

  View details for PubMedID 12368280

• Mucus secretion from single submucosal glands of pig - Stimulation by carbachol and vasoactive intestinal peptide JOURNAL OF BIOLOGICAL CHEMISTRY Joo, N. S., Saenz, Y., Krouse, M. E., Wine, J. J. 2002; 277 (31): 28167-28175

  Abstract

  Secretion rates of >700 individual glands in isolated tracheal mucosa from 56 adult pigs were monitored optically. "Basal" secretion of 0.7 +/- 0.1 nl x min(-1) gland(-1) was observed 1-9 h post-harvest but was near zero on day 2. Secretion to carbachol (10 microm) peaked at 2-3 min and then declined to a sustained phase. Peak secretion was 12.4 +/- 1.1 nl x min(-1) gland(-1); sustained secretion was approximately one-third of peak secretion. Thapsigargin (1 microm) increased secretion from 0.1 +/- 0.05 to 0.7 +/- 0.2 nl x min(-1) gland(-1); thapsigargin did not cause contraction of the trachealis muscles. Isoproterenol and phenylephrine (10 microm each) were ineffective, but vasoactive intestinal peptide (1 microm) and forskolin (10 microm) each produced sustained secretion of 1.0 +/- 0.5 and 1.7 +/- 0.2 nl x min(-1) gland(-1), respectively. The density of actively secreting glands was 1.3/mm(2). Secretion to either carbachol or forskolin was inhibited (approximately 50%) by either bumetanide or HCO(3)(-) removal and inhibited approximately 90% by the combined treatments. Mucus secreted in response to carbachol or forskolin was acidic by approximately 0.2 pH units relative to the bath and remained acidic by approximately 0.1 pH units after bumetanide. The strong secretory response to vasoactive intestinal peptide, the acidity of [cAMP](i)-stimulated mucus, and its inhibition by bumetanide were unexpected.

  View details for DOI 10.1074/jbc.M202712200

  View details for Web of Science ID 000177189800074

  View details for PubMedID 12011087

• A phase II, double-blind, randomized, placebo-controlled clinical trial of tgAAVCF using maxillary sinus delivery in patients with cystic fibrosis with antrostomies HUMAN GENE THERAPY Wagner, J. A., Nepomuceno, I. B., Messner, A. H., Moran, M. L., Batson, E. P., DiMiceli, S., Brown, B. W., Desch, J. K., Norbash, A. M., Conrad, C. K., Guggino, W. B., Flotte, T. R., Wine, J. J., Carter, B. J., Reynolds, T. C., Moss, R. B., Gardner, P. 2002; 13 (11): 1349-1359

  Abstract

  tgAAVCF, an adeno-associated cystic fibrosis transmembrane conductance regulator (CFTR) viral vector/gene construct, was administered to 23 patients in a Phase II, double-blind, randomized, placebo-controlled clinical trial. For each patient, a dose of 100,000 replication units of tgAAVCF was administered to one maxillary sinus, while the contralateral maxillary sinus received a placebo treatment, thereby establishing an inpatient control. Neither the primary efficacy endpoint, defined as the rate of relapse of clinically defined, endoscopically diagnosed recurrent sinusitis, nor several secondary endpoints (sinus transepithelial potential difference [TEPD], histopathology, sinus fluid interleukin [IL]-8 measurements) achieved statistical significance when comparing treated to control sinuses within patients. One secondary endpoint, measurements of the anti-inflammatory cytokine IL-10 in sinus fluid, was significantly (p < 0.03) increased in the tgAAVCF-treated sinus relative to the placebo-treated sinus at day 90 after vector instillation. The tgAAVCF administration was well tolerated, without adverse respiratory events, and there was no evidence of enhanced inflammation in sinus histopathology or alterations in serum-neutralizing antibody titer to adeno-associated virus (AAV) capsid protein after vector administration. In summary, this Phase II trial confirms the safety of tgAAVCF but provides little support of its efficacy in the within-patient controlled sinus study. Various potentially confounding factors are discussed.

  View details for Web of Science ID 000177015400009

  View details for PubMedID 12162817

• Natural animal models of human genetic diseases. Methods in molecular medicine Wine, J. J., Dean, M., Glavac, D. 2002; 70: 31-46

  View details for PubMedID 11917533

• Evidence that CFTR channels can regulate the open duration of other CFTR channels: Cooperativity JOURNAL OF MEMBRANE BIOLOGY Krouse, M. E., Wine, J. J. 2001; 182 (3): 223-232

  Abstract

  CFTR channels mediate secretion and absorption in epithelia, and cystic fibrosis is caused by their malfunction. CFTR proteins are members of the ABC transporter family and are complexly regulated by phosphorylation and nucleosides; they also influence other channel activity. Do CFTR molecules also influence one another? Cooperativity has been observed among other channels and has been suggested for CFTR. Therefore, we looked for evidence of cooperativity among CFTR channels using three independent approaches. All three methods provided evidence for cooperativity in CFTR gating. We estimated mean open times, independent of the number of channels in the patch, in multi-channel patches and showed that, on average, they increased as channel number increased. We observed many trials having larger than expected variances, consistent with cooperative gating. We also measured deviations from binomial statistics, which revealed cooperativity and further indicated that its magnitude is underestimated to an unknown extent because of masking that occurs when CFTR channel populations within a single patch have heterogeneous open probabilities. Simulations showed that the observed departures from binomial statistics were too large to have arisen by chance. The evidence that CFTR P(o) increases with channel density has important functional implications.

  View details for Web of Science ID 000170542300007

  View details for PubMedID 11547345

• Optical method for quantifying rates of mucus secretion from single submucosal glands AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Joo, N. S., Wu, J. V., Krouse, M. E., Saenz, Y., Wine, J. J. 2001; 281 (2): L458-L468

  Abstract

  We describe an optical method to quantify single- gland secretion. Isolated tracheal mucosa were mounted at the air-Krebs interface and coated with oil. Gland secretions formed spherical bubbles that were digitally imaged at intervals, allowing rates of secretion to be calculated. We monitored 340 glands in 54 experiments with 12 sheep. Glands secreted basally at low rates (0.57 +/- 0.04 nl x min(-1) x gland(-1), 123 glands) in tissues up to 9 h postharvest and at lower rates for up to 3 days. Carbachol (10 microM) stimulated secretion with an early transient and a sustained or oscillating phase. Peak secretion was 15.7 +/- 1.2 nl x min(-1) x gland(-1) (60 glands); sustained secretion was 4.5 +/- 0.5 nl x min(-1) x gland(-1) (10 glands). Isoproterenol and phenylephrine (10 microM each) stimulated only small, transient responses. We confirmed that cats have a large secretory response to phenylephrine (11.6 +/- 3.7 nl x min(-1) x gland(-1), 12 glands), but pigs, sheep, and humans all have small responses (<2 nl x min(-1)m x gland(-1)). Carbachol-stimulated peak secretion was inhibited 56% by bumetanide, 67% by HCO replacement with HEPES, and 92% by both. The distribution of secretion rates was nonnormal, suggesting the existence of subpopulations of glands.

  View details for Web of Science ID 000169637600021

  View details for PubMedID 11435221

• Submucosal gland secretions in airways from cystic fibrosis patients have normal [Na+] and pH but elevated viscosity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jayaraman, S., Joo, N. S., Reitz, B., Wine, J. J., Verkman, A. S. 2001; 98 (14): 8119-8123

  Abstract

  Fluid and macromolecule secretion by submucosal glands in mammalian airways is believed to be important in normal airway physiology and in the pathophysiology of cystic fibrosis (CF). An in situ fluorescence method was applied to measure the ionic composition and viscosity of freshly secreted fluid from airway glands. Fragments of human large airways obtained at the time of lung transplantation were mounted in a humidified perfusion chamber and the mucosal surface was covered by a thin layer of oil. Individual droplets of secreted fluid were microinjected with fluorescent indicators for measurement of [Na(+)], [Cl(-)], and pH by ratio imaging fluorescence microscopy and viscosity by fluorescence recovery after photobleaching. After carbachol stimulation, 0.1--0.5 microl of fluid accumulated in spherical droplets at gland orifices in approximately 3--5 min. In gland fluid from normal human airways, [Na(+)] was 94 +/- 8 mM, [Cl(-)] was 92 +/- 12 mM, and pH was 6.97 +/- 0.06 (SE, n = 7 humans, more than five glands studied per sample). Apparent fluid viscosity was 2.7 +/- 0.3-fold greater than that of saline. Neither [Na(+)] nor pH differed in gland fluid from CF airways, but viscosity was significantly elevated by approximately 2-fold compared to normal airways. These results represent the first direct measurements of ionic composition and viscosity in uncontaminated human gland secretions and indicate similar [Na(+)], [Cl(-)], and pH to that in the airway surface liquid. The elevated gland fluid viscosity in CF may be an important factor promoting bacterial colonization and airway disease.

  View details for Web of Science ID 000169744200085

  View details for PubMedID 11427704

  View details for PubMedCentralID PMC35477

• HCO3- transport in relation to mucus secretion from submucosal glands. JOP : Journal of the pancreas Joo, N. S., Krouse, M. E., Wu, J. V., Saenz, Y., Jayaraman, S., Verkman, A. S., Wine, J. J. 2001; 2 (4): 280-284

  Abstract

  The role of HCO(3)(-) transport in relation to fluid secretion by submucosal glands is being studied in sheep, pigs, cats and humans. Optical methods have been developed to measure secretion rates of mucus volume from single glands with sufficient temporal resolution to detect differences in minute-by-minute secretion rates among glands. The ionic composition and viscoelastic properties of the uncontaminated gland mucus are measured with a combination of ratiometric fluorescent indicators, ion-selective microelectrodes, FRAP, and a miniaturized, magnetic force viscometer. Sheep glands secreted basally at low rates, showed small, transient responses to alpha- and beta-adrenergic agonists, and large responses to a cholinergic agonist, carbachol. Peak rates and temporal patterns of responses to carbachol differed markedly among glands. To assess the contribution of HCO(3)(-) transport to gland secretion, we either inhibited Na(+)/K(+)/2Cl(-) cotransporter (NKCC) with bumetanide or replaced HCO(3)(-) with HEPES and gassed with O(2). Bumetanide caused a small, non-significant inhibition of basal secretion, but removal of HCO(3)(-)/CO(2) significantly reduced basal secretion almost by half. Both bumetanide and removal of HCO(3)(-)/CO(2) reduced carbachol-stimulated secretion significantly, with HCO(3)(-) removal having the larger effect: a reduction to 33% of control (P<0.01). The remaining secretory response to carbachol was nearly eliminated by bumetanide. Sheep mucus pH measured with ion selective electrodes was about 0.4 log more acidic than the bath. In humans, we observed the same pattern of responses to agonists and antagonists as in sheep, and observed a mucus pH of 7.0 using 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). We hypothesize that HCO(3)(-) transport is important in the formation of mucus secretion, but that most HCO(3)(-) is scavenged before the final mucus appears at the duct opening. Cystic fibrosis transmembrane conductance regulator's (CFTR) best understood function is as an anion channel, but increasing attention has been given to its role in HCO(3)(-) transport. By analogy with organ-specific CFTR effects on Cl(-) transport, it seems likely that the relative importance of CFTR in HCO(3)(-) transport will also vary across organs. Because lung disease is by far the greatest cause of mortality among people with cystic fibrosis, it is important to determine how loss of CFTR function causes lung disease. We are testing the hypothesis that loss of CFTR alters serous cell secretion in the lungs, and the corollary that such loss contributes to cystic fibrosis (CF) lung disease. CFTR is highly expressed in serous cells of submucosal glands and the Calu-3 serous cell model secretes HCO(3)(-). Human gland serous cells grown in culture and tested for fluid secretion under open circuit conditions showed reduced fluid secretion to all mediators. However, submucosal glands are complex organs containing at least 4 distinct regions and at least that many cell types, making it difficult to predict the consequences on whole-organ function from experiments with individual cell types. Therefore, we have resurrected long-neglected methods for studying whole-gland function, and have attempted to improve them in a variety of ways. We are refining these methods and increasing our understanding of gland function by studying tracheal glands from sheep, pigs and cats. As human tissues become available, they are studied with the best methods presently available. The key questions now being asked are: Is mucus secretion from submucosal glands altered in cystic fibrosis? If so, how is it altered and how does it contribute to CF lung disease? Answering the last question will require an understanding of how glands interact with other regions of the lung. In the context of this meeting, we present preliminary data on the role of HCO(3)(-) in gland mucus secretion.

  View details for PubMedID 11875272

• Cystic fibrosis: The 'bicarbonate before chloride' hypothesis CURRENT BIOLOGY Wine, J. J. 2001; 11 (12): R463-R466

  Abstract

  The specific effects of some mutations that cause cystic fibrosis suggest that reduced HCO(3)(-) transport is the key to understanding cystic fibrosis pathology. But there is a puzzling discrepancy between measures of CFTR-mediated chloride conductance in expression systems and the sweat chloride values of patients.

  View details for Web of Science ID 000169612900007

  View details for PubMedID 11448786

• Cystic fibrosis transmembrane conductance regulator gating requires cytosolic electrolytes JOURNAL OF BIOLOGICAL CHEMISTRY Wu, J. V., Joo, N. S., Krouse, M. E., Wine, J. J. 2001; 276 (9): 6473-6478

  Abstract

  Cystic fibrosis transmembrane conductance regulator (CFTR), which causes cystic fibrosis when nonfunctional, is an anion channel and a member of the ATP binding cassette superfamily. After phosphorylation, CFTR gates by binding and hydrolyzing ATP. We show that CFTR open probability (P(o)) also depends on the electrolyte concentration of the cytosol. Inside-out patches from Calu-3 cells were transiently exposed to solutions of 160 mm salt or solutions in which up to 90% of the salt was replaced by nonionic osmolytes such as sucrose. In lowered salt solutions, CFTR P(o) declined within 1 s to a stable lower value that depended on the electrolyte concentration, (K(1/2) approximately 80 mm NaCl). P(o) was rapidly restored in normal salt concentrations without regard to the electrolyte species. Reducing external electrolytes did not affect CFTR P(o). The same results were obtained when CFTR was stably phosphorylated with adenosine 5'-O-(thiotriphosphate). The decrease in P(o) resulted entirely from an increase in mean closed time. Increasing ATP levels up to 20-fold did not counteract the effect of low electrolytes. The same effect was observed for CFTR expressed in C127 cells but not for a different species of anion channel. Cytosolic electrolytes are an unsuspected, essential cofactor for CFTR gating.

  View details for Web of Science ID 000167261000055

  View details for PubMedID 11112782

• Comprehensive mutation screening in a cystic fibrosis center PEDIATRICS Wine, J. J., Kuo, E., Hurlock, G., Moss, R. B. 2001; 107 (2): 280-286

  Abstract

  The identities of a cystic fibrosis (CF) patient's CFTR mutations can influence therapeutic strategies, but because >800 CFTR mutations exist, cost-effective, comprehensive screening requires a multistage approach. Single-strand conformation polymorphism and heteroduplex analysis (SSCP/HA) can be an important part of mutation detection, but must be calibrated within each laboratory. The sensitivity of a combined commercial-SSCP/HA approach to genotyping in a large, ethnically diverse US center CF population has not been established.We screened all 27 CFTR exons in 10 human participants who had an unequivocal CF diagnosis including a positive sweat chloride test and at least 1 unknown allele after commercial testing for the 70 most common mutations by SSCP/HA. These participants were compared with 7 participants who had negative sweat tests but at least 1 other CF-like symptom meriting complete genotyping.For the 10 CF participants, we detected 11 of 16 unknown alleles (69%) and all 4 of the known alleles (100%), for an overall rate of 75% inpatients not fully genotyped by conventional 70 mutation screen. For 7 participants with negative sweat tests, we confirmed 1 identified mutation in 14 alleles and detected 3 additional mutations. Mutations detected in both groups included 7 missense mutations (S13F, P67L, G98R, S492F, G970D, L1093P, N1303K) and 9 deletion, frameshift, nonsense or splicing mutations (R75X, G542X, DeltaF508, 451-458Delta8 bp, 5T, 663DeltaT, exon 13 frameshift, 1261+1G-->A and 3272-26A-->G). Three of these mutations were novel (G970D, L1093P, and 451-458Delta8 bp(1)). Thirteen other changes were detected, including the novel changes 1812-3 ins T, 4096-278 ins T, 4096-265 ins TG, and 4096-180 T-->G.When combined with the 70 mutation Genzyme test, SSCP/HA analysis allows for detection of >95% of the mutations in an ethnically heterogeneous CF center population. We discuss 5 possible explanations that could account for the few remaining undetected mutations.

  View details for Web of Science ID 000166714000025

  View details for PubMedID 11158459

• CFTR activation raises extracellular pH of NIH/3T3 mouse fibroblasts and C127 epithelial cells JOURNAL OF MEMBRANE BIOLOGY Luckie, D. B., Singh, C. N., Wine, J. J., Wilterding, J. H. 2001; 179 (3): 275-284

  Abstract

  Cystic Fibrosis (CF) is caused by mutations in the gene for CFTR, a cAMP-activated anion channel found in apical membranes of wet epithelia. Since CFTR is permeable to HCO3- and changes in extracellular fluid composition may contribute to CF lung disease, we investigated possible differences in extracellular pH (pHo) between CFTR-expressing and control cell lines. The Cytosensor Microphysiometer was used to study forskolin-stimulated extracellular acidification rates in CFTR-expressing and control mouse mammary epithelial (C127) and fibroblast (NIH/3T3) cell lines. Forskolin, which activates CFTR via raised cAMP, caused decreased extracellular acidification of CFTR-expressing NIH/3T3 and C127 cells by 15-35%. By contrast, forskolin caused increased extracellular acidification of control cells by 10-20%. Ionomycin, which may activate CFTR via PKC, also elicited this decreased extracellular acidification signal only in cells expressing CFTR. In control experiments, dideoxyforskolin had no effect on the acidification rates and osmotic stimuli were shown to equally stimulate all cell lines. These results suggest a role for CFTR in controlling pHo and complement recent evidence that HCO3- dependent epithelial secretion may be reduced in amount and altered in composition in CF.

  View details for Web of Science ID 000166972200007

  View details for PubMedID 11246424

• Novel Cystic Fibrosis mutation L1093P: functional analysis and possible Native American origin. Human mutation Yee, K., Robinson, C., Hurlock, G., Moss, R. B., Wine, J. J. 2000; 15 (2): 208-?

  Abstract

  A novel mutation was detected using single-strand conformation polymorphism and heteroduplex analysis in a cystic fibrosis subject of mixed ancestry. Mutation 3410T-->C in exon 17b caused the novel missense mutation L1093P; the other chromosome has mutation N1303K. The 31-year-old subject is pancreatic insufficient, had an FEV(1) score that was 33% of normal prior to a heart/lung transplant, and sweat chloride values of 116 and 95 mM when tested at ages 1 and 11. Functional analysis using forskolin-stimulated efflux of (125)I in HEK cells transfected with an ABCC7 construct harboring the L1093P mutation confirmed that cAMP-mediated anion efflux was abnormal, but some function was preserved. Analysis of parental DNA established that N1303K was of English origin, while L1093P was of Greek, Irish or Native American (Cherokee) origin. Given the intensive screening for CF mutations in European populations, we hypothesize that L1093P is of Native American origin. Hum Mutat 15:208, 2000.

  View details for PubMedID 10649505

• Screening methods for cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in non-human primates Life Sciences Conference 1998 Signalling Concepts in Life Sciences Glavac, D., Ravnik-Glavac, M., Potocnik, U., Dean, M., Wine, J. SPRINGER. 2000: R12–R13

  Abstract

  We report here a comparison of isotopic and non-isotopic conformation analysis approach, for screening genomic DNA for coding variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A large pool of non-human primates was tested in order to detect naturally occuring CFTR carriers, for future testing of gene therapy of cystic fibrosis. We screened 25 of 27 CFTR exons in over 1,000 animals. We have detected numerous missense mutations and single nucleotide polymorphisms. We found that both methods are highly efficient for detection of variations in DNA sequence, but the non-radioactive approach is faster, less expensive and in some cases more sensitive.

  View details for Web of Science ID 000084572000005

  View details for PubMedID 10653125

• Screening methods for cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in non-human primates. Pflugers Archiv : European journal of physiology Glavac, D., Ravnik-Glavac, M., Potocnik, U., Dean, M., Wine, J. 2000; 439: r012-r013

  Abstract

  We report here a comparison of isotopic and non-isotopic conformation analysis approach, for screening genomic DNA for coding variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A large pool of non-human primates was tested in order to detect naturally occuring CFTR carriers, for future testing of gene therapy of cystic fibrosis. We screened 25 of 27 CFTR exons in over 1,000 animals. We have detected numerous missense mutations and single nucleotide polymorphisms. We found that both methods are highly efficient for detection of variations in DNA sequence, but the non-radioactive approach is faster, less expensive and in some cases more sensitive.

  View details for DOI 10.1007/s004240000072

  View details for PubMedID 28176056

• Two novel mutations in a cystic fibrosis patient of Chinese origin HUMAN GENETICS Wagner, J. A., Vassilakis, A., Yee, K., Li, M., Hurlock, G., Krouse, M. E., Moss, R. B., Wine, J. J. 1999; 104 (6): 511-515

  Abstract

  Cystic fibrosis is rare in non-Caucasian populations, and in such populations little is known about the spectrum of mutations and polymorphisms in the CFTR gene. We studied a 23-year-old patient of Chinese ethnicity with sweat chloride values of 104 mM/l, pancreatic sufficiency, an FEV1 60% of normal, sputum cultures positive for Staphylococcus aureus and Burkholderia cepacia, and a history of allergic bronchopulmonary aspergillosis. Genetic screening for 31 common CFTR mutations was negative, leading us to search for unknown mutations using single-strand conformation polymorphism and heteroduplex analysis (SSCP/HA). Two novel mutations were detected. In exon 4, a deletion of 8 bp (451458, deltaGCTTCCTA) causes a frameshift and immediately creates a stop codon. In exon 16, mutation 3041G-->A causes the missense change G970D. Functional analysis using an isotopic flux assay indicated that the G970D mutation retains partial function; western blotting indicated that the protein is glycosylated. The patient is heterozygous for the common polymorphisms (2694T/G) in exon 14a and (GATT)6/7 in intron 6a, indicating that these variants arose in ancestors common to Caucasians and Chinese.

  View details for Web of Science ID 000081854000012

  View details for PubMedID 10453741

• Safety and biological efficacy of an adeno-associated virus vector cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus 11th Annual North American Cystic Fibrosis Conference Wagner, J. A., Messner, A. H., Moran, M. L., DAIFUKU, R., Kouyama, K., Desch, J. K., Manley, S., Norbash, A. M., Conrad, C. K., Friborg, S., Reynolds, T., Guggino, W. B., Moss, R. B., Carter, B. J., Wine, J. J., Flotte, T. R., Gardner, P. JOHN WILEY & SONS INC. 1999: 266–74

  Abstract

  The host immune response and low vector efficiency have been key impediments to effective cystic fibrosis transmembrane regulator (CFTR) gene transfer for cystic fibrosis (CF). An adeno-associated virus vector (AAV-CFTR) was used in a phase I dose-escalation study to transfer CFTR cDNA into respiratory epithelial cells of the maxillary sinus of 10 CF patients.A prospective, randomized, unblinded, dose-escalation, within-subjects, phase I clinical trial of AAV-CFTR was conducted.Ten patients with previous bilateral maxillary antrostomies were treated.Safety, gene transfer as measured by semiquantitative polymerase chain reaction (PCR), and sinus transepithelial potential difference (TEPD) were measured.The highest level of gene transfer was observed in the range of 0.1-1 AAV-CFTR vector copy per cell in biopsy specimens obtained 2 weeks after treatment. When tested, persistence was observed in one patient for 41 days and in another for 10 weeks. Dose-dependent changes in TEPD responses to pharmacologic intervention were observed following treatments. Little or no inflammatory or immune responses were observed.AAV-CFTR administration to the maxillary sinus results in successful, dose-dependent gene transfer to the maxillary sinus and alterations in sinus TEPD suggestive of a functional effect, with little or no cytopathic or host immune response. Further study is warranted for AAV vectors as they may prove useful for CFTR gene transfer and other in vivo gene transfer therapies. A prospective, randomized, double-blind, placebo-controlled, within-subjects, phase II clinical trial of the effect AAV-CFTR on clinical recurrence of sinusitis will determine the clinical efficacy of AAV gene therapy for CF.

  View details for Web of Science ID 000078432500017

  View details for PubMedID 10890777

• The genesis of cystic fibrosis lung disease JOURNAL OF CLINICAL INVESTIGATION Wine, J. J. 1999; 103 (3): 309-312

  View details for Web of Science ID 000078581400002

  View details for PubMedID 9927490

• Maxillary sinusitis as a surrogate model for CF gene therapy clinical trials in patients with antrostomies JOURNAL OF GENE MEDICINE Wagner, J. A., Nepomuceno, I. B., Shah, N., Messner, A. H., Moran, M. L., Norbash, A. M., Moss, R. B., Wine, J. J., Gardner, P. 1999; 1 (1): 13-21

  Abstract

  Assessing the biological activity and clinical efficacy of gene therapy is critically important in cystic fibrosis (CF). It is widely accepted that clinical testing using surrogate markers including pulmonary function will be useful in assessing clinical efficacy. One problem with pulmonary surrogate markers of CF disease is the large number of patients and length of time required to demonstrate clinical efficacy. An alternative to pulmonary testing of new CF treatments is use of the maxillary sinuses as a surrogate model of CF lung disease. Using CF sinusitis as a surrogate model for testing clinical efficacy of new treatments is attractive because CF upper respiratory disease is similar to the lower respiratory disease with respect to electrophysiology and microbiology.Sinusitis recurrence in untreated sinuses was analyzed during a prospective, randomized, unblinded, dose-escalation, within-subjects, phase I clinical trial of the adeno-associated virus mediated cystic fibrosis transmembrane conductance regulator (AAV-CFTR) gene transfer.Clinical symptoms combined with sinus endoscopy proved useful in the diagnosis of unilateral and bilateral sinusitis recurrence. Sinusitis recurred at a rate of 45% during one month of follow-up. IL-8 concentration rose in sinus fluids from affected sinuses. Bacterial cultures and increased sinus leukocytes corroborated recurrent sinusitis. Sinus CT scans were also useful in diagnosing recurrent sinusitis in this surrogate model of CF infectious exacerbations.CF sinusitis as a surrogate for lung disease is particularly well-suited for phase II clinical trials of gene transfer agents, with the potential for measuring clinical efficacy in relatively small numbers of patients over relatively short periods of time.

  View details for Web of Science ID 000083463500002

  View details for PubMedID 10738581

• Efficient and persistent gene transfer of AAV-CFTR in maxillary sinus LANCET Wagner, J. A., Reynolds, T., Moran, M. L., Moss, R. B., Wine, J. J., Flotte, T. R., Gardner, P. 1998; 351 (9117): 1702-1703

  View details for Web of Science ID 000074088900015

  View details for PubMedID 9734891

• A phase I/II study of tgAAV-CF for the treatment of chronic sinusitis in patients with cystic fibrosis HUMAN GENE THERAPY Wagner, J. A., Moran, M. L., Messner, A. H., DAIFUKU, R., Conrad, C. K., Reynolds, T., Guggino, W. B., Moss, R. B., Carter, B. J., Wine, J. J., Flotte, T. R., Gardner, P. 1998; 9 (6): 889-909

  View details for Web of Science ID 000073201700014

  View details for PubMedID 9581911

• Genomic DNA sequence of Rhesus (M-mulatta) cystic fibrosis (CFTR) gene MAMMALIAN GENOME Wine, J. J., Glavac, D., Hurlock, G., Robinson, C., Lee, M., Potocnik, U., Ravnik-Glavac, M., Dean, M. 1998; 9 (4): 301-305

  Abstract

  Cystic fibrosis is a common human genetic disease caused by mutations in CFTR, a gene that codes for a chloride channel that is regulated by phosphorylation and cytosolic nucleotides. As part of a program to discover natural animal models for human genetic diseases, we have determined the genomic sequence of CFTR in the Rhesus monkey, Macaca mulatta. The coding region of rhesus CFTR is 98.3% identical to human CFTR at the nucleotide level and 98.2% identical and 99.7% similar at the amino acid level. Partial sequences of flanking introns (5582 base pair positions analyzed) revealed 91.1% identity with human introns. Relative to rhesus intronic sequence, the human sequences had 27 insertions and 22 deletions. Primer sequences for amplification of rhesus genomic CFTR sequences are provided. The accession number is AF013753 (all 27 exons and some flanking intronic sequence).

  View details for Web of Science ID 000072628000007

  View details for PubMedID 9530627

• Evidence that Calu-3 human airway cells secrete bicarbonate AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Lee, M. C., Penland, C. M., Widdicombe, J. H., Wine, J. J. 1998; 274 (3): L450-L453

  Abstract

  The Calu-3 cell line is being investigated as a model for human submucosal gland serous cells. In a previous investigation of basal short-circuit current (Isc) in Calu-3 cells, high levels of bumetanide-insensitive basal Isc (approximately 60 microA/cm2) were measured in cells grown at an air interface. Basal Isc was reduced only 7% by bumetanide, and the largest component of basal Isc required both Cl- and HCO3- in the bathing solutions. Because Isc could be partially inhibited by basolateral 4,4'-dinitrostilbene-2,2'-disulfonic acid and because the only known apical exit pathway for anions is the cystic fibrosis transmembrane conductance regulator, which has a relatively poor conductance for HCO3-, it was concluded that most basal Isc is HCO3(-)-dependent Cl- secretion [M. Singh, M. Krouse, S. Moon, and J. J. Wine. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L690-L698, 1997]. We have now measured isotopic fluxes of 36Cl- and 22Na+ across short-circuited Calu-3 cells and found that virtually none of the basal Isc is Cl- secretion or Na+ absorption. Thus, in contrast to the earlier report, we conclude that the major component of basal Isc is HCO3- secretion. Stimulation recruits primarily Cl- secretion, as previously proposed.

  View details for Web of Science ID 000072415800019

  View details for PubMedID 9530182

• Calcium-stimulated Cl- secretion in Calu-3 human airway cells requires CFTR AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Moon, S., Singh, M., Krouse, M. E., Wine, J. J. 1997; 273 (6): L1208-L1219

  Abstract

  Human airway serous cells secrete antibiotic-rich fluid, but, in cystic fibrosis (CF), Cl(-)-dependent fluid secretion is impaired by defects in CF transmembrane conductance regulator (CFTR) Cl- channels. Typically, CF disrupts adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion but spares Ca(2+)-mediated secretion. However, in CF airway glands, Ca(2+)-mediated secretion is also greatly reduced. To determine the basis of Ca(2+)-mediated Cl- secretion in serous cells, we used thapsigargin to elevate intracellular Ca2+ concentration ([Ca2+]i) in Calu-3 cells, an airway cell line bearing some similarities to serous cells. Cells were cultured using conventional and air interface methods. Short-circuit current (Isc) and transepithelial conductance (Gte) were measured in confluent cell layers. Thapsigargin stimulated large, sustained changes (delta) in Isc and Gte, whereas forskolin stimulated variable and smaller increases. delta Isc was decreased by basolateral bumetanide, quinidine, barium, or diphenylamine-2-carboxylate (DPAC) but was unaffected by high apical concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 4,4'-dinitrostilbene-2,2'-disulfonic acid, and calixarene. Isc was measured after permeabilizing the basolateral membrane and establishing transmembrane ion gradients. Unstimulated apical membranes displayed high Cl- conductance (GCl) that was decreased by DPAC but not by DIDS. Apical GCl could be increased by elevating intracellular cAMP concentration but not [Ca2+]i. We conclude that CFTR channels are the exclusive GCl pathway in the apical membrane and display approximately 60% of maximum conductance at rest. Thus elevated [Ca2+]i increases K+ conductance to force Cl- through open CFTR channels. We hypothesize that loss of CFTR channels causes diminution of cholinergically mediated gland secretions in CF.

  View details for Web of Science ID 000071106700014

  View details for PubMedID 9435576

• Disruption of monolayer integrity enables activation of a cystic fibrosis ''bypass'' channel in human airway epithelia NATURE MEDICINE Xia, Y. F., HAWS, C. M., Wine, J. J. 1997; 3 (7): 802-805

  Abstract

  Cystic fibrosis (CF) is a genetic disease characterized by marked reduction in Cl- conductance across many epithelia. Two kinds of Cl- channels have been associated with CF. One channel, termed the cystic fibrosis transmembrane conductance regulator (CFTR), is directly coded by the CF gene. The other channel is an outwardly rectifying depolarization induced Cl- channel (ORDIC) that is distinguished from other outwardly rectifying chloride channels (ORCCs) because its activity is induced most reliably by patch excision and depolarization. An issue in current CF research is whether ORDIC channels are indirectly activated by CFTR to contribute a significant portion of apical membrane Cl- conductance in airway cells. We now show that ORDIC channels are readily activated in patches excised and depolarized from isolated cells, but are rarer or refractory to activation in patches from the apical membranes of confluent human airway epithelia. These findings have important implications for proposed therapies that would bypass the CFTR conductance by activating ORDIC channels.

  View details for Web of Science ID A1997XG76700045

  View details for PubMedID 9212112

• Most basal I-sc in Calu-3 human airway cells is bicarbonate-dependent Cl- secretion AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Singh, M., Krouse, M., Moon, S., Wine, J. J. 1997; 272 (4): L690-L698

  Abstract

  Serous cells secrete antibiotic-rich fluid, but secretion is impaired in cystic fibrosis. We are investigating Calu-3 cells as a serous cell model. Basal short-circuit current (I[SC]) in Calu-3 cells grown at air interface had a basal I(SC) approximately six times larger than submerged cultures (69 +/- 22 vs. 11 +/- 10 microA/cm2). Basal I(SC) in either condition was reduced only 7 +/- 5% by bumetanide and was unaffected by apical amiloride, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), or calixarene but was reduced 77 +/- 18% by N-phenylanthranilic acid. Three transport mechanisms accounted for almost all basal I(SC). The largest component is HCO3(-)-dependent Cl- secretion. Replacement of Krebs-Henseleit solution with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solution and changing gassing from 95% O2-5% CO2 to air reduced the basal I(SC) by 61 +/- 10%. Acetazolamide decreased basal I(SC) by 33 +/- 6%, whereas acetazolamide + basolateral DNDS eliminated 42-58% of the bumetanide-insensitive basal I(SC). Neither DNDS nor acetazolamide had any effect when applied in HCO3(-)-free solution. Apical phlorizin, a blocker of Na+-glucose cotransport, eliminated one-half of the remaining I(SC). Cl- replacement with gluconate eliminated all I(SC) except the phlorizin-sensitive component. Unlike basal I(SC), 80 +/- 24% of stimulated I(SC) was inhibited by bumetanide. Thus basal and stimulated secretions are mediated by different mechanisms.

  View details for Web of Science ID A1997WT60100017

  View details for PubMedID 9142943

• Cystic fibrosis transmembrane conductance regulator and adenosine triphosphate - Response SCIENCE Reddy, M. M., Quinton, P. M., HAWS, C., Wine, J. J., Grygorczyk, R., Tabcharani, J. A., Hanrahan, J. W., Gunderson, K. L., Kopito, R. R. 1997; 275 (5304): 1325-1325

  View details for Web of Science ID A1997WK64400049

• Cell-volume regulation: P-glycoprotein - A cautionary tale CURRENT BIOLOGY Wine, J. J., Luckie, D. B. 1996; 6 (11): 1410-1412

  Abstract

  P-glycoprotein turns out not to be 'VSOAC', a known channel activated by cell swelling; it does seem to influence cell-volume recovery after swelling, but the physiological importance of this effect is presently unclear.

  View details for Web of Science ID A1996VT11900021

  View details for PubMedID 8939588

• Swelling and Ca2+-activated anion conductances in C127 epithelial cells expressing WT and Delta F508-CFTR JOURNAL OF MEMBRANE BIOLOGY Xia, Y., Krouse, M. E., Fang, R. H., Wine, J. J. 1996; 151 (3): 269-278

  Abstract

  CFTR is a chloride channel that is required for fluid secretion and salt absorption in many exocrine epithelia. Mutations in CFTR cause cystic fibrosis. CFTR expression influences some ion channels, but the range of channels influenced, the mechanism of the interaction and the significance for cystic fibrosis are not known. Possible interactions between CFTR and other ion channels were studied in C127 mouse mammary epithelial cell lines stably transfected with CFTR, delta F508-CFTR, or vector. Cell lines were compared quantitatively using an 125I efflux assay and qualitatively using whole-cell patch-clamp recording. As expected, 125I efflux was significantly increased by forskolin only in the CFTR line, and forskolin-stimulated whole-cell currents were time- and voltage independent. All three lines responded to hypotonic challenge with large 125I efflux responses of equivalent magnitude, and whole-cell currents were outwardly rectified and inactivated at positive voltages. Unexpectedly, basal 125I efflux was significantly smaller in the delta F508-CFTR cell line than in either the CFTR or control cell lines (P < 0.0001), and the magnitude of the efflux response to ionomycin was largest in the vector cell line and smallest in the cell line expressing delta F508-CFTR (P < 0.01). Whole-cell responses to ionomycin had a linear instantaneous I-V relation and activated at depolarizing voltages. Forskolin responses showed simple summation with responses to ionomycin or hypotonic challenge. Thus, we found no evidence for interactions between CFTR and the channels responsible for swelling-mediated responses. Differences were found in basal and ionomycin-stimulated efflux, but these may arise from variations in the clonally selected cell lines that are unrelated to CFTR expression.

  View details for Web of Science ID A1996UQ30800007

  View details for PubMedID 8661514

• Delta F508-CFTR channels: Kinetics, activation by forskolin, and potentiation by xanthines AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY HAWS, C. M., Nepomuceno, I. B., Krouse, M. E., Wakelee, H., Law, T., Xia, Y., Nguyen, H., Wine, J. J. 1996; 270 (5): C1544-C1555

  Abstract

  Trafficking, activation, and kinetics of delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR were compared in stably transduced C127I mouse mammary epithelial cells. Western blots detected a small amount of fully glycosylated delta F508-CFTR Efflux of 125I was stimulated by forskolin with the same mean effective concentration (EC50; approximately 0.5 microM) for CFTR and delta F508-CFTR cells, but the maximum response was reduced more than fivefold and its latency increased approximately threefold in delta F508-CFTR cells. In delta F508-CFTR cells, 3-isobutyl-1-methylxanthine (IBMX; EC50 = 1.45 microM) and 8-cyclopentyl-1,3-dipropylxanthine (CPX; EC50 = 58 microM) increased the peak forskolin-stimulated efflux rate approximately 2.5-fold and decreased the time to peak. A sevenfold increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels accompanied potentiation of forskolin-induced 125I efflux by IBMX but not by CPX. Elevation of intracellular cAMP increased linear voltage-independent whole cell currents 30-fold in CFTR and 4-fold in delta F508-CFTR cells; the response rate in delta F508-CFTR cells was much slower. Single-channel currents were detected in 57 of 68 cell-attached patches from forskolin-prestimulated CFTR cells vs. 6 of 35 patches in delta F508-CFTR cells. Mean number of active channels per patch was 4.1 for CFTR [open probability (Po) = 0.34] and 0.2 for delta F508-CFTR (Po = 0.11). The lower Po of delta F508-CFTR resulted from an approximately threefold longer mean interburst interval. We estimate that forskolin-stimulated chloride conductance of delta F508-CFTR C127I cells is < 5% of CFTR cells. CPX is approximately 25-fold more potent than IBMX in potentiating delta F508-CFTR and may operate by a mechanism other than elevation of cAMP.

  View details for Web of Science ID A1996UJ81400034

  View details for PubMedID 8967457

• Doxorubicin selection for MDR1/P-glycoprotein reduces swelling-activated K+ and Cl- currents in MES-SA cells AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Luckie, D. B., Krouse, M. E., Law, T. C., Sikic, B. I., Wine, J. J. 1996; 270 (4): C1029-C1036

  Abstract

  To test the hypothesis that P-glycoprotein enhances swelling currents through regulation of volume-sensitive Cl- channels [recently termed VSOAC (volume-sensitive osmolyte and anion channel)], a human uterine sarcoma cell line (MES-SA) and its doxorubicin-selected counterpart (Dx5) were studied. P-glycoprotein mRNA and protein levels were detected only in Dx5 cells. However, whole cell patch-clamp experiments showed that swollen Dx5 cells (n = 5) produced smaller VSOAC currents than MES-SA cells (n = 4; 106 +/- 26 pA/pF vs. 232 +/- 76 pA/pF at 90 mV). In radioisotopic efflux experiments, both swelling-activated 125I (Cl-) currents (n = 15) and 86Rb (K+) currents (n = 8) were found to be two-to fourfold smaller in the Dx5 (high P-glycoprotein) cells. Inhibitors of P-glycoprotein showed no specificity for the doxorubicin-selected cells (Dx5). Dideoxyforskolin (100 microM) blocked swelling-activated 125I efflux equally in both cell lines, whereas 100 microM verapamil had no effect. Thus, in this cell line, selection for P-glycoprotein expression is associated with reduced swelling currents. These findings suggest that P-glycoprotein expression does not directly facilitate VSOAC.

  View details for Web of Science ID A1996UD60600008

  View details for PubMedID 8928730

• Failure of the cystic fibrosis transmembrane conductance regulator to conduct ATP SCIENCE Reddy, M. M., Quinton, P. M., HAWS, C., Wine, J. J., Grygorczyk, R., Tabcharani, J. A., Hanrahan, J. W., Gunderson, K. L., Kopito, R. R. 1996; 271 (5257): 1876-1879

  Abstract

  The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel regulated by protein kinase A and adenosine triphosphate (ATP). Loss of CFTR-mediated chloride ion conductance from the apical plasma membrane of epithelial cells is a primary physiological lesion in cystic fibrosis. CFTR has also been suggested to function an an ATP channel, although the size of the ATP anion is much larger than the estimated size of the CFTR pore. ATP was not conducted through CFTR in intact organs, polarized human lung cell lines, stably transfected mammalian cell lines, or planar lipid bilayers reconstituted with CFTR protein. These findings suggest that ATP permeation through the CFTR is unlikely to contribute to the normal function of CFTR or to the pathogenesis of cystic fibrosis.

  View details for Web of Science ID A1996UC77800050

  View details for PubMedID 8596959

• Clinical protocol: AAV-CFTR for the treatment of chronic sinusitis in CF patients. Wagner, J. A., Moran, M. L., Kouyama, K., Moss, R. B., Wine, J. J., Gardner, P. NATURE PUBLISHING GROUP. 1996: PII63–PII63

  View details for Web of Science ID A1996TX00100182

• Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation JOURNAL OF BIOLOGICAL CHEMISTRY Sato, S., Ward, C. L., Krouse, M. E., Wine, J. J., Kopito, R. R. 1996; 271 (2): 635-638

  Abstract

  The common delta F508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) interferes with the biosynthetic folding of nascent CFTR polypeptides, leading to their retention and rapid degradation in an intracellular compartment proximal to the Golgi apparatus. Neither the pathway by which wild-type CFTR folds nor the mechanism by which the Phe508 deletion interferes with this process is well understood. We have investigated the effect of glycerol, a polyhydric alcohol known to stabilize protein conformation, on the folding of CFTR and delta F508 in vivo. Incubation of transient and stable delta F508 transfectants with 10% glycerol induced a significant accumulation of delta F508 protein bearing complex N-linked oligosaccharides, indicative of their transit to a compartment distal to the endoplasmic reticulum (ER). This accumulation was accompanied by an increase in mean whole cell cAMP activated chloride conductance, suggesting that the glycerol-rescued delta F508 polypeptides form functional plasma membrane CFTR channels. These effects were dose- and time-dependent and fully reversible. Glycerol treatment also stabilized immature (core-glycosylated) delta F508 and CFTR molecules that are normally degraded rapidly. These effects of glycerol were not due to a general disruption of ER quality control processes but appeared to correlate with the degree of temperature sensitivity of specific CFTR mutations. These data suggest a model in which glycerol serves to stabilize an otherwise unstable intermediate in CFTR biosynthesis, maintaining it in a conformation that is competent for folding and subsequent release from the ER quality control apparatus.

  View details for Web of Science ID A1996TP88900009

  View details for PubMedID 8557666

• Cystic fibrosis: How do CFTR mutations cause cystic fibrosis? CURRENT BIOLOGY Wine, J. J. 1995; 5 (12): 1357-1359

  View details for Web of Science ID A1995TL10200009

  View details for PubMedID 8749384

• SELECTION FOR MDR1 P-GLYCOPROTEIN ENHANCES SWELLING-ACTIVATED K+ AND CL- CURRENTS IN NIH/3T3 CELLS AMERICAN JOURNAL OF PHYSIOLOGY Luckie, D. B., Krouse, M. E., Harper, K. L., Law, T. C., Wine, J. J. 1994; 267 (2): C650-C658

  Abstract

  The relationship between multidrug resistance (MDR) P-glycoprotein expression and swelling-activated Cl- and K+ conductance was investigated in mouse NIH/3T3 fibroblasts and their colchicine-selected counterparts (COL1000, high P-glycoprotein). Whole cell patch-clamp and isotopic flux experiments confirmed that swelling-activated Cl- currents were induced by 20-30% bath dilution only in the MDR-expressing cell line. However, at bath dilutions > 30%, both cell lines developed Cl- currents that reached similar large magnitudes at higher dilution levels. Thus the apparent absolute difference in cell lines at lower dilutions is due to a shift in the response curve relating hypotonicity to Cl- conductance. At all dilutions and in both cell lines, the swelling-activated Cl- currents were outwardly rectifying, active at negative cell voltages, and inactivated at positive voltages. Verapamil (100 microM) and 1,9-dideoxyforskolin (100 microM), which inhibit P-glycoprotein drug transport, did not significantly inhibit the swelling-activated Cl- conductance efflux in the COL1000 cells also showed a leftward shift in the response curve to hypotonicity. These results indicate that response curve to hypotonicity. These results indicate that colchicine-selection for increased P-glycoprotein expression did not lead to the expression of swelling-activated Cl- channels, but instead enhanced a step in the pathway from bath dilution to regulatory volume decrease that is common to both K+ and Cl- channels.

  View details for Web of Science ID A1994PC43900038

  View details for PubMedID 7915492

• DISSOCIATION OF DEPOLARIZATION-ACTIVATED AND SWELLING-ACTIVATED CL- CHANNELS AMERICAN JOURNAL OF PHYSIOLOGY Krouse, M. E., HAWS, C. M., Xia, Y. F., Fang, R. H., Wine, J. J. 1994; 267 (2): C642-C649

  Abstract

  In many cells, patch excision and depolarization induce outwardly rectifying Cl- channels (ORDIC channels) whose function and normal mode of regulation are unknown. One possible function is the mediation of swelling-activated Cl- conductance, because in many cells rectifying Cl- currents are activated by cell swelling. However, swelling-activated Cl- channels in some epithelia have larger conductances than ORDIC channels and inactivate more rapidly, although both have similar anion selectivity and are blocked by stilbenes. Thus it has not been possible to determine whether the two types of channel current arise from distinct proteins or alternate states of a single protein. We studied 14 cell lines and found 2 lines, C127 mouse mammary epithelial cells and IEC-6 rat intestinal crypt cells, with very low levels of ORDIC channels. However, despite the near absence of ORDIC channels in these rodent cells, a large swelling-activated Cl-conductance was demonstrated by whole cell, efflux, and single-channel methods. Thus it is likely that ORDIC and swelling-activated channel currents arise from different channel proteins.

  View details for Web of Science ID A1994PC43900037

  View details for PubMedID 8074197

• CFTR IN CALU-3 HUMAN AIRWAY CELLS - CHANNEL PROPERTIES AND ROLE IN CAMP-ACTIVATED CL- CONDUCTANCE AMERICAN JOURNAL OF PHYSIOLOGY HAWS, C., Finkbeiner, W. E., Widdicombe, J. H., Wine, J. J. 1994; 266 (5): L502-L512

  Abstract

  Calu-3, a cell line derived from a lung adenocarcinoma, forms tight junctions, expresses cystic fibrosis transmembrane conductance regulator (CFTR), and secretes Cl- in response to adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents. Anion conductance of Calu-3 cells was assessed with isotopic flux and patch-clamp methods at 22 degrees C. Iodide efflux was increased by cAMP-elevating agents and brief trypsin treatment. A 7.1 +/- 0.4-pS voltage-independent Cl- channel with linear current-voltage relation was the most common channel observed in cell-attached recordings and was identified as CFTR on the basis of shared features with recombinant CFTR. In unstimulated cells, the mean minimum number of active CFTR channels per patch was 1 +/- 1 (n = 12), increasing to 6 +/- 8 (n = 40) after stimulation with cAMP-elevating agents or after brief trypsin treatment. Channel closure after excision was biexponential with tau 1 approximately 4 s and tau 2 approximately 79 s; typically channels were open continuously until closing permanently. In 11 of 12 excised patches, channels were reactivated by exposure to cAMP-dependent protein kinase (PKA) plus ATP. Efficacy of reactivation was inversely related to the duration from excision to addition of PKA. Channels were blocked by 20-40 microM 5-nitro-2-(3-phenylpropylamino)benzoate on cytosolic but not external side. Active CFTR channels were recorded in 83% of total patches. Other types of Cl- channels were observed in 5 of 52 (10%) cell-attached patches and in 17 of 34 (50%) excised patches, including an outwardly rectifying channel in 2 patches. CFTR channels are the predominant pathway for cAMP-stimulated Cl- conductance in Calu-3 cells; the long open times in the absence of ATP are not explained by present models of CFTR activation.

  View details for Web of Science ID A1994NP99500091

  View details for PubMedID 7515579

• CALU-3 - A HUMAN AIRWAY EPITHELIAL-CELL LINE THAT SHOWS CAMP-DEPENDENT CL- SECRETION AMERICAN JOURNAL OF PHYSIOLOGY Shen, R. Q., Finkbeiner, W. E., Wine, J. J., Mrsny, R. J., Widdicombe, J. H. 1994; 266 (5): L493-L501

  Abstract

  Of 12 cell lines derived from human lung cancers, only Calu-3 cells showed high transepithelial resistance (Rte) and increases in short-circuit current (Isc) in response to mediators. Calu-3 cells formed polarized monolayers with tight junctions and Rte of approximately 100 omega.cm2. Baseline Isc was approximately 35 microA/cm2 and was increased by approximately 75 microA/cm2 on elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) by isoproterenol. Flux studies showed that the increase in Isc was due to Cl- secretion. Forskolin and permeant analogues of cAMP also increased Isc. Consistent with the presence of cAMP-dependent Cl- secretion, immunoprecipitation demonstrated the presence of the cystic fibrosis transmembrane conductance regulator (CFTR). Bradykinin, methacholine, trypsin, and histamine all transiently (15-30 s) elevated Isc, probably by increasing intracellular Ca concentration. Experiments in which the basolateral membrane was permeabilized with nystatin indicated that CFTR was substantially activated under baseline conditions and that Ca-activated Cl- channels were absent from the apical membrane. We anticipate that Calu-3 cells will prove useful in the study of Cl- secretion and other functions of human airway epithelial cells.

  View details for Web of Science ID A1994NP99500090

  View details for PubMedID 7515578

• CFTR AND OTHER CL- CHANNELS IN HUMAN AIRWAY CELLS 19th Seiriken Conference on Cl(-) Channel - Molecular and Cellular Physiology Wine, J. J., Finkbeiner, W. E., HAWS, C., Krouse, M. E., Moon, S., Widdicombe, J. H., Xia, Y. F. CENTER ACAD PUBL JAPAN. 1994: S199–S205

  Abstract

  CFTR is the major Cl- channel in several epithelia, but its importance in airway cells and its interactions with other channels are still poorly understood. We studied the role of CFTR and other Cl- channels in two well-differentiated human airway cell lines--16HBE and Calu-3. Both lines form tight junctions, and the Calu-3 line exhibits large currents in the Ussing chamber that represent Cl- secretion. Apical membrane Cl- conductance in both cell lines in mediated primarily and possibly exclusively by CFTR, because (1) CFTR channels are almost the only anion channels observed in cell-attached patches from confluent cells, (2) whole-cell Cl- current has a linear, time-independent signature, and (3) blockers of many Cl- channels, such as DIDS, do not block Isc. Outwardly rectifying (ORDIC) channels were identified in both airway cell lines. Activation of ORDIC channels in Calu-3 cells required 3 conditions: (1) stimulation with isoproterenol, (2) excision and depolarization, and (3) prior treatment of the cell to disrupt tight junctions. To further assess the physiological significance of ORDIC channels, we compared cell lines which have > 10-fold difference in functional expression of ORDIC channels. ORDIC channel expression was not associated with swelling-activated Cl- currents.

  View details for Web of Science ID A1994QA44600037

  View details for PubMedID 7752526

• Cystic fibrosis: ion channels and transmembrane transporters. Current biology Wine, J. J. 1993; 3 (2): 118-120

  View details for PubMedID 15335809

• STILBENES STIMULATE T84 CL- SECRETION BY ELEVATING CA-2+ AMERICAN JOURNAL OF PHYSIOLOGY Brayden, D. J., Krouse, M. E., Law, T., Wine, J. J. 1993; 264 (2): G325-G333

  Abstract

  Basolateral but not apical application of 10-200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to T84 monolayers produced a transient increase in short-circuit current (Isc), followed by a sustained inhibition. 4,4'-Dinitrostilbene-2,2'-disulfonic acid (DNDS) had no effect. The increase in Isc produced by DIDS represents Cl- secretion and appears to result from Ca2+ elevation, because in all respects except time course the response to DIDS mimicked the response to the Ca(2+)-elevating agent thapsigargin. Fura-2 measurements established that thapsigargin elevates Ca2+ in T84 cells, but Ca2+ responses to DIDS could not be established directly because DIDS absorbs strongly at the critical wavelengths. Responses to DIDS and thapsigargin were 1) blocked by bumetanide; 2) not blocked by basolateral Ba2+; 3) completely nonadditive; 4) strongly synergistic with basal levels of Isc or with Isc increases produced by elevating adenosine 3',5'-cyclic monophosphate (cAMP; with forskolin) or guanosine 3',5'-cyclic monophosphate (with heat-stable enterotoxin); and 5) reversibly abolished by removal of basolateral Ca2+. Interactions between Ca2+ and cAMP-elevating agents strongly support a model of Cl- secretion in which apical Cl- conductance is activated by cyclic nucleotides but not by Ca2+ while basolateral K+ channels are activated by Ca2+. In contrast with this mechanism, whole cell patch-clamp recordings of nonconfluent T84 cells indicated that DIDS and other Ca(2+)-elevating agents stimulated an increase in Cl- conductance. Thus increases in cytosolic free Ca2+ in nonconfluent T84 cells activate conductances that differ from those in confluent monolayers.

  View details for Web of Science ID A1993KN68000075

  View details for PubMedID 8447416

• CFTR CHANNELS IN IMMORTALIZED HUMAN AIRWAY CELLS AMERICAN JOURNAL OF PHYSIOLOGY HAWS, C., Krouse, M. E., Xia, Y. F., Gruenert, D. C., Wine, J. J. 1992; 263 (6): L692-L707

  Abstract

  The cystic fibrosis (CF) gene codes for CF transmembrane regulator (CFTR), a small-conductance linear Cl- channel, but numerous studies have identified a larger conductance, rectifying Cl- channel as the adenosine 3',5'-cyclic monophosphate (cAMP)-regulated channel that is defective in airway cells. We examined Cl- conductance in a bronchial epithelial cell line that expresses CFTR, 16HBE14o-, (CFTR+) and in an airway cell line that does not, 9HTEo-/S, (CFTR-). Ionomycin or hypotonic Ringer increased iodide efflux from both cell lines; however, forskolin increased iodide efflux or whole cell Cl- currents only in CFTR+ cells. Forskolin-stimulated whole cell currents were linear, voltage independent, and blocked by iodide. Cell-attached and outside-out patches from confluent CFTR+ but not CFTR- cells revealed 6-pS channels having linear current-voltage relations, permselectivity Cl > I (partial block by external iodide), and little or no inhibition by 5-nitro-2-(3-phenylpropylamino)-benzoate. The number of active channels per patch increased from 0.6 to 3.0 after forskolin. Channels closed after excision with tau = 4 s, but activity could be prolonged with ATP or protein kinase A plus ATP. Channels were modeled with one open and four closed states and show apparent cooperativity in gating. Rectifying Cl- channels previously implicated in CF were not seen in cell-attached recordings from either cell line but were abundant in excised patches from both cell lines. Thus CFTR channels are the pathway for cAMP-mediated Cl- conductance in these human airway cells, Ca2+ and swelling-induced channels do not require CFTR, and CFTR-cells display a CF phenotype.

  View details for Web of Science ID A1992KF37500077

  View details for PubMedID 1282304

• THE BASIC DEFECT IN CYSTIC-FIBROSIS TRENDS IN BIOCHEMICAL SCIENCES Widdicombe, J. H., Wine, J. J. 1991; 16 (12): 474-477

  Abstract

  Recent evidence strongly suggests that the cystic fibrosis gene product (CFTR) is a Cl- channel. Its properties, however, differ from those of a 30-50 pS outwardly rectifying channel previously implicated as defective in cystic fibrosis. It is still uncertain whether the pleiotropic effects of the CF defect, such as increased airway Na+ absorption and mucus sulfation, are secondary to reduced Cl- conductance, or reflect additional functions of CFTR.

  View details for Web of Science ID A1991GW01300010

  View details for PubMedID 1723552

• SWELLING-INDUCED AND DEPOLARIZATION-INDUCED CL- CHANNELS IN NORMAL AND CYSTIC-FIBROSIS EPITHELIAL-CELLS AMERICAN JOURNAL OF PHYSIOLOGY SOLC, C. K., Wine, J. J. 1991; 261 (4): C658-C674

  Abstract

  Cl- currents induced by cell swelling were characterized at the whole cell and single-channel levels in primary cultures of normal and cystic fibrosis (CF) epithelial cells and in the T84 cell line. Currents recorded in normal and CF cells were indistinguishable. At 22-24 degrees C with isotonic CsCl in the pipette, initial whole cell outward current density at 100 mV in unswollen cells was 2-4 pA/pF. The current density increased with time during whole cell recording up to 100 pA/pF in isotonic solutions and up to 200 pA/pF in a hypotonic bath, though values typically ranged between 10 and 70 pA/pF. Currents were outwardly rectifying, active at negative voltages, started to inactivate above approximately 40 mV, and were blocked by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Single Cl- channels (approximately 50 pS near 0 mV) with an outwardly rectifying current-voltage relation were recorded in cell-attached and outside-out patches from swollen cells. The channels were mostly open at negative voltages and inactivated at positive voltages with a voltage dependence similar to the whole cell currents. Channel activity decreased rapidly (channel rundown) after seal formation. After swelling-induced channel activity had ceased, outwardly rectifying, depolarization-induced Cl- channels (ORDIC channels) were activated in some patches. The swelling-induced and ORDIC single-channel currents were similar, but some consistent differences were observed. ORDIC channels were often closed at resting voltages (-70 to -50 mV), while swelling-induced channels were always open in this voltage range. In addition, ORDIC channels started to inactivate at more positive voltages (approximately 90 vs. approximately 50 mV), rectified more, and had smaller conductances (approximately 25 pS near 0 mV), shorter mean open durations (approximately 70 vs. approximately 350 ms), and more open-channel noise than swelling-induced channels. The two types of currents might arise from separate channel proteins or from a single channel molecule in different states.

  View details for Web of Science ID A1991GK86700015

  View details for PubMedID 1656769

• METHOD FOR RAPID EVALUATION OF TOPICALLY APPLIED AGENTS TO CYSTIC-FIBROSIS AIRWAYS AMERICAN JOURNAL OF PHYSIOLOGY Wine, J. J., KING, V. V., LEWISTON, N. J. 1991; 261 (2): L218-L221

  Abstract

  The proposal is to target a single maxillary sinus for treatment with agents designed to reverse or ameliorate the cystic fibrosis (CF) defect in airway mucosa, with the opposite sinus serving as a control. Selected CF patients have undergone maxillary antrostomy and antibiotic lavage to help relieve severe pulmonary disease and chronically impacted and infected sinuses. After treatment, the mucosa in the maxillary sinuses of these patients are accessible and can be bathed with fluids introduced via the stomas with procedures that restrict the fluid to a single sinus. The ability of an agent to reverse mucosal pathology can therefore be determined easily with the mucosa of the contralateral sinus serving as a control. Electrophysiological properties, amounts and composition of fluid and mucus, immune functions, and bacterial colonization can be measured accurately and repeatedly. The consistent observation that sinus involvement in CF is near universal and bilaterally symmetric offers a unique opportunity for a simultaneous within-subject, double-blinded control paradigm. This approach should speed evaluation of any agent designed to improve airway mucosal function.

  View details for Web of Science ID A1991GC25100098

  View details for PubMedID 1872413

• CYSTIC-FIBROSIS GENE-EXPRESSION IS NOT CORRELATED WITH RECTIFYING CL- CHANNELS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ward, C. L., Krouse, M. E., Gruenert, D. C., Kopito, R. R., Wine, J. J. 1991; 88 (12): 5277-5281

  Abstract

  Cystic fibrosis (CF) involves a profound reduction of Cl- permeability in several exocrine tissues. A distinctive, outwardly rectifying, depolarization-induced Cl- channel (ORDIC channel) has been proposed to account for the Cl- conductance that is defective in CF. The recently identified CF gene is predicted to code for a 1480-amino acid integral membrane protein termed the CF transmembrane conductance regulator (CFTR). The CFTR shares sequence similarity with a superfamily of ATP-binding membrane transport proteins such as P-glycoprotein and STE6, but it also has features consistent with an ion channel function. It has been proposed that the CFTR might be an ORDIC channel. To determine if CFTR and ORDIC channel expression are correlated, we surveyed various cell lines for natural variation in CFTR and ORDIC channel expression. In four human epithelial cell lines (T84, CaCo2, PANC-1, and 9HTEo-/S) that encompass the full observed range of CFTR mRNA levels and ORDIC channel density we found no correlation.

  View details for Web of Science ID A1991FR44800044

  View details for PubMedID 1711224

• BETA-ADRENERGIC SWEAT RESPONSES IN CYSTIC-FIBROSIS HETEROZYGOTES WITH AND WITHOUT THE DELTA-F508 ALLELE PEDIATRIC RESEARCH Johnson, J. P., Louie, E., LEWISTON, N. J., Wine, J. J. 1991; 29 (6): 525-528

  Abstract

  Cystic fibrosis (CF) causes early death for most homozygotes, yet has a carrier frequency among Caucasians of about 4-5%, suggesting a heterozygote advantage. The major defect in the CF gene is a three-base deletion leading to loss of a phenylalanine residue at position 508 (delta F508) that accounts for about 68% of CF alleles in the North American population; the remaining 32% appears to consist of a large assortment of mutations. Sweat secretion in response to beta-adrenergic stimulation is completely lacking in CF homozygotes and is reduced to 1/2 normal in heterozygotes. To determine if this secretory process is affected by different CF alleles, we used the polymerase chain reaction technique with DNA obtained from peripheral leukocytes to determine retrospectively the presence or absence of the delta F508 allele in 20 CF heterozygotes for whom sweat responses to beta-adrenergic stimulation had previously been determined. Twelve of 20 subjects (60%) were positive for the delta F508 mutation. The variance in sweat responses was not reduced in the delta F508 group relative to the non-delta F508 group, but a gender/allele interaction was noted.

  View details for Web of Science ID A1991FN05500001

  View details for PubMedID 1650939

• BASIC ASPECTS OF CYSTIC-FIBROSIS CLINICAL REVIEWS IN ALLERGY Wine, J. J. 1991; 9 (1-2): 1-28

  View details for Web of Science ID A1991GK55600002

  View details for PubMedID 1715811

• PEPTIDE POTENTIATION OF CALCIUM-CHANNEL ACTIVITY CAN BE SEASONALLY VARIABLE JOURNAL OF EXPERIMENTAL BIOLOGY Bishop, C. A., Krouse, M. E., Wine, J. J. 1991; 156: 607-610

  View details for Web of Science ID A1991FC07500038

  View details for PubMedID 1646851

• CYSTIC-FIBROSIS, THE CFTR, AND RECTIFYING C1- CHANNELS INTERNATIONAL WORKSHOP ON THE IDENTIFICATION OF THE CYSTIC FIBROSIS GENE : RECENT PROGRESS AND NEW RESEARCH STRATEGIES Wine, J. J., Brayden, D. J., Hagiwara, G., Krouse, M. E., Law, T. C., Muller, U. J., SOLC, C. K., Ward, C. L., Widdicombe, J. H., Xia, Y. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1991: 253–272

  Abstract

  The human genetic disease cystic fibrosis is caused by a single defective gene on chromosome 7 that codes for a 1480 amino acid protein called the cystic fibrosis transmembrane conductance regulator (CFTR). The defect causes a profound reduction of Cl- permeability in several tissues, which in turn impairs salt absorption and fluid secretion. A 25-80 pS, rectifying Cl- channel has been targeted as the exclusive or primary channel affected in CF. However, we have found no evidence for significant activation or spontaneous activity of this channel in cell-attached patches of normal lymphoblasts or dog tracheal cells. However, in dog tracheal cells, we find lower conductance, linear Cl- channels that are spontaneously active in unstimulated cells and may show increased activity in stimulated cells. Attempts to correlate the expression of mRNA for the CFTR protein in various types of cells with the presence of the rectifying Cl- channel show a lack of correlation: i.e., depolarization-activated rectifying Cl- channesl have been found in excised, inside-out patches from all cell types that we have examined to date, but the CFTR mRNA has so far only been detected in a subset of epithelial cells.

  View details for Web of Science ID A1991BT84Z00025

  View details for PubMedID 1719763

• PEPTIDE COTRANSMITTER POTENTIATES CALCIUM-CHANNEL ACTIVITY IN CRAYFISH SKELETAL-MUSCLE JOURNAL OF NEUROSCIENCE Bishop, C. A., Krouse, M. E., Wine, J. J. 1991; 11 (1): 269-276

  Abstract

  The activity of 2 types of Ca2+ channels (38 and 14 pS in 137 mM Ba2+) in the plasma membrane of the crayfish tonic flexor muscle is modulated by the peptide proctolin. This peptide serves as a cotransmitter in 3 of the 5 excitatory tonic flexor motoneurons and greatly enhances tension after depolarization by the conventional neurotransmitter. Proctolin alone has no effect on these channels, but renders them capable of sustained activity following depolarization. After depolarization induces activity, 5 x 10(-9) M proctolin increases the open probability of the larger channel up to 50-fold due to a marked decrease in the mean channel closed time. There is also at least a 4-fold increase in the percentage of patches with active channels for the large channel and a 2-fold increase for the small channel. Proctolin modulation appears to occur via an intracellular messenger, possibly cAMP. The peptide's effect on channel activity is dose dependent in a manner that parallels its effect on tension. These results indicate that the activation of these channels and the resulting influx of Ca2+ into the muscle fiber play a role in the potentiation of tension in this muscle.

  View details for Web of Science ID A1991ER41700021

  View details for PubMedID 1702465

• CHLORIDE CHANNELS IN CYSTIC-FIBROSIS PATIENTS SCIENCE Wine, J. J., SOLC, C. K. 1990; 247 (4939): 222-222

  View details for Web of Science ID A1990CH52000041

  View details for PubMedID 2152986

• AMINE AND PEPTIDE MODULATION OF A VOLTAGE-SENSITIVE, PLASMA-MEMBRANE CA-2+-CHANNEL IN CRAYFISH SKELETAL-MUSCLE SYMP ON FRONTIERS IN CRUSTACEAN NEUROBIOLOGY Bishop, C. A., Krouse, M. E., Wine, J. J. BIRKHAUSER VERLAG. 1990: 381–387

  View details for Web of Science ID A1990BQ64V00046

• IS REGULATION OF A CHLORIDE CHANNEL IN LYMPHOCYTES AFFECTED IN CYSTIC-FIBROSIS SCIENCE Hagiwara, G., Krouse, M., Muller, U., Wine, J. 1989; 246 (4933): 1049-1049

  View details for Web of Science ID A1989CA77600046

  View details for PubMedID 2479979

• PATCH-CLAMP STUDY OF CULTURED HUMAN SWEAT DUCT CELLS - AMILORIDE-BLOCKABLE NA+ CHANNEL PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY JORIS, L., Krouse, M. E., Hagiwara, G., Bell, C. L., Wine, J. J. 1989; 414 (3): 369-372

  Abstract

  The reabsorptive duct of the eccrine sweat gland has a large transepithelial conductance consisting mainly of a high conductance to Cl- and a smaller, amiloride-blockable Na+ conductance (Bijman and Frömter 1986; Quinton 1986). Cells have been cultured from sweat ducts and their properties previously studied in Ussing chambers (Pedersen 1988) and with microelectrodes (Jones et al. 1988). We have now studied the ion channels present in excised, inside-out patches of human cultured sweat duct cells, and find a marked predominance of linear, 15 pS, amiloride-blockable, low selectivity, Na+ channels. Such channels were seen in 54/92 (59%) of the patches, with up to 7 channels recorded in a single patch. Other channel types were seen at much lower densities. The prevalence of an amiloride-blockable Na+ channel in cultured duct cells clearly distinguishes these cells from cultured sweat gland secretory cells, which lack such a channel.

  View details for Web of Science ID A1989AD67800019

  View details for PubMedID 2550886

• ION CHANNELS IN NORMAL HUMAN AND CYSTIC-FIBROSIS SWEAT GLAND-CELLS AMERICAN JOURNAL OF PHYSIOLOGY Krouse, M. E., Hagiwara, G., Chen, J., LEWISTON, N. J., Wine, J. J. 1989; 257 (1): C129-C140

  Abstract

  Single-channel patch-clamp techniques were used to study the population of apical membrane ion channels in cultured sweat gland secretory cells from normal and cystic fibrosis subjects. Four types of anion channels and two types of cation channels were found. At physiological voltages, anion channels had chord conductances of 10, 18, 24, and greater than 200 pS. All had linear current-voltage relations except the 24 pS channel, which showed outward rectification. Cation channels had chord conductances of 5 and 18 pS, were linear, and were nonselective for a variety of cations. Channel types and proportions were equivalent in control, cystic fibrosis, and cystic fibrosis heterozygote cells. Beyond showing that the distribution of channel types remains unchanged in cystic fibrosis cells, the data provide a basis for comparison with cells cultured under different conditions, with other cell types, and with native tissues.

  View details for Web of Science ID A1989AF82900017

  View details for PubMedID 2473651

• FEEDFORWARD AFFERENT EXCITATION OF PERIPHERAL INHIBITORS IN THE CRAYFISH ESCAPE SYSTEM JOURNAL OF NEUROPHYSIOLOGY Takahata, M., Wine, J. J. 1987; 58 (6): 1452-1467

  Abstract

  1. Each abdominal ganglion of the crayfish contains peripheral inhibitors of the fast flexor muscles. These flexor inhibitors (FIs), which can effectively inhibit tension development in the tailflip powerstroke muscles, are excited by a delayed central pathway from the same giant axons which trigger escape (33). The FIs also received sensory input, which increases in efficacy in the more posterior segments (4), but until now neither the origin of the input nor its central pathways had been well described. We have used intracellular recording and staining techniques to investigate the afferent input onto the two telson flexor inhibitors (F16 and F17), which receive more powerful sensory input than any of their anterior homologs (4). 2. Both F16 and F17 showed a delayed (3.7 ms) compound postsynaptic potential (PSP), which peaked at long latency when any afferent nerve in the abdomen was stimulated. The amplitude of these slow PSPs waned rapidly with repeated stimulation at 1 Hz and was increased by hyperpolarization and decreased by depolarization of the FI. The PSPs are most likely to be mediated chemically, via polysynaptic pathways. 3. When any afferent nerve from the telson was stimulated, both telson FIs showed an additional fast-rising, short-latency (1.4 ms) PSP, which preceded the slow component. This fast component was not produced by afferent nerves innervating any region other than the telson. The fast PSPs of the two FIs were similar, but in F16 the fast component was always subthreshold, whereas in F17 it often elicited an impulse at short latency. 4. The amplitude of the fast component was not affected by changing the membrane potential of the FIs, suggesting electrical transmission. In spite of its short latency, the fast component is unlikely to be mediated monosynaptically, since it was variably present even in the same animal, and occlusion was observed when any two of the four telson nerves that evoked the response were stimulated simultaneously. 5. Although occlusion was seen among responses produced by stimulating afferents from any source, the responses summated linearly with the compound excitatory postsynaptic potential evoked in FI by the lateral giant escape command axons. Thus at least two separate suprathreshold pathways converge onto the telson FIs.

  View details for Web of Science ID A1987L438200015

  View details for PubMedID 3437338

• HYPOSECRETION OF BETA-ADRENERGICALLY INDUCED SWEATING IN CYSTIC-FIBROSIS HETEROZYGOTES PEDIATRIC RESEARCH BEHM, J. K., Hagiwara, G., LEWISTON, N. J., Quinton, P. M., Wine, J. J. 1987; 22 (3): 271-276

  Abstract

  In order to determine if expression of the cystic fibrosis gene can be detected in heterozygotes, we determined sweat responses induced by local stimulation with cholinergic and beta-adrenergic agents for 20 heterozygotes, 19 age- and sex-matched controls, and five subjects with cystic fibrosis. Active sweat glands were counted and sweat droplets were collected in constant bore capillaries and measured optically. Each subject was tested two to six times. The central finding was that the sweat response of carriers was significantly lower than controls to beta-adrenergic stimulation (p = 0.0013, two-tailed t test; p less than 0.02, Mann-Whitney U), while cystic fibrosis homozygotes did not sweat at all. In contrast, the cholinergic sweat responses did not differ between carriers and controls. For both groups the correlation between cholinergic and beta-adrenergic sweating was positive, but a linear regression of beta-adrenergic sweat responses as a function of cholinergic sweat responses yielded slopes that were significantly different for the two groups. The ratio of beta-adrenergic to cholinergic sweating was plotted for each subject; the mean ratio of the carriers was approximately half of the mean for the controls (p = 0.0002 using t test or p less than 0.002 using the Mann-Whitney U). Our results confirm previous studies and provide new evidence that carriers have, on average, a beta-adrenergically stimulated secretory response that is significantly reduced relative to the control response.

  View details for Web of Science ID A1987J768700006

  View details for PubMedID 2889182

• PHYSIOLOGICAL CONSEQUENCES OF A PEPTIDE COTRANSMITTER IN A CRAYFISH NERVE MUSCLE PREPARATION JOURNAL OF NEUROSCIENCE Bishop, C. A., Wine, J. J., Nagy, F., OSHEA, M. R. 1987; 7 (6): 1769-1779

  Abstract

  The pentapeptide proctolin is colocalized with a conventional, conductance-increasing neurotransmitter in 3 of 5 excitatory motoneurons that innervate a posture-related tonic flexor muscle of the crayfish. It is released from these neurons in response to nerve impulses. Nanomolar concentrations of proctolin superfused on the tonic flexor muscle act postsynaptically to potentiate tension generated by a given level of depolarization. Proctolin alone has no detectable effect on muscle tension, nor does it alter the resting membrane potential of the muscle. Proctolin produces no detectable effect on the EPSPs of the 1 proctolinergic motoneuron that was examined. Neurally released proctolin can be selectively depleted from severed motor axons following prolonged, low-frequency stimulation; EPSPs reflecting conventional transmitter release are unaltered by this procedure. After proctolin depletion, tension generated by the motoneuron is greatly reduced. Taken together, these results indicate that the peptide secondary transmitter in this neuromuscular preparation is an important contributor to the magnitude of tension generated by the motoneuron, but since its effect is dependent on the depolarizing EPSPs of the conventional neurotransmitter, it does not contribute to the temporal aspects of tension generation. These aspects are controlled exclusively by the conventional neurotransmitter.

  View details for Web of Science ID A1987H789700018

  View details for PubMedID 3598647

• FREQUENCY CODING OF WATERBORNE VIBRATIONS BY ABDOMINAL MECHANOSENSORY INTERNEURONS IN THE CRAYFISH, PROCAMBARUS-CLARKII JOURNAL OF COMPARATIVE PHYSIOLOGY A-NEUROETHOLOGY SENSORY NEURAL AND BEHAVIORAL PHYSIOLOGY Plummer, M. R., Tautz, J., Wine, J. J. 1986; 158 (6): 751-764

  Abstract

  Nine identified interneurons that originate in the 6th abdominal ganglion were studied with intracellular techniques while activating the receptors presynaptic to them with coherent water vibrations of precisely controlled amplitude and frequency. Each of the interneurons showed a characteristic response to different stimulus frequencies that was consistent from animal to animal. As a first approximation, the cells were categorized as low pass, broad band, and high pass interneurons. Two interneurons classified as low pass interneurons (LPIs) have low thresholds to waterborne vibrations below 100 Hz, are inhibited by stimuli above 100 Hz, and respond maximally to 30 Hz stimuli. Three interneurons classified as broad band interneurons (BBIs) respond maximally to stimuli from 30-60 Hz, but also respond well to oscillations as low as 1 Hz and as high as 80 Hz. This class is heterogeneous, spanning the range between low pass and high pass interneurons. Two interneurons classified as high pass interneurons (HPIs) have very high thresholds to water oscillations below 6 Hz. They respond best to 60 Hz oscillations, above which their responsiveness sharply declines, although they continue to respond weakly up to 400 Hz. Two other neurons, also classified as HPIs, responded with relatively few spikes to the stimuli we used. As a result, they do not show a clear peak responsiveness to a particular stimulus frequency.

  View details for Web of Science ID A1986C948900002

  View details for PubMedID 3735163

• SEGMENTAL DIFFERENCES IN PATHWAYS BETWEEN CRAYFISH GIANT-AXONS AND FAST FLEXOR MOTONEURONS JOURNAL OF NEUROPHYSIOLOGY Miller, L. A., Hagiwara, G., Wine, J. J. 1985; 53 (1): 252-265

  Abstract

  We have used electrophysiological techniques to document segmental differences in the pathways between the giant, escape command axons, lateral giants (LG) and medial giants (MG), and the nongiant, fast flexor (FF) motoneurons. We found no difference in the input from LG and MG axons to FF motoneurons in the posterior (4th and 5th) ganglia. Since flexor motor output in these segments would be inconsistent with the LG-evoked behavior pattern, this finding was puzzling. Electromyographic (EMG) recordings during escape responses by intact unrestrained animals confirm that the FF muscles innervated by the posterior ganglia are not excited during LG-mediated tailflips, but are excited during MG-mediated tailflips. In the 2nd and 3rd ganglia, the command axons fire the FF motoneurons with high probability, in part via electrical excitatory postsynaptic potentials (EPSPs) from premotor neurons, the segmental giants (SG). In the 4th and 5th ganglia, the equivalent pathway is much less effective. Single, directly elicited impulses in SGs in ganglia 2 and 3 fire their respective FF motoneurons with high probability, while those in ganglia 4 and 5 rarely fire FF motoneurons. The command axons fire the SGs reliably in all segments. The amplitude of the SG-evoked EPSP in FF motoneurons is significantly smaller in posterior vs. anterior ganglia. For technical reasons, we are unable to present conclusive evidence on ganglionic variations in FF-motoneuron thresholds. The FF motoneurons receive additional excitatory input from intersegmental interneurons recruited by the command neurons. Motoneurons in ganglia 4 and 5 are excited by large interneurons that do not synapse on motoneurons in ganglia 2 and 3, but this additional input is not sufficient to compensate for the weaker effect of SG input. Unlike the all-or-none segmental differences demonstrated previously for the LG-to-motor giant pathway (24), the SG-to-FF pathway changes gradually, retains significant though subthreshold strength in posterior ganglia, and is common to both LGs and MGs. These features provide opportunities for variation in the spatial patterning of flexion and in the resulting escape trajectories.

  View details for Web of Science ID A1985ABS0300017

  View details for PubMedID 3973660

• NEUROPEPTIDE PROCTOLIN IN POSTURAL MOTONEURONS OF THE CRAYFISH JOURNAL OF NEUROSCIENCE Bishop, C. A., Wine, J. J., OSHEA, M. 1984; 4 (8): 2001-2009

  Abstract

  The neuropeptide transmitter candidate proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) was associated with three of the five excitatory motoneurons innervating the tonic flexor muscles of the crayfish abdomen. Proctolin immunohistochemical staining occurred in cell bodies and axons of these three identified neurons. Stained axon terminals were detected across the entire tonic flexor muscle. Bioassay of extracts of the tonic flexor muscles indicated the presence of 370 fmol of proctolin/muscle or 670 fmol/mg dry weight. Bioactivity was eliminated in muscles in which the tonic flexor motor root was cut 2 months prior to extraction and in muscle extracts pre-incubated with proctolin antiserum. High pressure liquid chromatography purification of tissue extract indicated that all bioactivity in the crude extract was due to authentic proctolin. Our findings suggest that these three cells function as peptidergic motoneurons. A precedent for this is the proctolin-containing postural motoneuron of the cockroach.

  View details for Web of Science ID A1984TE46400008

  View details for PubMedID 6206210

• PLASTICITY OF NON-GIANT FLEXION CIRCUITRY IN CHRONICALLY CUT ABDOMINAL NERVE CORDS OF THE CRAYFISH, PROCAMBARUS-CLARKII JOURNAL OF PHYSIOLOGY-LONDON Lee, M. T., Wine, J. J. 1984; 355 (OCT): 661-675

  Abstract

  We have investigated the pattern of neuronal activity involved in the gradual return of sensory-evoked abdominal flexions in crayfish with chronically transected nerve cords. Recordings were made from eight types of identified neurone that mediate phasic abdominal movements, in a preparation consisting of the isolated abdominal nerve cord and tailfan. Responses of the cells to pinches and dorsiflexions of the tailfan were compared in two groups of animals: animals whose cords had been cut at the thoracic-abdominal junction 4-17 weeks earlier (chronic preparations), and animals whose cords had been cut at the same site either just before the experiment or up to 6 days earlier (acute preparations). Sensory stimuli produced bursts of spikes in 73% of the fast flexor motoneurones impaled in chronic preparations, but never fired these neurones in acute preparations. However, fast flexor motoneurones in both preparations were fired with approximately equal frequency by single impulses in the giant axons, suggesting that the firing thresholds of these motoneurones had not changed. Sensory stimuli also caused spiking in the extensor inhibitor and the flexor inhibitor in chronic preparations; in contrast, responses in the fast extensor motoneurones were always subthreshold and occasionally hyperpolarizing. None of these cells was fired by similar stimuli in acute preparations. Neurones restricted to the giant axon pathways (lateral, medial, segmental and motor giants) were silent during sensory-evoked flexor discharges in chronically transected cords. Flexor discharges were accompanied by intense activity in non-giant axons recorded from the dorsal cord. Two identified, non-giant interneurones with axons in the dorsal cord were substantially depolarized but never fired by sensory input in chronic preparations. Sensory-evoked firing in the fast flexor motoneurones was not abolished by removal of the posterior stump of the nerve cord at the transection site. About 20% of chronic preparations generated cyclic motor output in response to unpatterned sensory stimulation. The pattern of motor activity that develops in chronically transected cords resembles that seen in normal crayfish during non-giant tailflips. Because cord transection permanently isolates the abdomen from rostral neural centres normally required for the generation of such tailflips, the return of co-ordinated motor output in chronically cut cords may result from the sensory activation of non-giant circuitry within the abdominal nervous system.

  View details for Web of Science ID A1984TN67700044

  View details for PubMedID 6238160

• LATERAL INHIBITION MEDIATED BY A NON-SPIKING INTERNEURON - CIRCUIT PROPERTIES AND CONSEQUENCES FOR BEHAVIOR JOURNAL DE PHYSIOLOGIE Reichert, H., Plummer, M. R., Wine, J. J. 1983; 78 (8): 786-792

  View details for Web of Science ID A1983RN48600014

• IDENTIFIED NON-SPIKING LOCAL INTERNEURONS MEDIATE NON-RECURRENT, LATERAL INHIBITION OF CRAYFISH MECHANOSENSORY INTERNEURONS JOURNAL OF COMPARATIVE PHYSIOLOGY Reichert, H., Plummer, M. R., Wine, J. J. 1983; 151 (3): 261-276

  View details for Web of Science ID A1983QY05000002

• COORDINATION OF LATERAL GIANT AND NON-GIANT SYSTEMS IN CRAYFISH ESCAPE BEHAVIOR JOURNAL OF COMPARATIVE PHYSIOLOGY Reichert, H., Wine, J. J. 1983; 153 (1): 3-15

  View details for Web of Science ID A1983RP72100001

• MECHANOSENSORY INTEGRATION IN THE CRAYFISH ABDOMINAL NERVOUS-SYSTEM - STRUCTURAL AND PHYSIOLOGICAL DIFFERENCES BETWEEN INTERNEURONS WITH SINGLE AND MULTIPLE SPIKE INITIATING SITES JOURNAL OF COMPARATIVE PHYSIOLOGY Sigvardt, K. A., Hagiwara, G., Wine, J. J. 1982; 148 (2): 143-157

  View details for Web of Science ID A1982PG64000001

• NEURAL MECHANISMS FOR SERIAL ORDER IN A STEREOTYPED BEHAVIOR SEQUENCE NATURE Reichert, H., Wine, J. J. 1982; 296 (5852): 86-87

  View details for Web of Science ID A1982ND47700053

  View details for PubMedID 7199621

• SEGMENTAL GIANT - EVIDENCE FOR A DRIVER NEURON INTERPOSED BETWEEN COMMAND AND MOTOR NEURONS IN THE CRAYFISH ESCAPE SYSTEM JOURNAL OF NEUROPHYSIOLOGY Roberts, A., Krasne, F. B., Hagiwara, G., Wine, J. J., Kramer, A. P. 1982; 47 (5): 761-781

  Abstract

  1. The giant command neurons for tailflip escape behavior in crayfish have been thought to excite the nongiant fast flexor (tailflip producing) motor neurons (FFs) via monosynaptic connections. We show here that excitation of FFs instead occurs via a bilateral pair of segmental giant neurons (SGs) interposed between the command axons and FFs in each segment. 2. Anatomically, the SGs appear to make numerous contacts with ipsilateral command axons and FFs and fewer contacts contralaterally. In contrast, the command axons have only sparse direct connections to the FFs. An SG has an axon in the ipsilateral first ganglionic root and may be a modified swimmeret motor neuron. 3. Each SG is depolarized well beyond threshold by the firing of an ipsilateral command axon and is depolarized to near threshold by the firing of a contralateral command axon. The synapses between command axons and SGs are electrical and probably rectifying. 4. Each FF is excited to a level near firing threshold by the SG ipsilateral to its axon and is excited weakly by the contralateral SG. The synapses between SGs and FFs are electrical and nonrectifying. 5. Variations in excitatory postsynaptic potentials (EPSPs) recorded in FFs during prolonged, high-frequency firing of the command axons can be accounted for by refractoriness of SG spikes, as opposed to refractoriness of dendritic branch spikes as had previously been delivered. 6. These findings illustrate the limitations of conventional tests for monosynapticity. 7. The functional significance of having driver neurons interposed between command neurons and motor neurons is discussed.

  View details for Web of Science ID A1982NP25500001

  View details for PubMedID 7086468

• LOCAL INTERNEURONS IN THE TERMINAL ABDOMINAL-GANGLION OF THE CRAYFISH JOURNAL OF COMPARATIVE PHYSIOLOGY Reichert, H., Plummer, M. R., Hagiwara, G., Roth, R. L., Wine, J. J. 1982; 149 (2): 145-162

  View details for Web of Science ID A1982PW30200001

• CRAYFISH ESCAPE BEHAVIOR - NEURO-BEHAVIORAL ANALYSIS OF PHASIC EXTENSION REVEALS DUAL SYSTEMS FOR MOTOR CONTROL JOURNAL OF COMPARATIVE PHYSIOLOGY Reichert, H., Wine, J. J., Hagiwara, G. 1981; 142 (3): 281-294

  View details for Web of Science ID A1981LT49400001

• INTERNEURONS BETWEEN GIANT-AXONS AND MOTO-NEURONS IN CRAYFISH ESCAPE CIRCUITRY JOURNAL OF NEUROPHYSIOLOGY Kramer, A. P., Krasne, F. B., Wine, J. J. 1981; 45 (3): 550-573

  Abstract

  1. Crayfish giant fibers are generally believed to generate tailflip movements by means of direct connections to two classes of phasic flexor muscle motoneurons, the motor giants (MoGs) and the nongiant fast flexor motoneurons (FFs). It is shown here that the giants also stimulate a network of interneurons that make connections with the FFs. 2. This network includes an intraganglionic neuron, the segmental giant (SG), in each abdominal hemisegment and a number of intersegmental neurons, two of which (I2 and I3) were studied in detail. 3. The SGs are driven reliably by the giant fibers and they in turn drive the FFs of their hemisegment about as effectively as do the giant fibers themselves; it is possible that the giant fibers excite the FFs mainly by way of the SGs. The SGs also have an efferent first root axon whose peripheral targets we have been unable to determine. 4. I2 and I3 originate in the second and third abdominal ganglia, respectively, and descend to the last ganglion. In their ganglia of origin they are reliably driven by the giant fibers and by the SGs. In addition, I2 weakly excites I3 and both receive weak, apparently direct, excitatory input from FFs as well as less direct excitatory and inhibitory input from unidentified afferent sources. Both weakly excite most FFs in ganglia behind the one in which they originate. This excitation adds to that produced directly by giant fibers and SGs and, we believe, is sometimes decisive in causing FF firing. Their firing also causes inhibition involved in suppressing effects of reafference, as do the giant fibers themselves. 5. I3 strongly excites the motoneurons of certain tail fan muscles (the ventral and posterior telson flexors). However, the contraction of these muscles would be maladaptive during some giant fiber-mediated tailflips. Accordingly, when the giant fibers, which always recruit I3, fire, they cause an inhibition of the motoneurons that nullifies the excitatory input from I3. At a formal level this means that the giants, viewed as command neurons, not only drive but also alter or modulate the subordinate motor pattern-generating network that they control. 6. Tailflips that are less stereotyped than those mediated by giant fibers are known to occur without participation of the giants. It is suggested that the presence of complex circuitry mediating between giant fibers and FFs may be related to the use of portions of this circuitry as well as the FFs themselves in production of nongiant tailflips.

  View details for Web of Science ID A1981LH57400011

  View details for PubMedID 7218014

• CRAYFISH ESCAPE BEHAVIOR .1. STRUCTURE OF EFFERENT AND AFFERENT NEURONS INVOLVED IN ABDOMINAL EXTENSION JOURNAL OF COMPARATIVE PHYSIOLOGY Wine, J. J., Hagiwara, G. 1977; 121 (2): 145-172

  View details for Web of Science ID A1977EE00900001

• EXTRINSIC MODULATION OF CRAYFISH ESCAPE BEHAVIOR JOURNAL OF EXPERIMENTAL BIOLOGY Krasne, F. B., Wine, J. J. 1975; 63 (2): 433-450

  Abstract

  Extrinsic systems were shown to control the excitability of the neurones which mediate tail-flip escape in the crayfish. Restraint suppresses the escape mediated by giant fibres and some, but not all, categories of non-giant mediated escape; autotomy of claws increases the excitability of non-giant mediated escape without affecting the lateral giant reflex. The effects of restraint on the lateral giant reflex result from inhibition rather than reduced facilitation. The inhibition descends from thoracic and higher levels, and the lateral giant escape command neurone appears to be its primary target. Inhibition may serve to shift the control of escape behaviour from short latency 'reflex' systems to more flexible 'voluntary' ones which can produce responses at times most opportune for successful escape.

  View details for Web of Science ID A1975AU80100013

  View details for PubMedID 1202134

• HABITUATION AND INHIBITION OF CRAYFISH LATERAL GIANT FIBER ESCAPE RESPONSE JOURNAL OF EXPERIMENTAL BIOLOGY Wine, J. J., Krasne, F. B., Chen, L. 1975; 62 (3): 771-782

  Abstract

  1. Decrement of the lateral giant fibre escape response was studied in intact, restrained, crayfish and in those with the ventral nerve cord transected at the thoracic-abdominal level. 2. Taps (delivered at rates of 1 per 5 min to the abdomen) depressed responsiveness to about 50% of its inital value in 10 trials, for both intact and operated animals. 3. With additional stimulation, responsiveness dropped to near zero for both groups. Recovery was negligible 2 h later, but nearly complete after an additional 24 h rest. 4. Protection against response decrement in this situation was obtained by directly activating the cord giant fibres 30 msec prior to the tactile stimulus. The directly-elicited giant fibre spikes which follow the tactile stimulus do not influence the course of response decrement. 5. The results establish the decrement as centrally mediated habituation, and minimize the role of receptor alterations or descending neuronal influences in the behavioural change. 6. A comparison is made between the properties of hibituation and the homosynaptic depression of afferent to interneurone synapses that is presumed to be the physiological mechanism of habituation in this situation.

  View details for Web of Science ID A1975AG48700019

  View details for PubMedID 1206352