Effect of in vivo corticosteroids on Na+ transport across airway epithelia

B. R. Grubb and R. C. Boucher

Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have investigated the role in vivo of mineralocorticoid and glucocorticoid hormones in regulating the rate of electrogenic amiloride-sensitive Na+ absorption across murine airway tissue studied in vivo (nasal potential difference) and in vitro (Ussing chambers). We found that elevating the plasma aldosterone concentration 10-fold (low-Na+ diet) had no significant effect on amiloride-sensitive Na+ absorption across tracheal or nasal epithelia. High doses of dexamethasone for 1 wk likewise did not change the rate of amiloride-sensitive Na+ absorption across airway epithelia. In contrast, both hormonal manipulations elevated the rate of colonic Na+ absorption. Furthermore, adrenalectomy (both normal and cystic fibrosis mice) also failed to alter Na+ absorption across airway epithelia. We conclude that, in vivo, neither the mineralocorticoid nor the glucocorticoid hormones significantly regulate the rates of amiloride-sensitive electrogenic Na+ absorption across airway epithelia in the adult mouse.

aldosterone; dexamethasone; adrenalectomy; mouse; cystic fibrosis

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

DESPITE THE LIKELIHOOD THAT the volume and composition of airway surface liquid (ASL) is important for mucociliary clearance and airways defense, little is known about the chronic regulation of this liquid layer. The depth and composition of ASL are thought to be regulated by the rates of ion transport and the linked osmotic volume flow. In most airway epithelia, the amiloride-sensitive epithelial Na+ channel is an important element that regulates the rate of absorptive volume flow and the ionic composition of ASL.

Little is known about the physiological regulation of the epithelial Na+ channel (ENaC) in airway epithelia. In the genetic disease cystic fibrosis (CF), the rate of Na+ absorption is markedly upregulated in vivo and in vitro across airway epithelia in both human (20, 21, 23) and mouse proximal airways (15). It has been shown that the product of the normal CF gene [cystic fibrosis transmembrane conductance regulator (CFTR)] tonically inhibits amiloride-sensitive Na+ absorption across airway epithelia (31), and, in CF, absent or defective CFTR is associated with an elevated rate of Na+ absorption (31).

In the renal distal tubules and colonic epithelia, both mineralocorticoid (aldosterone) and glucocorticoid hormones [dexamethasone (DMS)] markedly stimulate amiloride-sensitive Na+ absorption (see Ref. 11 for a review) in a variety of species, including mice (13) and humans (19, 29). In times of Na+ deficit, epithelial Na+ channels in both the kidney and colonic epithelia are recruited under the influence of aldosterone to scavenge Na+ that may otherwise be excreted from the body. In contrast, the ASL functions to maintain an environment optimized for ciliary function and airway defense. Thus the amiloride-sensitive Na+ channel might be expected to be regulated differently in airway epithelia.

Although it has been demonstrated that both the glucocorticoid and mineralocorticoid hormones upregulate amiloride-sensitive Na+ absorption in cultured airway epithelia (7, 8, 24, 26), almost no data are available on the in vivo effect of these hormones on Na+ absorption across airway epithelia. Therefore, the purpose of this investigation was to determine the in vivo effect of mineralocorticoid (raised by low-Na+ diet) and glucocorticoid (DMS subcutaneous injection) hormones on the amiloride-sensitive Na+ absorption across nasal and tracheal epithelia of normal mice studied in vivo and in vitro. The effect of adrenalectomy (resulting in very low levels of adrenocortical hormones) on amiloride-sensitive Na+ absorption across the airway epithelia of both normal and CF mice was also determined in Ussing chamber airway preparations.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Both CF (cftrtm1unc) and normal littermates (heterozygous for CFTR) were studied (8 wk and older, both sexes). All mice were hybrids of four strains: BAL/c, DBA/2, C57BL/6, and 129/SvEv. Details of mouse care have previously been published (12). Mice were euthanized with 100% CO2, and the nasal, tracheal, and distal colonic epithelia were removed as previously described (13-15).

In vitro tissue study. The airway tissues were mounted on small-aperture Ussing chambers (0.025 cm2), and distal colonic epithelia were mounted on chambers having an aperture surface area of 0.25 cm2. Tissues were bathed bilaterally in Krebs-Ringer bicarbonate buffer having the following composition (in mM): 140 Na+, 120 Cl-, 5.2 K+, 1.2 Mg2+, 1.2 Ca2+, 2.4 HPO2-4, 0.4 H2PO-4, and 25 HCO-3. Some tissues were studied in bilateral Cl--free Ringer solution (see Ref. 12 for details). All tissues were allowed to equilibrate for 30 min after mounting before basal bioelectric measurements were taken. The tissues were studied under short-circuit current (Isc) conditions for the duration of the experiment. A constant voltage pulse (1 mV, 1 s duration) was applied to the tissue every minute. Potential difference (PD) and resistance were calculated by Ohm's law from the changes in Isc in response to the voltage pulse. After the basal bioelectric measurements were made, amiloride (10-4 M) was added to the luminal bath. Inhibition of the Isc by amiloride was used as an index of the magnitude of electrogenic Na+ absorption (see RESULTS). Unless otherwise indicated, all drugs and chemicals were purchased from Sigma (St. Louis, MO).

Nasal PD. Mice were anesthetized with Avertin for the nasal electrical PD measurements, as previously described (15). Mice used for nasal PD measurements were allowed to recover for 1 day before euthanasia and tissue removal for Ussing chamber studies.

Elevated hormone levels. To elevate serum aldosterone levels, mice (normal mice only) were placed on a low-Na+ diet (Purina Mills, Richmond, IN) for 14 days as previously described (13). To elevate glucocorticoid levels, a group of mice were injected subcutaneously with the synthetic glucocorticoid hormone DMS (Elkins-Sinn, Cherry Hill, NJ) (600 µg/100 g twice per day) for 7-8 days before study.

Adrenalectomy. Mice, both normal and CF, were anesthetized with Avertin (15) and were shaved, and a small incision was made just behind the ribs through the cutaneous and muscle layer. A small retractor was inserted to hold the muscle layers open to aid in the visualization of the adrenal gland. The vessels supplying the adrenal gland were crushed, the gland was removed, muscle layers were sutured with 5-0 silk, and the skin incision was closed with wound clips. The procedure was then repeated on the opposite side. All mice tolerated the procedure well. After adrenalectomy (ADX), the mice drank Colyte (13) (0.75% Na+) to which 2.5% glucose was added.

Because levels of adrenocortical hormones were still measurable in many of these mice 7 days following ADX (see Table 1), additional mice were adrenalectomized and then treated with aminoglutethimide (50 mg/kg twice per day for 3 days and then 100 mg/kg twice per day for an additional 4 days before study). This drug inhibits the synthesis of steroid hormones and reduces residual corticosteroid secretion following ADX.

                              
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Table 1.   Murine corticosteroid levels

Hormone assay. Blood was collected by cardiac puncture in heparinized syringes immediately after euthanasia. Plasma was separated by centrifugation for both the aldosterone and corticosterone assays. Corticosterone was determined using an RIA kit (I125; ICN Biomedical, Costa Mesa, CA). Although the detection limit of the kit is reported to be 10 ng/ml, we found that we could reproducibly measure the hormone to 5 ng/ml. Aldosterone was also determined using a I125 RIA kit (detection limit 6 pg/ml; Biodata, Cortland Manor, NY). All assays were run in duplicate and the results averaged.

Statistics. Results are shown as means ± SE, with the number of mice studied shown (n). If only two groups were being compared, a Student's t-test was used. If more than two groups were compared, an ANOVA was used, and a Student-Newman-Keuls test was used for multiple comparison testing among groups. In the event of nonhomogeneity of variance, a Kruskal-Wallis one-way ANOVA on ranks was used where indicated.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Low-Na+ diet. To determine the effects of elevating the serum aldosterone concentration on the rate of electrogenic Na+ absorption across nasal and tracheal airway epithelia, normal mice were placed on a low-Na+ diet for 2 wk before study. This protocol elevated the serum aldosterone levels 10-fold (Table 1). However, neither the basal bioelectric properties (Table 2) nor the amiloride-sensitive Isc (Fig 1, A and B) were significantly altered across the tracheal or nasal epithelia in response to raised serum aldosterone levels. Although the high aldosterone levels did not appear to influence the amiloride-sensitive Na+ absorption across airway epithelia, we have previously shown that mice on a low-Na+ diet exhibit a significantly enhanced amiloride-sensitive Isc across the distal colonic epithelia compared with mice on a normal diet (13).

                              
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Table 2.   Basal bioelectric parameters


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Fig. 1.   Amiloride-sensitive short-circuit current (Isc; that portion of the basal Isc sensitive to amiloride) from normal murine tracheal (A) or nasal (B) epithelia studied in vitro. Ctl, tissue from control animals receiving no treatment (n = 11 trachea, n = 10 nasal); high aldo, tissue from low-Na+ mice having high aldosterone levels (n = 9 trachea and nasal); DMS Rx, dexamethasone-treated mice (n = 6); ADX, tissue from adrenalectomized mice (n = 5 trachea and nasal). Data are means ± SE. As there was nonhomogeneity of variance among the groups in B, a Kruskal-Wallis one-way ANOVA on ranks was used for data analysis.

We have previously shown that the magnitude of the basal PD measured in vivo across the murine nasal epithelium is an index of the rate of electrogenic Na+ absorption across this tissue (15). Furthermore, both basal and amiloride-sensitive in vivo nasal PDs correlate with amiloride-sensitive Isc measured in the Ussing chambers (15). Thus nasal PDs were also measured for normal mice and for mice on the low-Na+ diet. Again, neither the basal PDs nor the change in PD in response to amiloride differed between mice on a normal diet and mice on a low-Na+ diet (Table 3), consistent with the data from freshly excised tissues, indicating that raised serum aldosterone levels do not increase the rate of electrogenic Na+ absorption across the nasal epithelium (Fig. 1B).

                              
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Table 3.   In vivo nasal electrical PDs (normal mice)

Glucocorticoid hormone. To determine whether the synthetic glucocorticoid hormone DMS increases electrogenic Na+ absorption in normal murine airways, mice were injected with DMS for 7 days and were then studied. This hormone also failed to affect the basal bioelectric parameters across either tracheal or nasal epithelia (Table 2). Furthermore, no significant effect of this hormone on the amiloride-sensitive Isc across either tracheal or nasal epithelia was observed (Fig. 1A and B). It should be noted that, especially in the case of nasal epithelia, the mean basal Isc and amiloride-sensitive Isc of the DMS and high-aldosterone (low-Na+ diet) groups are approximately one-third to one-half the values of the control group. The data in the control group exhibited some variability, which may have contributed to the failure to detect a significant difference between the groups if indeed one were present. However, based on findings from cultured airway epithelia (see DISCUSSION), it would be expected that the amiloride-sensitive Isc in the DMS and high-aldosterone groups would be greater rather than less than that in the control group. Congruent with the in vitro nasal data, the amiloride-sensitive nasal PD in vivo was also not altered by DMS treatment (Table 3). The basal in vivo PD of DMS-treated mice was significantly less than the PD of the control mice. In contrast, the distal colonic epithelia of normal mice treated with DMS exhibited a significant amiloride-sensitive Isc (74.6 ± 19 µA/cm2, n = 6) compared with colonic epithelia from normal, untreated mice that exhibit no amiloride-sensitive Isc (0 ± 0, n = 6). Therefore, the DMS dosing protocol was effective in elevating the rate of electrogenic Na+ absorption across the murine distal colonic epithelia but appeared to be completely ineffective in raising amiloride-sensitive Na+ absorption across airway epithelia.

Adrenalectomy. To determine the effect of lowering the endogenously produced mineralocorticoid (aldosterone) and glucocorticoid (corticosterone) hormones on electrogenic Na+ absorption across airway epithelia, mice were adrenalectomized and studied 7-8 days following surgery. CF mice, which, as we previously have shown, exhibit a significantly increased rate of amiloride-sensitive Na+ absorption across nasal epithelia, were also studied. As an index of the efficacy of the surgical procedure, corticosterone levels were measured in a group of normal and CF mice receiving no treatment and were compared with mice 7 days following ADX. We chose to measure corticosterone levels rather than aldosterone, as only 30 µl of plasma were required for a duplicate corticosterone assay, whereas 100 µl of plasma were required for the aldosterone assay. Because both hormones are produced by the adrenal cortex (and accessory adrenal tissue), reduction in the level of corticosterone as a result of ADX is a good index of the change in plasma level of aldosterone.

We found that untreated CF mice exhibited a significantly higher level of plasma corticosterone than untreated normal animals (Table 1). ADX significantly reduced the corticosterone levels in both groups of mice. However, the steroid hormone level was still significantly greater than zero in both groups of animals following ADX (Table 1). This hormone was undoubtedly produced by the accessory adrenal tissue that is present to varying degrees in different strains of mice (17). Because this accessory adrenal tissue is very difficult to remove surgically, we chose to use a chemical means to suppress hormone production from the accessory adrenal tissue (28). Therefore, aminoglutethimide was administered for 7 days following ADX. This drug protocol was completely effective in reducing the serum corticosterone to nondetectable levels in the normal mice (Table 1). Although the CF mice still exhibited detectable corticosterone levels, the levels were very low (Table 1). ADX (normal animals) mice for which bioelectric data are shown are those mice that had nondetectable corticosterone levels and CF mice are those that had the very low steroid hormone levels (both groups = ADX + aminoglutethimide).

In the normal mice, ADX plus aminoglutethimide affected neither the basal bioelectric parameters (Table 2) nor the amiloride-sensitive Isc in tracheal or nasal epithelia (Fig. 1, A and B). The treated CF mice also did not exhibit reductions in the Isc of tracheal or nasal epithelia following ADX plus aminoglutethimide (Fig. 2, A and B) compared with untreated CF mice. Note that the magnitude of amiloride-sensitive Isc across the CF nasal epithelia is significantly larger than that exhibited by normal mice (Figs. 1B and 2B), as we have previously reported (15).


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Fig. 2.   Amiloride-sensitive Isc from cystic fibrosis tracheal (A) and nasal (B) epithelia studied in vitro; n = 6 for control cystic fibrosis tracheas and n = 5 for nasal epithelia; n = 4 for ADX tracheas and nasal epithelia; and n = 8 for tracheal and nasal epithelia from the high aldo (low-Na+ diet) mice.

In addition to blocking electrogenic Na+ absorption, amiloride has been shown to hyperpolarize the epithelial apical membrane (1, 4), thus increasing the driving force for Cl- secretion. Thus it can be argued that the change in Isc in response to amiloride may not solely reflect a change in the rate of Na+ absorption. Therefore, an additional group of CF nasal epithelia (ADX + aminoglutethimide) were studied in bilateral Cl--free Ringer. Although CF mice do not exhibit a cAMP-mediated Cl- secretory response in nasal epithelia, they secrete Cl- via the Ca2+-mediated Cl- secretory pathway and are thus capable of electrogenic Cl- secretion (15). In these preparations, the amiloride-sensitive Isc (129.3 ± 69 µA/cm2, n = 3) did not differ significantly from paired nasal preparations from the same mice studied in Cl--replete Krebs buffer (135.3 ± 21, n = 3). In addition, we have previously shown that murine nasal (15) and tracheal (14) epithelia (both normal and CF) incubated in Krebs buffer exhibited amiloride-sensitive Isc not significantly different from preparations incubated in bilateral Cl--free Ringer solution. Therefore, in normal and CF murine airway epithelia, even when studied in Cl--replete Krebs buffer, the amiloride-sensitive Isc appears to be an accurate index of electrogenic Na+ absorption.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Electrogenic Na+ absorption appears to be the major ion actively transported across human (4, 23) airway epithelia and murine nasal (15) and bronchial (unpublished observations) epithelia. In addition, in a variety of other species, electrogenic Na+ absorption appears to be the major ion actively absorbed across all airway epithelia (3, 25, 32). Therefore, regulation of airway Na+ transport is predicted to be important in homeostasis of ASL.

In both gut and airway tissue, the amiloride-sensitive Na+ channel has been shown to be comprised of three subunits (alpha , beta , gamma ) (5, 6). Direct evidence for the role of the subunits in amiloride-sensitive Na+ transport across airway epithelia comes from the finding that mice in which the alpha -ENaC gene (codes for alpha -subunit) has been disrupted by gene targeting [alpha -ENaC(-/-)] lose all amiloride-sensitive Isc across tracheal epithelia (18). The importance of electrogenic Na+ absorption in clearance of lung liquid at birth was demonstrated when alpha -ENaC(-/-) mice were found to die in the early postnatal period due in part to a failure to clear lung liquid (18). There is evidence that the mineralocorticoid and glucocorticoid hormones regulate ENaC subunit expression in colonic epithelia (27), but evidence for hormonal regulation of ENaC expression and function in pulmonary epithelia of the adult is conflicting (24, 27).

In cultured airway epithelia (adult human, canine, bovine), glucocorticoid and mineralocorticoid hormones have been shown to elevate electrogenic Na+ absorption (7, 8, 26, 33). In cultured human nasal and bronchial epithelia, a 24-h exposure to aldosterone, hydrocortisone, or DMS increased the amiloride-sensitive Na+ absorption two- to fourfold (7). This study also revealed that cultured human airway epithelia had a greater sensitivity to aldosterone (ED50 = 0.1 µM) than to the other two hormones (ED50 = 25 µM for hydrocortisone and 50 µM for DMS). Another recent report also noted that aldosterone significantly increased amiloride-sensitive Na+ absorption across cultured human airway epithelia (33). In cultured canine tracheal epithelia (primarily a Cl- secretory tissue), a 2-day exposure to aldosterone increased the baseline Isc by 55% and the amiloride-sensitive Isc similarly (8). In cultured bovine tracheal epithelia, DMS, but not aldosterone exposure (18 h to 8 days) significantly increased the amiloride-sensitive Isc (26).

When native airway epithelia were studied in Ussing chambers, however, exogenous corticosteroid hormones failed to alter the basal or amiloride-sensitive Isc of these airway preparations. Addition of exogenous aldosterone to native rabbit tracheal preparations failed to alter the basal or amiloride-sensitive Isc when tissues were studied 4 h after hormonal addition (2). In contrast, when aldosterone was exogenously added to rabbit colonic preparations in vitro, an increase in the basal and amiloride-sensitive Isc was detectable within 2 h (10). In another Ussing chamber study, a 6-h DMS exposure failed to alter the basal Isc or PD of canine and feline native tissue tracheal preparations (30).

There are few reported studies investigating the in vivo administration of either glucocorticoid or mineralocorticoid hormones on electrogenic Na+ absorption across airway epithelia. When adult rats were placed on a low-Na+ diet for 15 days (to elevate aldosterone) or given DMS exogenously (2 days) and tissue removed, there was no increase in either mRNA or protein expression for any of the three Na+ channel subunits in the airway epithelia (27). However, if the rats were adrenalectomized and tissue studied 7 days after the surgery, there was a significant decrease both at the mRNA and protein levels of all three subunits in the airways (27).

Data from the present study suggest that, in vivo, electrogenic Na+ absorption across adult murine airway epithelia is not regulated by the corticosteroid hormones. Neither tracheal nor nasal epithelia from normal mice treated exogenously with DMS or placed on a low-Na+ diet to stimulate aldosterone production exhibit any change in amiloride-sensitive Isc (or amiloride-sensitive PD in vivo) as a result of these hormonal manipulations. However, the colonic epithelia of the hormone-treated mice clearly exhibited an increase in electrogenic Na+ absorption. Our data are consistent with a recent study that reported that nasal epithelia from rabbits receiving a low-Na+ diet for 1 wk exhibited bioelectrical properties that were not different from nasal epithelia from rabbits on a control diet (9). These authors concluded that aldosterone is not responsible for long-term regulation of Na+ absorption across rabbit airway epithelia.

Although elevation of aldosterone or DMS failed to alter electrogenic Na+ absorption across the airway epithelia of mice, based on data in rats (27) we had speculated that electrogenic Na+ absorption across airway epithelia may be downregulated by decreasing the endogenous corticosteroid hormones to very low levels. Thus groups of normal and CF mice were adrenalectomized and studied 7 days later. The normal adrenalectomized mice, having nondetectable corticosterone levels, exhibited amiloride-sensitive Na+ absorption that was not significantly different from tissue from control mice. CF mice, which exhibit a significantly elevated rate of electrogenic Na+ absorption across nasal epithelia (15), also showed no change in amiloride-sensitive Na+ absorption following ADX. In the rat, ADX reduces the expression of both the message and protein levels of the three ENaC subunits in airway tissue, but function was not studied (27). We did not measure the ENaC mRNA or protein responses to ADX in murine airway epithelia, but, if changes did occur, our functional data suggest that sufficient subunit protein remains to allow normal rates of Na+ absorption.

Little data are available on the role of in vivo corticosteroids in human airway epithelia. However, there are two recent reports in which the effect of an elevated aldosterone concentration (33) and topical DMS administration (16) were studied in vivo in human nasal epithelia by the PD technique. Patients on a low-Na+ diet (6 days) exhibited a significant increase in the transepithelial amiloride-sensitive PD compared with controls. However, the magnitude of the difference in the amiloride-sensitive PD between controls and low-Na+ subjects was small [2.2 mV (<10%)] (33) compared with the magnitude of the increase in amiloride-sensitive PD across colonic epithelia as a result of aldosterone stimulation [40-60 mV (19)]. In another study, DMS was applied topically to human nasal epithelia every 15 min for 8 h, and nasal PD was measured at hour 8. In this study, while the basal PD was significantly elevated in the treated nasal epithelia, there was no significant effect of DMS treatment on amiloride-sensitive PD (16). Similarly (22), when spironolactone was given to human patients to block the aldosterone receptors, it significantly reduced the rectal PD but failed to alter nasal PD (22), suggesting that electrogenic Na+ absorption was unaltered across airway epithelia. Therefore, as found in the present investigation, these studies suggest that there is little, if any, influence of the corticosteroid hormones on electrogenic Na+ absorption across human airway epithelia in vivo.

In conclusion, we find no evidence in adult murine airway epithelia that in vivo alterations of either the mineralocorticoid or glucocorticoid hormones regulate amiloride-sensitive Na+ absorption across airway epithelia. These data are consistent with the hypothesis that Na+ transport by pulmonary tissue is not involved in the overall salt balance of the organism. We speculate that, if indeed alterations in the rate of transport of this cation are important in ASL homeostasis, rapid regulation of Na+ transport by an as yet unidentified factor is necessary.

    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Specialized Center of Research Grant HL-42384-10 (Project 1A and Core C).

    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: B. R. Grubb, Cystic Fibrosis/Pulmonary Research and Treatment Center, 7011 Thurston-Bowles Bldg., CB# 7248, The Univ. of North Carolina, Chapel Hill, NC 27599-7248.

Received 16 January 1998; accepted in final form 8 April 1998.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(1):C303-C308
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