INTRODUCTION

TOP ABSTRACT INTRODUCTION CHLORIDE-CHANNEL ACTIVATION AND... CONCLUSIONS REFERENCES

BASOLATERAL ADDITION of the selective ₂-agonist terbutaline increases amiloride-sensitive short-circuit current (I_sc) and net Na absorption as determined by transepithelial flux experiments across adult rat alveolar epithelial cells under short-circuit conditions (4, 9, 17, 18). The time course of terbutaline action on I_sc exhibited an initial rapid decrease in current followed by a time-dependent increase in I_sc that reached a new steady-state plateau that was ~40% greater than the basal I_sc (4). This sustained increase in current was inhibited by the apical addition of amiloride. The magnitude of the amiloride-sensitive I_sc observed after terbutaline stimulation closely agreed with the terbutaline-stimulated net Na flux across the monolayer, providing strong support for the conclusion that terbutaline increases transepithelial Na absorption and that amiloride-sensitive Na channels are involved in the process. In a subsequent study (9), terbutaline was shown to increase net transepithelial Cl absorption as reflected by an increase in the apical-to-basolateral unidirectional Cl flux. The flux experiments were performed under short-circuit conditions (transepithelial potential held at 0 mV) with symmetric physiological saline solutions bathing both the apical and basolateral surfaces of the epithelium. Under these conditions, the increase in Cl transport must have resulted from activation of a transcellular pathway for Cl absorption. Thus terbutaline stimulates active NaCl absorption across monolayers of adult rat alveolar epithelial cells, but the mechanisms responsible for transcellular Cl transport were not identified in these earlier investigations.

	CHLORIDE-CHANNEL ACTIVATION AND REGULATION OF ALVEOLAR SODIUM TRANSPORT

TOP ABSTRACT INTRODUCTION CHLORIDE-CHANNEL ACTIVATION AND... CONCLUSIONS REFERENCES

Terbutaline and cAMP activate apical membrane Cl channels in cultured alveolar epithelial cells. In a recent study, Jiang et al. (8) demonstrated that the addition of 2 µM terbutaline to the basolateral surface of cultured adult rat alveolar epithelial cells bathed on both sides with DMEM-Ham's F-12 medium produced changes in I_sc that were similar to those previously reported by Cheek et al. (4). Figure 1A shows the biphasic effects of terbutaline on I_sc, beginning with a rapid decrease in current followed by a time-dependent increase in I_sc that was blocked by amiloride. The initial decrease in I_sc was clearly apparent (but smaller in magnitude) when the monolayer was pretreated with apical amiloride, but no secondary increase in current was detected in the presence of amiloride (Fig. 1B). Cl replacement experiments abolished the initial decrease in I_sc produced by the basolateral addition of terbutaline, and no significant increase in amiloride-sensitive current was detected in the absence of Cl (Fig. 2). Similar results were obtained when monolayers were treated with 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP). These results suggested the presence of a terbutaline- and cAMP-activated electrogenic Cl transport pathway that could potentially mediate Cl absorption across the monolayer.

View larger version (6K): [in this window] [in a new window]   Fig. 1.   Representative short-circuit current (Isc) tracings showing effects of basolateral administration of terbutaline (2 µM) on Isc with and without pretreatment with apical amiloride (20 µM). Monolayer filters (4.5 cm2) were mounted in Ussing chambers and bathed on both apical and basolateral sides with identical serum-free DMEM-Ham's F-12 medium. A: addition of terbutaline produced a rapid decrease in Isc followed by a slow increase back to initial Isc. Addition of amiloride blocked most of remaining Isc. B: in presence of amiloride, addition of terbutaline produced a rapid sustained decrease in Isc without a secondary recovery phase.

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Representative Isc tracings showing effects of basolateral administration of terbutaline (2 µM) on Isc under Cl-free conditions. Monolayers (4.5 cm2) were mounted in Ussing chambers and bathed with identical Cl-free Ringer solution on both apical and basolateral sides. No response was produced by terbutaline, whereas addition of amiloride (20 µM) to apical solution produced a sustained decrease in Isc.

It is worth noting that comparison of current traces from monolayers bathed in serum-free DMEM-Ham's F-12 medium and Cl-free saline solution indicates a reduction in basal I_sc under Cl-free conditions (Fig. 2). This reduced basal current is due to the absence of amino acids (normally present in DMEM-Ham's F-12 medium) and is not the result of basal Cl secretion. Replacement of DMEM-Ham's F-12 medium with Cl-containing Ringer solution produces a decrease in basal I_sc similar in magnitude to that observed in Cl-free solution. Addition of the same free amino acids present in DMEM-Ham's F-12 medium to the apical solution produces a concentration-dependent increase in basal current for monolayers bathed in either Cl-containing Ringer solution or Cl-free solution. This result is consistent with the presence of an electrogenic amino acid cotransport system in the apical membrane that contributes to the basal I_sc (8).

To characterize terbutaline-regulated transport pathways present in the apical membrane, we used the pore-forming antibiotic amphotericin to perforate the basolateral membrane, thus eliminating it as a resistive barrier to the movement of monovalent ions. The basolateral and apical surfaces of the monolayer were bathed with either intracellular solution (in mM: 120 potassium methanesulfonic acid, 10 mM NaCl, 20 mM KHCO₃, 0.7 MgSO₄, 0.3 KH₂PO₄, 3 mM calcium gluconate, and 30 mannitol) or DMEM-Ham's F-12 medium, respectively. The apical membrane voltage was stepped through a series of command potentials ranging between +70 and 70 mV in 10-mV increments before and after treatment with either terbutaline or 8-CPT-cAMP. The current-voltage relationships for the terbutaline-activated and cAMP-activated conductances (Fig. 3A) were nearly linear, and the reversal potentials were 28.4 ± 2.7 and 26.3 ± 2.5 mV, respectively. To confirm that Cl was the permeant ion, increasing the Cl concentration in the intracellular solution from 10 to 20 to 35 mM produced a shift in reversal potential to more depolarized voltages as the Cl concentration gradient decreased, indicating that Cl was the current-carrying ion (Fig. 3B). The anion selectivity of the terbutaline-activated current was examined by performing a series of bi-ionic experiments with Br, I, and thiocyanate and measuring the shift in reversal potential produced in the presence of each of these replacement anions in the apical solution. The selectivity sequence was found to be thiocyanate > Br > Cl > I, similar to that previously reported for the cystic fibrosis transmembrane conductance regulator (CFTR) (1). As a control for the bi-ionic experiments, the current-voltage relationship for the terbutaline-activated current was measured under symmetrical Cl concentration conditions in the presence of an outwardly directed K concentration gradient and an inwardly directed Na concentration gradient. The reversal potential for the terbutaline-activated current was not significantly different from zero, again indicting that Cl was the current-carrying ion and that terbutaline does not alter apical membrane Na or K conductance. Finally, three known Cl-channel blockers [5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), glibenclamide, and diphenylamine-2-carboxylic acid (DPC)] produced a concentration-dependent block of the terbutaline-activated current with the following order of potency: NPPB (IC₅₀ = 12 mM) > glibenclamide (IC₅₀ = 110 mM) > DPC (IC₅₀ = 640 mM) (Fig. 4).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Current-voltage relationships for terbutaline- and 8-(4-chloro-phenylthio)-cAMP (8-CPT-cAMP)-activated conductance in apical membrane. Experiments were performed with amphotericin-perforated monolayers (4.5 cm2) mounted in Ussing chambers and bathed with potassium methanesulfonic acid (KMeSO4)-Ringer solution on basolateral side and serum-free DMEM-Ham's F-12 medium on apical side. A: current-voltage plot for terbutaline- and 8-CPT-cAMP-activated currents, with mean reversal potentials of 28.42 ± 2.68 and 26.32 ± 2.50 mV, respectively. Terbutaline (2 µM) and 8-CPT-cAMP (100 mM) were applied to basolateral bathing solution after pretreatment of monolayers with apical amiloride (20 µM). B: increasing basolateral Cl concentration ([Cl]i) produced a shift in terbutaline-sensitive reversal potential. Experiments were performed with amphotericin-perforated monolayers mounted in Ussing chambers and bathed with KMeSO4-Ringer solution on basolateral side and serum-free DMEM-Ham's F-12 medium on apical side. Changes in [Cl]i were achieved by replacing KMeSO4 with equimolar KCl. Terbutaline (2 µM) was applied to basolateral bathing solution after pretreatment of monolayers with apical amiloride (20 µM). Reversal potentials were plotted against log of basolateral [Cl]i values. Linear regression analysis was used to fit data (R = 0.983).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effects of Cl-channel blockers on terbutaline-activated current. Experiments were performed with amphotericin-perforated monolayers (4.5 cm2) mounted in Ussing chambers and bathed with KMeSO4-Ringer solution on basolateral side and serum-free DMEM-Ham's F-12 medium on apical side. Monolayer voltage was clamped at 0 mV. 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), glibenclamide, and diphenylamine-2-carboxylic acid (DPC) were added to apical solution. A: representative tracing of effect of apical glibenclamide on terbutaline-activated current. B: concentration-response relationship for NPPB, glibenclamide, and DPC inhibition of terbutaline-sensitive current. IC50 values for NPPB, glibenclamide, and DPC were 12, 110, and 640 µM, respectively.

Recently, cAMP-activated Cl currents were observed in monolayer cultures of adult rabbit alveolar epithelial cells (13). Stimulation with cAMP increased the I_sc. This response was blocked by basolateral treatment with bumetanide, suggesting that cAMP stimulates Cl secretion. In contrast to these results from rabbit monolayers, terbutaline- and cAMP-stimulated I_sc responses in adult rat alveolar cell monolayers were unaffected by basolateral bumetanide treatment and the direction of the current response was not consistent with stimulation of Cl secretion.

Terbutaline and cAMP do not directly increase the amiloride-sensitive Na conductance. An interesting observation from the Cl replacement experiments shown in Fig. 2 was the absence of any increase in amiloride-sensitive I_sc after treatment with terbutaline. Similar results were obtained when monolayers were stimulated with 8-CPT-cAMP under Cl-free conditions. These results suggested that terbutaline and cAMP do not directly activate apical membrane Na channels. To test this further, we again used amphotericin to perforate the basolateral membrane so that we could investigate the effects of terbutaline on apical membrane Na conductance. The basolateral and apical surfaces of the monolayer were bathed with intracellular solution and DMEM-Ham's F-12 medium, respectively, and the apical membrane voltage was clamped at 0 mV. Under these conditions, basolateral addition of terbutaline produced a decrease in current without the secondary increase that was previously observed in nonpermeable monolayers (Fig. 5). The lack of an increase in amiloride-sensitive current after stimulation with terbutaline again supported the conclusion that amiloride-sensitive Na channels were not activated in response to -adrenergic stimulation. In addition, examination of the current-voltage relationship for amiloride-sensitive Na channels in the apical membrane (Fig. 6) showed that the reversal potential was 46.5 mV, indicating high selectivity for Na over K (12.5:1), and no significant difference in either the conductance or reversal potential could be detected after terbutaline stimulation. The results shown in Fig. 6 represent difference currents calculated from the current-voltage relationships measured before and after amiloride treatment. Thus the results of these experiments support the conclusion that the increase in Na absorption produced by terbutaline is not the result of direct activation of amiloride-sensitive Na channels present in the apical membrane.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Representative Isc tracings showing effects of basolateral addition of terbutaline (2 µM) followed by apical addition of amiloride (20 µM; A) or apical addition of amiloride (20 µM) followed by basolateral addition of terbutaline (2 µM; B). Experiments were performed with amphotericin-perforated monolayers (4.5 cm2) mounted in Ussing chambers and bathed with KMeSO4-Ringer solution on basolateral side and serum-free DMEM-Ham's F-12 medium on apical side. Voltage across monolayers was clamped at 0 mV. C: terbutaline (T)-activated and amiloride (A)-sensitive current responses obtained from amphotericin-perforated monolayers voltage clamped at 0 mV. Pretreatment with amiloride did not significantly affect magnitude of terbutaline-activated current (15.29 ± 3.38 µA and 14.06 ± 4.26 µA). In addition, pretreatment with terbutaline did not significantly affect amiloride-sensitive current (7.63 ± 0.92 and 6.63 ± 1.23 µA).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Current-voltage relationship for apical membrane amiloride-sensitive Na conductance. Experiments were performed with amphotericin-perforated monolayers (4.5 cm2) mounted in Ussing chambers and bathed with KMeSO4-Ringer solution on basolateral side and serum-free DMEM-Ham's F-12 medium on apical side. Amiloride (20 µM) was applied to apical bathing solution, and terbutaline (2 µM) was applied to basolateral bathing solution. Mean reversal potentials for amiloride-sensitive current in absence (control) and presence of terbutaline were 46.47 ± 3.44 and 49.58 ± 4.29 mV, respectively.

Role of Cl-channel activation in stimulation of transepithelial Na absorption. We propose the following model to explain -adrenergic stimulation of Na and Cl absorption in cultured adult rat alveolar epithelial cells (Fig. 7). Both Na and Cl channels are present in the apical membrane, and in the absence of adrenergic agonists, Na channels are constitutively open and Cl channels are closed. Na influx through amiloride-sensitive Na channels maintains the apical membrane at a voltage that is more depolarized than the reversal potential of apical membrane Cl channels. Adrenergic-receptor stimulation with terbutaline or treatment with cell-permeant analogs of cAMP activate Cl channels without directly increasing apical membrane Na conductance. This increase in apical membrane Cl permeability rapidly decreases the I_sc after adrenergic stimulation, consistent with Cl influx across the apical membrane in response to an inwardly directed electrochemical gradient. Electrogenic Cl influx by this mechanism hyperpolarizes the apical membrane and increases the driving force for Na uptake through amiloride-sensitive Na channels. This is consistent with the time-dependent, amiloride-inhibitable increase in I_sc that follows the rapid decrease in current produced by terbutaline. This secondary phase of the terbutaline response is eliminated if the monolayers are pretreated with amiloride and is not observed at all in amphotericin-permeable monolayers where hyperpolarization is prevented by voltage clamping the apical membrane at 0 mV. Thus stimulation of amiloride-sensitive Na absorption by terbutaline or 8-CPT-cAMP is dependent on an increase in driving force produced by electrogenic Cl uptake mediated by apical membrane Cl channels. The idea that Cl absorption can involve apical Cl channels serving as uptake pathways for Cl was first proposed in human sweat duct epithelial cells where activation of CFTR resulted in Cl influx across the apical membrane (16). More recently, CFTR was shown to directly mediate Cl absorption and transepithelial fluid absorption in cultured bovine tracheal epithelial cells (20).

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Cell model showing proposed mechanisms for Na and Cl transport across adult rat alveolar epithelial cells. PKA, protein kinase A, AC, adenylyl cyclase; G, G protein.

An interesting result from our studies of alveolar epithelial cell monolayers is that terbutaline and 8-CPT-cAMP do not increase apical membrane Na conductance. This contrasts with the results of previous experiments with dissociated adult rat alveolar epithelial cells and fetal alveolar epithelial cells where cAMP and adrenergic stimulation increase the open probability of amiloride-sensitive Na channels without changes in single-channel conductance (11, 12, 19). One possible explanation for the discrepancy between our studies and a previous report on adult alveolar epithelial cells (12) may be related to differences in the regulation of epithelial Na channels (ENaC) in dissociated cells compared with cells in monolayers. Dissociated cells lose their polarity once tight junction complexes have been disrupted. This can alter the cell surface distribution of Na channels and possible regulatory proteins, resulting in different responses to second messengers such as cAMP. It is worth noting that association of regulatory proteins with ENaC subunits may be essential for conferring cAMP sensitivity to Na channels in epithelial cells. This idea is supported by the observation that injection of -ENaC, -ENaC, and -ENaC subunit cRNAs into Xenopus oocytes results in expression of amiloride-sensitive Na channels that are inhibited by protein kinase C activation but do not respond to increases in intracellular cAMP (2).

Mechanism of terbutaline stimulation of alveolar fluid absorption. It has been previously shown that adrenergic agonists increase fluid absorption across the rat alveolar epithelium and that this increase is inhibited if the lungs are infused with amiloride (5, 15, 18). This result supports the idea that stimulation of amiloride-sensitive Na absorption establishes the necessary osmotic driving force required for fluid removal from the alveoli (3, 6, 7, 14). The proposed mechanism to account for enhanced Na absorption is an increase in apical membrane Na permeability resulting from cAMP-mediated increases in the open probability of amiloride-sensitive Na channels. We suggest that there is another potential explanation consistent with results of our monolayer experiments with adult rat alveolar epithelial cells. Preliminary in vivo studies of fluid absorption in adult rats indicate that infusion of the Cl-channel blocker NPPB into rat lungs blocks terbutaline-stimulated increases in fluid absorption (10). Basal amiloride-sensitive fluid absorption was unaffected by treatment with NPPB. The lack of effect of NPPB on basal fluid absorption indicates that NPPB does not block amiloride-sensitive Na channels and is not toxic to the alveolar epithelium under in vivo conditions. These results suggest a role for Cl-channel activation in increasing amiloride-sensitive fluid absorption in adult rats. At the present time, it is not possible to confirm whether any increase in Na-channel activation takes place under in vivo conditions so we do not exclude the possibility that terbutaline may directly activate Na channels under in vivo conditions as well.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION CHLORIDE-CHANNEL ACTIVATION AND... CONCLUSIONS REFERENCES

The original goal of our studies with alveolar epithelial monolayers was to understand the mechanism of adrenergic receptor-stimulated Cl absorption previously observed by Kim et al. (9) using a similar preparation of rat alveolar epithelial cells. Our experiments indicate that selective -adrenergic-receptor agonists such as terbutaline activate apical membrane Cl channels with functional and pharmacological properties similar to those of CFTR. Our results indicate that adult rat alveolar epithelial cells absorb Cl from the apical medium by a mechanism analogous to that proposed for human sweat duct and bovine airway epithelial cells. This mechanism is interesting in that it involves an electrogenic influx pathway for Cl. Cl uptake is possible as a result of depolarization produced by Na influx through amiloride-sensitive Na channels present in the apical membrane. The mechanism for Cl efflux across the basolateral membrane is presently unknown but may involve an electroneutral transporter such as KCl cotransport or basolateral Cl channels. Our results indicate that an important secondary effect of Cl-channel activation is to increase transepithelial Na absorption. We propose that this is due to an increase in electrical driving force for Na uptake across the apical membrane through amiloride-sensitive Na channels and does not involve any increase in apical membrane Na permeability. Preliminary data from in vivo experiments provide pharmacological evidence to suggest that Cl-channel activation by terbutaline also occurs in intact adult rat lungs and that this activation is coupled to an increase in amiloride-sensitive fluid absorption. We suggest that activation of a transcellular pathway for Cl absorption that couples to and enhances the rate of transepithelial Na absorption provides an efficient mechanism for alveolar solute absorption and increases the osmotic driving force for fluid absorption across the alveolar epithelium.