INTRODUCTION

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THE ALVEOLAR EPITHELIUM is directly exposed to variations in alveolar O₂ tension. At sea level, with normal ventilation, alveolar O₂ partial pressure (PA_O2) is ~100 mmHg. However, a decrease in PA_O2 is observed in many physiological or pathological conditions. For example, during ascent to high altitude, a decrease in PA_O2 occurs in direct relation to the decline in barometric pressure. Also, alveolar hypoxia may be the consequence of hypoventilation related to either a central nervous disorder, obstructive airway diseases, or pulmonary edema from heart failure or acute lung injury. Although the alveolar epithelium is directly exposed during hypoxia, little information has been obtained about the effects of low O₂ tension on alveolar epithelial cell functions. In many tissues, maintenance of normal cell function during hypoxia depends on the ability of the cells to develop adaptive strategies to overcome O₂ deprivation (10). Tolerance to hypoxia has been assumed to be the result of the ability of the cell to cope with a decrease in available energy due to the limitation of oxidative phosphorylation. Adaptive strategies take place to accommodate for the available supply of ATP. Cells increase anaerobic glycolysis by upregulating glycolytic enzymes and decreasing the activity of some proteins that are ATP consuming, such as Na⁺-K⁺-ATPase. In addition, each organ may respond to hypoxia by regulating expression of specific genes that control organ-specific functions. For instance, the pulmonary circulation responds to hypoxia by vasoconstriction and remodeling and the kidney responds by production of erythropoietin.

Evidence from both in vivo and in vitro studies indicates that lung cells are relatively tolerant to hypoxia (16, 26, 36). Neither the ultrastructural characteristics nor cell viability is altered by severe and prolonged O₂ deprivation. The alveolar epithelium consists of two cell types: large, flat, squamous alveolar type I (ATI) cells and cuboidal type II (ATII) cells. These cells form a tight barrier that severely restricts the passage of lipid-insoluble molecules between the alveolar and interstitial spaces. ATII cells are more numerous and have a number of critical functions. First, they serve as progenitors of ATI cells during development as well as in the reparative phase of the alveolar epithelium after injury. In addition, they produce and store surfactant, a complex mixture of phospholipids and specific apoproteins. Also, ATII cells participate in active reabsorption of alveolar fluid because of their ability to transport Na⁺ from the apical to the basolateral surface with water following passively, probably across both ATI and ATII cells. ATII cells express membrane transport proteins located at either the apical or the basolateral membrane surface. A variety of membrane proteins are involved in vectorial Na⁺ transport (17). Na⁺ enters ATII cells through different pathways. Taken as a whole, the results of in vivo and in vitro studies indicate that epithelial Na⁺- and cation-selective channels, located at the apical membrane, represent the main pathway for Na⁺ entry. Under normal conditions, other Na⁺-entry pathways, such as Na⁺-glucose, Na⁺-phosphate, Na⁺-amino acid, or Na⁺-K⁺-Cl symports and Na⁺/H⁺ antiport, contribute only a small fraction of vectorial or net total Na⁺ influx. Na⁺ is extruded at the basolateral surface through the ouabain-sensitive Na⁺-K⁺-ATPase. In addition to Na⁺ transport proteins, ATII cells express membrane proteins used for the supply of substrates required for cell metabolism. Under basal conditions, ATII cells display a high metabolic rate and use glucose for cellular oxidative metabolism, synthesis of surfactant, growth, and differentiation (32). Intracellular transport of glucose is mainly accomplished by membrane-associated glycoproteins termed glucose transporters. In ATII cells, Western blotting and RNase protection assay analyses have identified the presence of proteins and mRNAs encoding for the Na⁺-independent D-glucose transporters GLUT-1 and GLUT-4 (29). It is likely that Na⁺ cotransporters (Na⁺-phosphate, Na⁺-amino acid, and Na⁺-glucose) contribute, at least in part, to supply substrate for surfactant synthesis.

Most of the studies that have examined the effects of O₂ on transport proteins in alveolar epithelial cells have focused on hyperoxia. However, more recently, several groups have studied the effect of hypoxia on alveolar epithelial transport. This brief review will summarize the results of these studies and also discuss possible cellular and molecular mechanisms responsible for the hypoxia-induced gene regulation of glucose and ion transport proteins.

	EFFECTS OF HYPOXIA ON ACTIVITY AND EXPRESSION OF THE GLUCOSE TRANSPORTERS

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Several studies have indicated that lung tissue and alveolar epithelial cells tolerate hypoxia remarkably well. For example, after 48 h of exposure to 10% O₂ hypoxia, the alveolar epithelium was not damaged and maintained a normal energy status (37). In vitro, ATII cells exposed to 0% O₂ survived at least 24 h without cellular damage and maintained an ATP content close to that in normoxic cells (23). It is likely that the maintenance of near-normal ATP levels results from the hypoxia-induced increase in anaerobic glycolysis, since in vitro studies showed that the decline in PO₂ upregulates the activity and expression of glycolytic enzymes and increases lactate production (33). In ATII cells, the first limiting step in glucose utilization is transport across the membrane because glucose concentration is low and the activity of glycolytic enzymes largely exceeds the rate of glucose utilization. Glucose transport in alveolar epithelial cells takes place through different pathways. On the basis of in vivo and in vitro studies, glucose may enter alveolar epithelial cells by a Na⁺-independent, carrier-mediated process (5). Molecular studies demonstrated that the ubiquitous glucose transporter GLUT-1, which is responsible for basal glucose uptake in most tissues, is predominant in ATII cells and was unaffected by the time in culture (29). The presence of an apical Na⁺-dependent D-glucose transporter is based on the results of in vivo studies. Phlorizin, a specific inhibitor of this transporter, decreases the movement of D-glucose across the alveolar epithelium of fluid-filled lungs (38) and reduces alveolar liquid clearance in perfused and ventilated rat (1), although the same effect does not occur in rabbits (34). The results are less consistent in vitro. In isolated ATII cells, Kerr et al. (14) reported that glucose uptake was dependent on external Na⁺, suggesting the presence of Na⁺-dependent glucose transport in ATII cells. However, other groups were unable to demonstrate its presence in cultured ATII cells using a glucose analog, -methyl glucopyranoside, which is specific for transport by the Na⁺-dependent glucose carrier (5 , 20). This discrepancy may be explained by the loss of expression of the Na⁺-dependent glucose cotransporter, SGLT-1, in culture (29).

Ouiddir et al. (23) clearly demonstrated that glucose transport is regulated by O₂ deprivation in ATII cells. Exposure of cultured ATII cells to hypoxia (0% or 5% ambient O₂) results in time- and O₂ concentration-dependent increases in glucose influx, measured by the uptake of deoxy-D-glucose, a glucose analog that is transported by the facilitative glucose transporter. This stimulation of glucose influx is associated with an increase in the levels of GLUT-1 mRNA measured by RNase protection assay and an increase in GLUT-1 protein by Western blotting. The time course for the hypoxia-induced increase in deoxy-D-glucose influx, with no substantial change before 6 h of hypoxia, the prevention of this effect by cycloheximide, an inhibitor of translation, and the parallel recovery during reoxygenation in deoxy-D-glucose uptake and the level of GLUT-1 mRNA support a causative link among these three events. Comparison of ATII cells with other hypoxia-tolerant cells showed that they resemble lung endothelial cells (15) but differ from heart and skeletal muscle, in which upregulation of glucose transport occurs in the first hour with no change in the GLUT-1 mRNA level (4). In ATII cells, Na⁺-dependent glucose transport is weakly expressed during normoxia, and Ouiddir et al. (23) showed that it was not stimulated by hypoxia.

The O₂-sensing mechanisms whereby hypoxia regulates the GLUT-1 mRNA levels in ATII cells result from both a reduced O₂ concentration per se and inhibition of oxidative phosphorylation (23). This distinction has been shown by studies with specific chemical agents that mimic the actions of the different components of the hypoxic response (2). Most hypoxia-regulated genes involve a ferroprotein sensor, and an important characteristic of this system is that the inducible response to hypoxia is mimicked by exposure to particular transition metals. For instance, cobalt chloride, in the presence of O₂, simulates the effect of lowered O₂ concentration, since it substitutes for O₂ in the heme protein. In normoxic ATII cells, the fact that GLUT-1 mRNA levels and glucose uptake are increased by cobalt chloride strongly supports the hypothesis that upregulation of the GLUT-1 gene is dependent on O₂ deprivation itself. Moreover, inhibition of oxidative phosphorylation by sodium azide in ATII cells is as effective as hypoxia for stimulating glucose transport and increasing GLUT-1 mRNA levels. Thus, in ATII cells, hypoxia-induced GLUT-1 gene upregulation results from two different mechanisms, as previously reported in a cell clone derived from hepatoma (2). However, the temporal and spatial contribution of these two mechanisms in hypoxia-induced upregulation of GLUT-1 mRNA level in ATII cells has not been determined.

Because GLUT-1 responds to hypoxia and to inhibition of oxidative phosphorylation, it is likely that its regulation depends on a complex array of cis-acting elements and transcription factors (6). Recent studies have clearly demonstrated that hypoxia-inducible GLUT-1 expression is critically dependent on the binding of a nuclear protein, hypoxia-inducible factor 1 (HIF-1), to the hypoxia response DNA element upstream from the GLUT-1 gene (2, 6). HIF-1 is a heterodimer composed of HIF-1 and HIF-1 subunits, both of which are members of the basic-helix-loop-helix Per Arn_t sim (PAS) (bHLH/PAS) family of proteins (31). HIF-1 is a protein unique to HIF-1, whereas HIF-1 is identical to the aryl hydrocarbon nuclear translocator that can dimerize with several other proteins, including the aryl hydrocarbon receptor, and other bHLH/PAS proteins, such as endothelial PAS protein 1 (EPAS-1). Both HIF-1 and HIF-1 are instrumental in activating the GLUT-1 gene in response to O₂ deprivation (12). Several lines of evidence indicate that HIF-1 is present in lung cells. HIF-1 mRNA is expressed in mouse and rat lung homogenate in normoxic conditions, and its expression depends on O₂ concentration (39). HIF-1 protein has been detected in a human alveolar epithelial cell line, A549, derived from an adenocarcinoma (41). In a recent study, Ouiddir et al. (23), using a GLUT-1 probe containing a sequence from the rat GLUT-1 enhancer that mediates hypoxia-inducible transcription, demonstrated by an electrophoretic mobility shift assay that rat alveolar epithelial cells exhibited HIF-1 DNA binding activity during hypoxic conditions as well as after cobalt chloride treatment. Surprisingly, at variance with most cells in which HIF-1 DNA binding is induced by O₂ deprivation with low or undetectable activity in normoxia (30), ATII cells expressed a constitutive level of HIF-1 DNA binding. The presence of high constitutive levels of HIF-DNA binding has already been reported in some cells such as pulmonary arterial smooth muscle cells (41), J1 embryonic stem cells (12), and v-Src-transformed rat fibroblasts (13), but its significance is not clearly understood. One possibility is that cultured cells exposed to normoxic atmosphere are in relatively hypoxic conditions due to the presence of overlying culture medium. The second possibility is that the high basal HIF-1 DNA binding may be adaptive in cells with increased metabolic demands and, in this case, a signal transduction pathway that was not hypoxia driven could upregulate HIF-1 expression in a cell-specific manner. Finally, it cannot completely be excluded that the nuclear protein that bound to the probe is not HIF-1 but an HIF-like factor. EPAS is an hypoxia-inducible factor that exhibits similar characteristics to HIF-1 dimerization, DNA binding, and transcriptional activation (28, 40). EPAS is less ubiquitously expressed than HIF-1 and mainly regulates the transcription of vascular endothelial growth factor (7). In ATII cells, EPAS may be a good candidate, since 1) EPAS-1 is more active in normoxia and less inducible by hypoxia than HIF-1 (40) and 2) EPAS mRNA transcript is expressed in mouse alveolar epithelium at a higher level than HIF-1 mRNA (7). Further studies are required to determine whether EPAS is involved in the transcriptional regulation of the GLUT-1 gene.

	EFFECT OF HYPOXIA ON NA+ TRANSPORT PROTEINS IN ALVEOLAR EPITHELIUM

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The question of whether hypoxia can regulate Na⁺ transport proteins has only been addressed recently. Amiloride-sensitive Na⁺ channels are the major pathway for apical Na⁺ entry in alveolar cells and may be the rate-limiting step in alveolar transepithelial Na⁺ transport (17). The presence of Na⁺ channels in the apical membrane of adult rat ATII cells was first established by patch-clamp studies (18). Two types of Na⁺ channels have been identified: 1) an amiloride-sensitive Na⁺ channel that is moderately selective (pNa/pK > 7), has a high conductance (25 pS), and exhibits relatively low affinity for amiloride (1 µM) (42); and 2) an amiloride-insensitive, nonselective (pNa/pK = 1) voltage-independent Ca²⁺-activated cation channel (8). Recent molecular studies demonstrated that rat adult ATII cells express mRNA transcripts encoding the three subunits , ,  of the rat epithelial Na⁺ channel (rENaC) (19, 25). The major functional role of ENaC in transepithelial Na⁺ transport can be best appreciated by considering that -ENaC-deficient mice died 40 h after birth from failure to clear their perinatal alveolar fluid (11).

Several studies indicate that hypoxia alters Na⁺ transport in ATII cells (Table 1). Exposure of ATII cell monolayers to severe hypoxia (0% and 3% O₂) inhibits dome formation in vitro (25) and results in a decrease in Na⁺ channel activity as measured by amiloride-sensitive ²²Na⁺ influx (16, 25). Planès et al. (25) have evaluated the effect of hypoxia and hypoxia reoxygenation in ATII cells on both the expression and activity of rENaC. Analysis of mRNA transcripts of ENaC subunits by RNase protection assay showed that the decrease in amiloride-sensitive Na⁺ uptake paralleled the fall in the -, -, and -ENaC subunit mRNA levels with a similar time course. The effect occurred after a 3-h exposure, and a maximal decrease was observed after 12 h of hypoxia. For longer exposure times (>12 h), a decrease in amiloride-sensitive Na⁺ uptake and mRNA levels was associated with a decline in the rate of -rENaC protein synthesis, suggesting a causative link between these three events. Moreover, during reoxygenation, the progressive recovery in Na⁺ channel activity paralleled the recovery in -, -, -rENaC mRNA levels and normalization of -rENaC protein, indicating that de novo protein synthesis of rENaC subunits was necessary to restore Na⁺ channel activity after prolonged hypoxia and that protein synthesis required the restoration of adequate rENaC mRNA levels (Fig. 1). For a short exposure time (3-h exposure), reduced channel activity occurred at a time when only -rENaC mRNA was decreased, whereas - and -rENaC mRNA levels were unchanged. One explanation is that a reduced amount of -rENaC mRNA led to insufficient production of -rENaC subunit protein and subsequently accounts for reduced functional activity, suggesting that -rENaC subunit is necessary for the correct processing of Na⁺ channel proteins at the cell surface. The relationship of ENaC subunits to the functional effect on Na⁺ uptake is an area of intense investigation (3). Although several lines of evidence suggest that hypoxia downregulates ENaC gene expression by a transcriptional effect, other mechanisms may be involved in regulation, including translational and posttranslational events, including decreased efficiency in the translation of rENaC mRNA or in apical membrane trafficking of rENaC subunits, abnormal degradation or internalization of the channel protein, and hypoxia-induced modification of intracellular signals.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effect of hypoxia and hypoxia reoxygenation on mRNA expression of -subunit (A), -subunit (B), and -subunit (C) of rat epithelial Na+ channel (rENaC) in alveolar type II (ATII) cells. Rat ATII cells were exposed after 4 days in culture to either normoxia (21% O2; ) or hypoxia (0% O2; ) for 3, 6, 12, and 18 h or to hypoxia followed by reoxygenation (18 h of 0% O2 + 24 or 48 h of 21% O2). At the end of exposure, RNase protection assays were performed on cell lysates and -, -, and -rENaC subunits were quantified using an Instant Imager. Data were normalized for the corresponding actin signal for each lane. Results are expressed as the unitless ratio of -, -, and -rENaC subunit mRNA to actin mRNA and are means ± SE of 3-6 independent experiments. * Significantly different from normoxic control value (P > 0.05). [Modified from Ref. 25, with permission.]

In conjunction with apical Na⁺ channels, basolateral Na⁺-K⁺-ATPase represents the major protein involved in transepithelial Na⁺ transport by alveolar cells, and its functional activity is usually tightly coupled with apical Na⁺ entry to ensure efficient vectorial Na⁺ transport. The effect of hypoxia on Na⁺-K⁺-ATPase activity has been evaluated using in vivo and in vitro studies (16, 25, 36). On the whole, these studies have demonstrated that hypoxia decreased Na⁺-K⁺-ATPase activity (Table 1). In vivo, in lung tissue preparations and ATII cells obtained from rats exposed to 10% O₂ for 48 h, Na⁺-K⁺-ATPase hydrolytic activity was decreased (36). In A549 cells, with features of ATII cells, exposure to 3% O₂ (PO₂ of 21 Torr) induced a dramatic decrease of Na⁺-K⁺-ATPase, which was apparent and maximal after 1 h of hypoxia (16). In SV40-transformed rat ATII cells, 0% and 5% O₂ impair Na⁺-K⁺-ATPase activity after 6 h (26). Finally, in primary cultures of ATII, Planès et al. (25) reported that hypoxia (0% O₂) induced a time-dependent decrease of Na⁺-K⁺-ATPase activity, estimated by ouabain-sensitive ⁸⁶Rb influx. This effect paralleled the change in Na⁺ channels activity after 3 and 6 h of hypoxia, with a maximal effect after 18 h of hypoxia. The question of whether the hypoxia-induced decrease in Na⁺-K⁺-ATPase activity matches with the change in Na⁺-K⁺-ATPase gene expression has been raised in the latter study (25). RNase protection assays showed that, in primary culture of rat ATII cells, the levels of mRNA transcripts encoding ₁- and ₁-Na⁺-K⁺-ATPase were reduced (Fig. 2). Because the inhibition of mRNA levels paralleled the decrease of Na⁺-K⁺-ATPase activity and these effects were fully reversed by reoxygenation of the cells for 48 h, downregulation of Na⁺-K⁺-ATPase mRNA transcripts by O₂ deprivation may account for the decrease in Na⁺-K⁺-ATPase activity. However, translational or posttranslational events could not be excluded.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effect of hypoxia and hypoxia reoxygenation on mRNA expression of 1-subunit (A) and 1-subunit (B) of Na+-K+-ATPase in ATII cells. Rat ATII cells were exposed after 4 days in culture to either normoxia (21% O2; ) or hypoxia (0% O2; ) for 3, 6, 12, and 18 h or to hypoxia followed by reoxygenation (18 h of 0% O2 + 24 or 48 h of 21% O2). At the end of exposure, RNase protection assays were performed on cell lysates and 1- and 1-Na+-K+-ATPase subunits were quantified using an Instant Imager. Data were normalized for the corresponding actin signal for each lane. Results are expressed as the unitless ratio of 1- and 1-subunit mRNA to actin mRNA and are means ± SE of 3-6 independent experiments. * Significantly different from normoxic control value (P > 0.05). [Modified from Ref. 25, with permission.]

These results raise the question of whether gene expression for Na⁺ channels and Na⁺-K⁺-ATPase is regulated by ambient PO₂ in ATII cells. In support of this hypothesis, others have shown that in ATII cells an increase in O₂ tension upregulates the level of ENaC mRNA transcripts: the transfer of fetal distal lung epithelial (FDLE) cells in culture from 3% O₂ to higher O₂ concentrations upregulates -, -, and -rENaC mRNA subunits as well as Na⁺ channel activity (24). Also, one recent in vivo study demonstrates enhanced expression of -ENaC and amiloride-sensitive epithelial transport at the time of birth when alveolar O₂ tension increases (9). Exposure of rats to sublethal hyperoxia (85% O₂) increased the -rENaC mRNA level in ATII cells (42). In addition, it has been recently reported that ₁- and ₁-Na⁺-K⁺-ATPase mRNA subunit levels both increased in ATII cells from rats exposed to hyperoxia (21). The mechanism whereby O₂ tension regulates Na⁺ channels and Na⁺-K⁺-ATPase gene expression is not clear. A transcriptional activation of ENaC by increased O₂ concentration has been suggested in FDLE cells. Rafii et al. (27) demonstrated that O₂ induced an increase in ENaC expression associated with nuclear factor (NF)-B activation that was blocked by a superoxide scavenger. These studies are consistent with an identification of NF-B transcription binding sites in the -ENaC promoter region (22). In addition to the potential role for an O₂-responsive element in the ENaC promoter, it is also possible that there are other genes that are O₂ responsive and alter ENaC expression through their expressed protein or metabolic products.

Whatever the mechanism, the hypoxia-induced downregulation of Na⁺ transport proteins in vitro may have important pathophysiological implications in vivo. Indeed, in some humans, a decrease in ambient O₂ tension at high altitude can be associated with development of pulmonary edema, although the initial cause of the edema may be related to altered hemodynamics or an increase in microvascular permeability (35). On the basis of in vitro studies, the decreased expression and activity of Na⁺ transport proteins in ATII cells may slow the rate of reabsorption of alveolar edema. Recently, Suzuki et al. (36) reported a decrease in alveolar clearance in rats exposed to hypoxia.

	FUTURE DIRECTIONS

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Genes regulated by hypoxia in the lung can serve as physiological signals, but how this occurs is incompletely understood. Therefore, basic mechanisms by which low O₂ tension directly activates or downregulates membrane transport proteins in the alveolar epithelium need to be explored. Studies of gene regulation by hypoxia need to address several issues.

What is the functional significance of hypoxia-induced downregulation of Na⁺ transport proteins? The downregulation may result in adaptive mechanisms to limit cellular demand for ATP. What are the mechanisms of O₂ sensing in ATII cells and the signal transduction pathways? It is usually thought that O₂ sensing in cells involves a heme protein. It will be of interest to determine what kind of heme protein is involved and the role of reactive O₂ species in the transduction of this information.

What is the role of the transcriptional factors such as HIF and/or EPAS in the response to hypoxia? To answer this question, in vitro and in vivo experiments need to be performed with an inducible HIF or EPAS negative dominant expressed in alveolar epithelial cells. For Na⁺ transport proteins, it will be of value to define the structure of the promoter region and to determine the location of the inducer-responsive elements and transcription factor binding sites. Finally, the functional effects of hypoxia on gene expression of transport proteins in alveolar epithelial cells will need to be evaluated with in vivo studies.