Behm, J. K., G. Hagiwara, N. J. Lewiston, P. M. Quinton and J. J. Wine (1987).
"Hyposecretion of beta-adrenergically induced sweating in cystic fibrosis heterozygotes."
Pediatr Res 22(3): 271-6.  [Full Text]

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.

Hagiwara, G., M. Krouse, U. Muller and J. Wine (1989).
"Is regulation of a chloride channel in lymphocytes affected in cystic fibrosis?"
Science 246(4933): 1049-50.

Joris, L., M. E. Krouse, G. Hagiwara, C. L. Bell and J. J. Wine (1989).
"Patch-clamp study of cultured human sweat duct cells: amiloride- blockable Na+ channel."
Pflugers Arch 414(3): 369-72.

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 Fromter 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.

Krouse, M. E., G. Hagiwara, J. Chen, N. J. Lewiston and J. J. Wine (1989).
"Ion channels in normal human and cystic fibrosis sweat gland cells."
Am J Physiol 257(1 Pt 1): C129-40.

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.

Wine, J. J. and C. K. Solc (1990).
"Chloride channels in cystic fibrosis patients."
Science 247(4939): 222.

Bishop, C. A., M. E. Krouse and J. J. Wine (1991).
"Peptide potentiation of calcium channel activity can be seasonally variable."
J Exp Biol 156: 607-10.

Johnson, J. P., E. Louie, N. J. Lewiston and J. J. Wine (1991).
"Beta-adrenergic sweat responses in cystic fibrosis heterozygotes with and without the DF508 allele."
Pediatr Res 29(6): 525-8.

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.

Solc, C. K. and J. J. Wine (1991).
"Swelling-induced and depolarization-induced C1-channels in normal and cystic fibrosis epithelial cells."
Am J Physiol 261(4 Pt 1): C658-74.

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.

Ward, C. L., M. E. Krouse, D. C. Gruenert, R. R. Kopito and J. J. Wine (1991).
"Cystic fibrosis gene expression is not correlated with rectifying Cl- channels."
Proc Natl Acad Sci U S A 88(12): 5277-81.

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.

Widdicombe, J. H. and J. J. Wine (1991).
"The basic defect in cystic fibrosis."
Trends Biochem Sci 16(12): 474-7.

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.

Wine, J. J. (1991).
"Cystic fibrosis. The mutant protein responds."
Nature 354(6354): 503-4.

Wine, J. J. (1991).
"Basic aspects of cystic fibrosis." [PDF]
Clin Rev Allergy 9(1-2): 1-28.

Wine, J. J., D. J. Brayden, G. Hagiwara, M. E. Krouse, T. C. Law, U. J. Muller, C. K. Solc, C. L. Ward, J. H. Widdicombe and Y. Xia (1991).
"Cystic fibrosis, the CFTR, and rectifying Cl- channels."
Adv Exp Med Biol 290: 253-69.

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.

Wine, J. J., V. V. King and N. J. Lewiston (1991).
"Method for rapid evaluation of topically applied agents to cystic fibrosis airways."
Am J Physiol 261(2 Pt 1): L218-21.

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.

Haws, C., M. E. Krouse, Y. Xia, D. C. Gruenert and J. J. Wine (1992).
"CFTR channels in immortalized human airway cells."
Am J Physiol 263(6 Pt 1): L692-707.

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.

Wine, J. J. (1992).
"No CFTR: are CF symptoms milder?"
Nat Genet 1(1): 10.

Wine, J. J. and S. C. Silverstein (1992).
"Cystic fibrosis. ATP and chloride conductance."
Nature 360(6399): 18.

Brayden, D. J., M. E. Krouse, T. Law and J. J. Wine (1993).
"Stilbenes stimulate T84 Cl- secretion by elevating Ca2+."
Am J Physiol 264(2 Pt 1): G325-33.

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.

Wine, J. J. (1993).
"Cystic fibrosis. Indictment of pore behaviour."
Nature 366(6450): 18-9.

Haws, C., W. E. Finkbeiner, J. H. Widdicombe and J. J. Wine (1994).
"CFTR in Calu-3 human airway cells: channel properties and role in cAMP- activated Cl- conductance."
Am J Physiol 266(5 Pt 1): L502-12.  [Full Text]

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.

Krouse, M. E., C. M. Haws, Y. Xia, R. H. Fang and J. J. Wine (1994).
"Dissociation of depolarization-activated and swelling-activated Cl- channels."
Am J Physiol 267(2 Pt 1): C642-9.  [Full Text]

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.

Luckie, D. B., M. E. Krouse, K. L. Harper, T. C. Law and J. J. Wine (1994).
"Selection for MDR1/P-glycoprotein enhances swelling-activated K+ and Cl- currents in NIH/3T3 cells."
Am J Physiol 267(2 Pt 1): C650-8. [Full Text]

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.

Shen, B. Q., W. E. Finkbeiner, J. J. Wine, R. J. Mrsny and J. H. Widdicombe (1994).
"Calu-3: a human airway epithelial cell line that shows cAMP-dependent Cl- secretion."
Am J Physiol 266(5 Pt 1): L493-501. [Full Text]

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.

Wine, J. J., W. E. Finkbeiner, C. Haws, M. E. Krouse, S. Moon, J. H. Widdicombe and Y. Xia (1994).
"CFTR and other Cl- channels in human airway cells."
Jpn J Physiol 44(Suppl 2): S199-205.

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.

Wine, J. J. (1995).
"Cystic fibrosis: How do CFTR mutations cause cystic fibrosis?"
Curr Biol 5(12): 1357-9.

Haws, C. M., I. B. Nepomuceno, M. E. Krouse, H. Wakelee, T. Law, Y. Xia, H. Nguyen and J. J. Wine (1996).
"DF508-CFTR channels: kinetics, activation by forskolin, and potentiation by xanthines."
Am J Physiol 270(5 Pt 1): C1544-55. [Full Text]

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.

Luckie, D. B., M. E. Krouse, T. C. Law, B. I. Sikic and J. J. Wine (1996).
"Doxorubicin selection for MDR1/P-glycoprotein reduces swelling- activated K+ and Cl- currents in MES-SA cells."
Am J Physiol 270(4 Pt 1): C1029-36.

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.

Reddy, M. M., P. M. Quinton, C. Haws, J. J. Wine, R. Grygorczyk, J. A. Tabcharani, J. W. Hanrahan, K. L. Gunderson and R. R. Kopito (1996).
"Failure of the cystic fibrosis transmembrane conductance regulator to conduct ATP."
Science 271(5257): 1876-9.

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.

Sato, S., C. L. Ward, M. E. Krouse, J. J. Wine and R. R. Kopito (1996).
"Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation."
J Biol Chem 271(2): 635-8.

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.

Wine, J. J. and D. B. Luckie (1996).
"Cell-volume regulation: P-glycoprotein--a cautionary tale."
Current Biol 6(11): 1410-2.

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.

Xia, Y., M. E. Krouse, R. H. Fang and J. J. Wine (1996).
"Swelling and Ca2+-activated anion conductances in C127 epithelial cells expressing WT and DF508-CFTR."
J Membr Biol 151(3): 269-78.

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.

Moon, S., M. Singh, M. E. Krouse and J. J. Wine (1997).
"Calcium-stimulated Cl- secretion in Calu-3 human airway cells requires CFTR."
Am J Physiol 273(6 Pt 1): L1208-19.  [Full Text]

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.

Singh, M., M. Krouse, S. Moon and J. J. Wine (1997).
"Most basal I(SC) in Calu-3 human airway cells is bicarbonate-dependent Cl- secretion."
Am J Physiol 272(4 Pt 1): L690-8.  [Full Text]

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.

Wine, J. J. (1997).
"A sensitive defense: salt and cystic fibrosis."
Nat Med 3(5): 494-5.

Xia, Y., C. M. Haws and J. J. Wine (1997).
"Disruption of monolayer integrity enables activation of a cystic fibrosis "bypass" channel in human airway epithelia."
Nat Med 3(7): 802-5.

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.

Lee, M. C., C. M. Penland, J. H. Widdicombe and J. J. Wine (1998).
"Evidence that Calu-3 human airway cells secrete bicarbonate."
Am J Physiol 274(3 Pt 1): L450-3.  [Full Text]

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.

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

Wagner, J. A., T. Reynolds, M. L. Moran, R. B. Moss, J. J. Wine, T. R. Flotte and P. Gardner (1998).
"Efficient and persistent gene transfer of AAV-CFTR in maxillary sinus."
Lancet 351(9117): 1702-3.

Wine, J. J., D. Glavac, G. Hurlock, C. Robinson, M. Lee, U. Potocnik, M. Ravnik-Glavac and M. Dean (1998).
"Genomic DNA sequence of Rhesus (M. mulatta) cystic fibrosis (CFTR) gene."
Mamm Genome 9(4): 301-5.

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).

Wagner, J. A., A. H. Messner, M. L. Moran, R. Daifuku, K. Kouyama, J. K. Desch, S. Manley, A. M. Norbash, C. K. Conrad, S. Friborg, T. Reynolds, W. B. Guggino, R. B. Moss, B. J. Carter, J. J. Wine, T. R. Flotte and P. Gardner (1999).
"Safety and biological efficacy of an adeno-associated virus vector- cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus."
Laryngoscope 109(2 Pt 1): 266-74.

OBJECTIVE: 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. STUDY DESIGN: A prospective, randomized, unblinded, dose-escalation, within-subjects, phase I clinical trial of AAV-CFTR was conducted. PATIENTS: Ten patients with previous bilateral maxillary antrostomies were treated. MAIN OUTCOME MEASURES: Safety, gene transfer as measured by semiquantitative polymerase chain reaction (PCR), and sinus transepithelial potential difference (TEPD) were measured. RESULTS: 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. CONCLUSION: 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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Wagner, J. A., A. Vassilakis, K. Yee, M. Li, G. Hurlock, M. E. Krouse, R. B. Moss and J. J. Wine (1999).
"Two novel mutations in a cystic fibrosis patient of Chinese origin."
Hum Genet 104(6): 511-5.

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.

Wine, J. J. (1999).
"The genesis of cystic fibrosis lung disease."
J Clin Invest 103(3): 309-12. FULL TEXT

It is remarkable that the two hypotheses of CF airway disease that dominate the present research field
are focused on defects in absorption. Most symptoms of CF such as meconium ileus, loss of pancreatic function, degeneration of the vas deferens, thickened cervical mucus, and failure of adrenergically mediated sweating are attributed to CFTR's role in Cl–-driven fluid secretion. In human lungs the highest levels of CFTR occur in serous cells of submucosal glands and in scattered, non-ciliated surface cells in more distal airways that may also be serous-like. Gland serous cells are abundant sources of bactericidal and antifungal compounds that probably are important for mucosal defenses. Animal and cell culture models of these poorly accessible cells indicate that CFTR is critical for glandular fluid secretion driven by Cl– and HCO3–, and predict that the loss of gland function will exacerbate whatever absorptive defect is eventually found.

The controversy about ASL composition has benefited the CF community by focusing research onto the most malign consequence of CF disease. It has reinforced the need for scrupulous reporting of cell culture properties, for continued development of better model systems, and for novel approaches to in vivo measurements. It has emphasized that molecular/cellular methods are not sufficient for understanding many human diseases — we neglect organ system physiology at our peril. Finally, individuals directly affected by CF can take comfort in the knowledge that the insights produced by these studies are spawning a host of innovative treatments for CF lung disease, including increased use of inhaled antibiotics and
renewed approaches to enhance CF lung clearance.

Glavac, D., M. Ravnik-Glavac, U. Potocnik, M. Dean and J. Wine (2000).
"Screening methods for cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in non-human primates."
Pflugers Arch 439(3): R12-3.

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.

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

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. 

Wine, J. J., E. Kuo, G. Hurlock and R. B. Moss (2001).
"Comprehensive Mutation Screening in a Cystic Fibrosis Center."
Pediatrics 107(2): 280-286. [Full Text]

Objectives and Background. 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. Study Design. 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. Results. 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. Conclusion. 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.

Wu, J. V., N. S. Joo, M. E. Krouse and J. J. Wine (2001).
"Cystic Fibrosis Transmembrane Conductance Regulator Gating Requires Cytosolic Electrolytes."
J Biol Chem 276(9): 6473-6478. [Full text]

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.

Luckie, D. B., C. N. Singh, J. J. Wine and J. H. Wilterding (2001).
“CFTR activation raises extracellular pH of NIH/3T3 mouse fibroblasts and C127 epithelial cells.”
J Membr Biol 179(3): 275-84. [Full Text]

 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.

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 µl of fluid accumulated in spherical droplets at gland orifices in 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 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. 

Joo, Nam Soo,  Mauri E. Krouse, Jin V. Wu, Yamil Saenz, Sujatha Jayaraman, Alan S. Verkman, Jeffrey J. Wine  (2001)  "HCO3- Transport in Relation to Mucus Secretion from Submucosal Glands"
J. of the Pancreas (Online) 2(4 Suppl):280-284.  [Full Text]

The role of HCO3- 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 HCO3- transport to gland secretion, we either inhibited Na+/K+/2Cl- cotransporter (NKCC)
with bumetanide or replaced HCO3- with HEPES and gassed with O2. Bumetanide caused a small,
non-significant inhibition of basal secretion, but removal of HCO3-/CO2 significantly reduced basal secretion
almost by half. Both bumetanide and removal of HCO3-/CO2 reduced carbachol-stimulated secretion
significantly, with HCO3- 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 HCO3- transport is important in
the formation of mucus secretion, but that most HCO3- is scavenged before the final mucus appears at the
duct opening.

   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.  Full Text

    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. min^-1. gland^-1, 123 glands) in tissues up to 9 h post-harvest and at lower rates for up to 3 days. Carbachol (10 mM) stimulated secretion with an early transient and a sustained or oscillating phase. Peak secretion was 15.7 +/- 1.2 nl. min^-1. gland^-1 (60 glands); sustained secretion was 4.5 +/- 0.5 nl. min^-1. gland^-1 (10 glands).  Isoproterenol and phenylephrine (10 mM each) stimulated only small, transient responses. We confirmed that cats have a large secretory response to phenylephrine (11.6 +/- 3.7 nl. min^-1. gland^-1, 12 glands), but pigs, sheep, and humans all have small responses (<2 nl. min^-1. 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 not normal, suggesting the existence of subpopulations of glands.

 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.min^-1 gland(-1) was observed 1-9 h post-harvest but was near zero on day 2. Secretion to carbachol (10 mm) peaked at 2-3 min and then declined to a sustained phase. Peak secretion was 12.4 +/- 1.1 nl.min^-1 gland^-1; sustained secretion was approximately one- third of peak secretion. Thapsigargin (1 mm) increased secretion from 0.1 +/- 0.05 to 0.7 +/- 0.2 nl.min^-1 gland^-1; thapsigargin did not cause contraction of the trachealis muscles. Isoproterenol and phenylephrine (10 mm each) were ineffective, but vasoactive intestinal peptide (1 mm) and forskolin (10 mm) each produced sustained secretion of 1.0 +/- 0.5 and 1.7 +/- 0.2 nl.min^-1 gland^-1, respectively. The density of actively secreting glands was 1.3/mm². 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.

 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.

    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 micro l chamber above the apical surface of epithelial cells on a Costar filter with a small hole leading to an oil-filled reservoir. After priming 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-HCO3(-) buffer at 37 degrees C. Basal Jv was 2.7 +/- 0.1 micro l/cm(2)/hr (n=42), and TEP was -9.2 +/- 0.6 mV (n=33); both measures were reduced to zero by ouabain (n=6). Jv/TEP were stimulated 64/59% by 5 micro M forskolin (n=10), 173/101 % by 1 mM 1-EBIO (n=5), 213/122 % by 333 nM thapsigargin (n=5), and 520/240 % by combined forskolin and thapsigargin (n=6). Basal Jv/TEP were inhibited to 82/63 % with 10 micro M bumetanide (n=5), 71/82% with 100 micro M acetazolamide (n=5), and 47/56% with 600 micro M glibenclamide (n=4). Basal Jv/TEP were 52/89 % of control values after HCO3(-) replacement with HEPES (n=16). The net [HCO3(-)] of the secreted fluid was close to bath (25 mM) except when stimulated with forskolin or VIP, when it increased (~80 mM). These results validate the use of the VG apparatus and provide the first direct measures of Jv in Calu-3 cells.

 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.

 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 (VIP) or forskolin, which increase [cAMP]i. This defect is attributed to gland serous cells, which express CFTR. Calu-3 cells, which mimic many features of serous cells, secrete Cl(-) and HCO3(-), with HCO3(-) 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 HCO3(-) in response to forskolin. Unexpectedly, HCO3(-) secretion continued in response to K(+)-channel openers, with Cl(-) secretion being added to it. Secretion of HCO3(-) from hyperpolarized cells occurs via the conversion of CO2 to HCO3(-), and is reduced by about 50% with acetazolamide. A gap between the base equivalent current and ISC was observed in all experiments, and was traced to secretion of H(+) via a ouabain-sensitive, K(+)-dependent process (H(+)-K(+)-ATPase?), which partially neutralized the secreted HCO3(-). The conjoint secretion of HCO3(-) 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.

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 CFTR and Kir present, we observed 79 CFTR 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 to 50 mV; these magnitudes and their ratio of ~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 mM 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.

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