Water permeability measured between the airspace and vasculature in intact sheep and mouse lungs is high. More than 95% of the internal surface area of the lung is lined by alveolar epithelial type I cells. The purpose of this study was to test whether osmotic water permeability (P f ) in type I alveolar epithelial cells is high enough to account for the high P f of the intact lung. P f measured between the airspace and vasculature in the perfused f luid-filled rat lung by the pleural surface f luorescence method was high (0.019 ؎ 0.004 cm͞s at 12°C) and weakly temperature-dependent (activation energy 3.7 kcal͞mol). To resolve the contributions of type I and type II alveolar epithelial cells to lung water permeability, P f was measured by stopped-f low light scattering in suspensions of purified type I or type II cells obtained by immunoaffinity procedures. In response to a sudden change in external solution osmolality from 300 to 600 mOsm, the volume of type I cells decreased rapidly with a half-time (t 1/2 ) of 60-80 ms at 10°C, giving a plasma membrane P f of 0.06-0.08 cm͞s. P f in type I cells was independent of osmotic gradient size and was weakly temperature-dependent (activation energy 3.4 kcal͞mol). In contrast, t 1/2 for type II cells in suspension was much slower, Ϸ1 s; P f for type II cells was 0.013 cm͞s. Vesicles derived from type I cells also had a very high P f of 0.06-0.08 cm͞s at 10°C that was inhibited 95% by HgCl 2 . The P f in type I cells is the highest measured for any mammalian cell membrane and would account for the high water permeability of the lung.Rapid water movement between the airspace and blood compartments of the lung is important in normal physiological processes such as the transition from an intrauterine to air environment and in pathological processes such as the formation and resolution of pulmonary edema. Water movement also may be important in maintaining lung water homeostasis. Between the air and the vasculature there are epithelial, interstitial, and endothelial compartments. The alveolar epithelium, which covers more than 99% of the internal surface area of the lungs (1), is comprised of a monolayer of two morphologically distinct types of cells, type I cells and type II cells. The very thin cytoplasmic extensions of type I cells cover 95-98% of the surface area of the lung (2). Type II cells, which cover the remaining 2-5% of the alveolar surface, are cuboidal cells best known for their ability to synthesize, secrete, and recycle components of pulmonary surfactant. The interstitial compartment varies considerably in thickness; at its thinnest, the alveolar epithelium is separated from capillary endothelium only by a fused basement membrane (3). Intercellular tight junctions between alveolar epithelial cells are thought to provide a tight barrier between the air and blood compartments of the lung (4). Based on these anatomic considerations, we postulated that alveolar epithelial type I cells might play an important role in water transport.Recent data indicate t...

A B STRA CT A surface fluorescence method was developed to measure transalveolar transport of water, protons, and solutes in intact perfused lungs. Lungs from c57 mice were removed and perfused via the pulmonary artery (~2 mi/min). The airspace was filled via the trachea with physiological saline containing a membrane-impermeant fluorescent indicator (FITC-dextran or aminonapthalene trisulfonic acid, ANTS). Because fluorescence is detected only near the lung surface due to light absorption by lung tissue, the surface fluorescence signal is directly proportional to indicator concentration. Confocal microscopy confirmed that the fluorescence signal arises from fluorophores in alveolijust beneath the pleural surface. Osmotic water permeability (t f) was measured from the time course of intraalveolar FITC-dextran fluorescence in response to changes in perfusate osmolality. Transalveolar Pf was 0.017 _+ 0.001 cm/s at 23~ independent of the solute used to induce osmosis (sucrose, NaC1, urea), independent of osmotic gradient size and direction, weakly temperature dependent (Arrhenius activation energy 5.3 kcal/mol) and inhibited by HgC12. Pf was not affected by cAMP activation but was decreased by 43% in lung exposed to hyperoxia for 5 d. Diffusional water permeability (Pd) and Pf were measured in the same lung from intraalveolar ANTS fluorescence, which increased by 1.8-fold upon addition of 50% I)20 tO the perfusate. Pd was 1.3 • 10 -~' cm/s at 23~ Transalveolar proton transport was measured from FITC-dextran fluorescence upon switching perfusate pH between 7.4 and 5.6; alveolar pH half-equilibrated in 1.9 and 1.0 rain without and with HCO3-, respectively. These results indicate high transalveolar water permeability in mouse lung, implicating the involvement of molecular water channels, and establish a quantitative surface fluorescence method to measure water and solute permeabilities in intact lung.

. Regulation of heme oxygenase-1 by nitric oxide during hepatopulmonary syndrome. Am J Physiol Lung Cell Mol Physiol 283: L346-L353, 2002. First published March 29, 2002 10.1152/ajplung.00385.2001.-During hepatopulmonary syndrome caused by liver cirrhosis, pulmonary endothelial nitric oxide (NO) synthase (NOS) expression and NO production are increased. Increased NO contributes to the blunted hypoxic pressor response (HPR) during cirrhosis and may induce heme oxygenase-1 (HO-1) expression and carbon monoxide (CO) production, exacerbating the blunted HPR. We hypothesized that NO regulates the expression of HO-1 during cirrhosis, contributing to hepatopulmonary syndrome. Cirrhosis was induced in rats by common bile duct ligation (CBDL). Rats were studied 2 and 5 wk after CBDL or sham surgery. Lung HO-1 expression was elevated 5 wk after CBDL. Liver HO-1 was increased at 2 wk and remained elevated at 5 wk. In catheterized rats, the blunted HPR was partially restored by HO inhibition. Rats treated with the NOS inhibitor N G -nitro-L-arginine methyl ester for the entire 2-or 5-wk duration had normalized HO-1 expression and HPR. These data provide in vivo evidence for the NO-mediated upregulation of HO-1 expression and support the concept that hepatopulmonary syndrome is multifactorial, involving not only NO, but also HO-1 and CO. carbon monoxide; cirrhosis; endothelial nitric oxide synthase; pulmonary vasoreactivity; calcium-activated potassium channels ENDOTHELIUM-DERIVED NITRIC OXIDE (NO) produced by the enzymatic activity of endothelial nitric oxide synthase (eNOS) has well-characterized actions as a vasodilator (33). Once produced, NO is freely diffusible and enters vascular smooth muscle cells (VSMCs) to activate soluble guanylate cyclase and produce guanosine 3Ј,5Ј-cyclic monophosphate (cGMP) (26,33). In pulmonary artery (PA) VSMCs, increased cGMP activates a cGMPsensitive kinase, which phosphorylates a calciumdependent potassium (K Ca ) channel leading to hyperpolarization and vasodilation (7,26,33). Although most of the NO production in the vascular endothelium is due to eNOS, some studies suggest that the two other isoforms of NOS, inducible NOS and neuronal NOS, may also be present in the vasculature and contribute to NO production (20,29,30).Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in the oxidative degradation of heme to biliverdin, releasing equimolar amounts of carbon monoxide (CO) and iron (5). Biliverdin is subsequently reduced to bilirubin by biliverdin reductase (5). CO, a gaseous messenger similar to NO, shares many properties with NO, including activation of guanylate cyclase, signal transduction, and gene regulation and may mediate important cellular functions (34).In addition to its action as a vasodilator, NO can regulate the expression of a variety of genes. In particular, there is solid evidence that NO regulates the expression of HO-1 (1, 4, 15). For example, treating aortic smooth muscle cells with the NO donor spermine NONOate (SNN) increases HO-1 gene transcription, resul...

Three members of the water channel (aquaporin) family are expressed in adult rat lung: CHIP28 (AQP-1), MIWC (AQP-4), and AQP-5. Because water channels may be important in the clearance of fluid from the newborn lung, the expression of water channels just before and after birth was investigated using the ribonuclease (RNAse) protection assay. RNA was isolated from lungs, brain, and heart of prenatal rats (fetal days F19, F20, and F21) and postnatal rats (days +1, +2, +5, +7, +21, and adult). Transcript expression was measured relative to a beta-actin control by quantitative densitometry. Whereas beta-actin mRNA expression was nearly constant over time, distinct expression patterns were observed for the three water channels. CHIP28 mRNA expression rose slowly from days F19 to +1, then strongly at day +2, and remained elevated over the first week. MIWC mRNA was weakly expressed prenatally, but strongly increased just after birth. AQP-5 mRNA increased slowly and monotonically between days F20 and +7. These patterns contrasted sharply with the developmental expression of CHIP28 in heart, which decreased over time, and MIWC in brain. Immunocytochemistry showed CHIP28 protein expression in capillary endothelia and MIWC in airway epithelia by day +1; quantitative immunoblot analysis showed increased CHIP28 protein expression over time. These findings are consistent with a role of lung water channels in perinatal fluid clearance; however, proof of physiologic significance will require functional measurements of air space-capillary water permeability.

The chronic role of nitric oxide (NO), independent of prostaglandin synthesis, in the primary peripheral vasodilation, increased glomerular filtration rate (GFR), and renal plasma flow (RPF) in normal pregnancy remains to be defined. The purpose of the present study was to chronically inhibit NOS to return systemic vascular resistance (SVR), cardiac output (CO), GFR, and RPF to nonpregnant values. Pregnant rats received the nitric oxide synthase (NOS) inhibitor, nitro-L-arginine methyl ester (L-NAME), orally from gestational days 7 through 14. Results were compared with nonpregnant and untreated pregnant rats. At 14 days gestation, CO significantly increased in pregnant vs. nonpregnant rats (187 +/- 17 vs. 125 +/- 10 ml/min, P < 0.05) as SVR decreased (0.64 +/- 0.08 vs. 1.08 +/- 0.08 mmHg. ml(-1). min, P < 0.05) and mean arterial pressure was unchanged (117 +/- 5 vs. 125 +/- 2 mmHg, not significant). Pregnant rats also demonstrated increased GFR (3,015 +/- 33 vs. 2,165 +/- 136 microl/min, P < 0.01) and RPF (7,869 +/- 967 vs. 5,507 +/- 290 microl/min, P < 0.05) vs. nonpregnant rats. L-NAME-treated pregnant rats had values for CO (118 +/- 7 ml/min), SVR (1.09 +/- 0.07 mmHg. ml(-1). min), GFR (2,264 +/- 150 microl/min), and RPF (5,777 +/- 498 microl/min), which were no different than nonpregnant animals. In summary, similar to human pregnancy, primary peripheral vasodilation occurs early in rat pregnancy. Furthermore, the hyperdynamic circulation and glomerular hyperfiltration of normal rat midterm pregnancy can be chronically reversed by NOS inhibition. These findings suggest a role for endothelial damage and decreased NO in the pathogenesis of preeclampsia.

Cirrhosis is typically associated with a hyperdynamic circulation consisting of low blood pressure, low systemic vascular resistance (SVR), and high cardiac output. We have recently reported that nonspecific inhibition of nitric oxide synthase (NOS) with nitro-L-arginine methyl ester reverses the hyperdynamic circulation in rats with advanced liver cirrhosis induced by carbon tetrachloride (CCl(4)). Although an important role for endothelial NOS (eNOS) is documented in cirrhosis, the role of neuronal NOS (nNOS) has not been investigated. The present study was carried out to specifically investigate the role of nNOS during liver cirrhosis. Specifically, physiological, biochemical, and molecular approaches were employed to evaluate the contribution of nNOS to the cirrhosis-related hyperdynamic circulation in CCl(4)-induced cirrhotic rats with ascites. Cirrhotic animals had a significant increase in water and sodium retention. In the aorta from cirrhotic animals, both nNOS protein expression and cGMP concentration were significantly elevated compared with control. Treatment of cirrhotic rats for 7 days with the specific nNOS inhibitor 7-nitroindazole (7-NI) normalized the low SVR and mean arterial pressure, elevated cardiac index, and reversed the positive sodium balance. Increased plasma arginine vasopressin concentrations in the cirrhotic animals were also repressed with 7-NI in association with diminished water retention. The circulatory changes were associated with a reduction in aortic nNOS expression and cGMP. However, 7-NI treatment did not restore renal function in cirrhotic rats (creatinine clearance: 0.76 +/- 0.03 ml. min(-1). 100 g body wt(-1) in cirrhotic rats vs. 0.79 +/- 0.05 ml. min(-1). 100 g body wt(-1) in cirrhotic rats+7-NI; P NS. ). Taken together, these results indicate that nNOS-derived NO contributes to the development of the hyperdynamic circulation and fluid retention in cirrhosis.

Rats with liver cirrhosis exhibit the hepatopulmonary syndrome composed of blunted hypoxic pulmonary vasoconstriction and arterial hypoxemia. The purpose of this study was to investigate the roles of nitric oxide (NO) and endothelin-1 (ET-1) in the blunted hypoxic pressor response (HPR) in rats with common bile duct ligation (CBDL). Lungs from CBDL rats exhibited markedly blunted HPR, increased endothelial NO synthase (NOS) protein expression, and decreased ET-1 mRNA and peptide expression. The blunted HPR was not reversed by sequential NOS and soluble guanylyl cyclase inhibition by nitro-L-arginine and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), respectively, or by NOS inhibition combined with ET-1 addition. The blunted HPR was not due to a generalized inability to vasoconstrict because perfusion pressure was equally elevated by increased perfusate KCl in CBDL and sham lungs. After KCl vasoconstriction, HPR was potentiated and did not differ between CBDL and sham lungs. Blunted HPR was also completely restored in CBDL lungs treated with nitro-L-arginine, ODQ, and the Ca(2+)-activated K(+) channel blockers apamin and charybdotoxin. These results indicate that although CBDL-induced liver cirrhosis is associated with increased NO and decreased ET-1 in the lung, the blunted HPR is a result of additional factors and appears to involve Ca(2+)-activated K(+) channel activation.

Transport of water between the capillary and airspace compartments in lung encounters serial barriers: the alveolar epithelium, interstitium, and capillary endothelium. We previously reported a pleural surface fluorescence method to measure net capillary-to-airspace water transport. To measure the osmotic water permeability across the microvascular endothelial barrier in intact lung, the airspace was filled with a water-immiscible fluorocarbon. The capillaries were perfused via the pulmonary artery with solutions of specified osmolalites containing a high-molecular-weight fluorescent dextran. An increase in perfusate osmolality produced a prompt decrease in surface fluorescence due to dye dilution in the capillaries, followed by a slower return to initial fluorescence as capillary and lung interstitial osmolality equilibrate. A mathematical model was developed to determine the osmotic water permeability coefficient (Pf) of lung microvessels from the time course of pleural surface fluorescence. As predicted, the magnitude of the prompt change in surface fluorescence increased with decreased pulmonary artery perfusion rate and increased osmotic gradient size. With raffinose used to induce the osmotic gradient, Pf was 0.03 cm/s at 23 degrees C and was reduced 54% by 0.5 mM HgCl2. Temperature dependence measurements gave an Arrhenius activation energy (Ea) of 5.4 kcal/mol (12-37 degrees C). The apparent Pf induced by the smaller osmolytes mannitol and glycine was 0.021 and 0.011 cm/s (23 degrees C). Immunoblot analysis showed approximately 1.4 x 10(12) aquaporin-1 water channels/cm2 of capillary surface, which accounted quantitatively for the high Pf. These results establish a novel method for measuring osmotically driven water permeability across microvessels in intact lung. The high Pf, low Ea, and mercurial inhibition indicate the involvement of molecular water channels in water transport across the lung endothelium.