RAPID COMMUNICATION
cAMP-dependent absorption of chloride across airway epithelium

S. N. Uyekubo, H. Fischer, A. Maminishkis, B. Illek, S. S. Miller, and J. H. Widdicombe

Children's Hospital Oakland Research Institute, Oakland 94609; School of Optometry and Department of Molecular and Cell Biology, University of California, Berkeley 94720; and Cardiovascular Research Institute, University of California, San Francisco, California 94143

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Elevated levels of Na and Cl in airway surface liquid may play a major role in the airway pathology of cystic fibrosis (CF) (J. J. Smith, S. M. Travis, E. P. Greenberg, and M. J. Welsh. Cell 85: 229-236, 1996) and could be caused by block of transcellular Cl absorption due to lack of a functional CF transmembrane conductance regulator (CFTR). To test for transcellular absorption of Cl across non-CF epithelium, we studied how fluid absorption was affected by the opening and closing of Cl channels. Forskolin (an activator of CFTR) tripled fluid absorption across primary cultures of bovine tracheal epithelium but had no effect on human cells. However, in both species, fluid absorption was markedly inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, a blocker of CFTR. Microelectrode studies suggested that the magnitude of the absorptive response to forskolin in bovine cells depended on the size of an inwardly directed electrochemical driving force for Cl movement across the apical membrane. Patch-clamp measurements of bovine cells revealed CFTR in the apical membrane and a cAMP-activated, inwardly rectifying Cl channel in the basolateral membrane. We conclude that a significant fraction of absorbed Cl passes transcellularly in bovine tracheal epithelial cultures, with CFTR as the path of entry in the apical membrane and a novel cAMP-activated Cl channel as the exit route in the basolateral membrane. Our data further indicate that a similar pathway may exist in non-CF human tracheal epithelium.

adenosine 3',5'-cyclic monophosphate; cystic fibrosis; airway surface liquid; epithelial ion transport

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THERE IS CURRENTLY considerable controversy as to the salt content in human airway surface liquid (ASL) and its alteration in cystic fibrosis (CF). One report (10) concluded that the Na and Cl levels in ASL are both lower to similar extents than those in plasma and are unaltered in CF. Another report (19) stated that in both CF and normal ASLs the Na and Cl levels are similar to those in plasma. By contrast, several groups (5, 6, 14) recently found Na and Cl concentrations in normal ASL to be less than those in plasma but elevated to plasma levels or above in CF. Strong indirect evidence for an abnormally high salt content of ASL in CF was provided by Smith et al. (25), who found 1) that the killing ability of natural antibiotics secreted by the surface epithelium was reduced in CF ASL, 2) that the antibiotic activity of normal ASL was reduced by increasing its salt content, and 3) that the bactericidal activity of CF ASL was increased to normal by lowering the levels of NaCl.

Human airway epithelia absorb NaCl and water (13, 17, 37). If a substantial fraction of the absorbed Cl follows a transcellular pathway involving the CF transmembrane conductance regulator (CFTR), then malfunction or absence of this channel should promote elevated levels of NaCl in ASL. Jiang et al. (13) earlier reported that cAMP-elevating agents induced fluid secretion across cultures of human airway epithelium. However, the cell sheets studied had been pretreated with amiloride, which, by hyperpolarizing the apical membrane, is known to induce or increase Cl secretion (1, 32). Without amiloride pretreatment, Willumsen et al. (32), studying human nasal epithelial cultures, reported that Cl is statistically at equilibrium across the apical membrane and above equilibrium across the basolateral membrane. In a fraction of the tissues studied, therefore, the electrochemical driving forces presumably favored net entry of Cl across the apical membrane. There is a Cl conductance in the basolateral membrane of human nasal epithelium (32). Thus it is possible that the simultaneous opening of Cl channels in apical and basolateral membranes could increase salt and water absorption across at least some sheets of human airway epithelium.

Accordingly, in this study, we measured transepithelial fluid flows and tested the effects of activating CFTR with forskolin, a cAMP-elevating agent, and of inhibiting CFTR with 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) (8). Microelectrode studies were used to determine whether there was a correlation between transepithelial volume flow and the driving force for Cl movement across the apical membrane. Finally, because changes in transcellular Cl movement must involve coordinate regulation of transport pathways in both the apical and basolateral membranes, we also sought to identify and characterize cAMP-dependent Cl-permeant pathways in both membranes.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Tracheal epithelial cells were cultured as previously described (20, 33). In brief, strips of epithelium were pulled off from the underlying tissues and treated with protease overnight. The following morning, epithelial cells were dispersed and plated onto collagen-coated Costar inserts (1-cm2 area, 0.44-µm pore size; Corning Costar, Cambridge, MA). Culture medium was added to the outside of the insert only ("air-interface feeding"). The time at which tight junctions formed was revealed by cessation of fluid leakage across the cells to the inside of the insert. Cells were studied from 5 to 10 days later. Human airway epithelial cells grown on Costar inserts are less differentiated than those grown on Vitrogen gels (33, 35). Unfortunately, transepithelial volume flows cannot be measured on cells grown on Vitrogen gels because of swelling and shrinkage of the gel, which is several millimeters thick. However, it was recently discovered that coculture with human airway fibroblasts produces epithelial sheets of pseudostratified histology, with both cilia and mucus granules that closely resemble both native tissue and cultured cells grown on Vitrogen (W. E. Finkbeiner and J. H. Widdicombe, unpublished data). Therefore, in our cultures of human airway epithelial cells, fibroblasts were grown on the underside of the filters, opposite the epithelial cells. To do this, airway fibroblasts were obtained from trypsinization of primary airway gland cultures (34) at passages 8-10 and plated at 104 cells/cm2 by placing a drop of cell suspension on the underside of an inverted insert. After 4 h, the insert was placed right side up in its original multiwell, and epithelial cells were plated on the inside.

Transepithelial fluid flows were measured with a capacitance probe technique (9) in which a sheet of epithelium (0.5-cm2 exposed area) was mounted between two water-impermeable Kel-F half-chambers. On each side of the tissue, capacitance probes were introduced through small ports on the chamber surface and measured the voltage signals produced by changes in capacitance between the probe tips and the menisci of the underlying fluids. In the absence of leaks, the change in capacitance at one probe should be equal and opposite to that at the other. This technique has an accuracy of ~0.25 µl · cm-2 · h-1. Entry and exit ports allowed for solution changes. Potential difference (PD)-sensing bridges built into each half-chamber permitted continuous monitoring of transepithelial voltage (Vt), which was referenced to the apical bath, i.e., is given as positive values in the text. Current-passing bridges delivered pulses of known magnitude, and transepithelial resistance (Rt) was calculated from the resulting voltage deflections.

Our Ussing chamber techniques are standard and have been described in detail elsewhere (33). For flux studies, we used short-circuited tissue pairs from the same culture studied simultaneously. Radioisotopes (36Cl and 22Na) were added to the serosal side of one tissue and the mucosal side of its pair at a final level of 1 µCi/ml. Three control samples (2 or 3 ml) were taken at 10-min intervals from the downhill reservoir (volume 10 ml). Fresh nonradioactive medium was readded immediately after sampling. Forskolin (10-5 M) was added to both sides of the tissues, and three more samples were taken at 10-min intervals. Finally, NPPB was added (100 µM, mucosal bath), and three more samples were taken. This time course was selected, in part, because it was similar to that used in the experiments on volume flow. The short-circuit current (Isc) and Rt in the middle of each flux period were recorded. The averages of the three values for the control, the forskolin, and the forskolin plus NPPB periods were used for data analysis. Samples (50 µl) were taken periodically from the hot side of the tissues. Total counts (36Cl and 22Na) were determined with a scintillation counter, and counts from 22Na were determined with a gamma-counter. Standards containing 22Na alone revealed the relative counting efficiency of 22Na on the beta- and gamma-counters. Knowing this, the 22Na counts on the gamma-counter could be converted to 22Na counts on the scintillation counter. This value was then subtracted from the total counts on the scintillation counter to yield the counts from 36Cl. The fluxes are expressed as microequivalents per square centimeter per hour.

Ussing chamber studies in which the basolateral membrane was permeabilized by the alpha -toxin of Staphylococcus aureus were as previously described by Illek et al. (12). The mucosal face of the tissues was bathed in conventional HEPES-buffered Krebs-Henseleit solution containing 140 meq/l of Cl, which was maintained at 37°C and bubbled with 100% O2. The serosal chamber contained a low Cl medium, in which Cl was reduced to 30 meq/l by replacement with gluconate. The other components of the low and high Cl media were identical except that ATP (5 mM) was added to the serosal bath.

The recording setup and perfusion system for microelectrode measurements have been previously described (15). The area of perfused tissue was 0.07 cm2. Calomel electrodes in series with Ringer solution-agar bridges were used to measure Vt, and the signals from the intracellular microelectrodes were referenced to either the apical or basal bath to measure the apical and basolateral membrane potentials (Va and Vb, respectively), where Vt = Vb - Va. Conventional microelectrodes were made from fiber-filled borosilicate glass tubing (0.5-mm ID, 1-mm OD; Omega Dot Glass, Bargaintown, NJ). They were backfilled with 150 mM KCl and had resistances of 100-170 MOmega . Bipolar current pulses of 4-µA amplitude and 3-s duration were passed across the tissue at 30-s intervals. The resulting change in Vt divided by 4 µA gives Rt. The ratio of the change in Va to the change in Vb gives a, the ratio of apical (Ra) to basolateral membrane (Rb) resistance (a = Delta Va/Delta Vb = Ra/Rb).

Cell-attached and excised patch-clamp measurements were made on single cultured cells on the day after plating or on freshly isolated bovine airway epithelial cells. Cells maintained their polarization for several hours after isolation as judged by the strict confinement of beating cilia to one end of the cells. Dispersed cells were immobilized with a holding pipette, and patch pipettes were sealed onto the unciliated basolateral membrane. All recordings were done in a perfused, open, and heated (37°C) chamber on the stage of a microscope as previously described (4). The composition of the bath and pipette solution in the cell-attached mode was (in mM) 145 N-methyl-D-glucamine (NMDG) chloride, 1.7 CaCl2, 1 MgCl2, 10 NMDG-HEPES, and 25 glucose, pH 7.4. In the excised mode, the bath contained (in mM) 145 NMDG-Cl, 1.7 MgCl2, 10 NMDG-HEPES, 25 glucose, and 0.1 MgATP, pH 7.4. Under these conditions, Cl is the principal current carrier in both the cell-attached and excised modes. Potentials are reported as negative pipette potentials.

Tests of significant differences between means (P < 0.05) were performed with Student's paired t-test.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Transepithelial volume flows. Jiang et al. (13) previously showed that forskolin induces fluid secretion across cultures of human tracheal epithelium that have been pretreated with amiloride. However, amiloride is known to hyperpolarize the apical membrane, thereby favoring Cl secretion (1, 32). When forskolin was added to open-circuited bovine tracheal cell cultures that had not received amiloride, it markedly stimulated fluid absorption (Fig. 1). In five similar experiments, forskolin significantly increased the volume flow (Jv) from 6.0 ± 3.3 to 15.0 ± 3.4 µl · cm-2 · h-1, significantly depolarized Vt from 67 ± 2 to 33 ± 6 mV (basal side positive), and significantly decreased Rt from 430 ± 100 to 160 ± 20 Omega  · cm2.


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Fig. 1.   Effects of forskolin and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) on fluid transport and electrical properties of bovine cultures. A: volume flow (Jv). B: transepithelial voltage (Vt; ) and resistance (Rt; black-diamond ). Solid horizontal bars, periods during which probes were removed and chambers were perfused with fresh Ringer solution. Volume flows were not recorded during this time. Open bars, drug application protocol, A, drug added to apical medium; A+B/L, drug added to both apical and basolateral sides. After control change of drug-free Ringer solution, forskolin increased Jv and decreased Vt and Rt. Increase in Jv was inhibited by apical NPPB (100 µM), which increased Rt and hyperpolarized and then depolarized Vt.

In the fluid transport experiments on bovine tracheal epithelium, baseline Vt ranged from 40 to 100 mV, and there was a significant correlation (R = 0.66; P = 0.037; n = 10 tissues) between baseline Vt and the change in fluid absorption induced by forskolin. The best least squares linear regression predicted increases in fluid absorption in response to forskolin of 5 µl · cm-2 · h-1 at Vt of 60 mV and 12 µl · cm-2 · h-1 at 100 mV. The same curve predicted that the response to forskolin should be zero at ~35 mV.

NPPB, a blocker of CFTR, had a biphasic response on Jv, Vt, and Rt when added after forskolin. Ten minutes after the addition of NPPB (the first point at which Jv measurements had been resumed in all tissues), Jv had declined from 13.2 ± 3.2 to 7.0 ± 1.7 µl · cm-2 · h-1. During this 10-min period, Vt initially increased and then decreased (see Fig. 1). Ten minutes after the addition of NPPB, the equivalent Isc (Ieq = Vt/Rt) was not significantly changed (179 ± 31 µA/cm2 before vs. 150 ± 29 µA/cm2 after). After the initial rapid decrease in Jv, NPPB caused exponential changes in Jv, Vt, and Rt, with half-time values of ~20 min. After a 1-h exposure, Jv had declined by -10.0 ± 2.2 µl · cm-2 · h-1, Rt had significantly increased from 160 ± 20 to 320 ± 60 Omega  · cm2, and Vt had declined from 33 ± 6 to 21 ± 4 mV. During this period, Ieq significantly declined from 150 ± 29 to 66 ± 11 µA/cm2.

A typical record of fluid transport for human cells is shown in Fig. 2. Although forskolin slightly increased fluid absorption in this experiment, this was not a consistent finding, and, on average, human cells showed no significant changes in Jv in response to 10 µM forskolin (4.1 ± 0.8 µl · cm-2 · h-1 before vs. 4.1 ± 0.9 µl · cm-2 · h-1 after; n = 6 cell sheets). Baseline Vt and Rt in these experiments were 37 ± 17 mV and 710 ± 290 Omega  · cm2, respectively, and were not significantly altered by forskolin. However, in all experiments, NPPB given after forskolin reduced fluid absorption to a level not significantly different from zero (0.1 ± 2.1 µl · cm-2 · h-1). Approximately 80% of this reduction was accomplished during the first 10 min of exposure to NPPB (Fig. 2), without a significant change in Ieq. As for bovine cultures, prolonged exposure to NPPB caused a slow decrease in Vt (from a preexposure level of 28 ± 9 to 12 ± 8 mV) and an increase in Rt (from 510 ± 140 to 700 ± 250 Omega  · cm2).


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Fig. 2.   Effects of forskolin followed by NPPB on Jv (A), Vt (B, ) and Rt (B, black-diamond ) of human cultures. Solid horizontal bars, periods during which probes were removed and chambers were perfused with fresh Ringer solution. Volume flows were not recorded during this time. Open bars, drug application protocol. After control change of Ringer solution, forskolin slightly increased Jv. Apical NPPB reduced fluid absorption to less than baseline value. Changes in Vt and Rt resembled those for bovine cells.

Ussing chambers. The mechanisms of action of forskolin on ion transport across bovine tracheal epithelium were investigated with Ussing's Isc technique. Five short-circuited tissues had a baseline Isc of 117 ± 37 µA/cm2. Amiloride significantly decreased Isc to 50 ± 14 µA/cm2. Forskolin added in the continued presence of amiloride increased steady-state Isc by 28 ± 9 µA/cm2. Bumetanide added next decreased Isc by 42 ± 6 µA/cm2.

Without amiloride pretreatment, forskolin had variable effects on Isc. In some tissues, it produced a sustained increase in Isc of up to 80%. In other tissues, a transient forskolin-induced increase in Isc of ~2-min duration was followed by a return to preexposure levels. In some cases, forskolin caused small sustained decreases in Isc. The mean value for Isc 10 min after the addition of forskolin was not significantly different from the mean value immediately before addition. However, all tissues responded to forskolin with increases in total conductance (GT = 1/Rt) of 1.68 ± 0.30 mS/cm2 (from 7.09 ± 1.00 to 8.77 ± 1.15 mS/cm2; n = 12).

Radiotracer studies are summarized in Table 1. Under control conditions, the sum of the net Cl secretion and the net Na absorption (4.98 ± 1.04 µeq · cm-2 · h-1) was not significantly different from the Isc (4.94 ± 1.14 µeq · cm-2 · h-1). Forskolin produced significant increases in both unidirectional Cl fluxes without a change in the unidirectional or net fluxes of Na. The average Isc during the 30-min period of exposure to forskolin was not significantly different from the Isc during the preceding 30-min control period.

                              
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Table 1.   Effects of forskolin on ion fluxes across short-circuited bovine tracheal epithelium

Flux data also showed that the primary effect of NPPB was an inhibition of active Cl transport. Thus NPPB added to the apical bath after forskolin significantly reduced Isc from 5.26 ± 0.71 to 2.61 ± 0.26 µeq · cm-2 · h-1, and GT from 8.77 ± 1.15 to 6.46 ± 0.99 mS/cm2 but had only small insignificant effects on the unidirectional or net Na fluxes (-32 to +5%).1 The flux of Cl from mucosa to serosa was also unaltered by NPPB (-6%), but the unidirectional flux from serosa to mucosa was significantly reduced (from 6.79 ± 0.72 to 4.31 ± 0.38 µeq · cm-2 · h-1). The change in net Cl flux of 2.15 ± 0.37 µeq · cm-2 · h-1 (from -2.37 ± 0.93 to -0.14 ± 0.64 µeq · cm-2 · h-1) was also significant.

In the presence of a transepithelial concentration gradient for Cl, the basolateral membrane of bovine tracheal epithelial cells was permeabilized by the alpha -toxin of Staphylococcus aureus. Subsequent addition of cAMP (50 µM in the serosal medium) caused Isc to increase from 65 ± 8 to 152 ± 8 µA/cm2 and GT to increase from 4.97 ± 0.96 to 8.92 ± 0.73 mS/cm2.

Microelectrodes. In experiments on eight different bovine cell cultures, Vt, Va, and Vb were 74 ± 9, 2 ± 9 (range +40 to -20 mV), and -72 ± 5 mV, respectively; Rt and a were 259 ± 21 Omega  · cm2 and 3.8 ± 0.5, respectively. Va and Vt were significantly correlated (R = 0.83; P = 0.011; n = 8 cultures), with large Vt values being associated with relatively positive Va values. The best least squares regression of Va on Vt had a gradient of -0.75 ± 0.21. Figure 3 shows that the addition of forskolin hyperpolarized Va, reduced Vt, and decreased both a and Rt.


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Fig. 3.   Microelectrode recording from bovine tracheal epithelium illustrating effects of forksolin. Forskolin hyperpolarized apical membrane potential (Va; A) and depolarized basolateral membrane potential (Vb; A), causing a large decrease in transepithelial potential difference (TEP; B). Rt decreased as did apical-to-basolateral membrance resistance ratio (Ra/Rb). This is representative of 3 similar recordings.

Patch clamping. Cell-attached patches on the upper surface of single cultured bovine or human cells revealed forskolin-activated anion channels, with the characteristic conductance and nonrectifying current-voltage relationship of CFTR (data not shown). In bovine cells, CFTR was found in 4 of 11 successful seals, with from 5 to >20 channels/patch. In human cells, CFTR was present in 3 of 15 patches, with 1-2 channels/patch.

When cell-attached patch-clamp measurements were made on the nonciliated, basolateral pole of bovine cells, we found a Cl current that was increased at least 10-fold by forskolin (Fig. 4A). Voltage-pulse protocols showed this current to be inwardly rectifying and not voltage activated (Fig. 4B), with instantaneous currents (measured at 10 ms) being not significantly different from steady-state currents (at 800 ms; Fig. 4B). When membrane patches were excised into 100 µM MgATP, channels were inactivated, with a half-time of ~1 min, allowing recording from single channels (Fig. 4C). In symmetrical Cl solution, these were inwardly rectifying, with a slope conductance of 35.0 ± 1.5 pS at -100 mV and 2.5 ± 0.5 pS at +100 mV (Fig. 4D). The open probability of this channel in excised recordings was not affected by voltage (open probability = 0.80 ± 0.04 pS at -100 mV and 0.66 ± 0.07 pS at +100 mV; n = 3 patches). Channels with the biophysical characteristics of CFTR were not found on the basolateral pole.


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Fig. 4.   Identification of a cAMP-activated Cl channel in basolateral membrane of bovine epithelium. A: cell-attached patch-clamp recording on basolateral pole of a freshly isolated polarized ciliated cell. Holding potential is -75 mV; bath and pipette contained symmetrical N-methyl-D-glucamine chloride solutions. Multiple channels were stimulated by addition of 10 µM forskolin to the bath. B: voltage-pulse protocol before and after forskolin stimulation in cell-attached mode. Holding potential was clamped from +100 to -100 mV in 25-mV steps. C: recordings from excised basolateral membrane patch at +75 (top) and -75 (bottom) mV. At +75 mV, 2 channels were present. C, closed level; O1 and O2, open levels of channels 1 and 2, respectively. D: single-channel current-voltage relationship of excised channel.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Effects of forskolin on fluid transport and transepithelial electrical properties. Ussing chamber studies have shown that short-circuited airway epithelium can actively secrete Cl and actively absorb Na (26). It has been suggested therefore that the volume of ASL may depend, in part, on the balance between these two active transport processes (26). However, the Cl secretion seen under short-circuit conditions may not be present under open-circuit conditions because the presence of a lumen negative Vt creates a shunt current that will depolarize Va and decrease or reverse the electrochemical driving force for Cl exit across the apical membrane (28, 29).

We reasoned that if Cl was not being actively secreted under open-circuit conditions, then opening of the apical membrane CFTR after elevation of cAMP with forskolin should fail to induce fluid secretion. In fact, forskolin increased fluid absorption across open-circuited bovine tracheal cell cultures by ~2.5-fold. If the increased fluid absorption elicited by forskolin involved opening of the apical membrane CFTR, then it should be inhibited by NPPB, a blocker of CFTR. This agent initially increased Vt, a result that is consistent with 1) a block of apical membrane Cl channels and 2) an inwardly directed electrochemical gradient for Cl across the apical membrane. Ten minutes after the addition of NPPB, transepithelial fluid movement was halved. At this time, the Ieq was not changed, suggesting 1) that our open-circuited tissues were not actively secreting Cl and 2) that the effects of NPPB were not due to inhibition of active Na absorption. After the initial rapid decrease in Jv, NPPB caused further slow decreases in Jv and Vt and an increase in Rt. These changes were accompanied by a significant decrease in Ieq and are best explained by an inhibition of active Na absorption secondary to a block of basolateral K channels (11).

In some human cell cultures, forskolin slightly increased fluid absorption (Fig. 2). However, on average, it was without effect on baseline Jv, Vt, or Rt. This negative result is perhaps best explained by assuming that CFTR is maximally activated under baseline conditions, a conclusion reached by Shen et al. (23) and Yamaya et al. (35) using a variety of other techniques. Importantly, however, forskolin did not stimulate fluid secretion as Jiang et al. (13) reported for tissues pretreated with amiloride. NPPB given after forskolin reduced fluid absorption to a level not significantly different from zero.

Mechanism of action of forskolin and NPPB. There are only two likely mechanisms for the stimulation of fluid absorption by forskolin. First, forskolin could act by stimulation of active Na absorption as described for cAMP-elevating agents in alveolar epithelium (22). Alternatively, it could increase absorption of salt and water by increasing the permeability of the tissue to the counteranions that accompany the actively transported Na. Similarly, NPPB could act via a specific inhibition of apical membrane CFTR or via a nonspecific action on active Na absorption.

Radiotracer studies on short-circuited tissues in Ussing chambers showed definitively that the primary actions of forskolin and NPPB were on Cl permeability rather than on active Na absorption. Thus forskolin produced significant increases in both unidirectional Cl fluxes without a change in the unidirectional or net fluxes of Na. In open-circuited tissues, the failure of forskolin to alter Ieq also argues against stimulation of active Na absorption by this drug. In the first 30 min of exposure to NPPB, Isc, net Cl flux, and the unidirectional flux of Cl from serosa to mucosa were reduced to similar extents, but the unidirectional and net fluxes of Na were unchanged. Thus, although long-term exposure to NPPB has multiple effects, the initial action is an inhibition of transcellular Cl pathways.

If forskolin acts by increasing transcellular Cl permeability, then there should be cAMP-activated Cl channels in both apical and basolateral cell membranes. Patch-clamp studies (see The molecular basis of Cl absorption) showed that this was indeed the case for single cells. Three additional approaches provided evidence for cAMP-activated Cl channels in the apical membrane of confluent, polarized cell sheets. In conventional Ussing chamber experiments, we first blocked Na absorption with amiloride. As well as blocking Na absorption, amiloride hyperpolarizes Va, thereby increasing the driving force for the secretion of Cl (31). In the continued presence of amiloride, the addition of forskolin increased Isc, and this increase was abolished by bumetanide. These results confirm earlier reports (20, 21) that indicated that short-circuited bovine tracheal epithelium is capable of cAMP-mediated active secretion of Cl and that its apical membrane therefore contains cAMP-activated Cl channels. In other studies, we imposed a transepithelial Cl concentration gradient and permeabilized the basolateral membrane with the alpha -toxin of Staphylococcus aureus. Under these conditions, forskolin increased both Isc and GT. These results do not formally rule out the presence of a cAMP-activated conductance in the tight junctions. However, two findings (Illek and Widdicombe, unpublished data) make this unlikely. First, forskolin did not increase fluxes of the paracellular marker [3H]mannitol across permeabilized tissues. Second, in the presence of a mucosal-to-serosal Cl concentration gradient across nonpermeabilized tissues, forskolin failed to alter Isc or GT. Further evidence for cAMP-dependent apical membrane Cl conductance was obtained with microelectrodes, which showed that forskolin hyperpolarized Va while reducing Vt (see Fig. 3). This result is expected for opening of apical membrane Cl channels; opening of apical membrane Na channels would produce the opposite effects, a depolarization of Va and an increase in Vt. (The forskolin-mediated decreases in a and Rt do not distinguish between opening of apical membrane Na or Cl channels.) Thus the effect of cAMP on apical membrane Cl conductance predominates over any effect on Na conductance.

Driving forces for Cl absorption. Stimulation of fluid absorption by forskolin and inhibition by NPPB suggest that bovine tracheal epithelial cells have a transcellular route for absorption of Cl, involving CFTR in the apical membrane. Furthermore, the threefold stimulation of fluid absorption by forskolin suggests that the ratio of transcellular to paracellular Cl absorption is at least 2:1 under conditions of elevated intracellular cAMP. However, for such transcellular absorption to occur, the electrochemical driving forces for Cl must be inward across the apical membrane and outward across the basolateral membrane. We used microelectrodes to determine whether this was indeed the case. In experiments on eight different bovine cell cultures, Va averaged 2 mV (range +40 to -20 mV), and Vb was ~70 mV. At the extracellular Cl concentration of 120 mM, these values of Va and Vb predict that the net electrochemical driving force for Cl will be inward across the apical membrane and outward across the basolateral membrane for all intracellular Cl concentrations between 8 and 129 mM. Intracellular Cl activity (aiCl) of airway epithelia ranges from 30 to 50 mM (24, 27, 30).

In our fluid transport studies on bovine tracheal epithelium, we found a significant correlation between baseline Vt and the change in fluid absorption induced by forskolin. Furthermore, microelectrodes revealed that Va and Vt were also significantly correlated, with large Vt values being associated with relatively positive Va values, in a relationship that was ~1:1. A depolarized Va will increase the electrical driving force for Cl entry. Therefore, when apical membrane Cl channels are activated by forskolin, tissues with higher Vt values will show greater increases in absorption of Cl and water.

The monotonic relationship of Va on Vt for bovine tracheal epithelium was obtained over a range of Vt from 40 to 100 mV. These Vt values are greater than is generally found across airway epithelia from other species. However, Welsh et al. (28) also reported a monotonic relationship between Va and Vt for primary cultures of canine tracheal epithelium over a Vt range of 5 to 45 mV. In the present studies, we did not measure membrane potentials in human cells. However, in cultures of human nasal epithelium with a Vt of 10 mV, Cl was in equilibrium across the apical membrane (32). Our human tracheal cultures had a Vt of 37 mV. In vivo, nasal PDs average ~25 mV (16, 18). In the trachea, main stem bronchus, bronchus intermedius, and lobar bronchus, they average 20 mV, declining to 14 mV in segmental bronchi (16). Given that the voltage-sensing probe used for in vivo PD measurements may damage the epithelium, these represent minimum values. Therefore, if the expected monotonic relation between Va and Vt holds for human airway cells, then the Va of our cultures and of native epithelium should be more positive than the equilibrium value, and the electrochemical driving force for Cl will be inward across the apical membrane. Direct measurements of Va and aiCl are needed to test this hypothesis.

The molecular basis of Cl absorption. If forskolin stimulates fluid absorption by increasing transcellular Cl permeability, a cAMP-activated route for Cl flow must be present in both the apical and basolateral membranes. Several indirect lines of evidence indicated the presence of a cAMP-activated Cl conductance in the apical membrane (see Mechanism of action of forskolin and NPPB). The inhibitory effects of NPPB suggest that this Cl conductance is CFTR, consistent with the findings of others (8, 23) that CFTR provides an important, perhaps predominant, Cl conductance in the apical membrane of airway epithelia. In addition, we demonstrated the presence of CFTR directly; cell-attached patches on the upper surface of single cultured bovine and human cells had forskolin-activated anion channels with the characteristic conductance and nonrectifying current-voltage relationship of CFTR.

Willumsen and Boucher (31) reported a minor Cl conductance in the basolateral membrane of human nasal epithelium but did not investigate its regulation. In this study, we made cell-attached patch-clamp measurements on the nonciliated, basolateral pole of bovine cells and found a Cl current that was increased at least 10-fold by forskolin (Fig. 4A). The channel responsible is probably not CFTR. It is inwardly rectifying with equal Cl concentration on both sides, whereas CFTR is nonrectifying (8). Furthermore, an immunocytochemical study (3) showed CFTR to be present in the apical membrane of human tracheal epithelium but undetectable in the basolateral membrane. The basolateral membrane anion channel described here shows some similarities to a Cl channel found in the basolateral membrane of the rabbit urinary bladder (7).

Are there enough basolateral Cl channels to carry the transcellular flux of Cl predicted from the volume flow measurements? Assuming that the patch occupies 10% of the basolateral membrane area, then the forskolin-induced current of 30 pA at a patch potential (-75 mV) equal to Vb (Fig. 4A) corresponds to ~10 peq · cell-1 · h-1. Assuming isotonic coupling of salt and water, the forskolin-activated volume flow of 10 µl · cm-2 · h-1 would correspond to a transcellular Cl flux of 1.5 µeq · cm-2 · h-1. If each apical membrane is a circle of 5-µm radius, then there are ~106 columnar cells/cm2 tracheal surface. Given the flux per square centimeter and the number of cells per square centimeter, the Cl movement across a single cell becomes ~1.5 peq · cell-1 · h-1. These calculations suggest that the cAMP-activated basolateral Cl conductance is more than adequate to accommodate the observed transcellular flow of Cl.

Physiological implications. Our results provide firm evidence for transcellular absorption of Cl across bovine tracheal epithelium. We showed that forskolin markedly stimulates fluid absorption and that this stimulation is reversed by NPPB. These initial results indicating a transcellular route for Cl absorption were strengthened by microelectrode measurements that demonstrated driving forces favoring Cl entry across the apical membrane and exit across the basolateral membrane. Finally, patch clamping revealed CFTR in the apical membrane and a different cAMP-activated Cl channel in the basolateral membrane.

Inhibition of fluid absorption across human tracheal cell cultures by NPPB is also consistent with transcellular Cl absorption involving CFTR. Indirect arguments suggested that the electrochemical gradient for Cl is inward across the apical membrane of our human cultures and of native human airway epithelium, although this suggestion will need to be tested with direct measurements of Va and of aiCl.

The route for Cl entry across the apical membrane appears to be CFTR, and, therefore, transcellular movement of Cl should be blocked in CF tissues. Thus the earlier finding by Jiang et al. (13) that CF tracheal cells absorb fluid indicated that Cl can be absorbed across the paracellular pathway. The exact amounts of Cl that pass trans- and paracellularly will depend on the degree of differentiation of the cell cultures and the activity of the Cl channels in the transcellular pathway. In poorly differentiated cultures with low levels of Cl-channel activity, paracellular Cl absorption will predominate, and the increased Na absorption in CF cultures (2) will elevate Vt and drive higher than normal levels of net Cl and fluid absorption as Jiang et al. (13) originally described. However, when the differentiation of the cells is increased (as reflected by increased amiloride-sensitive Isc), Zabner et al. (36) recently showed that non-CF cultures have substantially higher rates of fluid absorption than CF cultures. This is presumably due to increased numbers of Cl channels in both the apical and basolateral cell membranes and a switch in Cl absorption from predominantly paracellular to predominantly transcellular.

In conclusion, airway epithelia absorb NaCl and water. Much of the absorbed Cl travels via the transcellular pathway. In CF, absence of a functioning CFTR would block this permeation route and reduce absorption of NaCl from the airway lumen, thereby promoting elevated salt levels in ASL.

    ACKNOWLEDGEMENTS

We thank Dr. Walt Finkbeiner for providing the cultures of human tracheal epithelium and Cunrong Li-Yun for help with the volume flow measurements.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-42368 (to J. H. Widdicombe); National Eye Institute Grant EY-02205 (to S. S. Miller); and a grant from Cystic Fibrosis Research Inc. (to H. Fischer).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

1 As a check on the reliability of our Na flux determinations, in four tissue pairs, amiloride was added after forskolin and NPPB. It produced significant and virtually identical decreases in Isc, net Na flux, and mucosal-to-serosal Na flux of 1.53 ± 0.20, 1.50 ± 0.42, and 1.68 ± 0.39 µeq · cm-2 · h-1, respectively. Chloride fluxes were not altered.

Address for reprint requests: J. H. Widdicombe, Children's Hospital Oakland Research Institute, 747 52nd St., Oakland, CA 94609.

Received 4 February 1998; accepted in final form 17 September 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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