Alternate pathways for chloride conductance activation in normal and cystic fibrosis airway epithelial cells HSIAO-CHANG

CHAN,

JAY GOLDSTEIN,

AND DEBORAH

J. NELSON

Departments of Medicine and Neurology, University of Chicago, Chicago 60637; and Department of Medicine, University of Illinois, Chicago, Illinois 60612 Chan, Hsiao-Chang, Jay Goldstein, and Deborah J. Nelson. Alternate pathways for chloride conductance activation in normal and cystic fibrosis airway epithelial cells. Am. J. Physiol. 262 (CeLI Physiol. 31): C1273-C1283, 1992.-Using whole cell patch-clamp and perforated patch recording techniques on human cystic fibrosis (CF) and non-CF airway epithelial cells, we sought to determine whether a single Cl- conductance (G,,) could be modulated via different regulatory pathways or whether multiple conductances could be identified. Cl- current in both CF and non-CF cells was activated by cellular swelling as well as by an elevation in intracellular calcium ([Ca”+]i). While the adenosine 3’,5’-cyclic monophosphate (CAMP)-activated GoI was absent in CF cells, its activation in non-CF cells was only observed in the perforated patch configuration at lower temperatures (24°C) or infrequently in the whole cell configuration at elevated temperatures (33°C). Currents activated by all three regulatory pathways were sensitive to the Cl- channel blocker 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS). Further increases in current activation could be produced by cellular swelling after maximal Ca2+ or CAMP-induced current activation. Intracellular application of a peptide inhibitor of Ca2+ -calmodulin-dependent protein kinase selectively blocked the Ca2+ -dependent current activation while leaving the swelling-induced current increase intact. These results are consistent with the presence of multiple anion conductances in both CF and non-CF airway cells. The heterogeneity of the responses to the three regulatory stimuli, however, prevented the correlation of a specific anion conductance with a separate modulatory pathway based on characteristic voltagedependent kinetics and conductance. chloride secretion; calmodulin-dependent monophosphate

cell volume regulation; A23187; calciumprotein kinase; adenosine 3’,5’-cyclic

and composition of respiratory tract fluid is both driven and determined by the regulated Clconductance (G& in the apical membranes of airway epithelial cells. Cl- channel regulation has been studied extensively at the single channel level and can be enhanced via adenosine 3’,5’-cyclic monophosphate (CAMP) -dependent protein kinase (PKA) and protein kinase C (PKC) in normal but not in cystic fibrosis (CF) epithelia (13, 19, 21, 22, 33). Recently, it has been reported (6) that Cl- secretion assayed as lz51- efflux can be stimulated in canine tracheal epithelial cells via an elevation in internal Ca2+. The Ca2+-dependent increase in Cl- secretion appears to be independent of prostaglandin synthesis and an increase in the intracellular level of CAMP and suggests either two independent mechanisms for Cl- channel activation or multiple populations of Cl- channels, each responsive to a separate modulatory pathway. Supportive evidence for the existence of at least two independent GoI pathways stems from the observation that Ca2+ ionophore stimulates Cl- secretion in CF airway cells, which are defective in PKA-dependent channel activation (35). Recent whole

THE ELABORATION

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cell patch-clamp studies of normal and CF-derived epithelial cells have demonstrated that Cl- channel activation by Ca2+ is mediated indirectly by multifunctional Ca2+-calmodulin-dependent protein kinase (CaMK; 31, 38). Thus epithelial Cl- channels are the target of three multifunctional protein kinases: PKA, PKC, and CaMK. It is then of tantamount importance to determine whether the specificity of GoI activation in epithelial cells resides in the pathway by which the channel is activated or rather by the presence of multiple conductance pathways. We undertook these studies to compare Go1 activation in CF and non-CF airway epithelial cells via three separate modulatory pathways, namely CAMP, Ca2+, and cell swelling. Previous studies on secretory Cl- conductances in the Cl-secreting colonic epithelial cell line T84 demonstrated that CAMP, Ca2+, and cellular swelling activate Cl- conductances with different and characteristic biophysical properties, i.e., voltage-dependent conductance and time-dependent kinetics (7, 37). Data that we obtained from airway cells demonstrate 1) the additive effects of CAMP, Ca2+, and cellular swelling on whole cell Cl- currents; 2) the selective inhibition of the Ca2+-dependent response by Ca2+-calmodulin-dependent protein kinase inhibitory peptide, which left the swelling-induced current activation pathway intact; and 3) current activation induced by Ca2+ and swelling in CF cells, which are defective for CAMP-induced current activation, consistent with the conclusion that the three secretory pathways activate distinct Cl- conductances in airway as well as T84 cells. Secretagogue-activated whole cell Cl- currents in the airway cells differed considerably from those previously reported for the colonic tumor cell line T84. Our data describing stimulusdependent Cl- current activation in cultured airway cells indicate that multiple Cl- current types characterized by different current vs. voltage (I- V) relationships and time-dependent kinetics can be activated by a single modulatory stimulus. Airway cells studied represented a heterogeneous cell population, and any given airway cell could express multiple anion channel types. Current isolation, as has been reported for T84 cells, may be easier to achieve in that cell line, which is both more homogeneous and may have a simpler array of apical anion channels than airway cells. The studies that we report also revealed that although CF cells responded to both Ca2+ and swelling, their responses appeared to be different from that of non-CF cells; e.g., the magnitude of the secretagogue-dependent current expressing a linear I-V relationship was reduced in CF cells in response to both cellular swelling and an increase in intracellular calcium ([Ca2+]i). Although our studies demonstrate the presence of

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multiple pathways for Cl- permeation that are independently regulated and suggest potential targets for therapeutic intervention circumventing the CAMP-dependent conductance defect in CF, they do not establish channel distribution. Restoration of defective Cl- secretion in CF cells via alternative activation pathways will rest on the determination of a polarized channel distribution to apical membrane sites. MATERIALS

AND

METHODS

Preparation of primary cultures. CF nasal polyps were obtained from eight CF subjects. Nasal specimens were obtained from 16 non-CF subjects and were typically inferior turbinates. The methods for obtaining and enzymatically dispersing human airway epithelial cells were similar to those described by Coleman et al. (8). Briefly, freshly dissected specimens were placed in Eagle’s Ca2+- and Mg2+-free minimal essential media (MEM) (GIBCO, Grand Island, NY) containing 0.1% protease XIV (Sigma, St. Louis, MO), 100 pg/ml deoxyribonuclease (Sigma), 100 U/ml penicillin-streptomycin, 50 pg/ ml gentamicin, 50 ,ug/ml piperacillin, and 50 pg/ml tobramycin (Sigma). After incubation at 4°C for 48 h, 10% fetal bovine serum was added to the dissociation media to neutralize the protease. The dissociated cells were then washed with MEM and passed through a 60-pm nylon mesh filter. The filtered cells were collected by centrifugation at 125 g for IO min. Cells were plated on 35-mm tissue culture dishes (Corning, Corning, NY) coated with 0.02% human placental collagen (Sigma). Cells were maintained in a 1: 1 mixture of Dulbecco’s modified Eagle’s medium (4.5 g/l glucose) and Ham’s F-12 medium. Aliquots of the freshly dissociated cells were suspended in culture medium containing 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen for future use as previously described (28). When needed, the cells were thawed slowly in a 37°C water bath and resuspended in normal growth medium before plating. We noted no difference between the freshly isolated and frozen cells either in their growth in culture, or in their electrophysiological characterization. Electrophysiology. Current recordings were obtained using the whole cell patch-clamp technique as described by Hamill et al. (16) and Schoppa et al. (28), or the perforated patch recording technique as described by Horn and Marty (18). Recordings were performed at room temperature, unless stated otherwise, using a List EPC-7 patch-clamp amplifier (List Electronic, Darmstadt, FRG). Briefly, the dish with the cultured cells was placed in a chamber on the movable stage of an inverted Leitz microscope equipped with phase-contrast optics. Experiments performed at elevated temperature were carried out on a temperature-controlled stage (Brook, Lake Villa, IL). Recording pipettes were formed from soda lime glass (Blue-Dot Hematocrit Glass, Fisher, Pittsburg, PA) using a horizontal puller (model P-87, Sutter Instrument, San Rafael, CA). Pipette tips had a diameter of -0.5 pm. Current recordings were obtained from single nonconfluent cells. Bath perfusion of the cells was performed via a gravity feed superfusion system at a rate of 2 ml/min. Perforated patch recordings used the pore-forming antibiotic nystatin (Sigma). Nystatin-containing pipette solutions were routinely prepared fresh from stock solutions every 3 h. Nystatin stock solutions (50 mg/ml in DMSO) were stored at -4°C for at most 1 wk before use. The tip of the recording pipette was filled with nystatin-free solution; the pipette was then backfilled with nystatin-containing pipette solution at a final concentration of 200 pg/ml. After seal formation, series resistance values decreased with time and reached a steady level of ~40 MS2 between 5 and 45 min.

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Data acquisition. The voltage commands were provided via the output of a Metrabyte digital-to-analog converter; currents were sampled with a Data Translation DT2818 analog-to-digital converter and analyzed using a 386 personal computer. Current records were filtered at 1 kHz and sampled at 2 kHz unless stated otherwise. Currents were not leak or capacity corrected. Cell capacitance was measured by integrating the current during a lo-mV voltage step and subtracting a baseline established -15 ms after the step. The selectivity of the various secretagogue-activated currents among halide anions was determined in experiments in which bath N-methyl-D-glucamine (NMDG)Cl was replaced by equimolar NMDG-I or NMDG-Br. In those experiments in which the bathing solution was changed, the ground electrode was connected to the bath solution via an agar salt bridge to minimize electrode junction potentials. Where a multiple number of experiments were performed for a given experimental condition, data are expressed as means t SE, with the number of experiments in parentheses. Significance between two populations was determined using Student’s t test. Solutions. The bath solution contained (in mM) 140 NMDGCl, 2 CaCl,, 2 MgC12, and 10 N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid (HEPES) buffered to pH 7.3. The pipette solution contained (in mM) 40 NMDG-Cl, 100 NMDGglutamate, 5 ethylene glycol-his@-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA), 0.5 CaCl, (20 nM calculated free Ca2+), 2 MgC12, and 10 HEPES; pH = 7.2. ATP (1 mM) was added to the pipette solution in those experiments investigating the effects of Ca2+ and CAMP. ATP was excluded from the pipette solution in the volume-regulation studies. In the Ca2+- and CAMP-activation experiments, the pipette and bath solutions were isosmotic (280 t 10 mosM). These solutions did not give rise to spontaneous current increases, as has been reported by Worrell et al. (37) for T84 cells in similar studies. Solution osmolarities were monitored using a vapor pressure osmometer (model 5500, Wescor, Logan, UT). 4,4’-Diisothiocyanostilbene-2,2’-disulfonic acid (DIDS), calcium ionophore A23187, and the nonhydrolyzable CAMP analogue 8-(4-chlorophenylthio)-CAMP (C-CAMP) were purchased from Sigma. CaMK inhibitory peptide was a generous gift from Dr. H. Schulman, Stanford University. RESULTS

Basal whole cell currents. Voltage-clamp experiments were performed on nonconfluent, single cells that had been maintained in culture from 2 to 5 days. No significant difference in capacitance was observed between CF (51 t 4 pF, n = 29) and non-CF cells (48 t 2 pF, n = 46) over the same culture period. Whole cell currents were elicited by applying hyperpolarizing and depolarizing voltage pulses from a holding potential of -40 mV to potentials between -110 and +llO mV. Experiments were performed using pipette and bath solutions containing NMDG as a Na+ and K+ replacement, and Cl- as the major permeant ion. Experiments were performed in asymmetrical Cl- solutions (Nernst potential for Cl- in these studies was -31 mV), thereby allowing us to identify an increase in the leak conductance as a depolarizing shift in the zero current potential. A total of 51 CF and 79 non-CF cells were included in the study. In 32% of the CF and 59% of the non-CF cells examined, the initial current records taken 1 min after the establishment of the whole cell configuration exhibited a time-dependent component of outward current that could be observed at depolarizations >50 mV (Fig. 1A). In 74% of all the non-CF cells

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examined (n = 79), whole cell currents exhibited a decrease in amplitude with time (Fig. 1B). We observed an average decrease in peak current amplitude of 61 t 11 pA at -90 mV and 142 t 23 pA at 50 mV over a time period of 15 min. In contrast, only 45% of all the CF cells A lOOO- .

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Fig. 1. Comparison of time-dependent changes in Cl- current amplitude after establishing whole cell recording conformation in cystic fibrosis (CF) and non-CF (normal) cells. A: initial currents (1 min after establishment of whole cell conformation) from a non-CF cell were elicited by applying hyperpolarizing and depolarizing voltage pulses from a holding potential of -40 mV to potentials between -110 to +llO mV. B: currents recorded 15 min later; currents remained stable at this level. Experiments were performed on cells exposed to the N-methyl-D-glucamine (NMDG)-Cl bath solution for 4 h. Current “rundown” was less prominent in cells exposed to external NMDG-Cl for longer incubation periods. C: corresponding peak current-voltage relationship for the nonCF cell for currents depicted in A and B. Current-voltage relationship in C is representative of what was seen in 80% (63/79) of the non-CF cells and 57% (29/51) of the CF cells studied. Peak current was measured at 20 ms after onset of voltage pulse. Note the loss of outward rectification after current rundown. D: current-voltage relationship over the same time period for a CF cell (raw data not shown). Current-voltage relationship in D was observed in 20% (16/79) of non-CF cells and 43% (22/51) of CF cells examined.

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examined (n = 51) showed demonstrable time-dependent changes in membrane current, with an average decrease in current amplitude of 39 t 13 pA at -90 mV and 68 t 24 pA at 50 mV, values considerably lower than those observed in non-CF cells. The nonstimulated I-V relationship was outwardly rectifying in 80% of the non-CF cells (see Fig. 1C) and 57% of the CF cells examined. The remaining cells exhibited a linear I-V relationship. The magnitude of the initial current and the zero current potential of the initial recordings varied over a wide range from cell to cell. After 15 min equilibration, however, the zero current potential was -23 t 1 mV in non-CF cells and -24 t 1 mV in CF cells (theoretical Cl- equilibrium potential = -31 mV). The magnitude of both outward and inward current after equilibration was lower in the CF cells compared with non-CF cells (-65 t 6 and -95 t 8 pA at -90 mV, and 81 t 7 and 123 t 9 pA at 50 mV for CF and non-CF cells, respectively; P < O.Ol), indicating a somewhat lower basal Cl- permeability in CF cells. Cells were equilibrated in bath recording solution for periods of 30 min to 1 h at room temperature before each experiment in an attempt to minimize the presence of antecedent baseline currents [similar equilibration periods have been reported by Rich et al. (26)]. To control for time-dependent changes in baseline currents after establishing the whole cell configuration, all cells were equilibrated in whole cell mode for 15 min before studies on secretagogue-induced Gel. Volume-sensitive conductance. A volume-regulated GoI has been reported in human colonic T84 cells (37)) cultured human airway epithelial cells (24), and cell lines derived from normal and CF airway epithelia (31). We compared the magnitude of the volume-regulated Got in CF and non-CF airway cells. Current recordings were made before and after the sequential exposure of a cell, in either the whole cell or perforated patch configuration, to a bathing solution in which the osmolarity had been reduced from 280 to 180 mosM (Fig. 2A) by a decrease in the bath NMDG-Cl concentration. The response to osmotic challenge in both CF and non-CF cells was similar in both the whole cell and perforated patch configuration. In that data obtained using the two techniques were similar, no further distinction was made in the analysis. All cells studied, both CF and non-CF cells, responded to osmotic challenge with current activation. The volume-regulated current was outwardly rectifying (Fig. 2B) and inactivated during depolarizing voltage pulses to potentials greater than +50 mV in 67% of the CF cells (18 of 27) and 68% of non-CF cells (23 of 34) examined. Currents with a linear I-V relationship and time-independent kinetics were observed in the remaining cells. The volume-regulated current could be inhibited variably by the Cl- channel blocker DIDS (50 PM), as summarized in Fig. 2C. A significantly larger DIDS-induced current inhibition at +50 mV was seen for the outwardly rectifying currents (84 t 6%) n = 8) compared with inhibition seen for the linear currents (53 t 18%, n = 4)

Although Cl- currents in both CF (n = 18) and non-CF cells (n = 23) showed an similar increase in the magnitude of outwardly rectifying current in response to hypos-

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Fig. 3. Comparison of swelling-induced Cl-- current activation in CF and non-CF cells. Mean changes in peak current amplitudes at +50 mV induced by exposing CF and non-CF cells to hypotonic solutions are plotted as a function of their current-voltage characteristics (linear and rectified). Magnitude of swelling-induced current exhibiting a linear current-voltage relationship was attenuated in CF cells over that observed for non-CF cells.

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Fig. 2. Swelling-induced Cl- current activation and its inhibition by DIDS. A: current recordings before and after sequential exposure of a single voltage-clamped cell to a 90 mM NMDG-Cl (180 mosM) bathing solution. After current activation, cell was superfused with hypotonic solution containing 50 PM DIDS. B: corresponding current-voltage relationship measured 20 ms after onset of voltage pulse. C: DIDS inhibition of swelling-activated conductance as a function of the nature of the associated current-voltage relationship (linear or rectified) plotted as mean peak current amplitude at +50 mV before and after exposure of non-CF cells to hypotonic and DIDS solutions. Current inhibition in response to DIDS in the positive voltage range was significantly larger for rectified than for linear currents. It should be noted that ATP was not added to the pipette solution in the volume regulation studies.

motic challenge (679 t 125 and 640 t 117 pA at +50 mV for CF and non-CF, respectively), the magnitude of the swelling-induced current with a linear I-V in CF cells was reduced over that observed in non-CF cells (Fig. 3). The average increase in linear current amplitude at +50 mV in 9 CF cells was 574 t 187 pA, while the average increase in 11 non-CF cells was 1,840 t 399 pA (P < 0.02). There was no significant difference in capacitance between CF and non-CF cells (see above) that would account for the difference in the magnitude of the swelling-induced linear current. Current activation was reversible over a time

course of l-5 min upon restoration of the bath solution to isosmotic conditions. A depolarizing shift in the zero current potential of 10.93 t 1.36 mV (20 CF and non-CF cells) was observed after a 100 mosM reduction in bathing solution osmolarity (decrease in external Cl- concentration of 50 mM). This is close to the predicted shift of 11 mV for a perfectly selective Gcl, indicating the swellinginduced current was Cl- selective. To determine whether current activation after a reduction in bath osmolarity resulted from cellular swelling or a reduction in Cl- concentration, experiments were performed in which a fraction of the external NMDG-Cl was replaced with an isosmolar amount of sucrose. As shown in Fig. 4, a depolarizing shift in the reversal potential of 10 t 1.5 mV was obtained without associated current activation in experiments in which the Cl- concentration in the bath was lowered from 140 to 80 mM (n = 3). The experimentally observed shift in reversal potential was in good agreement with the predicted shift of 14 mV. Thus a change in cell volume rather than a decrease in external Cl- concentration appears to mediate current activation in response to a reduction in bath osmolarity. I I I

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Fig. 4. Cellular swelling and not a decrease in external Cl- produces Clcurrent activation. Current-voltage relationship obtained before and after exposure of the cell to an external solution in which Cl- concentration was lowered by replacing 60 mM NMDG-Cl with an isosmolar amount of sucrose. No current activation was observed after exposure of the cell to low Cl- solution, indicating that cellular swelling rather than a decrease in external Cl- was responsible for current activation. Reduction of bath Cl- (open squares) decreased outward current amplitude and produced a depolarizing shift in the zero current potential.

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The selectivity of the volume-regulated conductance among halide anions was I- > Br- > Cl-, as determined in experiments in which bath NMDG-Cl was replaced by equimolar NMDG-I or NMDG-Br. Iodide permeability (PI)/Pcl was 2.60 t 0.42, while P&PC1 was 1.56 t 0.09, as shown in Table 1. Ca2+-induced current activation. We studied Ca2+-dependent Gel activation in CF and non-CF cells after exposure to the Ca2+ ionophore A23187 and in the presence of elevated intracellular (pipette) Ca2+. A23187 (5-10 PM), in the presence of extracellular Ca2+, induced significant current activation in both CF and non-CF cells. When the ionophore was introduced in a Ca”+-free solution, no significant current increase was observed (n = 2), indicating that elevated intracellular Ca2+ via the Ca2+ ionophore was responsible for the current activation. Experiments performed on non-CF cells using pipette solutions containing an elevated free Ca2+ concentration of 200 nM resulted in a gradual increase in current across the voltage range after formation of the whole cell configuration. Maximal current levels were observed 10 to 15 min after membrane disruption. Mean peak current amplitude in the presence of 200 nM [Ca2+]i after current equilibration was 799 t 192 pA (n = 9) compared with the mean peak current of 123 t 9 pA at 50 mV (n = 79) in the presence of 20 nM [Ca2+];. Current measurements were obtained 15 min after establishment of the whole cell configuration for both [ Ca2+]i levels. The response of the airway cells to the Ca2+ ionophore varied considerably with respect to both time-dependent kinetics and peak I-V relationship. There was no apparent correlation between basal current amplitude or time -course and either the time-dependent kinetics, the I-V relationship, or DIDS sensitivity of the Ca2+-induced current, as can be seen in the three representative experiments in Fig. 5. Ca2+- induced currents could be elicited from both CF and non-CF cells, which showed either time-dependent activation, time-dependent inactivation, or time-independent kinetics in the depolarizing voltage range. A shift in the kinetic behavior of the Ca2+-induced currents could frequently be observed in single cells over time after activation, as shown in Fig. 6, A and B. In this experiment, current kinetics shifted from delayed activating to inactivating in 16 min after exposure to the ionophore. In some experiments, a shift in current kinetTable 1. Comparative chloride

conductance

halide permeability regulatory pathways.

Volume regulated (n =5) Ca2+ activated (n = 6) CAMP activated (n = 5)

of the three

I-

Br-

2.60t0.42

1.56t0.09 1 A30t0.04 1.43t0.17

2.60t0.31 1.51~0.11

Values are means t SE. Data were obtained from individual reversal potential measurements during ion substitution experiments. Experiments were performed by replacement of the bath solution containing 140 mM NMDG-Cl with an appropriate NMDG salt. The relative permeabilities were determined using the relationship Px/Pcl = [Cl-Ii/ [X-l, exp - (ERF/RT), where Px/Pcl is the permeability ratio of the test anion X relative to Cl-, and ER is the reversal potential. CAMP activation was performed using perforated patch recording techniques; all other experiments were performed in the whole cell configuration.

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its from delayed activating to time independent was also observed. The variability in current kinetic profiles could be due to 1) the presence of multiple anion channel types or 2) a shift in kinetic behavior for a single channel type as a function of either an increase in [Ca’+]i or a Ca2+dependent phosphorylation step. The Ca2+-activated Cl- current was inhibited by DIDS (500 PM), as shown in Fig. 6C. There was no difference in the magnitude of current inhibition produced by DIDS between those cells expressing a linear I-V (91 t 4% current inhibition, n = 4) and those expressing an outwardly rectifying I- V (97 t 4% current inhibition, n = 5). Both CF and non-CF airway cells responded to A23187, as is shown in Fig. 7. There was no significant difference in the amplitude of the Ca2+-sensitive current between CF and non-CF cells expressing an outwardly rectifying I-V relationship (324 t 74 pA at +50 mV, n = 8, and 327 t 91 pA, n = 6, respectively). However, the magnitude of the ionophore-induced current with a linear I-V was reduced in CF cells (108 & 21 pA at +50 mV, n = 3) compared with non-CF cells (1,424 t 165 pA, n = 4). It has been recently demonstrated that Cl- channel activation by Ca2+ is mediated by CaMK and that peptide inhibitors of either the kinase or calmodulin block the Ca2+-dependent activation of Cl- channels (31, 38). We used the CaMK inhibitory peptide, a synthetic peptide containing the autoinhibitory region of CaMK (residues 273-302 of the a-subunit of CaMK) in our experiments. When the inhibitory peptide was present in the pipette solution at a concentration of 20 PM, it did not inhibit the swelling-induced increase in Go1 (Fig. 8, A and B). The CaMK inhibitory peptide, however, inhibited the Ca2+ -dependent increase in current (Fig. 8C), indicating that the Ca 2+-dependent and volume-regulated Go1 appear to be separate. In three similar experiments, exposure of the cells to a hyposmolar solution resulted in a current increase of 706 t 329 pA despite the presence of the CaMK inhibitory peptide. Subsequent exposure of the cells to A23187 (10 PM) after recovery of the current to preswelling levels in isosmolar bathing solution in the presence of CaMK inhibitory peptide resulted in a current increase of only 57 t 21 pA. The additive nature of the two conductance pathways was demonstrated in experiments done in the absence of the inhibitory peptide. In three cells in which a maximal Ca2+-dependent current increase had been obtained, an additional increase in current magnitude (80% to 280%) was elicited by cell swelling. The relative halide permeability of the Ca2+-activated GoI was I- > Br- > Cl-, as given in Table 1. In these experiments, current activation was obtained in the presence of 10 PM A23187. The order of the anion permeability sequence was identical to that observed for volumeregulated Gel. Current modulation by cAA4P. Exposure of voltageclamped cells to the nonhydrolyzable CAMP analogue C-CAMP (0.5 and 1 mM) at room temperature in the whole cell recording mode failed to activate current in both CF cells (n = 12) and non-CF cells (n = 7). Therefore, current recordings were performed using the perforated patch recording technique, which has been reported

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Fig. 5. Heterogeneity of Ca2+-activated Cl- current and associated inhibition by Cl- channel blocker DIDS. Current recordings before and after exposure of 3 separate non-CF cells to the Ca2+ ionophore A23187 (10 PM) and DIDS (500 PM). A, B, and C represent whole cell currents with associated current-voltage relationships: solid diamonds, control currents; solid circles, currents after A23187 activation; open squares, currents in the presence of A23187 and DIDS.

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to reduce the dilution of cytoplasmic factors and the decay of membrane excitability commonly seen during conventional whole cell recording (18). In 15 of 21 nonCF cells in which perforated patch recordings were performed, an increase in current amplitude of 60-450% (198 + 50%) at +50 mV was observed in response to C-CAMP. YVe failed to observe a similar C-CAMP-dependent increase in current amplitude CF cells (n = 5) in perforated patch recordings. A comparison of C-cAMP-dependent current activation obtained from both whole cell and perforated patch recordings in CF and non-CF cells is presented in Fig. 9. Note that at room temperature, significant CAMP-dependent current activation was observed only in perforated patch experiments in non-CF cells. As has been reported for T84 cells (4, 7), CAMPinduced current activation could be elicited in the whole cell recording mode in non-CF airway cells at elevated temperature (33°C). However, the frequency of observing current activation under these conditions was only 15% (4 of 27 cells). The I- V relationship for the CAMP-activated current exhibited strong outward rectification in all of the perforated patch experiments (similar to that seen in Fig. 10). Of the 15 responding non-CF cells, 75% showed time-dependent current inactivation during depolarizing voltage pulses to potentials greater than +50 mV. The CAMP-dependent currents observed in the remaining cells were time-independent. CAMP-dependent current in the perforated patch experiments was inhibitable by DIDS (50 PM) (79 t lo%, n = 3). Currents induced by CAMP at elevated temperature in whole cell configuration exhibited a linear I-V and time-independent kinetics. In one experiment, perfusion of DIDS (50 PM) did not block the CAMP-activated current elicited in

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whole cell configuration. Further increases in current amplitude could be elicited by cell swelling in non-CF cells that had responded to CAMP in the perforated patch configuration as shown in Fig. 11. In those CF cells that did not respond to CAMP, current activation could be elicited by cellular swelling. These results suggest the presence of separate pathways for CAMP- and swelling-induced Go1 activation. The selectivity sequence among the halide anions for the CAMP-activated Go1 was determined only in the perforated patches (because of the low frequency of current activation in whole cell experiments) and was similar to that observed for the Ca 2+-dependent and volume-regulated conductance pathways. However, the relative halide permeability of the CAMP-dependent current differed considerably from both the volume and Ca2+-regulated pathway; PI/& was 1.51 t 0.11, and &J& was 1.43 t 0.17 (n = 5). DISCUSSION

Although long established that Cl- secretion in airway epithelia is mediated via a number of regulatory pathways (1, 5, 6, 35), the presence of multiple Cl- conductances has not been clearly shown. Multiple populations of Clchannels have been identified and characterized in a number of single-channel studies carried out on airway epithelial cells. Frizzell and co-workers (13, 14) identified Cl- channels of 20 and 50 pS in conductance in human airway epithelia both activated by [Ca2+]; and CAMP. It remains unclear as to why channel activation in the studies of Frizzell et al. (13,14) was obtained directly through elevation of Ca2+ at the internal surface of the membrane and was not observed in similar studies carried out by

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IN AIRWAY

0

a=

m

non-CF SEM

I

0

200

400

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200

Cl279

EPITHELIA

400

Time (ms)

B 4000

T

I--l n=3

0 .

I

n=4

n=8 Rectified

Linear

0

A23187

(16 min)

A23187

(9 min)

Fig. 7. Comparison of Ca2+- induced Cl- current activation in CF and non-CF cells. Mean changes in peak current at +50 mV induced by exposing CF and non-CF cells to A23187 (10 PM) are plotted as a function of their current-voltage characteristics (linear and rectified). Magnitude of Ca2+ -induced current exhibiting a linear current-voltage relationship was attenuated in CF cells over that observed for non-CF cells.

0

A

6

C

Control

180

4000

4OOOT)

2000

2oooi

mOs

f

-2000

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tI -60 Membrane

1 0 Potential

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(mV)

P b 3000

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3 E

2Q Y E a k 3

b

I-

t

2000

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s

CZI Control tZZA23187

-2000

.2000 0

2400

280 mOs 1OuM A23187

4000T

2 I

n=6

t

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D

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TIME 3000

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l--k-+ , 0

;

;

200

, 400

(ms)

I

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;

180

mOs

2000 1200

2 f

600

1000

i5 E

Control 10&M

A23187

0

s

0 Rectified

(n=5)

Linear

(n=4)

Fig. 6. Ca2+-induced Cl- current activation and its inhibition by DIDS. A: current recordings before and after sequential exposure of a single voltage-clamped cell to the Ca2+ ionophore A23187 (10 PM) and DIDS (500 PM). Note time-dependent change in current kinetics after exposure of the cell to A23187 (9 and 16 min). B: corresponding currentvoltage relationship. C: DIDS inhibition of Ca2+-activated currents (linear and rectified) plotted as mean peak current amplitude at +50 mV before and after exposure of non-CF cells to A23187 and DIDS solutions.

Welsh (33) and Clancy et al. (6). Shoemaker et al. (29) were able to differentiate between channels of 10, 20, 40, and 250 pS, each characterized with varying degrees of anion selectivity, in cells that had been preswollen in isotonic KC1 solutions. An outwardly rectifying Cl- channel with a conductance at 0 mV of between 20 and 50 pS that is activated by both CAMP-dependent PKA and PKC has been described in detail in a number of studies (13,19,21,22,33). More recently, Duszyk et al. (10) have described a 20 pS Cl- channel to be the most commonly found apical membrane channel in unstimulated human airway cells. Thus, although single channel studies adequately demonstrate the multiplicity of Cl- channels present in the apical membranes of airway epithelial cells, no strong correlation exists between activation pathwav

-1000

-2000

I

-120

-80

8

-40 MEMBRANE

I I

1 r

0

40

POTENTIAL

1 I

80

I

120

(mV)

Fig. 8. Ca2+ -activated but not volume-regulated current activation is inhibited after intracellular dialysis with CaMK inhibitory peptide. A: current recording made before exposure of the cell to a hypotonic bathing solution (180 mosM) (B). C: after recovery of the current to preswelling levels in isosmolar bathing solution (-5 min) currents were recorded in response to a solution containing 10 PM A23187. D: corresponding current-voltage curves for currents in A-C.

and specific channel type. We demonstrated that in both whole cell and perforated patch experiments, Cl--selective currents could be activated in human airway cells, both in CF and non-CF, through at least three separate regulatory pathways. However, we were unable to link a given regulatory stimulus with the activation of a single current type that could be characterized by specific kinetic properties, e.g., time dependence of current activation or inactivation, or by the nature of the I- V relationship as reported in T84 cells. This apparent lack of correlation could be due to 1) the

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Cl280

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IN AIRWAY

2000

A

CF

Control

EPITHELIA mM C-CAMP 1 lOOr

T

1500 0 Control tZ?ZI After CAMP I SEM

2 Q :1000 iiL

1 mM C-CAMP 180 mOs,

-r

5 0 500 B

Normal 3000

2

0

w-c

perf

w-c 1

I

perf

W-C/high

L

T 1

CF

NC

Fig. 9. Comparison of CAMP-induced Cl- current activation in whole cell vs. perforated patch recordings in CF and non-CF (NC) cells at room temperature. Results from whole cell experiments in non-CF cells at elevated temperature (33°C) are also plotted. Mean current amplitude at +50 mV was compared before and after exposure of voltage-clamped cells to the nonhydrolyzable membrane-permeant analogue of CAMP, 8-(4-chlorophenylthio)-CAMP (C-CAMP, 1 mM). Initial current amplitude was determined after current stabilization. Note that significant current activation was observed only in perforated patch experiments and whole cell experiments at elevated temperature in non-CF cells. Mean current amplitude is plotted only for C-CAMP responding cells in the perforated patch (15 of 21) and in the whole cell experiments carried out at elevated temperatures (4 of 27) on non-CF cells; mean current amplitude is plotted for all cells examined in the case of both whole cell and perforated patch experiments on CF cells and for the whole cell experiments carried out at room temperature on non-CF cells.

A

Control

6

2 1000 Q CI E 0 5 0

T 8

1mM

C-CAMP

Time

hs)

C

50 AM DIDS

1000

-1000 0

200

400

D 1 mM C-CAMP

800 2 -Q

Control 50~M

800

DIDS

0 -200 I

1

1 0

-60 Membrane

Fig. 10. DIDS inhibition recordings before (A) and 1 mM C-CAMP (B) and sponding current-voltage

Potential

1 60

I 120

(mV)

of CAMP-activated current. Perforated patch after sequential exposure of the non-CF cell to 1 mM C-CAMP + 50 PM DIDS (C). D: correrelationship for currents in A-C.

heterogeneity of the airway cell population, 2) the presence of multiple Cl- channel types in any given airway cell, which are not expressed in the colonic cell line, or 3) simultaneous activation of a number of regulatory/anion conductance pathways via a single secretory stimulus. CAMP-dependent Cl- current activation. Evidence

2000

U

& L 05

1000 0 I 0

!

Time

! 200

!

1 -1000 400

I 0

!

! 200

!

I 400

(ms)

Fig. 11. Additive effects of CAMP and cellular swelling on Cl- current activation in non-CF vs. CF cells. Perforated patch recordings before and after sequential exposure of CF (A) and normal cell (B) to C-CAMP (1 mM) and a dilute external NMDG-Cl(180 mosM) solution. Note that exposure of the CF cell to C-CAMP failed to induce an increase in Clconductance; significant current activation was seen after exposure of the cell to dilute NMDG-Cl solution. A decrease in solution osmolarity in the non-CF cell after C-CAMP current activation produced a further increase in Cl- conductance.

from single-channel studies has indicated that the regulation of apical Cl- channels via PKA and PKC is defective in CF cells (19,21, 22). Whole cell current activation in human airway cells via CAMP in our studies is consistent with data obtained in the single-channel studies; namely, CAMP-induced GoI was observed only in non-CF cells and not in CF cells. The fact that the CAMP-induced Get activation in non-CF cells could be elicited at room temperature only in perforated patch recordings and in whole cell configuration only at elevated temperatures suggests the PKA activity in the airway cells is tightly regulated and highly sensitive to cytoplasmic dialysis. The CAMP-activated currents reported recently for T84 cells (7), for cells from normal and CF-derived airway epithelial cell lines (31), for CF cells expressing recombinant cystic fibrosis transmembrane conductance regulator (CFTR) (9, 15, 26), as well as for nonepithelial cells expressing transfected with CFTR (20), all have a relatively linear I-V relationship. This is in contrast to the outwardly rectifying currents that we observed in our perforated patch studies. There are a number of possibilities that could account for the discrepancy: 1) a dialyzable cytoplasmic factor may block Cl- efflux from the cell (inward current) thereby creating the apparent current rectification similar to that described for the internal Mg2+ block of the inwardly rectifying K+ channel (23) and the ATP-sensitive K+ channel (ll), 2) the presence of an asymmetric Cl- gradient (lower internal Cl-), 3) multiple populations of Cl- channels with PKA-dependent activation, or finally, 4) rapid channel deactivation in the hyperpolarized voltage range. The final possibility, that of rapid channel deactivation, would imply that channel gating loses or significantly shifts its voltage dependence in the whole cell voltage-clamp experiments.

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It should be noted that an enhancement of single Clchannel current rectification by HEPES and related buffers has been demonstrated by Hanrahan and Tabcharani (17) in excised patches from the PANC-1 pancreatic cell line. In their studies, HEPES reduced the conductance of the channel by approximately one-half when present on the cytoplasmic side of the membrane. Interestingly, Sullivan et al. (30) have recently reported both linear and outwardly rectifying I- V relationships for the CAMP-activated current functionally expressed in Xenopus oocytes injected with shark rectal gland mRNA in the presence of a standard Cl- gradient. An alternative explanation for the rectification observed in the CAMP-activated current studies could be that C-CAMP is elevating Ca2+ in the airway cells in the perforated patch configuration and that the currents seen are, at least in part, Ca2+ mediated. This explanation, namely that a portion of the CAMP-activated current is Ca2+-mediated, is consistent with both the shape of the currents (presence of voltage-dependent inactivation, vide infra) and their block by DIDS, which is similar to that seen in the Ca2+-activation studies. Currents elicited by CAMP demonstrated voltage-dependent inactivation in 75% of the cells studied using the perforated patch technique. It should be noted that CAMP-evoked currents in the CFTR-expression studies of Rich et al. (26) and Anderson et al. (2), as well as Kartner et al. (20), failed to exhibit voltage-dependent inactivation in the depolarized range, a characteristic shared with the Cl-secreting T84 colonic cell line (7). A dialyzable, cytoplasmic factor may well variably contribute to the current inactivation seen in our perforated patch studies, a factor that is absent in the whole cell studies on the CFTR-transfected cells. The limited number of successful CAMP-activation experiments (4 of 27) that we were able to obtain in the whole cell configuration on the airway cells would support this conclusion. In those experiments, CAMP-dependent currents resembled those described for T84 cells with time-independent kinetics and a linear I- V relationship. Alternatively, multiple-channel populations with variable expression, or a single-channel population that is able to undergo kinetic transformations dependent on the phosphorylated state of the protein, may account for the voltage-dependent inactivation that was variably observed. Previous whole cell studies on T84 cells demonstrated DIDS insensitivity for the CAMP-activated Cl- current (4, 7). In contrast, we observed a significant DIDS inhibition of the CAMP-dependent current (66-lOO%, at +50 mV) in the perforated patch experiments on the airway cells. It is possible that CAMP-induced current activation in the airway cells is associated with concomitant changes in intracellular Ca2+ and/or volume regulation, thus simultaneously activating alternative conductance pathways that are DIDS sensitive. Ca2+-dependent Cl- current actiuation. In contrast to CAMP-dependent current activation, increases in GoI after an elevation in [Ca”‘]; via either the Ca2+ ionophore A23187 or elevated free Ca2+ in the pipette solution was observed in both CF and non-CF cells. These results are consistent with earlier studies which demonstrated that

IN AIRWAY

EPITHELIA

Cl281

Ca2+-dependent Cl- secretion (34,35) as well as ionomytin-induced current activation (26, 31) remains intact in CF airway epithelial cells. Recently, Kartner et al. (20) have expressed the CF gene product (CFTR) in nonepithelial invertebrate cells and have shown that these cells exhibited a new CAMP-stimulated anion permeability. They were also able to show that the CFTR-transfected cells failed to respond with an increase in anion permeability to the ionophore A23 187 stimulation, consistent with the notion that mutations in the CF gene do not influence the Ca2+ -activated anion pathway. Wagner et al. (31) have recently demonstrated in whole cell studies of transformed cell lines derived from normal and CF airway epithelia that intracellular application of activated CaMK and ATP activates a Cl- current similar to that activated by the Ca2+ ionophore ionomycin. Furthermore, they were able to demonstrate that peptide inhibitors of either the kinase or calmodulin itself blocked the Ca2+-dependent activation of the current. In the studies of Wagner et al. (31), both activation as well as inhibition of the Ca2+ -dependent current was observed in the normal as well as the CF cell lines. The demonstration that Ca2+-dependent current activation in airway epithelial cells occurs via CaMK was consistent with the earlier single-channel studies in which Cl- channel activation was not observed after exposure of the cytoplasmic side of the membrane to elevated Ca2+ (6, 33). The fact that CF airway cells retain the CaMK-dependent Cl- current regulation even though they lack PKAdependent current regulation suggests the existence of two separate conductance pathways. Consistent with the results of Wagner et al. (31), we were also able to inhibit Ca2+-dependent current activation in cells loaded with the CaMK inhibitory peptide. Cliff and Frizzell (7) observed that the Ca2+-activated Cl- current in T84 cells was outwardly rectified, showing delayed activation during depolarizing voltage pulses and inactivation during hyperpolarizing voltage pulses. In contrast to the characteristic conductance and kinetic properties of the Ca2+ -activated current reported for T84 cells, Ca2+ -dependent currents in airway cells exhibited variable conductance and kinetic profiles in both CF and non-CF airway cells. Both linear and outwardly rectifying currents were observed in CF and non-CF cells, but the magnitude of the linear current was attenuated in CF cells. In that all CF cells were derived from polypoid tissue and non-CF cells from turbinate tissue, the difference in current distribution could well represent a difference in channel expression between the two tissue subtypes, which are maintained in culture. Alternatively, the data are consistent with the conclusion that Ca2+-activated channels with a linear I-V may well be defectively regulated in CF. Volume-regulated Cl- current regulation. In addition to kinase-dependent Cl- activation, results from our experiments also demonstrate a volume-regulated conductance that is present in CF cells as well as in non-CF cells. A similar conductance has been reported for canine tracheal cells (24) and the colonic cell line T84 (37). It has been demonstrated in salivary acinar cells (12)) toad urinary bladder cells (36), and pituitary-derived GH4C1 cells (27)

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that changes in cell volume are associated with increases in [ Ca2+] i. Volume-sensitive GoI activation, however, does not appear to be dependent on CaMK activity in that we were able to successfully elicit an increase in Clcurrent in cells loaded with the CaMK inhibitory peptide after a decrease in bathing solution tonicity. Volume-regulated currents with both linear and outwardly rectifying I-V relationships were observed in CF and non-CF airway cells in response to hyposmotic challenge. As was observed for the Ca2+-dependent current, the magnitude of the swelling-induced current with a linear I- V relationship was also attenuated in CF cells. This data would suggest that either the two cell types, turbinate and polypoid, from which the non-CF and CF cells are derived differ in their current expression or that one of a multiplicity of Cl- channels associated with cellular swelling is downregulated in CF airway cells. Cross- talk between regulatory pathways. The observation that Cl- current in airway epithelial cells is the target of three multifunctional protein kinases, PKA, PKC, and CaMK, allows one to speculate on the relationship between the regulatory enzymes and Go1 activation. Data obtained from current modulation studies in the colonic cell line T84 (4, 7, 37) are consistent with the presence of three distinct Cl- conductances, each characterized by different conductive and kinetic properties as well as activation by specific stimuli, i.e., an elevation in CAMP, [Ca2+]i, and cell swelling. Although we were unable to link a given Go1 regulatory stimulus in airway epithelial cells with the activation of a characteristic current, stimuluslinked conductances in airway epithelia could, however, be distinguished based on the observations that 1) although CAMP-induced current activation was not observed in CF cells, they responded to both an elevation in [ Ca2+]i and cellular swelling; 2) further current activation could be elicited by cellular swelling in cells that had already responded to either an increase of [Ca2+]i or CAMP; and 3) CaMK inhibitory peptide blocked Ca2+ activation of Gcl while leaving the swelling-induced response intact. These observations suggest that the three regulatory pathways may indeed activate separate Cl- conductances in airway epithelial cells as reported in T84 cells. The heterogeneity of the current elicited in response to the regulatory stimuli in our studies also suggests the possibility that a multiplicity of Cl- conductances could be activated by a single regulatory stimulus. It has been reported that cellular swelling generates a number of intracellular signals including intracellular CAMP as well as [ Ca2+]i [see review by Watson (32)]. The component of the swelling-induced current with linear I-V observed in our studies could result from a predominant increase in intracellular CAMP. Similarly, the Ca2+-induced current with a linear I-V could represent a PKA/PKC-mediated current activation step, since Ca2+ and CAMP can act as synarchic messengers (25) and increases in [Ca2+]i can activate PKC directly. The attenuated magnitude of the swelling and Ca2+ -induced current expressing a linear I- V observed in the airway CF cells may simply be a reflection of the defective CAMP-dependent GoI in CF cells rather than a defect in the regulation of the Ca2+- or swelling-

IN AIRWAY

EPITHELIA

induced Gol. On the other hand, the CAMP-elicited currents in our perforated experiments could be contaminated with either swelling- or Ca2+-induced currents. This may explain the anion permeability sequence in our CAMP-elicited currents (I- > Br- > Cl-), which differs considerably from the relative halide permeability reported for CFTR [Br- > Cl- > I- (3)] expressed in a variety of cell types. In summary, we conclude that activation of Cl- current in airway epithelial cells occurs via at least three distinct regulatory pathways based on the observation of additive effect of different pathways and selective inhibition of different pathways as in the case of CAMP-insensitive CF cells or in the presence of specific peptide inhibitors of regulatory kinases. The fact that all three of the regulatory pathways studied do not appear to converge on a single membrane protein suggests that therapeutic modulation of alternate anion conductance pathways could be exploited as a possible means to circumvent the CAMPdependent regulatory defect present in CF. However, the restoration of defective Cl- secretion in CF cells via alternative activation pathways will rest on the determination of a polarized channel distribution to apical membrane sites. The authors thank Drs. H. Schulman for the CaMK inhibitory peptide, M. Haas for help in cell Palfrey for many helpful discussions throughout the This study was supported in part by a grant from Foundation through the Cystic Fibrosis Research versity of Chicago and by a grant from the Sprague Address for reprint requests: D. J. Nelson, Univ. Neurology, Hospital Box 425, 5841 S. Maryland 60637. Received

22 April

1991; accepted

in final

form

generous gift of the preparation, and C. tenure of this work. the Cystic Fibrosis Center at the UniFoundation. of Chicago, Dept. of Ave., Chicago, IL

11 December

1991.

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Findlay, I. The effects of magnesium upon adenosine triphosphate-sensitive potassium channels in a rat insulin-secreting cell line. J. Physiol. Lond. 391: 611-629, 1987. 12. Foskett, J. K., and J. E. Melvin. Activation of salivary secretion: coupling of cell volume and [Ca2+] in single cells. Science 11.

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