Hypoxia reduces airway epithelial sodium transport in rats

L. A. Tomlinson1,2, T. C. Carpenter1, E. H. Baker2, J. B. Bridges1, and J. V. Weil1

1 Cardiovascular-Pulmonary Research Laboratory, University of Colorado Health Sciences Center, Denver, Colorado 80262; and 2 Department of Pharmacology and Clinical Pharmacology, St. George's Hospital Medical School, London, United Kingdom SW17 ORE


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ascent to high altitude leads to pulmonary edema formation in some individuals. Recent laboratory evidence supports the hypothesis that hypoxia may impair the function of the alveolar epithelium and thus augment edema accumulation via reduced clearance of lung liquid. We investigated the effect of hypobaric hypoxia on epithelial sodium transport in adult Sprague-Dawley rats by measuring the nasal transepithelial potential difference (PD) as an index of airway sodium transport. Baseline PDs were similar to those previously reported in other species. Administration of amiloride resulted in a significant fall in nasal PD, as did ouabain administration for 24 h (-27.8 vs. -18.8 mV; P = 0.001; n = 5 rats). Exposure to hypobaric hypoxia (0.5 atm) for 24 h caused a significant fall in nasal PD (-23.7 vs. -18.8 mV; P = 0.002; n = 15 rats), which was not additive to the changes in nasal PD produced by amiloride or ouabain. We conclude that subacute exposure to moderate hypobaric hypoxia can inhibit sodium transport by the airway epithelium in rats.

transepithelial potential difference; ouabain


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ASCENT TO HIGH ALTITUDE leads to pulmonary edema formation in some individuals. Most previous work examining the mechanisms by which hypoxia may lead to the accumulation of fluid in the lung has focused on factors that alter the permeability of the pulmonary capillary endothelium and the tone of the pulmonary vascular bed. The occurrence of frank alveolar flooding associated with hypoxic exposure suggests, however, that hypoxia may also impair the function of the alveolar epithelium and thus augment edema accumulation via reduced clearance of lung liquid. Recent evidence derived from isolated lung and cell culture preparations supports this hypothesis (18, 19, 21), but an effect of moderate hypoxia on the function of the airway epithelium has not yet been demonstrated in vivo.

Alveolar liquid clearance is largely dependent on active sodium transport by airway epithelial cells. Epithelial sodium transport is determined both by amiloride-sensitive channels (sodium channels) located in the apical membrane and by Na-K-ATPases located in the basolateral membrane that pump sodium out of the cell in exchange for potassium. The Na-K-ATPase pumps create a chemical gradient for sodium, which is absorbed from the airway lumen into the cell through the apical sodium channels. There is some evidence that both of these elements can be affected by hypoxia. Exposure of cultured rat alveolar epithelial cells to severe hypoxia has been reported to decrease the expression and activity of both the apical sodium channels and the basolateral Na-K-ATPase (18, 19). In addition, exposure to hypoxia has been shown in an isolated rat lung preparation to decrease the rate of alveolar liquid clearance (21).

Active sodium absorption across the airway epithelium generates a lumen-negative potential difference (PD). This potential is easily accessible to study in vivo in the nasal mucosa where basal ion transport rates are similar to those found in the lower airways (4). PD measurements across the nasal mucosa have been used to quantify active sodium transport across the airways of many species including mouse, human, and sheep (3, 9, 11) and to determine the effect of diseases such as cystic fibrosis (1) or drugs such as diuretics (20) on airway ion transport.

In the present study, we investigated the effect of hypobaric hypoxia on airway epithelial sodium transport in vivo by measuring the transepithelial nasal PD as an index of airway sodium transport. Our results suggest that subacute exposure to moderate hypoxia inhibits sodium transport by the airway epithelium in rats.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental animals. Experimental animals (n = 24) were adult male Sprague-Dawley rats weighing 280-350 g purchased from a commercial vendor (Harlan Sprague Dawley, Indianapolis, IN). All animals were allowed to adjust to Denver's altitude (5,280 feet above sea level) for 1 wk before being studied. The animals were allowed standard chow and water ad libitum, and they were subjected to similar day-night light cycles. After completion of the planned studies, the rats were killed with an intraperitoneal injection of phenobarbital sodium (80 mg/kg).

Measurement of nasal PD. For measurement of the transepithelial nasal PD, the rats were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (12 mg/kg). The nasal PD was then measured between a 24-gauge catheter filled with Ringer solution inserted into a nostril and a butterfly needle filled with 1% Ringer agar inserted subcutaneously into the abdominal wall. The two electrodes were connected to calomel half cells (EDT Instruments, Kent, UK) by bridges containing a 1% Ringer agar solution. Measurements were made with a high-impedance voltmeter (Medical Physics Department, St. George's Hospital, London, UK) and recorded on a chart recorder (Kipp and Zonen, Delft, The Netherlands). Transepithelial PD measurements are lumen negative with respect to the submucosa. In this paper, depolarization of the epithelium, where the measured nasal PD becomes less negative, is referred to as a decrease or drop in PD.

The animals were allowed to recover from each anesthetic for at least 24 h before undergoing further testing. After administration of the drugs or exposure to hypobaric hypoxia, nasal PD measurements were repeated in each rat by repositioning the nasal electrode in the same nostril at the same depth as the prior measurement. All rats had measurement of the baseline nasal PD performed at Denver's altitude. The reproducibility of nasal PD recordings made with these techniques was confirmed in five rats by repeat nasal PD measurements made at least 24 h apart under baseline conditions.

Effect of amiloride. To determine the effects of blocking active sodium transport through apical sodium channels on the nasal PD, measurements were made before and after exposure of the nasal mucosa to amiloride in all rats (n = 24). Amiloride was administered to the nasal mucosa by nebulization of 10-3 M amiloride in Ringer solution over 2 min, a method previously used to block nasal apical sodium channels in mice (9). The nebulization was achieved with a Medic-aid compressor with an Acorn system 22 nebulizer attached to a mask. The mask was occluded with Parafilm, and a hole made so that it fit over both nostrils of the rats. Nasal PD was then recorded as described in Measurement of nasal PD immediately after treatment with amiloride.

Effect of ouabain. A subgroup of rats (n = 5) was used to study the effects of Na-K-ATPase inhibition on the nasal PD under normoxic conditions and to determine whether these effects were additive to the effects of amiloride. These animals underwent measurement of baseline nasal PD before and after amiloride application as in Effect of amiloride. They were then treated with ouabain for 24 h (1 mg · kg-1 · dose-1 intraperitoneally every 12 h) under normoxic conditions, after which the animals underwent a repeat nasal PD measurement, again before and after amiloride application.

Exposure to hypoxia. A group of rats (n = 15) was studied to test the hypothesis that exposure to the hypobaric hypoxic conditions of high altitude could alter epithelial function. These animals underwent measurement of baseline nasal PD and were then exposed to hypobaric hypoxia (barometric pressure = 383 Torr or 0.5 atm) for 24 h in a decompression chamber. Although blood gas measurements were not obtained in this study, a previous study (16) has reported that similar exposures to hypobaric hypoxia in rats result in a drop in the arterial PO2 value from 80 to 45 Torr and a drop in the arterial PCO2 value from 35 to 25 Torr. The animals were removed from the chamber, and hypoxia was maintained by immediate placement under normobaric hypoxic conditions (inspired O2 fraction = 0.1) until a repeat measurement of nasal PD at an interval of no more than 1 h after removal from the hypobaric chamber. To determine whether the effect of hypoxia on the measured PD was due to alterations in apical sodium channel activity, 9 of these 15 rats had nasal PD measurements before and after amiloride administration both before and after exposure to hypobaric hypoxia.

To determine whether the effect of hypoxia on the measured PD was due to alterations in Na-K-ATPase activity, nasal PD was measured in animals that received ouabain during exposure to hypoxia. The five rats previously treated with ouabain were allowed a 2-wk recovery period at Denver's altitude. They then were exposed to hypobaric hypoxia for 24 h, during which time they again received 12 hourly intraperitoneal injections of 1 mg/kg of ouabain. Nasal PD measurements were again performed before and after amiloride application as described in Exposure to hypoxia.

Statistical analysis. Results are means ± SE. Student's paired t-test was used to compare repeated measurements made within groups. Student's unpaired t-test was used to compare values between groups. A P value of <0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Baseline measurements of transepithelial nasal PD and response to amiloride. Nasal PD was measured in 24 rats at Denver's altitude. The maximum PD was -24.0 ± 0.7 mV. To determine whether active sodium absorption through apical sodium channels contributed to the generation of the measured nasal PD, the animals underwent a repeat measurement of the PD after amiloride application. The nasal PD fell significantly in response to amiloride to -13.2 ± 0.6 mV (P = 0.001).

Reproducibility of nasal PD measurements. To test the reproducibility of the nasal PD measurements, six rats had measurements of nasal PD before and after amiloride at Denver's altitude on two occasions separated by at least 24 h. In five of these rats, the effect of amiloride on the nasal PD was also determined on both occasions. None of the measured parameters differed significantly between measurements (Table 1).

                              
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Table 1.   Serial measurements of nasal epithelial PDs are reproducible under baseline conditions

Effect of ouabain on nasal PDs. To determine the extent to which active sodium transport by Na-K-ATPase pumps contributes to the generation of the nasal PD in rats, the nasal PD was measured in a subgroup of animals (n = 5) before and after ouabain treatment at Denver's altitude. To determine whether these effects were additive to the effects of amiloride, on each occasion the PD was measured both before and after the application of amiloride (Fig. 1). Intraperitoneal ouabain administration led to a significant decrease in nasal PD from -27.8 ± 1.0 to -16.8 ± 2.1 mV (P = 0.001). The PD after ouabain alone (-16.8 ± 2.1 mV) was not significantly different from the PD after amiloride alone (-16.1 ± 1.7 mV). However, amiloride application caused an additional fall in potential in ouabain-treated animals from -16.8 ± 2.1 to -12.8 ± 1.0 mV (P = 0.045).


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Fig. 1.   Both ouabain and amiloride alone decreased measured nasal potential differences (PDs), and combination of ouabain and amiloride produced a further decrease compared with either compound alone (n = 5 rats). * P < 0.001 vs. baseline. dagger  P < 0.05 vs. all other groups.

Effect of hypoxia on nasal PDs. Exposure to hypobaric hypoxia for 24 h resulted in a significant fall in measured nasal PDs. Fifteen rats underwent measurement of nasal PD before and after 24 h of exposure to hypobaric hypoxia. The maximum PD was -23.7 ± 0.8 mV at Denver's altitude and -18.8 ± 0.8 mV after hypobaric hypoxia (P = 0.002), and the maximum PD fell in 12 of the 15 animals (Fig. 2).


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Fig. 2.   Hypobaric hypoxia decreased nasal PD, although interindividual variation existed in the effect (n = 15 rats). Twelve of the fifteen animals tested showed a decrease in measured nasal PD after a 24-h exposure to hypoxia.

To determine whether the effect of hypoxia on the measured PD was due to alterations in apical sodium channel activity, the effect of amiloride on nasal PD was also determined before and after exposure to hypobaric hypoxia in nine of these rats (Fig. 3). Amiloride caused a significantly greater drop in the PD than hypoxia (11.9 ± 0.6 vs. 5.5 ± 1.3 mV; P < 0.001), and there was no additional decrease in PD in animals exposed to hypoxia and amiloride compared with those exposed to amiloride alone.


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Fig. 3.   Exposure to hypobaric hypoxia decreased nasal PD, and effect of hypoxia was not additive to effect of amiloride (n = 9 rats). * P < 0.01 vs. baseline. dagger  P = 0.001 vs. hypoxia alone.

Effect of ouabain during hypoxia. To determine whether the effect of hypoxia on the measured PD was due to alterations in Na-K-ATPase activity, nasal PD was measured in animals that received ouabain during exposure to hypoxia. The rats (n = 5) that previously received ouabain under normoxic conditions recovered at Denver's altitude for 2 wk after their first ouabain exposure. In a second experiment, they again received ouabain while exposed to hypobaric hypoxia for 24 h, followed by a repeat measurement of nasal PD before and after amiloride (Fig. 4). The maximum PD fell significantly from baseline (normoxia) in rats exposed to ouabain and hypobaric hypoxic conditions (-27.8 ± 1.0 vs. -21.3 ± 0.9 mV; P = 0.0001). The PD fell less, but not significantly less, after exposure to both ouabain and hypobaric hypoxia than after exposure to ouabain treatment alone (6.6 ± 0.4 vs. 11.0 ± 1.4 mV; P = 0.06). The PD in these rats exposed to ouabain and hypoxia was not different from the PD in animals exposed to hypoxia alone (-21.3 ± 0.9 vs. -18.8 ± 0.8 mV; P = 0.13).


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Fig. 4.   Exposure to ouabain combined with exposure to hypobaric hypoxia decreased nasal PD, but effect of hypoxia was not additive to that of ouabain alone (n = 5 rats). * P < 0.001 vs. baseline. NS, not significant.

Amiloride produced a further fall in PD in the rats exposed to ouabain and hypoxia, although this change did not reach significance (-21.3 ± 0.9 vs. -13.9 ± 3.1 mV; P = 0.06). The PD after exposure to hypoxia, ouabain, and amiloride was not significantly lower than the PD in the same animals exposed to ouabain and amiloride under normoxic conditions (-13.9 ± 3.1 vs. -12.8 ± 1.0 mV; P = 0.74).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have shown that rats, like humans and mice (9, 11), generate an electrical PD across the nasal mucosa that can be measured by a simple technique. The nasal PD in the rat is sensitive to inhibition by amiloride and ouabain and is therefore generated, at least in part, by active sodium absorption. Subacute exposure to moderate hypobaric hypoxia significantly decreases the magnitude of that PD, particularly the amiloride-sensitive component of the potential, suggesting that hypobaric hypoxic exposure impairs airway epithelial sodium transport.

Although measurement of the transepithelial nasal PD is a simple in vivo method of assessing ion transport across the respiratory epithelium, it requires interpretation in light of in vitro data. In humans, sodium absorption has been shown to be the predominant form of ion transport across respiratory epithelia under physiological conditions (2, 5, 10). Sodium absorption is driven by Na-K-ATPase transporters that pump sodium out of the cell into the interstitium in exchange for potassium, thus creating a gradient for sodium entry into the cell through apical sodium channels. Sodium absorption down this gradient creates a lumen-negative PD. Administration of amiloride blocks sodium absorption through the apical epithelial sodium channels and causes depolarization of the nasal epithelium. The transepithelial PD, however, is not completely abolished in the presence of amiloride, but rather, it is maintained by the induction of chloride secretion through apical chloride channels (4). In addition, the sodium transport characteristics of the nasal epithelium are thought to be similar to those of the distal air spaces (4). Although some variation in expression of the subunits of the epithelial sodium channel has been noted along the respiratory tree, the expression of epithelial sodium channel subunits has been detected at the mRNA and protein levels in rat tracheae, bronchi, and bronchioles (13), and Na-K-ATPase subunit mRNA is expressed in fetal rat bronchioles (17). Also, all three subunits of the epithelial sodium channels are expressed in the distal air spaces of the postnatal mouse (22) and rat (7) lungs as well as in rat and human nasal epithelia. Finally, in the human nasal mucosa, nasal PDs were recently shown to correlate with the expression of the gamma -epithelial sodium channel subunit (15).

Our results show that PD measurements can be made across the nasal mucosa of rats and that the measured potentials are of similar magnitude to those reported in humans (12). We were able to demonstrate, by comparing serial measurements, that nasal PD measurement in rats provides reproducible results, although some variability is clearly present, particularly in the amiloride-sensitive component of the PD. Possible reasons for this variation include catheter-induced damage to the nasal epithelium and, more likely, inconsistencies in the delivery of nebulized amiloride to the nasal mucosa of a spontaneously breathing rat.

Amiloride, which blocks epithelial sodium channels and directly inhibits apical sodium absorption, caused a fall in nasal PD in all rats. Ouabain, which inhibits basolateral Na-K-ATPase-pump activity, thus decreasing the concentration gradient for apical sodium entry into the cell, also caused a fall in PD in all treated animals. The effect of these sodium transport inhibitors on nasal PD measurements suggests that in rats, as in humans, at least part of this PD is generated by transepithelial sodium absorption. These results are consistent with in vitro studies (7, 13, 17) that have demonstrated the presence and activity of both sodium transporters in rat airways.

The fall in PD in normoxic rats treated with a combination of amiloride and ouabain was greater than the fall in PD after treatment with either amiloride or ouabain alone. This additive effect of the two agents can be explained if neither amiloride nor ouabain individually causes complete inhibition of active sodium transport at the concentrations used and they achieve greater inhibition of sodium transport when used in combination. Alternatively, only part of the sodium absorption will be inhibited by amiloride if sodium absorption also occurs through amiloride-insensitive cation channels, and ouabain would have an additive effect by reducing the driving sodium gradient for absorption through amiloride-insensitive sodium channels. In addition, if the doses of amiloride or ouabain used were high enough to cause epithelial damage, increases in epithelial permeability could have resulted in the observed fall in nasal PD. In particular, ouabain might have altered epithelial function through cellular ATP depletion. Direct toxic effects of these agents seem unlikely, however, because amiloride at similar concentrations has been shown to inhibit apical sodium channel activity in vitro and has been used in human and mouse studies without toxicity. Also, there were no outward signs of ouabain toxicity in treated animals nor were there any premature deaths. Finally, the physiological cause of the residual PD after inhibition of active ion transport by amiloride and ouabain was not determined by this study. A previous study (14) has demonstrated flux of chloride from the submucosa to the lumen in the rat trachea. It is possible that in rats, as in humans, induced chloride secretion accounts for the postamiloride, postouabain PD.

The major finding of this study was that exposure of rats to hypobaric hypoxia for 24 h induced a significant decrease in the measured transepithelial nasal PD. The effect of hypoxia was not additive to that of amiloride or ouabain separately or in combination, suggesting that hypoxia reduced nasal PDs by reducing transepithelial active sodium absorption. These results could be explained if hypobaric hypoxia inhibits apical sodium channel expression, downregulates Na-K-ATPase pumps, or reduces the activity of both transporters. These findings are consistent with in vitro studies that have shown that exposure to severe hypoxia reduces the activity and expression of both the apical sodium channels and the Na-K-ATPase pumps in cultured alveolar epithelial cells (18, 19). Our results could also be explained if hypoxia reduced the transepithelial PD by causing epithelial damage and increasing epithelial permeability. Hypoxia, however, only reduces the amiloride-sensitive, ouabain-sensitive change in PD and does not affect the residual PD. This finding suggests that hypoxia selectively alters active sodium absorption by the airway epithelium. Although prior in vitro studies support this hypothesis (18, 19, 21), our findings represent the first in vivo evidence that moderate hypoxia decreases airway epithelial sodium transport, a finding of potential importance in explaining the pathogenesis of some forms of pulmonary edema such as that at high altitude.

Finally, the release of an endogenous mediator that inhibits the Na-K-ATPase pump is one mechanism by which hypoxia could inhibit active sodium absorption. A soluble factor that inhibits Na-K-ATPase activity has been shown to be released from cultured rat alveolar epithelial cells after exposure to hypoxia (19) and an endogenous ouabain-like compound has been found to be increased in human plasma at high altitude (6, 8). Our studies in ouabain-treated rats suggest that responsiveness to ouabain may be quite variable. When rats were exposed to ouabain alone, the PD fell in some animals almost twice that seen in other animals (range 8.0-15.5 mV). When the same rats were then exposed to both ouabain and hypoxia, the changes in nasal PD tended to be less dramatic than the changes seen with ouabain alone. This tendency for the decreases in nasal PD to be smaller in animals exposed to ouabain and hypoxia than in those exposed to ouabain alone could reflect the action of an endogenous inhibitor of Na-K-ATPase producing a complementary reduction in the response to exogenous inhibition. Alternatively, the combination of ouabain and hypoxia may have altered epithelial permeability, although the finding that the nasal PD in animals exposed to the combination of ouabain, amiloride, and hypoxia was not different from the PD in normoxic animals exposed to ouabain and amiloride argues against this concept. It is also possible that the rats may have become partially resistant to the effect of ouabain due to prior recent exposure.

In summary, we have shown that subacute exposure to moderate hypobaric hypoxia alters measured nasal epithelial PDs in rats. Ion transport across the nasal epithelium is thought to be similar to transport across other regions of the respiratory tract epithelium. If hypoxia also alters sodium transport more distally in the lung, as in vitro evidence suggests, then a reduction in active sodium absorption across the alveolar epithelium may play a role in the accumulation of lung liquid and in the formation and maintenance of pulmonary edema in individuals susceptible to high-altitude pulmonary edema.


    ACKNOWLEDGEMENTS

L. Tomlinson was supported by a Wellcome Trust Student Elective Prize. T. Carpenter was supported by a fellowship grant from the Colorado/Wyoming Affiliate of the American Heart Association.


    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 and other correspondence: E. H. Baker, Dept. of Pharmacology and Clinical Pharmacology, St. George's Hospital Medical School, Cranmer Terrace, Tooting, London, UK SW17 ORE (E-mail: ebaker{at}sghms.ac.uk).

Received 9 November 1998; accepted in final form 16 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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