PERSPECTIVE
Modification of Sodium Transport and Alveolar Fluid Clearance by Hypoxia
Mechanisms and Physiological Implications

Karin M. Hardiman and Sadis Matalon

Departments of Physiology and Biophysics and Anesthesiology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama

In order for gas exchange to occur optimally, the alveoli must remain open and free from fluid. In utero, the fetal lung is filled with fluid which is removed shortly after birth, mainly because active reabsorption of sodium ions (Na⁺) across the alveolar epithelium creates an osmotic force favoring reabsorption of alveolar fluid (1, 2). The classic studies from Dr. Matthay's laboratory showing reabsorption of intratracheally instilled isotonic fluid or plasma from the alveolar spaces of adult anesthetized animals, and the partial inhibition of this process by amiloride and ouabain, implied that adult alveolar epithelial cells are also capable of actively transporting sodium (Na⁺) ions (reviewed in Ref. 3).

In the adult lung, active Na⁺ reabsorption plays an important role in limiting the degree of alveolar edema in pathologic conditions in which the alveolar epithelium has been damaged. For example, blocking Na⁺ transport increased lung water in rats exposed to hyperoxia (4). Conversely, intratracheal instillation of adenoviral vectors containing copies of the Na⁺,K⁺-ATPase genes increased survival of rats exposed to hyperoxia (5). Patients with acute lung injury who were able to concentrate alveolar protein (as a result of active Na⁺ reabsorption) had a better prognosis than those that did not (6, 7). Presently, it has not been definitely established whether or not active Na⁺ transport also plays an important role in maintaining the normal alveoli free of fluid.

Additional insight into the nature and regulation of transport pathways has been derived from electrophysiologic studies in freshly isolated and cultured alveolar type II (ATII) cells: Na⁺ ions diffuse passively into ATII cells through apically located amiloride-sensitive, amiloride- insensitive, and cGMP-gated cation channels with conductances of 4-25 pS (8, 9) and are extruded across the basolateral membranes by the ouabain-sensitive Na⁺,K⁺-ATPase (10). To preserve neutrality, chloride (Cl) ions move from the apical to the basolateral compartments either through the paracellular junctions and/or through chloride channels located in alveolar epithelial cells (11, 12). In situ hybridization studies identified the presence of two of the three subunits of the cloned epithelial Na⁺ channel (ENaC and ENaC) in the alveolar region of both fetal and adult lungs (13). Currently there is controversy as to whether ENaC per se or ENaC-type channels (i.e., channels with biophysical properties distinct from those of ENaC) are the main pathways for Na⁺ entry into ATII cells (14).

There have been numerous studies attempting to identify whether decreased alveolar fluid clearance (AFC) contributes to alveolar edema formation in a variety of pathophysiologic conditions. The results of several studies suggest that severe alveolar hypoxia results in decreased AFC and Na⁺ transport (see Table 1). This is of major interest because alveolar hypoxemia may be encountered in a variety of pathologic conditions including hypoventilation, obstructive lung disease, and ascent to high altitude (15).

To investigate the mechanisms responsible for the downregulation of AFC during hypoxia, Vivona and colleagues exposed rats to physiologic levels of hypoxia (8% O₂ for up to 24 h) and measured AFC in situ and ENaC and Na⁺,K⁺-ATPase mRNA and protein in isolated ATII cells (16). They reported that alveolar hypoxia decreased both the total and amiloride-sensitive portion of AFC and that these changes were ameliorated by intratracheal instillation of a ₂ agonist. Surprisingly, levels of -ENaC and ₁- and ₁-Na⁺,K⁺-ATPase in ATII cells remained unchanged. These findings are highly significant because not only do they provide new insight into the mechanism of edema formation during ascent to high altitude, but they also highlight a potential therapeutic strategy to decrease edema and improve gas exchange. Furthermore, the results of these studies clearly point out that it is not always possible to extrapolate changes in function from biochemical and molecular biology measurements.

The methodology of measuring AFC in vivo was introduced by Matthay and coworkers almost twenty years ago (17). In this set of experiments, AFC was measured in nonventilated rats with cardiac arrest. At first glance, this seems contradictory to the basic premise, namely that alveolar fluid clearance depends on the presence of an energy-requiring Na⁺,K⁺-ATPase. However, previous studies have shown that constant levels of AFC can be maintained for brief periods of time following cessation of ventilation and perfusion. Other studies have shown that Na⁺,K⁺-ATPase activity can be maintained at normal levels even in the presence of large decreases in cellular ATP levels (18). Fukuda and associates (19) reported that the lungs of dead mice developed a significant amount of interstitial edema, most likely due to the lack of pulmonary lymph flow, which inhibited AFC by ~ 30%. Vivona and colleagues avoided any possible artifacts by measuring AFC at various intervals and reporting AFC values within 30 min from death, well within its linear range. However, since multiple measurements of AFC are often very difficult to perform, especially in smaller animals, every effort should be made to measure AFC in properly ventilated animals with normal blood gases (20). If the experimental conditions such as the ones present here make this approach not plausible, then investigators must establish the linear range of AFC, as was done in this study.

The investigators also measured AFC in the presence and absence of amiloride and showed that hypoxia decreases the amiloride-sensitive portion of transport. At first glance the amiloride concentration used in this study (as in every other in vivo study) is quite high and several orders of magnitude the value required to inhibit Na⁺ transport across alveolar epithelial cells (21, 22). It has been proposed that amiloride is either cleared quickly across the alveolar epithelium or it loses its effectiveness due to binding to albumin. However, more recent measurements revealed significant concentrations (> 500 µM) of amiloride in the alveolar fluid 2 h after instillation of 1 mM amiloride (11). Furthermore, amiloride was equally effective in blocking sodium currents across oocytes injected with , and  rENaC in the presence or absence of albumin (unpublished observations of S. Matalon). A more likely explanation for the difference between in vivo and in vitro studies is that because the instillate is a small fraction of the total lung capacity, the effective concentration of amiloride in the alveolar space is much lower than in the instillate. Indeed, Olivera and coworkers (23) showed that low concentrations of amiloride (10 µM) inhibited most of Na⁺ transport across lungs filled with large amounts of fluid. It should be stressed that measurements of the amiloride- inhibitable fraction of AFC are essential in the proper interpretation of the data because a number of factors (including passive forces and movements of other ions) may affect the value of AFC.

As mentioned above, the authors provided provocative evidence that hypoxia decreases AFC in rats in the absence of any decrease in the mRNA and protein levels of ENaC or the ₁ and ₁ isoforms of the Na⁺,K⁺-ATPase in the ATII cells. Since terbutaline restored AFC to its control levels, it is very unlikely that the decrease in AFC was due to diminished lung ATP levels. Instead, there are various other explanations for these findings.

First, sodium transporters may have been damaged by reactive oxygen-nitrogen intermediates, the production of which is known to be increased in hypoxia (24). Suzuki and associates (25) showed that exposure of rats to 10% O₂ for 48 h decreased AFC and lung Na⁺,K⁺-ATPase hydrolytic activity. They also reported that terbutaline failed to increase AFC in the lungs of hypoxic rats despite the fact that it increased cAMP levels considerably. Results of various other studies (summarized in Table 1), indicate that more severe levels of hypoxia may decrease mRNA levels of Na⁺ transporters in alveolar epithelial cells.

Second, it is also possible that hypoxia may reduce trafficking of Na⁺ transporters from the cytoplasm to the membranes by unknown mechanisms. Various studies, using Western blotting and immunofluorescence techniques, have shown the existence of submembrane pools of ENaC protein which can be translocated to the membrane in response to various stimuli (26). The results of these studies suggest that trafficking of the ENaC protein to the membrane is an important regulatory process. In addition, Na⁺,K⁺-ATPase translocation to the plasma membrane from late endosomes is an important mechanism for Na⁺,K⁺-ATPase regulation in rat alveolar epithelial cells (27). A recent study demonstrated that terbutaline restored AFC in rat lungs ventilated with high tidal volume and this effect was prevented by colchicine (28), consistent with the idea that terbutaline promoted recruitment of Na⁺,K⁺-ATPase ₁ and ₁ subunits from intracellular pools to the basolateral membrane. Alternatively, or in addition, terbutaline may have increased Na⁺ transport by increasing the open probability of Na⁺ channels located in the apical membranes of ATII cells (29).

Clearly, additional studies are needed to elucidate the mechanisms involved. One approach is to isolate apical and basolateral proteins from ATII cells from these animals and quantify levels of Na⁺ transporters by Western blotting studies using ENaC and Na⁺,K⁺-ATPase antibodies. If trafficking is impaired, the amount of immunoreactive protein in the basolateral membranes of ATII cells should be lower than normal, despite the fact that total levels of Na⁺,K⁺-ATPase may remain unchanged. In addition, patch clamp measurements assessing the biophysical properties of Na⁺ channels in ATII cells isolated from normal and hypoxic rats, as well as measurements of Na⁺-K⁺-ATPase activity, will offer considerable insight as to whether these pathways have been damaged.

Finally, the conclusion that decreased AFC may be responsible, at least in part, for the development of pulmonary edema during ascent to altitude should be viewed with caution. Rats exposed to hypoxia had "mild interstitial edema" as shown by a slight (< 10%) increase in lung wet:dry weight ratios. Suzuki and colleagues (25) found no evidence of interstitial or alveolar edema in rats breathing 10% O₂ for 48 h. It needs to be demonstrated whether inhibition of transalveolar Na⁺ transport by intratracheal instillation of amiloride or its more potent analogs (4) may result in higher levels of pulmonary edema in hypoxia. Clearly, there is a sufficient amount of work to be done for many years to come.

	Footnotes

Address correspondence to: Sadis Matalon, Ph.D., Department of Anesthesiology, University of Alabama at Birmingham, 1530 3rd Avenue South, THT 940, Birmingham, AL 35294-0006. E-mail: Sadis.Matalon{at}ccc.uab.edu

(Received in original form August 23, 2001).

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