Hypocapnic but Not Metabolic Alkalosis Impairs Alveolar Fluid Reabsorption

    Division of Pulmonary and Critical Care Medicine, Northwestern University
    Medical Service, Veterans Affairs, Chicago Health Care System
    Department of Mathematics, Northeastern Illinois University, Chicago, Illinois
    Intensive Care Unit, "KAT" General Hospital, Athens University, Athens, Greece
    Departamento de Fisiopatologia, Facultad de Medicina, Universidad de la Republica, Montevideo, Uruguay

    ABSTRACT

    Acid-base disturbances, such as metabolic or respiratory alkalosis, are relatively common in critically ill patients. We examined the effects of alkalosis (hypocapnic or metabolic alkalosis) on alveolar fluid reabsorption in the isolated and continuously perfused rat lung model. We found that alveolar fluid reabsorption after 1 hour was impaired by low levels of CO2 partial pressure (PCO2; 10 and 20 mm Hg) independent of pH levels (7.7 or 7.4). In addition, PCO2 higher than 30 mm Hg or metabolic alkalosis did not have an effect on this process. The hypocapnia-mediated decrease of alveolar fluid reabsorption was associated with decreased Na,K-ATPase activity and protein abundance at the basolateral membranes of distal airspaces. The effect of low PCO2 on alveolar fluid reabsorption was reversible because clearance normalized after correcting the PCO2 back to normal levels. These data suggest that hypocapnic but not metabolic alkalosis impairs alveolar fluid reabsorption. Conceivably, correction of hypocapnic alkalosis in critically ill patients may contribute to the normalization of lung ability to clear edema.

    Key Words: alveolar epithelial cells  hypocapnic alkalosis  ion transport  Na,K-ATPase  pulmonary edema

    The resolution of pulmonary edema is the result of active sodium transport across the alveolar epithelium (1eC5). Sodium enters the alveolar epithelial cells via apically located sodium channels, and it is extruded via basolaterally located sodium pumps (Na, K-ATPases), which causes water to be reabsorbed from the alveolar space (4, 6eC10). In experimental models, it has been reported that alveolar fluid reabsorption (AFR) is decreased during acute hyperoxia (11), hypoxia (12), ventilator-associated lung injury (13), increased left atrial pressures (14), infection (15), and scorpion envenomation (16). In some of these models of lung injury, the impairment in AFR has been associated with decreased Na,K-ATPase activity and protein abundance at the basolateral membrane of alveolar epithelial cells (13, 16eC18).

    Acid-base disturbances and particularly hypocapnia can be observed during hyperventilation because it may occur during air travel, hiking at high altitudes, early asthma attack, diabetic ketoacidosis, or systemic inflammatory response syndrome (19eC22). The effect of hypocapnic alkalosis on alveolar fluid reabsorption as an index of alveolar epithelial function has not been elucidated. However, it has been reported that when AFR is impaired in patients with lung dysfunction, it is associated with worst outcomes (23).

    The present study examined the effects of hypocapnic alkalosis by changing the levels of CO2 in the pulmonary circulation of the isolated, perfused, fluid-filled rat lung model. In parallel experiments, we examined the effects of CO2 partial pressure (PCO2) changes on Na,K-ATPase activity and protein abundance at the plasma membranes of distal airspaces.

    METHODS

    Pathogen-free male Sprague-Dawley rats weighing 320 to 350 g were purchased from Harlan Sprague Dawley (Indianapolis, IN). All animals were provided food and water ad libitum and were maintained on a 12- to 12-hour lighteCdark cycle. Animals were handled according to National Institutes of Health guidelines and Institutional Animal Care and Use CommitteeeCapproved experimental protocols. A total of 46 rat lungs were studied.

    Isolated Perfused Lung Model

    The isolated lung preparation was performed as previously described (1, 11, 13, 14). Briefly, rats were anesthetized with intraperitoneal pentobarbital (50 mg/kg body weight) after which the lungs and heart were removed en bloc. The pulmonary artery and left atrium were catheterized and continuously perfused with a solution of 3% bovine serum albumin in buffered physiologic salt solution. Trace amounts of fluorescein isothiocyanateeClabeled albumin were also added to the perfusate to check capillary barrier integrity along the experiment. Arterial and venous pressures were set at 12 and 0 cm H2O, respectively, to avoid high hydrostatic pressure through alveolareCcapillary barrier. Lungs were also immersed in a "pleural" bath filled with the same bovine serum albumin solution, and the entire system was maintained at 37°C. The lungs were then instilled via the tracheal cannula with a 5-ml volume of the bovine serum albumin solution containing 0.1 mg/ml Evans blue dyeeClabeled (Sigma, St. Louis, MO) albumin, 0.02 e藽i/ml of 22Na+ (DuPont NEN, Boston, MA) and 0.12 e藽i/ml of [3H]mannitol (DuPont NEN).

    The amount of instilled Evans blue dye albumin remains constant during the experimental protocol, so any change in its concentration at a given time will reflect the change in the airspace volume. Differences in concentration of Evans blue dye albumin among samples taken from the instillate at the beginning and after a determined time reflect the amount of fluid that has been reabsorbed. The fraction of fluorescein isothiocyanate albumin that appears in the alveolar space during the experimental protocol was used to calculate the albumin flux from the pulmonary circulation into the alveolar space.

    Perfusate acid-base status was controlled and perfusing solution was monitored in real time during experimental course with a pH electrode located inside the circuit (Semimicro pH electrode, 476346; Corning, Inc., Corning, NY). Additional samples were taken every 15 minutes to measure levels of pH and perfusate gases using a blood gas analyzer (pHOx Plus Analyzer, StatProfile; Nova Biomedical Corp., Waltham, MA). Samples were quickly processed to avoid accidental degassing during measurement maneuvers. Changes on prefixed parameters (pH and/or PCO2) were immediately corrected by bubbling more or less CO2 and/or adding NaOH or HCl in the pulmonary circulation according to each experimental condition.

    Additional details on the method for making these measurements are provided in the online supplement.

    Specific Protocols

    Group A.

    We conducted experiments to determine baseline AFR over 1 hour in a control group of rat lungs in which the pH was maintained at 7.40 and PCO2 = 40 mm Hg (n = 5).

    Group B.

    Group B had three components as follows:

    B1. We investigated the effects of respiratory alkalosis for 1 hour on AFR (first group: pH = 7.50, with  PCO2 30 mm Hg; second group: pH = 7.60,  PCO2 20 mm Hg; third group: pH = 7.70,  PCO2 10 mm Hg; n = 5 in each group).

    B2. We studied the effects of hypocapnia ( PCO2 = 10, 20, and 30 mm Hg) while maintaining the pH at 7.40 for 1 hour (n = 5 in each group). The pH was maintained at 7.40 by adding HCl (0.1 normality) to the pulmonary circulation perfusate.

    B3. We assessed the effects of metabolic alkalosis while maintaining PCO2 at 40 mm Hg and pH = 7.70 (n = 5). The desired PCO2 was achieved by bubbling additional or less 10%CO2eC21%O2 mixture in the perfusate as needed, and alkaline pH was achieved with titration of NaOH (0.1 normality) during the experiment.

    Group C.

    We investigated the reversibility of the respiratory alkalosis effect on AFR by conducting 3-hour experiments: the pH and PCO2 were maintained at normal values (pH = 7.40,  PCO2 40 mm Hg) during the first hour. Respiratory alkalosis was established (pH = 7.70,  PCO2 20 mm Hg) during the second hour, and then the PCO2 and pH were returned to normal values (7.40/40) during the third hour (n = 5).

    Basolateral Plasma Membrane Isolation and Western Blotting

    Basolateral plasma membrane proteins were obtained from tissue collected from the distal 2 to 3 mm of right rat lungs after serial bronchoalveolar lavage and perfusion of the pulmonary artery, as previously described (24). Protein fractions enriched from the basolateral plasma membrane domain were produced from distal lung tissue and separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, as previously described (18, 24, 25). Immunodetection was achieved using a monoclonal antirat 1 Na,K-ATPase (Upstate Biotech, Inc., Uppsala, NY) primary antibody and a chemiluminescent detection system (PerkinElmer Life Sciences, Boston, MA). The bands were quantified by densitometric scanning (ImageJ 1.29X; National Institutes of Health, Bethesda, MD).

    Determination of Na,K-ATPase Activity

    Twenty micrograms of basolateral membrane protein was resuspended in 100 e of a high (Na+)/low (K+) reaction buffer (in mmol/L: 50 Tris-HCl, pH 7.4; 50 NaCl; 5 KCl; 10 Mg2Cl; 1 mmol/L ethyleneglycol-bis-[-aminoethyl ether]-N,N'-tetraacetic acid, 10 mmol/L Na2ATP, with [-32P]-ATP [3.3 nCi/e]). Triplicate samples were placed at eC20°C for 15 minutes before incubation for 15 minutes at 37°C. The reaction was terminated by addition of 5% trichloroacetic acid/10% charcoal and cooling to 4°C. The charcoal phase containing unhydrolyzed nucleotide was separated by centrifugation (12,000 x g for 5 minutes) and the liberated 32P quantified. Na,K-ATPase activity was calculated as the difference between the test samples (total ATPase activity) and samples assayed in reaction buffer with 2.5 mmol/L ouabain but devoid of Na+ and K+ (nonspecific ATPase activity). Results are expressed as nmol of Pi/mg of protein/minute.

    Data Analysis

    Data are expressed as means ± SEM; n represents the number of animals in each group. Data were compared using analysis of variance adjusted for multiple comparisons by the Tukey test. A p value of 0.05 or less was considered significant.

    RESULTS

    AFR

    As shown in Figure 1A, AFR was impaired when PCO2 levels were decreased, with corresponding increase in pH. The PCO2 levels were 40.8 ± 1.9 mm Hg for a pH of 7.40, 32.1 ± 1.4 mm Hg for a pH of 7.50, 25.2 ± 0.5 mm Hg for a pH of 7.60, and 14.6 ± 0.7 mm Hg for a pH of 7.70. To determine whether hypocapnia and/or pH caused the observed AFR impairment, additional experiments were conducted. As showed in Figure 1B, PCO2 was decreased, whereas the pH was maintained at 7.40 (PCO2 = 29.6 ± 1.5, 19.8 ± 2.0, and 13.2 ± 0.7 mm Hg). The AFR was impaired only when PCO2 was decreased to 13.2 and 19.8 mm Hg (but not 29.6 mm Hg). In contrast, in Figure 1C, in another set of experiments, the PCO2 was maintained at normal range (38.7 ± 4.8 mm Hg), whereas the pH was increased to 7.70 and the AFR was not impaired. However, when the PCO2 was reduced (14.6 ± 0.7 mm Hg) and pH maintained at 7.70, the AFR was reduced by 50%. We found a linear correlation between decreasing PCO2 levels and decreased AFR: r2 = 0.929 (Figure 2). As depicted in Figure 3, the effect of hypocapnia on AFR was reversible and, when PCO2 and pH were restored to normal values on the third hour of the experimental protocol, the AFR reverted to normal values (first hour: pH = 7.40, PCO2 = 41.67 ± 1.03 mm Hg; second hour: pH = 7.70, PCO2 = 14.8 ± 0.44 mm Hg; and third hour: pH = 7.40, PCO2 = 39.47 ± 0.35 mm Hg).

    Lung Epithelial Fluxes of Solutes during Alkalosis

    As shown in Table 1, hypocapnic alkalosis had a tendency to increase (but not significantly) passive mannitol and sodium fluxes. Neither hypocapnic nor metabolic alkalosis increased albumin flux across the lung epithelium.

    Na,K-ATPase Activity and Protein Abundance at the Basolateral Membranes

    Na,K-ATPase activity in lungs with hypocapnic alkalosis (PCO2  14 mm Hg, pH = 7.70) decreased as compared with control values (PCO2  40 mm Hg and pH = 7.40; Figure 4A). The decreased Na,K-ATPase activity correlated with a decrease in the Na, K-ATPase 1-subunit protein abundance at the basolateral membrane of hypocapnic alkalosis peripheral lung tissue, without changes in the total amount of protein (Figures 4B and 4C).

    DISCUSSION

    The clearance of pulmonary edema occurs via active sodium transport across the alveolar epithelial cells via apically located sodium channels and basolateral Na,K-ATPases, with water following isoosmotically the Na+ gradients (1eC5). It has been reported that AFR is an important factor contributing to the outcome of patients with respiratory failure (23, 26). Respiratory and metabolic alkalosis occurs in critically ill patients with different disorders, such as dehydration and early asthma, and in patients with central nervous system disorders (20eC22).

    Hypocapnia has been associated with adverse outcomes in mechanically ventilated patients with acute lung injury (20, 22). The injurious effects of hypocapnic alkalosis include bronchospasm, increased airway permeability, dysfunctional surfactant, and worsening intrapulmonary shunt (21, 27, 28). In the isolated lung model, hypocapnia caused lung injury during mechanical ventilation and after ischemia-reperfusion (28). Furthermore, in that study, hypocapnia resulted in increased capillary permeability (Kf,c) and lung weight because of increased fluid accumulation during hypocapnia (28). Our data provide further mechanistic insights on the effects of hypocapnia on alveolar epithelial function. We propose that persistence of the pulmonary edema could also be caused by hypocapnia (and not metabolic alkalosiseC)-mediated impairment in the lung ability to clear edema. We provide evidence that PCO2-mediated alkalosis impaired the lung ability to clear alveolar epithelial fluid, probably by inhibiting the Na,K-ATPase, and that these effects were reversible on normalization of the PCO2 (see Figures 1 and 3). We found that mild decreases in PCO2 to 30 mm Hg had no negative effects on AFR, whereas more significant hypocapnia (20 and 10 mm Hg) was deleterious to the alveolar epithelial function. Furthermore, alveolar epithelial function (assessed as fluid clearance from the lungs) was not altered by metabolic alkalosis (see Figure 1).

    It is yet unclear what mechanisms regulate how hypocapnia inhibits the Na+ pump and alveolar fluid clearance. There are several intracellular pathways that can be linked with CO2 metabolism that could have a role. One possible explanation could be from changes in intracellular pH, as has been previously suggested (29). For example, hypocarbic alkalinization in neutrophils requires HCO3eC influx as well as proton efflux through ZnCl2eCeCsensitive proton channels. In alveolar epithelial cells, net sodium movement from the apical epithelium to the interstitium and vascular space requires Na+-H+ exchange in the apical surface, and this step could be impaired if intracellular pH is alkaline (30). In the same context, CleC/HCO3eC exchange is activated during recovery from alkaline loads and may help maintain intracellular pH under normal conditions (31). Bicarbonate, also, has been involved as a modulator of soluble adenylyl cyclases, acting as an intracellular signaling molecule. This modulation has been described in nuclear processes, but the same rationale could be applied to other cAMP-mediated process (32) and HCO3eC/CO2 is a well recognized physiologic buffer system. Also, carbonic anhydrase, which is expressed in alveolar epithelial cells, could participate because of its action in CO2 hydration and alveolar elimination (33, 34).

    Hypocapnia can modify vascular reactivity (35), but it is not clear whether these effects may modulate AFR. For example, endothelin 1 (which can modify vascular reactivity) increases pulmonary edema formation and was associated with an impairment in alveolar fluid clearance and Na,K-ATPase activity (36). Moreover, ET-B receptoreCdeficient rats develop lung edema (37).

    We did not find changes in small or large solutes fluxes during hypocapnia or metabolic alkalosis as compared with control animals, suggesting that there were no large changes in alveolareCcapillary barrier during the relatively short term (60 minutes) of the experimental protocol. These results contrast to the report by Laffey and colleagues (28), who found an increase in microvascular permeability. However, these changes in permeability were found only after 3 hours of hypocapnic alkalosis.

    Hypocapnia is relatively common during hyperventilation, diabetic acidosis (38), or central nervous system disorders (39), and we believe that it is of relevance to understand that respiratory alkalosis can be harmful to the alveolar epithelium. Na,K-ATPase activity inhibition by G-protein coupled receptors, hypoxia, and models of lung injury has been reported to be because of its endocytosis from the plasma membrane to intracellular compartments (17, 18, 40). The present report provides evidence that hypocapnic alkalosis impairs AFR and inhibits Na,K-ATPase activity by promoting its endocytosis from the basolateral membrane into intracellular compartments.

    We provide evidence that severe hypocapnia of up to 20 mm Hg, but not metabolic alkalosis, impairs the function of the alveolar epithelial cell Na,K-ATPase. We reason that hyperventilation-hypocapnia may impair lung edema clearance and have deleterious effects on critically ill patients. Conceivably, correction of pH by normalizing CO2 might be a simple but significant supportive measure for patients with hypocapnia.

    This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

     These investigators contributed equally to this article.

    REFERENCES

    Rutschman DH, Olivera W, Sznajder JI. Active transport and passive liquid movement in isolated perfused rat lungs. J Appl Physiol 1993;75:1574eC1580.

    Saumon G, Basset G. Electrolyte and fluid transport across the mature alveolar epithelium. J Appl Physiol 1993;74:1eC15.

    Sznajder JI, Olivera WG, Ridge KM, Rutschman DH. Mechanisms of lung liquid clearance during hyperoxia in isolated rat lungs. Am J Respir Crit Care Med 1995;151:1519eC1525.

    Sznajder JI, Factor P, Ingbar DH. Lung edema clearance: role of Na+-K+-ATPase. J Appl Physiol 2002;93:1860eC1866.

    Dada LA, Sznajder JI. Mechanisms of pulmonary edema clearance during acute hypoxemic respiratory failure: Role of the Na,K-ATPase. Crit Care Med 2003;31:248eC252.

    Matalon S, O'Brodovich H. Sodium channels in alveolar epithelial cells: molecular characterization, biophysical properties, and physiological significance. Annu Rev Physiol 1999;61:627eC661.

    Crandall ED, Matthay MA. Alveolar epithelial transport: basic science to clinical medicine. Am J Respir Crit Care Med 2001;163:1021eC1029.

    Johnson MD, Widdicombe JH, Allen L, Barbry P, Dobbs LG. Alveolar epithelial type I cells contain transport proteins and transport sodium, supporting an active role for type I cells in regulation of lung liquid homeostasis. Proc Natl Acad Sci USA 2002;99:1966eC1971.

    Borok Z, Liebler JM, Lubman RL, Foster MJ, Zhou B, Li X, Zabski SM, Kim KJ, Crandall ED. Na transport proteins are expressed by rat alveolar epithelial type I cells. Am J Physiol Lung Cell Mol Physiol 2002;282:L599eCL608.

    Ridge KM, Olivera WG, Saldias F, Azzam Z, Horowitz S, Rutschman DH, Dumasius V, Factor P, Sznajder JI. Alveolar type 1 cells express the alpha2 Na,K-ATPase, which contributes to lung liquid clearance. Circ Res 2003;92:453eC460.

    Olivera WG, Ridge KM, Sznajder JI. Lung liquid clearance and Na, K-ATPase during acute hyperoxia and recovery in rats. Am J Respir Crit Care Med 1995;152:1229eC1234.

    Vivona MLM, Chabaud M, Friedlander MBG, Clerici C. Hypoxia reduces alveolar epithelial sodium and fluid transport in rats: reversal by beta-adrenergic agonist treatment. Am J Respir Cell Mol Biol 2001;25:554eC561.

    Lecuona E, Saldias F, Comellas A, Ridge K, Guerrero C, Sznajder JI. Ventilator-associated lung injury decreases lung ability to clear edema in rats. Am J Respir Crit Care Med 1999;159:603eC609.

    Saldias FJ, Azzam ZS, Ridge KM, Yeldandi A, Rutschman DH, Schraufnagel D, Sznajder JI. Alveolar fluid reabsorption is impaired by increased left atrial pressures in rats. Am J Physiol Lung Cell Mol Physiol 2001;281:L591eCL597.

    Davis IC, Sullender WM, Hickman-Davis JM, Lindsey JR, Matalon S. Nucleotide-mediated inhibition of alveolar fluid clearance in BALB/c mice after respiratory syncytial virus infection. Am J Physiol Lung Cell Mol Physiol 2004;286:L112eCL120.

    Comellas A, Pesce PLM, Azzam Z, Saldias FJ, Sznajder JI. Scorpion venom decreases lung liquid clearance in rats. Am J Respir Crit Care Med 2003;167:1064eC1067.

    Dada LA, Chandel NS, Ridge KM, Pedemonte C, Bertorello AM, Sznajder JI. Hypoxia-induced endocytosis of Na,K-ATPase in alveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-zeta. J Clin Invest 2003;111:1057eC1064.

    Azzam ZS, Dumasius V, Saldias FJ, Adir Y, Sznajder JI, Factor P. Na,K-ATPase overexpression improves alveolar fluid clearance in a rat model of elevated left atrial pressure. Circulation 2002;105:497eC501.

    Gardner WN. Hyperventilation. Am J Respir Crit Care Med 2004;170:105eC106.

    Garland JS, Buck RK, Allred EN, Leviton A. Hypocarbia before surfactant therapy appears to increase bronchopulmonary dysplasia risk in infants with respiratory distress syndrome. Arch Pediatr Adolesc Med 1995;149:617eC622.

    Laffey JG, Kavanagh BP. Hypocapnia. N Engl J Med 2002;347:43eC53.

    Amato MB, Barbas CS, Medeiros DM, Magaldi RB, Schettino GP, Lorenzi-Filho G, Kairalla RA, Deheinzelin D, Munoz C, Oliveira R, et al. Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome. N Engl J Med 1998;338:347eC354.

    Ware LB, Matthay MA. Alveolar fluid clearance is impaired in the majority of patients with acute lung injury and the acute respiratory distress syndrome. Am J Respir Crit Care Med 2001;163:1376eC1383.

    Dumasius V, Sznajder JI, Azzam ZS, Boja J, Mutlu GM, Maron MB, Factor P. Beta(2)-adrenergic receptor overexpression increases alveolar fluid clearance and responsiveness to endogenous catecholamines in rats. Circ Res 2001;89:907eC914.

    Hammond TG, Verroust PJ, Majewski RR, Muse KE, Oberley TD. Heavy endosomes isolated from the rat renal cortex show attributes of intermicrovillar clefts. Am J Physiol 1994;267:F516eCF527.

    Sznajder JI. Alveolar edema must be cleared for the acute respiratory distress syndrome patient to survive. Am J Respir Crit Care Med 2001;163:1293eC1294.

    Laffey JG, Kavanagh BP. Carbon dioxide and the critically ill—too little of a good thing Lancet 1999;354:1283eC1286.

    Laffey JG, Engelberts D, Kavanagh BP. Injurious effects of hypocapnic alkalosis in the isolated lung. Am J Respir Crit Care Med 2000;162:399eC405.

    Coakley RJ, Taggart C, Greene C, McElvaney NG, O'Neill SJ. Ambient pCO2 modulates intracellular pH, intracellular oxidant generation, and interleukin-8 secretion in human neutrophils. J Leukoc Biol 2002;71:603eC610.

    Nord EP, Brown SE, Crandall ED. Characterization of Na+-H+ antiport in type II alveolar epithelial cells. Am J Physiol 1987;252:C490eCC498.

    Nord EP, Brown SE, Crandall ED. Cl-/HCO3- exchange modulates intracellular pH in rat type II alveolar epithelial cells. J Biol Chem 1988;263:5599eC5606.

    Zippin JH, Farrell J, Huron D, Kamenetsky M, Hess KC, Fischman DA, Levin LR, Buck J. Bicarbonate-responsive "soluble" adenylyl cyclase defines a nuclear cAMP microdomain. J Cell Biol 2004;164:527eC534.

    Geers C, Gros G. Carbon dioxide transport and carbonic anhydrase in blood and muscle. Physiol Rev 2000;80:681eC715.

    Lien YH, Lai LW. Respiratory acidosis in carbonic anhydrase II-deficient mice. Am J Physiol 1998;274:L301eCL304.

    Gordon JB, VanderHeyden MA, Halla TR, Cortez EP, Hernandez G, Haworth ST, Dawson CA, Madden JA. What leads to different mediators of alkalosis-induced vasodilation in isolated and in situ pulmonary vessels Am J Physiol Lung Cell Mol Physiol 2003;284:L799eCL807.

    Comellas A, Azzam Z, Lecuona E, Briva A, Butti M, Pesce L, Sznajder JI. Endothelin-1 decreases Na,K-ATPase activity in alveolar epithelial cells and lung liquid clearance in rats. . Am J Resp Crit Care Med 2004;169(7):A707.

    Carpenter TS, Steudel S, Ozimek W, Colvin J, Stenmark KK, Ivy DD. Endothelin B receptor deficiency predisposes to pulmonary edema formation via increased lung vascular endothelial cell growth factor expression. Circ Res 2003;93:456eC463.

    Flanagan JF, Garrett JS, McDuffee A, Tobias JD. Noninvasive monitoring of end-tidal carbon dioxide tension via nasal cannulas in spontaneously breathing children with profound hypocarbia. Crit Care Med 1995;23:1140eC1142.

    Pfeffer JM. The aetiology of the hyperventilation syndrome: a review of the literature. Psychother Psychosom 1978;30:47eC55.

    Chibalin AV, Katz AI, Berggren PO, Bertorello AM. Receptor-mediated inhibition of renal Na(+)-K(+)-ATPase is associated with endocytosis of its alpha- and beta-subunits. Am J Physiol 1997;273:C1458eCC1465.