SPECIAL TOPIC
Alveolar Epithelial Ion and Fluid Transport
beta -Adrenoceptor-mediated control of apical membrane conductive properties in fetal distal lung epithelia

A. Collett, S. J. Ramminger, R. E. Olver, and S. M. Wilson

Lung Membrane Transport Group, Tayside Institute of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom


    ABSTRACT
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Distal lung epithelial cells isolated from fetal rats were cultured (48 h) on permeable supports so that transepithelial ion transport could be quantified electrometrically. Unstimulated cells generated a short-circuit current (Isc) that was inhibited (~80%) by apical amiloride. The current is thus due, predominantly, to the absorption of Na+ from the apical solution. Isoprenaline increased the amiloride-sensitive Isc about twofold. Experiments in which apical membrane Na+ currents were monitored in basolaterally permeabilized cells showed that this was accompanied by a rise in apical Na+ conductance (GNa+). Isoprenaline also increased apical Cl- conductance (GCl-) by activating an anion channel species sensitive to glibenclamide but unaffected by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). The isoprenaline-evoked changes in GNa+ and GCl- could account for the changes in Isc observed in intact cells. Glibenclamide had no effect upon the isoprenaline-evoked stimulation of Isc or GNa+ demonstrating that the rise in GCl- is not essential to the stimulation of Na+ transport.

alveolar ion transport; Ussing chambers; permeabilized epithelia


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INTRODUCTION
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THROUGHOUT FETAL LIFE the distal lung epithelia secrete fluid into the developing air spaces (31) and establish a distending pressure that is crucial to lung morphogenesis (11). However, this liquid must be removed from the lungs if the newborn infant is to breathe at birth. The absorption of this liquid occurs during the final stages of gestation and is dependent upon the active withdrawal of Na+ from the lung lumen, a process that can be controlled via beta -adrenoceptors (23, 30, 36-38). The means by which this control is achieved are not fully understood, but there is evidence that the process involves a rise in apical Na+ conductance (GNa+) (15, 20). It is also known that beta -adrenoceptor agonists can increase apical Cl- conductance (GCl-) in these cells, and it has been suggested that this may facilitate Na+ transport by increasing the driving force for Na+ entry (16, 29). It is, however, difficult to see how the ionic gradients that normally prevail in epithelial cells could allow Na+ transport to be controlled in this way (see Refs. 20, 44). To clarify the means by which beta -adrenoceptor agonists can control Na+ absorption in the distal lung epithelia, we now explore the effects of isoprenaline upon the conductive properties of the apical membrane.


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Isolation and culture of rat fetal distal lung epithelial cells. Fetuses removed from anesthetized (3% halothane), 20-day pregnant (term = 22 days) rats were immediately decapitated, and their lung tissue was collected into ice-cold, Ca2+- and Mg2+-free Hanks' balanced salt solution. The anesthetized animals were then killed (cervical dislocation/exsanguination) before regaining consciousness. The fetal lung tissue was chopped into pieces (<0.5 mm) and disaggregated using 0.2% trypsin/0.012% DNase (2 × 20 min, 37°C) followed by 0.1% collagenase/0.012% DNase [15 min, 37°C, both in Dulbecco's modified Eagle's medium (DMEM)]. Cells pelleted from the resultant digest were resuspended in DMEM containing 10% fetal calf serum and then incubated (1 h, 37°C) in a 75-cm2 culture flask. The supernatant was then gently decanted to separate nonadherent epithelial cells from fibroblasts and smooth muscle cells. After a second such fractionation, the nonadherent cells were resuspended in culture medium, plated (1.5 × 106 cells/cm2) onto Transwell-Col membranes (Costar, High Wycomb, UK) and incubated (37°C) in an atmosphere of water-saturated room air containing 5% CO2 and sufficient N2 to reduce partial pressure of oxygen (PO2) to the level found in the adult alveolar region (100 mmHg). Cells were incubated in PC-1 medium unless otherwise stated. This is a serum-free medium that contains defined amounts of the hormones/growth factors found in fetal calf serum, which is almost invariably added to media used to maintain epithelial cells in primary culture. Its exact composition, however, is regarded as commercially sensitive. After 24 h, we changed the medium and removed nonviable cells by gently washing each culture. The cells were then incubated for a further 24 h before being used in experiments. By this time, the cells had almost invariably (>90% of cell preparations) become integrated into epithelial sheets with transepithelial resistances (Rt) >200 Omega cm2 (see also Refs. 1, 37). Although the cells are isolated from fetal animals, previously published work (1, 33, 36) shows that maintaining these cells in an atmosphere that mimics the PO2 of the postnatal alveolar region (~100 mmHg) causes the development of a Na+-absorbing phenotype typical of the neonatal, rather than the fetal lung.

Measurement of Isc and the conductive properties of the apical membrane. Cultured epithelia were mounted in Ussing chambers where they were bathed with bicarbonate-buffered physiological salt solution (composition given in Solutions and chemicals) that was continually circulated with a gas mixture identical to that in which the epithelia had been incubated (PO2 = 100 mmHg). Initially, the cells were maintained under open-circuit conditions until the transepithelial potential difference (Vt) had stabilized (30-40 min). Vt was then clamped to 0 mV, and the current required to hold this potential [short-circuit current (Isc)] was digitized (4 Hz) and displayed on a computer screen while simultaneously being recorded to computer disk using a PowerLab computer interface and associated software (ADI Instruments, Hastings, UK). In some experiments, Rt was also monitored by observing the currents flowing in response to repeated, 1-mV excursions in Vt. The conductive properties of the apical membrane were explored by using polyene antibiotics (nystatin or amphotericin B) to permeabilize the basolateral plasma membrane. These compounds form pores in cholesterol-containing membranes that are permeable to Na+, K+, and Cl- but not to divalent cations or higher-molecular-weight substances and thus allow experimental control over the cytoplasmic [Na+], [K+], and [Cl-], whereas intracellular Ca2+ is regulated by the normal, physiological mechanisms. Because the pores formed by amphotericin B have a higher Cl- conductance than do those formed by nystatin (13, 21), the former compound was used to make measurements of GCl-.

To determine apical GNa+, cells bathed with the cytoplasm-like solution (Table 1) were exposed to basolateral nystatin (75 µM) to permeabilize this membrane. An inwardly directed Na+ gradient was then imposed upon the permeabilized preparations by selectively modifying the composition of the apical solution (Table 1). Under these conditions, the driving force for Na+ entry (VNa+) is determined by the difference between Vt (0 mV) and the equilibrium potential for Na+ (ENa+, 41.8 mV). GNa+ can thus be calculated using the expression GNa+ = Iamil/VNa+, in which Iamil is the change in apical membrane current (Iap) elicited by apical amiloride (10 µM). The apical membranes' GCl- was measured using an analogous approach in which the Iap was evoked by imposing an outwardly directed Cl- gradient (Table 1) in amphotericin B (100 µM)-permeabilized epithelia.

                              
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Table 1.   Final concentrations (in mM) of the principal anions and cations in the solutions used in to determine GNa+ and GCl-

The use of such pore-forming agents can be complicated by the fact that they can promote cell swelling under certain conditions. This problem can be particularly severe when external [Cl-] is high, as the pore-forming agent will allow internal [Cl-] to reach a level greater than that found in intact cells. This will promote cell swelling by providing an osmotic driving force for water entry, and this, in turn, could activate other membrane conductances. Moreover, as most intracellular anions are essentially immobile, exposing permeabilized cells to high external [Cl-] can allow a Donnan potential to be established across the permeabilized membrane, which will affect the driving force for ionic movement across the intact side of the epithelium. The design of the present study was thus influenced by a need to prevent internal [Cl-] rising outside its normal, physiological range, and so in all experiments involving permeabilized epithelia, most Cl- in the standard solution was isosmotically replaced by gluconate, a nominally impermeant anion (Table 1). Although we could not measure cell volume in our system, preliminary experiments showed that no spontaneous changes in GCl- conductance occurred over the time scale of the present experiments. We have thus assumed that the present measurements were not complicated by the activation of volume-sensitive conductances.

The technique used to measure GNa+ and GCl- relies upon exposing the cultured epithelia to asymmetrical ionic conditions, and, by definition, this will establish a liquid junction potential that, if uncompensated, would provide a driving force for the movement of ions. Immediately before each experiment, we therefore monitored the effects of imposing the appropriate ionic gradient upon the potential difference across a culture membrane bearing no cells. The adjustments needed to offset this potential were noted and applied during the subsequent experiments.

Quantification of Na+ pump capacity. The method used to determine the capacity of the basolateral Na+ pump is described elsewhere (21, 36), and so only brief details are presented here. Epithelial monolayers were mounted in Ussing chambers, bathed with the standard physiological solution (i.e., high Na+), and treated with apical amiloride (10 µM) to block the Na+ channels in this membrane, which was then permeabilized using nystatin (75 µM). This elicited a slowly developing rise in Isc attributed to the activity of the basolaterally located Na+ pump. The fall in Isc evoked by subsequently adding ouabain (1 mM) to the basolateral solution (Ipump) was then measured to provide an indicator of the Na+ extrusion capacity of this pump. Our previously published work (36) shows that 1 mM ouabain never entirely abolished the Isc recorded from nystatin-permeabilized cells, and it is therefore possible that the method may slightly underestimate the capacity of the basolateral pump. However, we are forced to accept this limitation as higher concentrations of ouabain caused loss of epithelial integrity.

Data analysis and experimental design. Data are presented as means ± SE, and values of n refer to the number of times a protocol was repeated using cells prepared from different litters. The control current was defined as that measured under basal conditions at the onset of the experiment, and control and experimental cells were age matched and derived from the same litters. The statistical significance of differences between mean values was assessed using Student's t-test. In studies of intact cells, positive Isc was defined as the current carried by cations moving from the apical to the basolateral compartments, whereas, in basolaterally permeabilized preparations, positive Iap is that carried by cations leaving the cytoplasm. These are standard electrophysiological conventions.

Solutions and chemicals. The standard physiological salt solution contained (in mM) 117 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, and 11 D-glucose, pH 7.3-7.4 when bubbled with 5% CO2. The K+-gluconate solution was prepared by isosmotically replacing Cl- in this standard solution with gluconate, whereas the K+-Cl- solution was prepared by replacing Na+ with K+. Both ionic substitutions were made in the K+-gluconate solution. The amount of calcium gluconate added to gluconate-containing solutions was raised to 11.5 mM to maintain Ca2+ activity despite gluconate's capacity to bind this cation. All solutions were bicarbonate buffered and continually bubbled with 5% CO2 to maintain pH. The minimal defined-composition serum-free (MDSF) medium used in some experiments consisted of a mixture (1:1) of DMEM/Ham's F-12 nutrient mix that contained bovine serum albumin (1.25 mg/ml), L-glutamine (2 mM), and nonessential amino acids (0.1%). Amiloride and isoprenaline were freshly prepared (10 mM in distilled water) on each experimental day, whereas stock solutions (10 mM) of ethylisopropyl amiloride (EIPA; RBI International, Gillingham, UK) and glibenclamide (Tocris, St. Albans, UK) were prepared in dimethyl sulfoxide, and benzamil (50 mM; Molecular Probes) was dissolved in distilled water containing 20% (vol/vol) methanol. These solutions were divided into aliquots and stored at -20°C. Appropriate experiments showed that the solvent vehicles had no effect upon the parameters under study. Cell culture reagents were purchased from Paisley Life Technologies (Paisley, UK), and general laboratory reagents were from Sigma Chemical (Poole, UK).


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Properties of intact epithelia. Apical amiloride (10 µM) caused the spontaneous Isc to fall rapidly (20-40 s) to a value that was 23.1 ± 6.6% (n = 7) of the control level, and the concentration required to exert half-maximal inhibition (IC50) was 0.47 ± 0.03 µM (Fig. 1A). EIPA and benzamil also caused ~80% inhibition of the basal Isc, and the IC50 values for these compounds were 3.3 ± 0.8 µM and 10.8 ± 1.8 nM, respectively (Fig. 1A). The rank order of potency amongst these Na+ channel antagonists was thus benzamil > amiloride > EIPA, a profile suggesting the involvement of selective Na+ channels in the generation of the basal Isc (4). These experiments also revealed an amiloride-resistant component to the spontaneous Isc, and previous work has shown that subsequent application of bumetanide causes very little further fall in the spontaneous current; the ionic basis of this current is thus unknown (34). Basolateral isoprenaline (10 µM) caused a slowly developing rise in Isc, although an initial, more rapid component to this response was usually evident (Fig. 1B, see also Refs. 36, 37). After 30-40 min stimulation with this drug, Isc had risen to a value that was 173.2 ± 10.3% of the control level. Subsequent addition of apical amiloride (10 µM) caused a rapid fall to a value that was only 34.1 ± 3.8% of the control current measured at the onset of the experiment (Fig. 1B). Further experiments were undertaken in which amiloride-pretreated cells were exposed to isoprenaline. Examination of the currents recorded at the onset of these experiments confirmed that amiloride caused ~80% inhibition of basal Isc, and, as anticipated, the response to isoprenaline was essentially abolished (94.7 ± 1.3% inhibition) by this drug (Fig. 1B).


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Fig. 1.   Properties of unstimulated and isoprenaline-stimulated cells. A: in each experiment short-circuit current (Isc) was recorded under control conditions, and aliquots of benzamil (n = 5), amiloride (Amil; n = 6), or ethylisopropyl amiloride (EIPA, n = 4) were then added to the apical solution to raise the concentration of the appropriate drug in a stepwise manner. At the end of experiments involving benzamil and EIPA, 100 µM amiloride was also added to this solution to abolish any residual amiloride-sensitive current. The amiloride-sensitive Isc measured in the presence of each concentration of the 3 drugs was then expressed as a fraction of the total amiloride-sensitive Isc generated by that preparation. The pooled data are plotted (means ± SE) against the concentration of antagonist used. Solid lines are sigmoid curves fitted to these data using a least squares regression procedure implemented in a commercially available software package (Grafit 4, Erithicus Software, Staines, UK). B: the changes in Isc evoked by exposing cultured epithelia (n = 12) to 10 µM basolateral isoprenaline followed by 10 µM apical amiloride (top trace, n = 7) or by exposing amiloride-pretreated cells (10 µM apical) to basolateral isoprenaline (Iso, bottom trace, n = 5). Traces show the mean current generated. Vertical bars denote SE.

Effects of basolateral Ba2+. We studied the effects of basolateral Ba2+, a cation that blocks epithelial K+ channels, in an attempt to explore the role of these channels in the response to beta -adrenoceptor agonists. The application of Ba2+ to unstimulated cells caused a rapid fall in spontaneous Isc, but this effect was transient, and Isc returned to its basal value within 10 min (Fig. 2). Subsequent application of isoprenaline also caused a small fall in Isc, which contrasted with the initial rapid rise seen under control conditions. Although the recorded current returned to its basal value after ~5 min, Isc failed to rise above this level during 30-40 min of exposure to isoprenaline (Fig. 2). Ba2+ thus abolishes the isoprenaline-evoked rise in Isc. Subsequent experiments in which we monitored Na+ pump capacity (see METHODS) in control and Ba2+-treated cells showed that this cation caused 40.2 ± 6.5% inhibition of Ipump (control, 10.9 ± 1.4 µA/cm2, Ba2+-treated, 6.5 ± 1.1 µA/cm2, P < 0.01, Student's paired t-test), indicating that Ba2+, a well-known inhibitor of epithelial K+ channels, causes substantial loss of Na+ pump capacity.


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Fig. 2.   Responses to Iso in Ba2+-treated cells. The trace shows the pooled data (means ± SE, n = 5) from experiments in which cultured distal lung epithelial cells were exposed to basolateral Ba2+ (3 mM) followed by Iso (10 µM).

Regulation of GCl-. Imposing an outwardly directed Cl- gradient upon basolaterally permeabilized cells evoked only small (1-2 µA/cm2) currents, demonstrating that GCl- is normally low (~30 µS/cm2). Application of DIDS or glibenclamide (both 100 µM) reduced Iap, establishing that this small conductance is due to the efflux of Cl- through channels sensitive to these compounds. Analysis of these data showed that the DIDS-sensitive and glibenclamide-sensitive components of GCl- were 10.7 ± 1.7 µS/cm2 (n = 4) and 11.8 ± 4.0 µS/cm2 (n = 4), respectively. Basolateral isoprenaline (10 µM) evoked a rise in Iap that occurred with no discernible latency, reaching a level that was ~15-fold greater than the basal value after ~20 s. Iap then fell to a plateau value two- to threefold above control (Fig. 3A). The application of glibenclamide (100 µM) to isoprenaline-stimulated preparations caused a rapid fall in Iap, and subsequent application of DIDS (100 µM) caused a further small fall (Fig. 3A). Analysis of these data indicated that isoprenaline augmented the glibenclamide-sensitive component of GCl- approximately fivefold (55.7 ± 7.2 µS/cm2, P < 0.05 vs. control data presented above) but had no significant effect upon the DIDS-sensitive component (17.1 ± 1.9 µS/cm2). Experiments in which cells were exposed to these Cl- channel blockers (100 µM) before stimulation with isoprenaline (10 µM) showed that glibenclamide caused ~80% inhibition of the peak response (Fig. 3, B and C) but that DIDS had no effect (Fig. 3C). Isoprenaline thus increases GCl- by selectively activating the glibenclamide-sensitive conductance. Further studies of basolaterally permeabilized cells (n = 4) showed that basolateral isoprenaline had no discernible effect upon Iap when both apical and basolateral [Cl-] were maintained at 10.3 mM. The isoprenaline-evoked changes in Iap are thus dependent upon the presence of the Cl- gradient and so reflect an increase in GCl- that would facilitate the flow of Cl- down its electrochemical gradient. To establish the extent to which this response is maintained during prolonged stimulation, cells were first exposed to isoprenaline while bathed with standard physiological salt solution, and the basolateral membrane was then permeabilized to allow the glibenclamide-sensitive component of GCl- to be measured. These experiments revealed an approximately fourfold stimulation of glibenclamide sensitive GCl- after 30-40 min of stimulation (control: 8.5 ± 2.4 µS/cm2; isoprenaline-stimulated: 34.3 ± 4.5 µS/cm2, n = 4, P < 0.01). Forskolin (100 µM), a drug that directly activates adenylate cyclase (40), caused changes in GCl- essentially identical to those seen during stimulation with isoprenaline (n = 4), and this response, in common with the response to isoprenaline, was inhibited by glibenclamide but unaffected by DIDS (Fig. 3D).


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Fig. 3.   Regulation of Cl- conductance (GCl-). A: apical membrane current recorded from basolaterally permeabilized distal lung epithelial cells exposed to an outwardly directed Cl- gradient and then stimulated by adding 10 µM Iso to the basolateral bath as indicated by the arrow. The apical membrane was then exposed to glibenclamide (Glib) followed by DIDS (both 100 µM), as indicated by the arrows. Throughout the experiment, transepithelial potential (Vt) was normally held at 0 mV, but the currents flowing in response to 1 mV excursions from this holding potential are shown to allow transepithelial resistance (Rt) to be monitored. B: current record obtained in an analogous experiment in which basolaterally permeabilized cells were exposed to apical Glib (100 µM) before being stimulated with basolateral Iso (10 µM). The cells were also exposed to 100 µM apical DIDS as indicated. C: mean increases in GCl- evoked by 10 µM Iso in control cells (n = 7) and in Glib- (n = 4) and DIDS-pretreated (n = 4) cells. D: forskolin-evoked (100 M) increases in GCl- recorded from control (n = 4), Glib- (n = 4), and DIDS-pretreated (n = 4) cells. Iap, apical membrane current. **P < 0.01; ***P < 0.005.

Regulation of GNa+. Experiments in which Iap was recorded from basolaterally permeabilized cells exposed to an inwardly directed Na+ gradient showed that isoprenaline had no effect over a 10- to 15-min period (n = 4). However, the isoprenaline-evoked rise in Isc develops over 30-40 min (Fig. 2), and so further experiments were undertaken in which cells were permeabilized and GNa+ was quantified once this response had become fully established. The data derived from intact cells (Fig. 4B) confirmed that isoprenaline evoked a slowly developing rise in Isc, and the corresponding measurements of GNa+ (Fig. 4, Aii and Bii) showed that this was accompanied by a rise (approximately twofold) in GNa+ (control: 60.0 ± 9.9 µS/cm2, n = 6; isoprenaline-stimulated: 138 ± 12.0 µS/cm2, n = 4, P < 0.01). We studied the effects of glibenclamide upon this response to explore the role of the cAMP-activated anion channels (16, 29). These studies showed that apical glibenclamide had no significant effect upon basal Isc (control Isc: 7.4 ± 1.4 µA/cm2; postglibenclamide Isc: 7.4 ± 1.7 µA/cm2, n = 4). Moreover, application of basolateral isoprenaline evoked a rise in Isc (3.5 ± 1.5 µA/cm2, n = 4) in the glibenclamide-treated cells that did not differ significantly from that seen in age-matched control cells at identical passage (3.6 ± 0.94 µA/cm2, n = 5). Once these responses to isoprenaline had become established, the control and glibenclamide-treated cells were basolaterally permeabilized so that GNa+ could be measured. This analysis showed that glibenclamide had no statistically significant effect upon this parameter (control: 138.4 ± 12.0 µS/cm2, n = 5; glibenclamide-treated, 143.1 ± 26.4 µS/cm2, n = 4). Thus glibenclamide, at a concentration that can cause >90% inhibition of the cAMP-evoked rise in GNa+, has no significant effect upon the overall response to isoprenaline.


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Fig. 4.   Effects of Iso upon Na+ conductance (GNa+). Cultured distal lung epithelial cells were initially bathed with the standard physiological salt solution, and Isc was monitored. Although Vt was normally held at 0 mV, brief excursions (1 mV) from this holding potential were regularly imposed so that Rt could be monitored. At the points indicated by the arrows [K+-gluconate solution (K-Gluc.)], aliquots of salt solution were withdrawn from the apical and basolateral baths and replaced with K-Gluc. to adjust [Na+], [Cl-], and [K+] to 11.5 and 10.3 mM, respectively. The basolateral membrane was then permeabilized by adding nystatin (Nys) to the salt solution, bathing this side of the cell layer, and the [Na+] was then raised to 55 mM to establish driving force for Na+ influx. The cells were then exposed to apical amiloride (10 µM) as indicated. A: representative currents recorded from unstimulated cells. B: representative currents recorded during stimulation with basolateral isoprenaline (10 µM). Each major panel is subdivided to include records of Isc, derived from intact cells (i), and Iap, which were obtained from the same cells after basolateral permeabilizations (ii).

Responses to isoprenaline in cells maintained in MDSF medium. Experiments in which the standard culture medium (PC-1) was replaced with the MDSF medium (see METHODS) used in a previous study of alveolar epithelial cells isolated from adult animals (16) showed that the cells still became incorporated into epithelial layers (Rt = 510 ± 27 Omega cm2) under these conditions. These cultured epithelia generated a spontaneous Isc (10.4 ± 0.5 µA/cm2) similar to that recorded from cells maintained in PC-1 medium. Apical amiloride (10 µM) elicited an immediate inhibition of this spontaneous current (83.4 ± 1.5% n = 6). Further studies of cells cultured under these conditions (n = 5; spontaneous Isc = 7.5 ± 0.6 µA/cm2; Rt = 389 ± 81 Omega cm2) showed that basolateral isoprenaline (10 µM) elicited only a barely discernible (Delta Isc < 0.5 µA/cm2), transient fall in Isc, so that the current measured after 20-30 min exposure to this drug (7.5 ± 1.4 µA/cm2) did not differ significantly from the initial control value. Although cells maintained in MDSF medium thus generate a spontaneous Isc that appears to be due largely to electrogenic Na+ absorption, this transport process does not appear to be subject to beta -adrenoceptor-mediated control under these conditions. This contrasts markedly with the consistent stimulation of Isc seen in cells maintained in medium PC-1 (Fig. 1).


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Properties of intact cells. Data obtained from cells maintained in medium PC-1 accord with the view that cultured distal lung epithelial cells spontaneously absorb Na+ from the apical solution and show that cAMP-coupled agonists elicit a slowly developing but substantial rise in the rate of transepithelial ion transport. This response was due to a stimulation of amiloride-sensitive Isc, and so these data confirm that the Na+ transport process underlying the basal Isc is subject to beta -adrenoceptor-mediated control (see also Refs. 3, 23, 26, 36-38). However, net Na+ absorption is not a feature of the fetal lung where the dominant ion transport process is the secretion of anions into the lung lumen (31). This discrepancy between the present observations and those made in the intact fetal lung almost certainly reflects the fact that the present study was undertaken using cells cultured at adult alveolar PO2 (100 mmHg) rather than at fetal PO2 (~23 mmHg). We (1, 36) and others (2, 33) have recently shown that this causes the development of the Na+-absorbing phenotype characteristic of the adult lung. Although the present study was undertaken using cells isolated from fetal animals, we believe that these cells expressed an adult phenotype when used in the experiments but cannot formally exclude the possibility that they may have adopted an intermediate phenotype.

Ba2+-evoked changes in Isc. It is well documented that basolateral K+ channels, which can be blocked by Ba2+, make an important contribution to the maintenance of membrane potential (Vm) in epithelial cells (32), and such channels could thus play an important role in epithelial Na+ transport by maintaining the driving force for Na+ entry (25, 27). The present study, in common with earlier data (27), showed clearly that Ba2+ abolishes the response to isoprenaline, but, in our hands, this cation caused only transient inhibition of basal Isc. This result is difficult to reconcile with the view that Ba2+ acts by selectively inhibiting K+ channels, and subsequent experiments showed that it also reduced the activity of the basolateral Na+ pump. Although this provides an alternative explanation for the inhibition of the response to isoprenaline, it cannot account for the transient inhibition of the basal Isc. High concentrations of Ba2+, in common with other alkaline earth ions, have the potential to interfere with the control of the internal free Ca2+ concentration ([Ca2+]i) (18, 19), and it is interesting, in this context, that Marunaka and colleagues (22) attribute the isoprenaline-evoked increase in Isc to a cAMP-evoked increase in intracellular [Ca2+]i. The inhibitory effect of Ba2+ that we now describe could, therefore, also involve a direct interaction with the beta -adrenoceptor-activated signal transduction pathway. The important point to emerge from these experiments is that Ba2+ exerts a number of effects under the present conditions, and so this cation cannot be used as a selective inhibitor of the K+ channels found in Na+-absorbing epithelia.

Conductive properties of the apical membrane. The small GCl- measured in unstimulated cells appears to be due to the presence of anion channels sensitive to DIDS and glibenclamide. Isoprenaline and forskolin elicited clear increases in GCl-, and these responses were inhibited by glibenclamide but not DIDS (see also Ref. 16), suggesting (see for example Refs. 10, 24, 41) that the currents elicited by cAMP-coupled agonists reflect the activation of anion channels formed by the cystic fibrosis transmembrane conductance regulator (CFTR), the gene product that is defective in cystic fibrosis (CF) (43). Support for this view came from parallel studies (O. G. Best, A. Collett, S. M. Wilson, and A. Mehta, unpublished data) in which apical membrane Cl- currents were characterized in two human airway epithelial cell lines. In wild-type cells (16HBE14o-), forskolin evoked changes in GCl- that were qualitatively similar to those described here. However, this response was absent from cells expressing a CF phenotype (CFBE41D-), which have only minimal levels of functional CFTR in the apical membrane. Our findings thus accord with the results of earlier, more detailed studies of cells isolated from adult animals that identified CFTR-like anion channels in distal lung epithelial cells (16, 29). However, in our hands, the cAMP-evoked increase in GCl- consisted of an initial peak followed by a slower, more sustained phase, whereas previous studies of anion currents flowing through CFTR-like channels have reported that cAMP causes a much more sustained response (12, 16). The reason for this discrepancy between these two studies is unknown but may reflect the different origin of the cells used in the two studies. However, CFTR can be subject to relatively rapid "rundown" in many experimental situations, and it has become clear that this process is controlled, at least in part, by Ca2+-dependent protein kinases (45). The cells used in the present study were cultured under conditions different to those used by Jiang et al. (16), and so it is therefore possible that the variations between the observed time courses may reflect slight differences in the basal level of protein kinase C activity. However, it is also possible that the increase in GCl- that we report may involve the activation of other anion channel species. It is interesting, in this context, that activation of CFTR has been shown to trigger the increased activity of other anion channels in epithelial cells and that this downstream effect is abolished when CFTR is blocked (9, 39). It is therefore possible that the ionic movements underlying the cAMP-evoked Cl- currents that we now describe may not occur exclusively via CFTR.

GNa+ was normally ~60 µS/cm2, and previous work has shown that this conductance reflects the presence of channels that discriminate clearly between Na+ and K+ (1), a situation very similar to that documented by Jiang et al.'s (16) study of cells isolated from adult animals. The channels underlying this apical conductance almost certainly correspond to the selective epithelial Na+ channels (ENaC) formed by the proteins encoded by the alpha -, beta -, and gamma -ENaC genes (5, 6, 14, 23). Experiments in which nystatin-treated cells were exposed to isoprenaline, in common with data presented by Jiang et al. (16), failed to provide evidence that these channels were subject to acute control via beta -adrenoceptor agonists. We found this surprising, because our studies of intact cells showed that the isoprenaline-evoked increase in Isc is evident within ~10 min and because a number of previous studies have suggested that cAMP-dependent agonists can increase GNa+ in distal lung epithelia (15, 20). To resolve this apparent contradiction, we undertook further experiments in which cells were stimulated with isoprenaline before the basolateral membrane was permeabilized. These studies showed clearly that GNa+ was higher than normal after stimulation with this drug. It is thus clear that cAMP-dependent agonists increase both GNa+ and GCl-, but, in the present study, the rise in GCl- was a much more rapid phenomenon than the rise in GNa+. It is possible, however, that rapid changes in GNa+ might occur in intact cells, since permeabilizing the basolateral membrane could allow the loss of a cytoplasmic component essential to the regulation of GNa+. Indeed, recent work has shown that ENaC can be rapidly controlled via changes in internal [Na+] and [Cl-] (8, 22), and it is very unlikely that such ionic control of GNa+ (8, 22) would be detected in studies of basolaterally permeabilized cells. In this experimental situation, transient changes in internal ionic composition may occur as apical conductances are activated, but the relatively large volume of the external solution implies that such changes would be rapidly compensated for by ions entering/leaving the cytoplasm via the permeabilized membrane (21). The present estimates of GNa+ were thus made under relatively constant ionic conditions.

Physiological effects of the changes in GNa+ and GCl-. The present estimates of GCl- and GNa+ were combined with previously reported values of Vm and internal [Cl-] and [Na+] to allow us to predict the Cl- and Na+ currents flowing across the apical membrane under basal conditions and after prolonged stimulation with isoprenaline. Although there is a driving force for Cl- entry in unstimulated cells, GCl- is so low that only a negligible (<0.5 µA/cm2) Cl- current can flow (Table 2). However, our analysis suggests that this would be accompanied by a substantial (~7 µA/cm2) inward Na+ current (Table 2) and thus predicts that Na+ absorption is the dominant electrogenic transport process in unstimulated cells. Stimulation with isoprenaline reverses the driving force for Cl- movement, suggesting that this drug may evoke the secretion of this anion, and so our analysis thus predicts a rapid but transient rise in Isc due to the secretion of Cl-, although it is difficult to anticipate the magnitude of this early current as Vm and internal [Cl-] are both subject to rapid change at this time (22, 42). During prolonged stimulation, however, GCl- remains relatively low, and so the total, inwardly directed Cl- current would amount to only ~1 µA cm-2 (Table 2). Most importantly, our model predicts that this initial transient would be followed by a slowly developing but persistent rise in Isc that is due, very largely (>90%), to increased Na+ absorption. There is thus excellent agreement between the observed and predicted responses, and so the changes in GCl- and GNa+ that we now report can explain the well-documented effects of isoprenaline upon these cells (23, 30, 36-38). Moreover, in A6 cells, a Na+-absorbing cell line derived from the amphibian kidney, cAMP-dependent agonists have been reported to elicit a biphasic response similar to that described above (7). This pattern of response may thus be a generalized feature of salt-absorbing epithelia.

                              
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Table 2.   Values of Vm, VNa+, VCl- and estimates of the appropriate apical membrane currents for unstimulated and isoprenaline-stimulated cells

Comparison with previous work. When stimulated with cAMP-coupled agonists, the adult cells used by Jiang et al. (16) displayed a rapid fall in Isc followed by a slow recovery toward the basal level consistent with a rapid stimulation of Cl- absorption superimposed upon a slowly developing stimulation of Na+ transport, a finding that accords with data from earlier isotope flux studies of such cells (17). The fetally derived cells used in the present study, however, responded to isoprenaline with a clear stimulation of Na+ transport that appeared to be accompanied by only a very small stimulation of Cl- secretion. As anion secretion is an important physiological feature of the fetal lung (31), it could be argued that the presence of this small secretory response implies that the cells have retained some features of the fetal lung.

There are thus clear physiological differences between the adult cells used by Jiang et al. (16) and the fetally derived cells used in the present study. However, the cells' origin is not the only difference between the two studies. It may also be relevant that the cells used by Jiang et al. (16) were cultured on permeable supports for 5-7 days in a simple, serum-free medium devoid of hormones/growth factors, whereas our cells were normally cultured for only 48 h in a medium containing many of the hormones and growth factors present in serum. The present study shows clearly that the isoprenaline-evoked stimulation of amiloride-sensitive Isc is suppressed when fetally derived cells are maintained in the medium used by Jiang et al. It is therefore possible that their failure to detect a rise in GNa+ may be due, at least in part, to the fact that the culture medium used lacks a component needed for beta -adrenoceptor-mediated control of alveolar Na+ transport.

Possible role of the CFTR-like channels. The present study shows that prolonged stimulation with cAMP-coupled agonists leads to a clear rise in GNa+, which implies increased Na+ entry despite a fall in VNa+ (Table 2). Because Jiang et al. (16) did not detect such control over GNa+, they concluded that cAMP-dependent agonists do not activate Na+ channels in alveolar epithelia. Instead, they suggested that such drugs might stimulate Na+ transport by activating the CFTR-like anion channels, hyperpolarizing the cell and thus increasing VNa+ (16). There are, however, conceptual problems with this model, some of which have been discussed by Widdicombe (44). For example, the analysis presented here suggests that VCl- is negative under resting conditions, and so, rather than hyperpolarizing the cell, a rise in GCl- would initially favor depolarization and a fall in VNa+ (Table 2).

Difficulties remain even if the estimates of Vm and [Cl-]i used here are rejected to allow the premise that increased GCl- might hyperpolarize the cell (16, 29). If it is accepted that [Na+]i normally lies between 12 and 25 mM, then ENa+ must be between 46 and 65 mV, and so, in resting cells, VNa+ will be between 80 and 100 mV. There is thus a substantial driving force for Na+ influx under resting conditions. If the rate of Na+ entry is to double with no change in GNa+, then VNa+ must rise to at least 160 mV, which will require a hyperpolarization of at least 80 mV. Changes in GCl- cannot be transduced into such large changes in Vm unless ECl- is at least 80 mV more negative than the resting Vm (i.e., approximately -120 mV). Examination of this problem using the Nernst equation shows that this condition can be satisfied only if internal [Cl-]i is ~1 mM, a value at least one order of magnitude lower that the values typically reported for mammalian epithelia. Moreover, the effect of selectively increasing GCl- upon Vm will be dependent upon the contribution that GCl- makes to the membrane's total ionic conductance. If the plasma membrane has substantial conductances to Na+ and K+, then the effect of changing GCl- will be smaller than described above. This implies that internal Cl- must be even lower (i.e., the ECl- must be even more negative) to allow Na+ transport to be controlled in the manner predicted by Jiang et al. (16).

In summary, the present study shows that, in cells derived from fetal animals but maintained under conditions that favor the development of an adult phenotype, the cAMP-dependent stimulation of Na+ transport reflects a rise in GNa+ rather than the parallel stimulation of GCl-. Nevertheless, the studies of adult cells (16) suggest that this response is dependent, in some way, upon the presence of external Cl-. It is interesting, in this context, that studies of the fetally derived cells have also shown that the isoprenaline-evoked stimulation of Isc is abolished if Cl- transport is blocked with a combination of bumetanide and DPC (35). In both experimental systems, the transport of Na+ and the transport of Cl- thus seem to be linked in some way. Interestingly, changes in [Cl-]i have recently been shown to influence epithelial Na+ channel activity in some systems (8, 22). It is therefore possible that the apparent coupling between Na+ and Cl- transport (16, 29) may, at least in part, be due to [Cl-]i-dependent control of GNa+.


    ACKNOWLEDGEMENTS

The authors are grateful to Helen Murphie for skilled technical help and to Sarah Inglis for many helpful comments and suggestions.


    FOOTNOTES

The authors are also grateful to the Wellcome Trust for the financial support (program grant no. 0548/Z/99/Z/JMW/CP/JF) that made this study possible.

Address for reprint requests and other correspondence: S. M. Wilson, Lung Membrane Transport Group, Tayside Institute of Child Health, Ninewells Hospital and Medical School, Univ. of Dundee, Dundee DD1 9SY, UK (E-mail: S.M.Wilson{at}Dundee.ac.uk).

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. Section 1734 solely to indicate this fact.

10.1152/ajplung.00142.2001

Received 24 April 2001; accepted in final form 5 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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