We examined whether hypoxic exposure in vivo would influence transalveolar fluid transport in rats. We found a significant decrease in alveolar fluid clearance of the rats exposed to 10% oxygen for 48 h. Terbutaline did not stimulate alveolar fluid clearance, and alveolar fluid cAMP levels were lower than those determined in normoxia experiment. Hypoxia did not influence the alveolar fluid lactate dehydrogenase levels, Evans blue dye fluid-to-serum concentration ratio, or lung wet-to-dry weight ratio, indicating no significant change in the permeability of alveolar-capillary barrier. Histological examination showed no significant fluid accumulation into the interstitium and the alveolar space. Hypoxia did not reduce lung ATP content; however, we found significant decrease in Na+-K+-ATPase hydrolytic activity in lung tissue preparations and isolated alveolar type II cells. Our data indicate that hypoxic exposure in vivo impairs transalveolar fluid transport, and this impairment is related to the decrease in alveolar epithelial Na+-K+-ATPase hydrolytic activity but is not secondary to the alteration of cellular energy source.
- alveolar fluid clearance
- alveolar type II cells
- β-adrenergic agonists
alveolar hypoxia during rapid ascent to high altitude has been considered to be one of the most important determinations of the occurrence of alveolar fluid accumulation, e.g., high-altitude pulmonary edema (1). Over the past decades, numerous attempts have been made to determine the impact of hypoxia on pulmonary circulation. Hypoxia causes pulmonary vasoconstriction (17, 32), and the uneven hypoxic pulmonary vasoconstriction may increase pulmonary capillary pressure and fluid filtration (46). Hypoxia may also activate some lung cells to release a wide variety of mediators, including arachidonate metabolites and cytokines (39), which are potentially able to increase pulmonary microvascular permeability.
Recently, several lines of evidence obtained from in vitro experiments have indicated that hypoxia impairs ion-transport properties of alveolar epithelial cells (20, 29, 30). Alveolar epithelium is currently considered to be not only a limited permeable barrier structure but also the most likely site for absorption of excess alveolar fluid in adults (24, 37). This transalveolar fluid transport plays an important role in keeping the alveolar spaces to be relatively fluid-free condition. Experimental studies have demonstrated that active Na+ transport system exists in alveolar epithelial cells, where Na+ enters into these cells at the apical surface via predominantly amiloride-sensitive Na+ channels and is pumped out of the cells by the basolateral Na+-K+-ATPase (2, 22, 23, 33). This vectorial Na+ transport allows fluid absorption from the alveolar space to the interstitium. Thus the intact Na+-K+-ATPase function in alveolar epithelial cells seems to be essential in opposing alveolar fluid accumulation. In fact, it has been shown in rat lungs subjected to ischemia and reperfusion injury that inhibition of Na+-K+-ATPase with ouabain results in a more significant pulmonary edema formation (18). Therefore, it can be speculated that hypoxia would decrease fluid absorption from the alveolar spaces if the impairment of alveolar epithelial Na+-K+-ATPase had occured in vivo. However, no direct evidence has been provided.
The purpose of this study was to examine whether hypoxic exposure in vivo would influence transalveolar fluid transport in rats. We evaluated transalveolar fluid transport as alveolar fluid clearance (AFC) by adapting the fluid-filled lung model. We also measured Na+-K+-ATPase hydrolytic activity in lung tissue preparations and isolated alveolar type II cells. Our data indicate that hypoxic exposure in vivo impairs transalveolar fluid transport, and this impairment is related to the decrease in alveolar epithelial Na+-K+-ATPase hydrolytic activity but is not secondary to the alteration of cellular energy source.
MATERIALS AND METHODS
Specific-pathogen-free male Sprague-Dawley rats weighing 320–350 g were used. All animals received humane care, in compliance with the guidelines from the University Committee on Animal Resources, Tohoku University, Japan. For the hypoxia experiments, rats were exposed to normobaric hypoxia for up to 72 h in a sealed chamber (140 × 60 × 60 cm), which was continuously flooded with hypoxic gas at 6 l/min. Three different oxygen concentration were examined: 15, 10, and 8% oxygen in nitrogen. For normoxia experiment, rats were exposed to room air in the same chamber. Every 12 h, sample gas was taken, and in the chamber was measured by using an automatic gas analyzer (ABL-300, Radiometer, Copenhagen, Denmark). in normoxia experiment was in the range of 155–160 Torr. Hypoxic gas containing 15, 10, and 8% oxygen resulted in a of 107–115, 75–82, and 55–62 Torr, respectively. For the control experiment, we used rats that were not taken into the chamber. Animals were allowed free access to food and water.
Rats were anesthetized outside the chamber by intraperitoneal injection of pentobarbital sodium (50 mg/kg), and the heart-lung blocks were removed from the thorax within 10 min. Once completely collapsed, the lungs were instilled with 5 ml of prewarmed albumin solution (in mM: 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 5d-glucose, 6 HEPES, and 4 g/dl bovine serum albumin, pH 7.1) and further inflated with 3 ml of air. This gave ∼7 cmH2O of airway pressure (data not shown). We have previously shown (43) that this airway pressure does not influence AFC. The lungs were then placed at an upright position in a humidified plastic box, and the box was incubated in a water bath at 37°C for 2 h. Alveolar fluid was drained into a small syringe and centrifuged at 5,000 rpm for 5 min at 4°C.
We determined albumin concentration in the supernatant of alveolar fluid by using an automatic analyzer (TBA-80FR, Toshiba, Tokyo, Japan). Similar to the previous studies (34, 36, 43), AFC was calculated by determining the increase of albumin concentration in the alveolar fluid as follows Equation 1where V represents the volume of albumin solution before instillation (i) and recovery (f) after 2 h of incubation. Fw is the water fraction obtained by subtracting albumin content. Vfis calculated from the following formula Equation 2where P represents albumin concentration.
To examine whether hypoxic exposure would alter the ability of alveolar epithelium in respond to β-adrenergic stimulation, AFC was also determined by using albumin solution containing 10−3 M terbutaline (Sigma Chemical, St. Louis, MO). Terbutaline has been shown to increase AFC in rats (9, 19, 34), sheep (3), and resected human lungs (34, 35). Alveolar epithelial cAMP production by β-adrenergic stimulation was assessed by determining alveolar fluid cAMP levels after 2 h of incubation. In brief, cAMP was extracted from the recovered alveolar fluid by incubation in boiling water for 10 min, followed by centrifugation at 12,000 rpm for 5 min at 4°C, and measured by using a cAMP assay kit (Cayman Chemical, Ann Arbor, MI). We also measured alveolar fluid lactate dehydrogenase (LDH) levels to assess possible damage of alveolar epithelial cells during hypoxic exposure.
Evans Blue Dye Fluid-to-Serum Concentration Ratio
Anesthetized rats received 30 mg/kg of Evans blue dye (0.05 mg/ml in sterilized saline) by injection into the inferior vena cava over a 1-min period through the abdominal incision. After 10 min, the chest was opened, and blood samples were collected by direct aspiration from the right ventricular cavity. Heart-lung blocks were removed, and the lungs were inflated with prewarmed albumin solution and incubated for 2 h as described above. We determined Evans blue dye concentration in the serum and the alveolar fluid by reading absorbance at 620 nm, and we calculated the fluid-to-serum concentration ratio to assess possible protein leak into the alveolar space during hypoxic exposure.
Lung Wet-to-Dry Weight Ratio
We determined lung wet-to-dry weight ratio corrected with lung hemoglobin concentration (28) to assess possible change in lung extravascular water content.
Lung ATP Content
Left lungs were removed from anesthetized rats and frozen in liquid nitrogen within 10 min. Next, the lung tissue was homogenized in 5 ml of 0.6 N perchloric acid by using a glass homogenizer (Iwaki Glass, Tokyo, Japan) at 1,000 rpm on ice and then centrifuged at 3,000 rpm for 20 min at 4°C. ATP concentration in the supernatant was determined enzymatically by using an ATP assay kit (Sigma Chemical) by reading the decrease in absorbance at 340 nm. Results were standardized to dry weight of the resulting pellet of whole lung homogenate.
Na+-K+-ATPase Hydrolytic Activity
Lung tissue preparations. Rats were anesthetized and ventilated through a tracheostomy. The chest was opened, and heparin sodium (200 U) was administrated directly into the right ventricular cavity. The inferior vena cava and descending aorta were dissected free at the diaphragm. The lungs were cleared of blood by flushing with 15 ml of perfusion solution (sucrose 250 mM, pH 7.4) at a pressure of 10 cmH2O at 24°C. Right lungs were removed and homogenized in 2.5 ml of homogenate buffer (in mM: 250 sucrose, 10 Tris, 1 EGTA, 1 dithiothreitol, 1 phenylmethylsulfonyl fluoride, pH 7.4) at 24,000 rpm in a rotor homogenizer (Physcotron; Nichi-On Irika, Tokyo, Japan) for three 20-s periods on ice. Whole lung homogenate was centrifuged at 1,000 gfor 15 min at 4°C. The supernatant was centrifuged at 9,500g for 10 min at 4°C, and then further centrifuged at 35,000 g for 30 min at 4°C to obtain the mixed plasma membrane fraction (6).
Alveolar type II cells. Alveolar type II cells were isolated by enzymatic tissue digestion with elastase (Worthington Biochemical, Freehold, NJ), and then purified by the differential adherence technique in rat IgG-coated plastic plates (11,13). The purity of the freshly isolated cells determined by alkaline phosphatase staining (12) was >85%. The viability of the isolated cells was determined by trypan blue staining. Approximately 10 × 106 cells were suspended in 250 ml of homogenate buffer and sonicated by using an ultrasound cell processor (UD-201, Tomy, Tokyo, Japan) with a 5-s burst at 60-W output on ice.
Enzyme assay. Protein concentration of lung tissue preparations and crude homogenate of alveolar type II cells were determined by the Bradford method (5). Na+-K+-ATPase hydrolytic activity was determined as ouabain-sensitive ATPase hydrolysis under the maximal velocity condition, as optimized previously (44). ATPase hydrolysis was monitored by the enzymatic detection of Pi (42). In brief, lung tissue preparations and crude cell homogenate containing 150–300 mg protein were preincubated in a final volume of 1 ml of assay buffer (in mM: 125 NaCl, 12.5 KCl, 12.5 MgCl2, 1.3 EGTA, 6 azide, 60 Tris, pH 7.4) in the presence and absence of 1 mM ouabain for 30 min at 37°C. ATPase hydrolysis was then initiated by adding 100 ml of stock ATP solution (50 mM) and terminated every 5 min by placing 200 ml of aliquots from the assay mixture into an ice-cold 50% trichlolacetate solution. Pi was separated by activated charcoal absorption and measured by using a Pi detection kit (Pi-S; Daiya-Itoron, Tokyo, Japan) by reading absorbance at 550 nm. Pi release was analyzed by linear regression in the function of sampling time, and Na+-K+-ATPase hydrolytic activity was calculated as the difference in the slopes of the regression lines obtained in the presence and absence of ouabain.
For light-microscopic examination, the lungs were removed from the thorax and inflation fixed with 10% Formalin at 10 cmH2O of airway pressure. Then the blocks were embedded in paraffin, sliced, and stained with hematoxylin and eosin. For electron microscopic examination, lung tissue (∼5 × 5 × 5 mm) was fixed in 4% glutaraldehyde and in 1% osmium tetraoxide. The lung tissue blocks were embedded in Epon, sliced, and stained with aranyl acetate and lead citrate.
The data are presented in the groups as means ± SD. Statistical analyses were performed by using analysis of variance and the Fisher’s exact test. We accepted P < 0.05 as indicating significant difference.
In the control experiment, during 2 h of incubation AFC was calculated to be 651 ± 86 ml (n = 6) (Fig.1). Exposure to 10% oxygen for 24 h did not change the AFC levels (656 ± 88 ml,n = 6); however, a significant decrease was observed at 48 h (414 ± 83 ml,n = 6) (Fig. 1). No further decrease in AFC was observed at 72 h (430 ± 46 ml,n = 6) (Fig. 1).
AFC in the rats exposed to 21% oxygen in the chamber for 48 h was 652 ± 93 ml (n = 6) (Fig.2). Exposure to 15% oxygen for the same period of time showed no significant decrease in AFC (596 ± 77 ml,n = 6) (Fig. 2). When rats were exposed to 8% oxygen, no animal survived for longer than 6 h (n = 4).
The effect of terbutaline on AFC was examined in rats exposed in the chamber to either 21% oxygen (normoxia experiment) or 10% oxygen (hypoxia experiment) for 48 h. In the hypoxia experiment, terbutaline did not stimulate AFC, whereas a 1.4-fold increase was observed in the normoxia experiment (Table 1). There was a significant difference in the alveolar fluid cAMP levels between normoxia and hypoxia experiments (Table 1).
There was a small increase in the alveolar fluid LDH levels during 2 h of incubation; however, no significant difference was observed both immediately after instillation (5 min) and at completion of incubation period (120 min) between normoxia and hypoxia experiments (Table2). There was also no significant difference in Evans blue dye fluid-to-serum concentration ratio or lung wet-to-dry weight ratio between normoxia and hypoxia experiments (Table2).
The lung ATP content in normoxia experiment was 15.1 ± 1.5 mmol/g dry lung (n = 5) (Fig.3). The lungs in hypoxia experiment (10% oxygen for 48 h) showed no decrease in the lung ATP content (19.0 ± 2.0 mmol/g dry lung, n = 5) (Fig. 3).
The Na+-K+-ATPase hydrolytic activity in lung tissue preparations was 4.49 ± 1.33 mmol Pi ⋅ h−1 ⋅ mg lung protein−1(n = 6) in the normoxia experiment (Fig. 4). In the hypoxia experiment (10% oxygen for 48 h), there was a significant decrease in the activity (2.44 ± 1.46 mmol Pi ⋅ h−1 ⋅ mg lung protein−1,n = 6) (Fig. 4).
The Na+-K+-ATPase hydrolytic activity in crude homogenate of alveolar type II cells was 1.96 ± 0.27 mmol Pi ⋅ h−1 ⋅ mg cell protein−1(n = 6) in the normoxia experiment (Fig. 5). In the hypoxia experiment (10% oxygen for 48 h), there was a significant decrease in the hydrolytic activity (1.25 ± 0.30 mmol Pi ⋅ h−1 ⋅ mg cell protein−1,n = 6) (Fig. 5). There was no significant difference between normoxia and hypoxia experiments in the viability of freshly isolated alveolar type II cells (92.3 ± 2.2%,n = 6, and 93.1 ± 3.2%,n = 6, respectively).
The lungs in the hypoxia experiment (10% hypoxia for 48 h) were macroscopically of normal appearance. Light-microscopic examinations showed no considerable fluid accumulation in the perivascular connective tissue and in the alveolar spaces. Electron microscopic examinations also showed no significant evidence for abnormalities in alveolar epithelial cells.
In the present study, we were able to provide the evidence indicating that hypoxic exposure in vivo impairs transalveolar fluid transport. We evaluated transalveolar fluid transport as AFC by adapting the fluid-filled lung model to isolated rat lungs. The isolated lung system without perfusion is currently considered to be a very useful and reliable tool to investigate transalveolar fluid and electrolytes transport. This system may be free from possible risk of edema formation due to the change in pulmonary vascular permeability and the difficulty of keeping microvascular pressure stable during artificial perfusion (34). We calculated the AFC in control and normoxia experiments to be ∼650 ml over 2 h of incubation (13% of volume of the instillated). This is similar to the levels determined in our previous study (24% over 4 h) (34), even though in vivo experiments have demonstrated higher AFC (14, 43). One of the possible explanations for this difference may be the fact that in the present study we instilled a larger volume of albumin solution (5 ml). Lung inflation with a larger volume of albumin solution may give relatively smaller surface area product of the alveoli per unit volume of instillate.
However, the fluid-filled lung model, using albumin concentration as an indicator, presents AFC in net values and does not measure unidirectional fluid transport separately. Thus AFC determined in this study might be influenced if hypoxic exposure increases pulmonary vascular permeability. Even though it has been demonstrated that hypoxia does not cause significant change in pulmonary microvascular permeability to plasma protein (4, 8), some limited data suggest that hypoxia might increase pulmonary vascular permeability in vivo (41) and in vitro (27). Our data, however, indicate that hypoxic exposure in vivo (10% oxygen for 48 h) caused no significant change in the permeability of alveolar-capillary barrier. First, there was no significant difference in alveolar epithelial LDH levels at 5 min and at completion of 2 h of incubation period between normoxia and hypoxia experiments. Second, we found no significant difference in Evans blue dye fluid-to-serum concentration ratio or lung wet-to-dry weight ratio between the normoxia and hypoxia experiments. Third, we were not able to demonstrate any considerable fluid accumulation in the interstitium and the alveolar space of the lungs in the hypoxia experiment by light microscopy. Finally, our electron microscopic examinations also showed no significant evidence of abnormalities in lung cells, including alveolar epithelial cells. Previous ultrastructural examinations have demonstrated that lung capillary endothelial cells and alveolar epithelial cells are highly resistant to hypoxia (26, 40). We believe, therefore, that the most likely interpretation of our AFC data is that hypoxic exposure in vivo impairs transalveolar fluid transport.
We found that the effect of hypoxia on AFC was dependent on exposure time as well as oxygen concentration. There was a significant decrease in AFC only in rats exposed to 10% oxygen for 48 and 72 h. No significant decrease in AFC was observed when rats were exposed to 15% oxygen. This seems to be somewhat different from the data obtained from in vitro experiments (20, 30). It has been reported that exposure to 3% oxygen caused significant decrease in ouabain-sensitive86Rb uptake, representing Na+-K+-ATPase pump activity, in A549 cells as early as 15 min into hypoxia, and further decrease is observed by exposing the cells to 1.5% oxygen (20). In isolated rat alveolar type II cells, 0% oxygen caused a time-dependent decrease in the pump activity over 18 h (30). However, these extremely low-oxygen or anoxia experiments cannot be applied to the in vivo studies. We found that no rats were able to survive for longer than 6 h in 8% oxygen.
It has been well known that AFC is stimulated by β-adrenergic agonists such as terbutaline (3, 9, 19, 34, 35). In this study, 1.4-fold increase in AFC was observed in the presence of 10−3 M terbutaline in normoxia experiment. However, we found that terbutaline does not stimulate AFC of the rats exposed to 10% oxygen for 48 h. This change in the ability of alveolar epithelium to respond to terbutaline may be in part related to the decrease in adenylate cyclase activity and/or the number of β-adrenergic receptors by hypoxia, as reported in other type of cells (21, 27). In fact, we found significantly lower alveolar fluid cAMP levels in the hypoxia experiment. We did not directly measured intracellular cAMP levels of the lung tissue. However, we believe that our measurement in alveolar fluid reflects the ability of cAMP production of alveolar epithelial cells. Instillate solution interacts predominantly with alveolar epithelial cells rather than with airway epithelial cells or other type of cells (38), and alveolar epithelial cells cover a dominated surface area of the alveoli (10).
Once it was clear that hypoxic exposure in vivo impaired transalveolar fluid transport, our next question was to examine whether this impairment was dependent on functional change of alveolar epithelial Na+-K+-ATPase. Because Na+ extrusion via Na+-K+-ATPase is an energy-dependent process (1 ATP for 3 Na+ extrusion), we first determined the effect of hypoxia on lung ATP content. It has been shown that hypoxic exposure (5% oxygen for 24 h) results in a 25% decrease of ATP content in alveolar type II cells (30). However, we were not able to show that hypoxic exposure in vivo (10% oxygen for 48 h) reduces lung cellular energy production. Conversely, there was a small but significant increase in lung ATP content in the hypoxia experiment. This may be due to the reoxygenation effect during surgical preparations performed outside the hypoxic chamber, even though this process lasted only 10 min. It has been shown that lung cells stimulate anaerobic glycolysis to maintain the optimal energy required for transalveolar fluid transport when oxidative phosphorylation is inhibited (38). Therefore, reoxygenation effect may stimulate energy production via oxidative phosphorylation and increase lung ATP content.
Because measurement of lung ATP content did not explain the decrease of AFC in the hypoxia experiment, we then decided to determine the activity of Na+-K+-ATPase in the lungs. We first determined Na+-K+-ATPase hydrolytic activity in the mixed plasma membrane obtained from whole lung homogenate. Our data indicate that hypoxia decreases lung Na+-K+-ATPase hydrolytic activity. However, the data do not imply the specific alteration of the alveolar epithelium. We then determined the Na+-K+-ATPase hydrolytic activity in crude homogenate of alveolar type II cells. Again, we were able to demonstrate a significant decrease in the Na+-K+-ATPase hydrolytic activity in the hypoxia experiment. Hypoxia did not influence the viability of isolated alveolar type II cells. Therefore, our data indicate that hypoxia impairs transalveolar fluid transport by decreasing alveolar epithelial Na+-K+-ATPase hydrolytic activity itself, but it does not change lung cellular energy production.
The exact cellular mechanism leading to the decrease in the alveolar epithelial Na+-K+-ATPase hydrolytic activity is still unclear. However, there might be some possible explanations. First, it is known that the Na+-K+-ATPase hydrolytic activity is inhibited by reactive oxygen species (16, 25). It has been reported previously (7) that hypoxic exposure increases plasma glutathione disulfide in rats, suggesting that hypoxia induces cellular oxidative stress in vivo. We have recently found the induction of oxidative stress in rat lungs as early as 24 h after hypoxic exposure (10% oxygen) in vivo (15). Second, hypoxia may also modulate the gene expression of ion transporters involved in transalveolar fluid transport. It has been shown in the isolated rat alveolar type II cells that hypoxia causes a significant decline of the Na+-K+-ATPase mRNA expression (30). This downregulation may be either a direct effect of hypoxia on Na+-K+-ATPase mRNA and/or a secondary effect of the impairment of Na+ channels. The pump activity of Na+-K+-ATPase is regulated in part by intracellular Na+ concentration (31). It has been shown that hypoxia causes a significant decrease in the expression of Na+ channel mRNAs and22Na+influx via Na+ channels of rat alveolar type II cells (20, 29). If hypoxia reduces Na+ entry via Na+ channels in vivo, this may decrease the Na+-K+-ATPase expression in alveolar epithelial cells. However, further studies are needed to clarify the impact of hypoxia on the function of the alveolar epithelial Na+ channels.
In summary, our data presented here demonstrate that hypoxic exposure in vivo impairs transalveolar fluid transport, and this impairment is related to the decrease in the alveolar epithelial Na+-K+-ATPase hydrolytic activity itself. The impairment of transalveolar fluid transport by alveolar hypoxia may influence the recovery process from pulmonary edema under hypoxic environment including high altitude.
Address for reprint requests: S. Suzuki, Dept. of Thoracic Surgery, Institute of Development, Aging, and Cancer, Tohoku Univ., 4–1 Seiryo-machi, Aoba-ku, Sendai, Japan 980-8575 (E-mail:).
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