cAMP activation of chloride and fluid secretion across the rabbit alveolar epithelium

Vance G. Nielsen1, Michael D. Duvall1, Manuel S. Baird1, and Sadis Matalon1,2,3

Departments of 1 Anesthesiology, 2 Physiology and Biophysics, and 3 Pediatrics, The University of Alabama at Birmingham, Birmingham, Alabama 35233

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Active Na+ transport by alveolar epithelial cells has been demonstrated to contribute significantly to alveolar fluid clearance. However, the contribution of transepithelial Cl- movement to the reabsorption of isosmotic fluid across the alveolar epithelium in vivo has not been elucidated. We hypothesized that Cl- transport could be increased across the alveolar epithelium in vivo and across cultured alveolar type II cells by agents that increase intracellular cAMP (e.g., forskolin). In studies where 5% albumin in sodium methanesulfonate (a Cl--free solution) was administered into the lung, forskolin administration significantly increased intracellular influx of Cl- and fluid into the alveolar space. In vitro studies with cultured rabbit alveolar type II cell monolayers in Ussing chambers demonstrated that elevations in intracellular cAMP increase short-circuit current by increasing both Cl- secretion and Na+ reabsorption. The cystic fibrosis transmembrane conductance regulator channel blocker glibenclamide and the loop diuretic bumetanide partially decreased the forskolin-induced increase in short-circuit current. These data may explain the failure of agonist that stimulated intracellular cAMP to increase alveolar fluid clearance in the rabbit. Moreover, the data suggest that in the event Na+ absorptive pathways are damaged, transepithelial Cl- secretion and the consequent intra-alveolar fluid influx may be upregulated.

amiloride; sodium transport; terbutaline; short-circuit current; alveolar type II cells; adenosine 3',5'-cyclic monophosphate

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE PRIMARY FUNCTION of the lung is gas exchange, which occurs by simple diffusion. The presence of fluid in the alveolar space increases the diffusion distances for O2 and CO2 and results in ventilation-perfusion mismatch, arterial hypoxemia, and hypercapnia. Thus the efficient removal of fluid (transudate or exudate) across the normal and injured alveolar epithelium is of primary importance for the maintenance of normal lung function. Active transport of Na+ across the alveolar epithelium creates an osmotic force, which results in the reabsorption of alveolar fluid isotonic to plasma (14). A study (13) across isolated alveolar type II (ATII) cells indicated that Na+ diffuses passively across the ATII cell apical membranes and down a favorable electrochemical gradient maintained by Na+-K+-ATPase, and then is actively transported across the basolateral membranes of these cells by ouabain-sensitive Na+-K+-ATPase. Various studies in humans and animals indicated that active Na+ reabsorption plays an important role in diminishing alveolar fluid and improving oxygenation in both patients with cardiogenic and noncardiogenic edema (15) and animals with hyperoxic lung injury (18, 27).

The demonstration that alveolar fluid clearance (AFC) in vivo and ex vivo is upregulated by agents that increase the concentration of intracellular cAMP (3, 6, 9, 21) has led to speculation that these agents may be useful in limiting alveolar edema in a variety of pathological conditions. Patch-clamp studies of freshly isolated ATII cells and a putative Na+-channel protein isolated from ATII cells and reconstituted in lipid bilayers indicated that agents that increase cAMP increase the open probability of amiloride-sensitive Na+ single channels without affecting their conductance (22, 28). Furthermore, cAMP increased the activity of ATII cell Na+-K+-ATPase by an Na+-independent mechanism (26).

It has been thought that Cl- transport across alveolar epithelial cells occurred passively through the paracellular junctions, driven by the existing electrochemical forces. However, recent evidence indicates that cultured rat ATII cells contain a cAMP-stimulated Cl- conductance in their apical membranes (11). Measurements of short-circuit currents (Isc) and Cl- fluxes across monolayers of rat ATII cells mounted in Ussing chambers were consistent with transient increases in transepithelial Cl- reabsorption by agents that increase cAMP (11, 12). However, the contribution of the transepithelial Cl- movement to the reabsorption of isosmotic fluid across the alveolar epithelium of intact animals has not been elucidated. It is conceivable that in some situations, especially when transepithelial Na+ reabsorption has been compromised by reactive species (10, 16), adult alveolar cells may secrete Cl-, which may cause a net influx of fluid into the alveolar space. Fetal distal lung epithelial cells secrete Cl- into the alveolar space in utero, which subsequently drives fluid secretion (17).

We thus performed a series of experiments to study the reabsorption of isotonic fluid instilled into the alveolar spaces of anesthetized rabbits. To investigate the contribution of Na+ and Cl- in this process, we performed measurements with 1) normal saline in the presence and absence of amiloride, which blocked the entry of Na+ across amiloride-sensitive Na+ channels, and 2) Cl--free solutions. These measurements were then repeated after intratracheal instillation of forskolin, an agent known to increase cAMP levels in ATII cells (19). We performed our experiments in rabbits because these animals due to their size are better suited than rats for sequential measurements of alveolar fluid. To elucidate the putative mechanisms, we also isolated ATII cells from the lungs of rabbits, cultured them until they formed tight monolayers, and then measured Isc before and after stimulation with forskolin or permeant analogs of cAMP. The results of these studies indicate that transcellular movement of both Na+ and Cl- contribute to the magnitude and direction of fluid fluxes across the alveolar epithelium after cAMP stimulation.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

The study was approved by the Animal Review Committee of the University of Alabama at Birmingham. All animals received humane care in compliance with the Principles of Laboratory Care formulated by the National Society for Medical Research and with the Guide for the Care and Use of Laboratory Animals prepared by the National Research Council (Washington, DC: Natl. Acad. Press, 1996).

Measurements of AFC in rabbits. Male New Zealand White rabbits (Myrtle's Rabbits, Thompson Station, TN) weighing 1.6-2.6 kg were allowed free access to food and water before experimentation. All rabbits were anesthetized with an intravenous injection of ketamine (10 mg/kg; Parke-Davis, Morris Plains, NJ) via a marginal ear vein. Anesthesia was maintained throughout the experimental procedure by the administration of inhaled 1% isoflurane (Abbott Laboratories, North Chicago, IL) carried in 99% O2. After a tracheotomy, a 3.5-mm-ID endotracheal tube and a modified 4F Fogarty catheter (American Edwards Laboratory, Irvine, CA) were placed into the trachea. The Fogarty catheter was modified by removal of the balloon tip and placement of a yellow suture bootie (Oxboro-Medical International, Ham Lake, MN) that was punctured on its distal end. Placement of this catheter ~9-10 cm into the trachea resulted in an airtight intubation of the right caudal lobe of the lung that was confirmed postmortem. Rabbits were ventilated with a Harvard Apparatus ventilator (model 683, Harvard Apparatus, Millis, MA). The tidal volume and rate were adjusted to yield a peak inspiratory pressure between 14 and 17 mmHg measured from within the endotracheal tube and an arterial PCO2 (PaCO2) of 32-45 Torr. Pancuronium bromide (0.3 mg · kg-1 · h-1; Elkins-Sinn, Cherry Hill, NJ) was administered intravenously to ensure relaxed chest wall muscle tone. Arterial pressure was monitored by placement of a 22-gauge central ear arterial catheter. All pressures were recorded on a Grass model 7D polygraph (Grass Instruments, Quincy, MA). All rabbits received a maintenance infusion of lactated Ringer at 4 ml · kg-1 · h-1, and esophageal temperatures were maintained at 38-39°C with a heating pad. A 15-min equilibration period followed completion of the surgical preparation.

After equilibration, 10 ml of 5% fat-free bovine serum albumin (Sigma) suspended in solutions with varying ionic compositions (see below) were instilled into the right caudal lobe over a 2-min period. The dead space in the modified Fogarty catheter was cleared by injection of 600 µl of 100% O2. The fluid in the right caudal lobe was subsequently withdrawn in 1-ml increments 1 and 2 h after instillation, a 300-µl aliquot of the bronchoalveolar lavage fluid (BALF) was removed for analysis, and the remaining fluid was reinstilled in the right caudal lobe. The 300-µl samples were centrifuged at 1,000 g for 10 min to pellet cells and debris, and the protein concentration was determined by modification of a spectrophotometric method (24).

AFC expressed as a percentage of total instilled volume (excluding the volume of albumin) was calculated from the following relationship, as described previously (7, 27): AFC = (1 - Ci/Ct)/0.95, where the variables Ci and Ct are, respectively, the protein concentrations at time 0 and at either 1 or 2 h. Time 0 was considered to be the end of the instillation. The osmolality of the instillate and the BALF samples was determined with a vapor pressure osmometer (model 5520, Wescor, Logan, UT). The pH, PaCO2, arterial PO2 (PaO2), and HCO-3 of arterial blood samples were determined after 15 min of equilibration and every 30 min thereafter throughout the experimental period. All blood gas samples were analyzed at 37°C with a blood gas analyzer (model 1306, Instrumentation Laboratory, Lexington, MA).

Four groups of rabbits (n = 12/group) were used to determine the effects of intratracheal cAMP instillation on AFC and its dependence on Na+ reabsorption. In the control group, 10 ml of 154 mM NaCl containing 5% BSA (NaCl-BSA) were instilled into the right lower lobe as described above. To investigate the contribution of Na+ reabsorption to AFC, we instilled NaCl-BSA containing 1 mM amiloride into a second group of rabbits. The concentration of amiloride in the instilled volume was determined by measuring the fluorescence intensity of alveolar samples at an excitation of 360 nm and emission of 415 nm (8). A third group of rabbits was instilled with NaCl-BSA containing 50 µM forskolin, and the fourth group was instilled with NaCl-BSA containing 50 µM forskolin and 1 mM amiloride.

The contribution of transepithelial Cl- transport in AFC was determined by instilling a solution in which NaCl was replaced with equimolar (154 mM) sodium methanesulfonate (pH 6.9-7.1, 301-304 mosmol). Cl- concentrations of the instillate and BALF samples were determined with a Cl- analyzer (model 925, Ciba Corning Diagnostics, Midfield, MA). To evaluate the effects of increased epithelial cAMP on AFC and Cl- influx into the alveolar space, a second group of rabbits (n = 8) was instilled with a similar Cl--free sodium methanesulfonate solution containing 50 µM forskolin. All rabbits were euthanized with an overdose of pentobarbital sodium (65 mg/kg; The Butler Company, Columbus, OH) after the 120-min instillate sample was obtained.

ATII cell isolation and culture. ATII cells were isolated from the lungs of young adult male New Zealand White rabbits (1.8-2.2 kg) as previously described (1). Briefly, the rabbits were anesthetized and killed by an intravenous pentobarbital sodium injection (100 mg/kg body wt). The lungs were ventilated with air, perfused with ~200 ml of cold phosphate-buffered saline, removed en bloc from the thoracic cavity, and lavaged with a balanced salt solution and buffered saline alternately to remove macrophages. Subsequently, the lungs were filled with Joklik's modified minimum essential medium (JMEM; 37°C) containing elastase (2 U/ml in JMEM; Worthington, Lakewood, NJ) and DNase. Proteolytic digestion was stopped after 30 min by the addition of cold (4°C) JMEM containing 10% heat-inactivated fetal bovine serum (FBS) and trypsin inhibitor (40 mg, soybean I-S; Sigma). The lungs were minced with dissecting scissors into small pieces, resuspended in the medium, and stirred for 20 min at 4°C. The suspension was filtered through nylon gauze of decreasing porosity (Tetko, Briarcliff Manor, NY) to remove debris. ATII cells were separated out of the crude cell suspension by discontinuous density-gradient centrifugation with Percoll (Sigma), and cell counts were determined with a hemocytometer. More than 85% of these cells were identified as ATII cells by the modified Papanicolaou stain or by a positive staining for alkaline phosphatase. Viability, quantified by trypan blue exclusion, was higher than 95%.

ATII cells were subsequently seeded onto 0.4-µm-pore, 0.33-cm2 Transwell tissue culture filters (Costar, Cambridge, MA) at a density of 1.5 × 106 cells/cm2 and maintained in JMEM containing 10% FBS, 0.1 µM dexamethasone, 100 µg/ml of penicillin, and 100 µg/ml of streptomycin at 37°C with 95% air-5% CO2. The medium was changed every other day after seeding.

Measurement of bioelectrical properties of ATII cells. Spontaneous potential difference (PD) and transepithelial resistance (Rt) across ATII cell monolayers were measured daily, beginning after 2 days in culture, with an Epithelial Voltohmeter equipped with chopstick-style electrodes (World Precision Instruments, Sarasota, FL). Only monolayers achieving Rt values >800 Omega  · cm2 within 4-6 days after seeding were used in our studies.

Confluent ATII cell monolayers were mounted into modified Ussing chambers, and their active ion transport properties were evaluated. The monolayers were bathed in a solution containing (in mM) 145 Na+, 5 K+, 125 Cl-, 1.2 Ca2+, 1.2 Mg2+, 25 HCO-3, 3.3 H2PO-4, and 0.8 HPO2-4 (pH 7.4) at 37°C gassed with 95% O2-5% CO2 (solution 1). Glucose (10 mM) and mannitol (10 mM) were added to the basolateral and apical solutions, respectively. We substituted glucose with mannitol in the apical solution to minimize the contribution of a putative sodium-glucose cotransporter to Na+ influx into ATII cells. A transepithelial voltage clamp (EC-825, Warner Instruments, Hamden, CT) was used for the continuous monitoring of Isc. After ATII cell monolayers were mounted in the Ussing chambers, the Isc was allowed to stabilize before each experiment was begun (~10 min). The Rt was determined with Ohm's law from the observed change in Isc resulting from 5-s square-voltage pulses (1 or 2 mV) imposed across the monolayer.

To test the hypotheses mentioned previously, different combinations of 1) 10 µM amiloride, 2) 10 µM forskolin or 100 µM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP), 3) 200 µM glibenclamide, or 4) 100 µM bumetanide were added to the medium. Amiloride and bumetanide were applied to the apical and basolateral sides of the cell monolayers, respectively, whereas the other compounds were applied to both sides of the monolayer.

Statistical analysis. All variables are expressed as means ± SE. Analysis of the effect of cAMP on transepithelial ion transport (in vivo) and AFC was conducted with one-way ANOVA. The Student-Newman-Keuls test was utilized for post hoc comparisons. Analysis of the effects of the different pharmacological interventions on heart rate, arterial blood pressure, arterial blood gas parameters, and peak inspiratory pressure was conducted with ANOVA. In vitro data were analyzed with an unpaired t-test. P < 0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effects of forskolin on AFC in the presence of NaCl. Forskolin administration resulted in a small increase in AFC at 60 and 120 min postinstillation compared with the control group, but this difference did not reach significance (Fig. 1). Rabbits administered amiloride had significantly decreased AFC compared with the control group at 60 and 120 min postinstillation (P < 0.05). Similarly, animals administered amiloride and forskolin had significantly decreased AFC compared with animals in the forskolin group at 60 and 120 min postinstillation (P < 0.05). At 60 min postinstillation, animals administered amiloride and forskolin had a significantly greater AFC than those administered amiloride (P < 0.05). These results indicate that the small increase in AFC after instillation of forskolin was due to reabsorption of Na+ through non-amiloride-inhibitable pathways. Figure 1B depicts the amiloride-sensitive fraction of AFC, which is the difference in group mean AFC values between the control and amiloride (and forskolin and forskolin with amiloride) groups.


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Fig. 1.   A: alveolar fluid clearance (AFC) at 1 and 2 h after instillation of 10 ml of 154 mM NaCl containing 5% albumin. Values are means ± SE. B: amiloride-sensitive fraction of AFC, which is difference in group mean AFC values between control and amiloride (and forskolin and forskolin + amiloride) groups. * P < 0.05 vs. control group. dagger  P < 0.05 vs. forskolin group. # P < 0.05 vs. amiloride group.

There was no significant difference in instillate amiloride concentration between the two groups administered amiloride. The values for the animals administered amiloride alone were 0.77 ± 0.01 and 0.68 ± 0.03 mM at 60 and 120 min postinstillation, respectively. The values for the animals administered amiloride and forskolin were 0.78 ± 0.01 and 0.66 ± 0.03 mM at 60 and 120 min postinstillation, respectively. Multiple regression analysis was performed, with the model including time, the presence or absence of forskolin, and amiloride concentration in the instilled alveolar fluid as independent variables and AFC as the dependent variable. The multiple regression coefficient was 0.64 (P < 0.0001), with amiloride concentration and the presence of forskolin significantly influencing AFC. Time did not significantly contribute to AFC in this model. The data are consequently depicted as the measured values of AFC vs. the concentration of amiloride in the instilled fluid in the presence or absence of forskolin (Fig. 2). There was a negative association between AFC and amiloride concentration in the instilled fluid in the presence (r = -0.52; P < 0.01) or absence (r = -0.62; P < 0.01) of forskolin.


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Fig. 2.   Association of AFC and bronchoalveolar lavage fluid (BALF) amiloride concentration. A: without forskolin. r = -0.62; P < 0.01. B: with forskolin. r = -0.52; P < 0.01. Lines, linear regression.

Effects of forskolin on AFC in the presence of Cl--free solutions. Rabbits administered sodium methanesulfonate were observed to have a significantly decreased AFC compared with the control group (i.e., rabbits that were instilled with NaCl) at 60 and 120 min postinstillation (P < 0.05; Fig. 3). Rabbits instilled with sodium methanesulfonate and forskolin demonstrated a negative AFC (i.e., fluid secretion into the alveolar space) at 60 min postinstillation and essentially zero AFC at 120 min. However, there was no significant difference in AFC between the two sodium methanesulfonate groups at 120 min postinstillation. Forskolin administration resulted in a significantly greater alveolar instillate Cl- concentration 60 min after instillation compared with animals administered sodium methanesulfonate alone (Fig. 3B). However, there were no significant differences in instillate osmolality between the groups at either time point (Fig. 3C).


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Fig. 3.   AFC (A), instillate Cl- concentration (B), and instillate osmolality (C) at 1 and 2 h after instillation of 10 ml of 154 mM sodium methanesulfonate (SMS) containing 5% albumin. * P < 0.05 vs. sodium methanesulfonate.

Hemodynamics and arterial blood gas. There were no significant differences in heart rate or arterial blood pressure between the groups at 60 and 120 min postinstillation (data not shown). Rabbits administered forskolin were noted to have a brief (2-4 min) period of hypotension (decrease in mean arterial pressure of ~30%) that was easily managed by elevation of the animals' hindquarters. There were no significant differences in any measured arterial blood gas parameter except PaO2. All rabbits exhibited a significant decrease in PaO2 after the instillation of fluid (PaO2 at 30 min postinstillation = 132 ± 16 mmHg; P < 0.05).

ATII cell monolayer studies. In the present study, ~50% of ATII cell monolayers had Rt values > 800 Omega  · cm2 between 4 and 6 days in culture. The Isc under basal conditions was 9.3 ± 0.5 µA/cm2. As shown in Fig. 4A, forskolin produced an early biphasic change in the Isc followed by a slow increase to a value that was 3.4 ± 0.2 µA/cm2 higher than baseline. Similar results were obtained after addition of CPT-cAMP (100 µM). Results of the various studies were pooled and mean Isc responses to forskolin and CPT-cAMP in ATII cells are shown in Table 1. Subsequent addition of amiloride (10 µM) to the apical bath significantly decreased the Isc from 12.6 ± 0.6 to 3.8 ± 0.3 µA/cm2. The residual Isc was then inhibited further by the cystic fibrosis transmembrane conductance regulator (CFTR) channel blocker glibenclamide (200 µM, apical and basolateral bath) and the loop diuretic bumetanide (100 µM, basolateral bath), indicating that at a fraction of the forskolin-induced increase in Isc was due to Cl- secretion.


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Fig. 4.   Short-circuit current (Isc) recordings from alveolar type II cell monolayers mounted in Ussing chambers. Addition of forskolin (Forsk) increases Isc either before (A) or after (B) amiloride (Amil) administration. Further addition of cystic fibrosis transmembrane conductance regulator channel blocker glibenclamide (Gliben) and/or loop diuretic bumetanide (Bumet) decreased Forsk-mediated Isc. Results of a typical experiment. I, II, III, and IV, various intervals, mean values of which are shown in Table 1.

                              
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Table 1.   CPT-cAMP-forskolin- and amiloride-induced changes in Isc of rabbit ATII cell monolayers

When ATII monolayers were first treated with amiloride, the Isc decreased by 6.5 ± 0.2 µA/cm2, which was significantly less than the amiloride-sensitive Isc after forskolin (8.8 ± 0.4 µA/cm2; Fig. 4, Table 1). Subsequent addition of forskolin resulted in a significant transient increase in Isc, which then stabilized at 2.5 ± 0.5 µA/cm2 higher than the preforskolin Isc values (Table 1). The resulting steady-state Isc was then partially inhibited by glibenclamide (Fig. 4B) and bumetanide (data not shown).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The bioelectric properties of the rabbit ATII cell monolayers in the present study are in good agreement with those in rats (4, 5, 10, 11). The fact that the Isc was decreased to ~20% of its control value after instillation of 10 µM amiloride into the apical bath (Fig. 4B) indicates that Na+ entered the apical membranes of rabbit ATII cells via amiloride-sensitive ion channels. This conclusion is further supported by our previous whole cell patch-clamp findings indicating the presence of amiloride-sensitive currents in rabbit ATII cells (12).

Immediately after the application of either forskolin or cAMP to both sides of the monolayers, there was a sharp increase in the Isc (Fig. 4). The fact that a similar increase in the Isc was observed after forskolin application in the presence of amiloride strongly implies that these agents stimulated Cl- secretion across the ATII cell monolayers. The rapid increase in the Isc likely reflects the efflux of Cl- from the cells across their apical membrane. The subsequent decrease would be expected as intracellular Cl- moves toward its equilibrium concentration. However, the elevated steady-state Isc after forskolin instillation in the presence of amiloride reflects the sustainable level of Cl- secretion by these cells. The sensitivity of the forskolin-induced Isc to both bumetanide and glibenclamide suggests that Cl- is loaded into these cells, at least in part, via a Na+-K+-2Cl- cotransporter in the basolateral membrane and exits across the apical membrane through the CFTR Cl--channel protein.

The role of Cl- in the cAMP response of rat ATII cells has been previously elucidated. In the study by Cheek et al. (5), an increase in intracellular cAMP resulted in a transient decrease in the Isc, followed by a sustained increase. The resulting steady-state Isc was partially inhibited by amiloride, suggesting that the cAMP response was due to increased Na+ absorption. However, in a subsequent study (12) from this group, isotopic flux measurements revealed that both Na+ and Cl- absorption were increased after cAMP. The results of Jiang et al. (11) indicated that treatment of rat ATII cells with terbutaline stimulated apical membrane Cl- channels, with functional and pharmacological properties similar to CFTR. They also concluded that Cl- influx into the cells couples with Na+ influx to increase net NaCl absorption without affecting the apical membrane Na+ conductance. Despite the qualitative differences among the aforementioned studies and ours, taken as a whole, the data indicate that agents that increase intracellular cAMP in ATII cells induce transepithelial Cl- fluxes.

Yue et al. (28) previously showed that agents that increase cAMP also increase the open probability and mean open time of Na+ channels in freshly isolated rat ATII cells. Furthermore, similar results were noted when protein kinase A was added along with ATP to the cytoplasmic site of inside-out patches of rat ATII cells (28) as well as to the trans (cytoplasmic) site of lipid bilayers containing an Na+-channel protein isolated from rabbit ATII cells (22). Protein kinase A also phosphorylated the rabbit ATII cell Na+-channel protein (2), a finding that has been previously associated with activation of Na+ channels (25). The significant increase in amiloride-sensitive Na+ Isc after elevation of intracellular cAMP is consistent with these previous data and suggest that cAMP increases Na+ absorption in this species. The failure of forskolin or terbutaline to increase AFC is likely due to the complexity of the cAMP response in stimulating both Cl- secretion and Na+ absorption. On the basis of these data, we anticipated that cAMP would have increased transepithelial Na+ absorption across rabbit ATII cell monolayers. Although, the mean amiloride-sensitive Isc after forskolin was increased over control cell monolayers, the differences were not significant. Taken in total, these in vitro data indicate that the cAMP-induced transport responses in rabbit ATII cells are complex and involve both Na+ and Cl- transport pathways.

Our in vivo measurements of AFC offer additional insight into the coupling of Na+ and Cl- movement across the alveolar epithelium. In the presence of normal saline, forskolin increased AFC by ~20%. However, this change was not significant and was brought about by a large increase in the amiloride-insensitive fraction of AFC. These data contrast with measurements of Isc in which forskolin resulted in a sustained increase in Isc. It is possible that this difference is due to nonspecific phenotypic changes in cultured ATII cells. More likely, it may simply reflect the higher sensitivity of Isc measurements where each filter was used as its own control.

Our data further demonstrate that removal of Cl- from the instillate reduces the AFC from 15 to ~0%. It is possible that the decrease in AFC may be due to the differences in size of Cl- (35 Da) and methanesulfonate (95 Da) molecules. However, this is unlikely because forskolin induced a net Cl- secretion into the alveolar space as shown by the doubling of the intra-alveolar Cl- concentration at 60 min, resulting in significant fluid influx into the alveolar space. In the presence of Cl-, Na+ reabsorption resumed as shown by the fact that AFC returned to zero at 120 min. Finally, the osmolality of the instilled alveolar fluid did not change significantly, indicating isotonic fluid reabsorption due to the uptake of both Na+ and Cl-.

One factor that could have confounded our in vivo AFC measurements was the administration of isoflurane, a halogenated anesthetic. Rats exposed to either isoflurane or halothane for 6 h were observed to have a decreased AFC compared with control animals (20). However, rats exposed to isoflurane for 2 h did not exhibit a significantly decreased AFC (20). Furthermore, the coadministration of 98% O2 or terbutaline abrogated the halothane-mediated decrease in AFC (20). In the present study, rabbits were coadministered 99% O2 with 1% isoflurane for ~2.5 h. Finally, the amiloride-sensitive fraction of AFC in the present study is similar to that reported by Smedira et al. (23). Consequently, although isoflurane administration may have influenced our observations, the short period of experimentation and the coadministration of 99% O2 likely decreased most isoflurane-mediated effects on AFC.

The teleological implications of our findings are potentially relevant to clinical scenarios of acute lung injury. The ability to clear alveolar fluid has been associated with improved morbidity in the setting of acute respiratory distress syndrome (15). The process of AFC may involve a balance between active Na+ absorption and Cl- secretion, and perturbations in active Na+ absorption by reactive O2-N2 intermediates (10, 16) may result in relatively unopposed Cl- secretion and resultant alveolar flooding. Indeed, if the concurrent pathophysiology includes a shock state (e.g., sepsis, hemorrhage), Cl- secretion may be upregulated by increased circulating catecholamines, further exacerbating alveolar fluid accumulation. The experiments involving sodium methanesulfonate would support the possibility of a clinical scenario of increased alveolar flooding after epithelial cAMP is increased via beta 2-adrenoreceptor agonism.

    ACKNOWLEDGEMENTS

This project was supported by National Heart, Lung, and Blood Institute Grants HL-31197 and HL-51173 and Office of Naval Research Grant N00014-97-1-0309. V. G. Nielsen is the recipient of American Heart Association (Southeast Affiliate) Grant-In-Aid 9810091SE. M. D. DuVall is a Parker B. Francis Fellow.

    FOOTNOTES

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.

Address for reprint requests: V. G. Nielsen, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 South 19th St., JT 845E, Birmingham, AL 35233-1924.

Received 11 June 1998; accepted in final form 9 September 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(6):L1127-L1133
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