Cyclic adenosine monophosphate (AMP) has numerous important effects on cell structure and function, but its role in endothelial cells is unclear. Since cyclic AMP has been shown to affect transmembrane transport, cell growth and morphology, cellular adhesion, and cytoskeletal organization, it may be an important determinant of endothelial barrier properties. To test this we exposed bovine pulmonary artery endothelial cell monolayers to substances known to increase cyclic AMP and measured their effect on endothelial permeability to albumin and endothelial cell cyclic AMP concentrations. Cholera toxin (CT), a stimulant of the guanine nucleotide binding subunit of adenylate cyclase, led to a concentration-dependent 2-6-fold increase in cyclic AMP which was associated with a 3-10-fold reduction in albumin transfer across endothelial monolayers. The effect was not specific to albumin as similar barrier-enhancing effects were also noted with an unrelated macromolecule, fluorescein isothiocyanate (FITC)-dextran (MW 70,000). Barrier enhancement with cyclic AMP elevation was also observed with forskolin, a stimulant of the catalytic subunit of adenylate cyclase. The temporal pattern of barrier enhancement seen with these agents paralleled their effects on increasing cyclic AMP, and the barrier enhancement could be reproduced by incubation with either dibutyryl cyclic AMP or Sp-cAMPS, cyclic AMP-dependent protein kinase agonists. Furthermore, the forskolin effect on barrier enhancement was partially reversed with Rp-cAMPS, an antagonist of cyclic AMP-dependent protein kinase. Since endothelial actin polymerization may be an important determinant of endothelial barrier function, we sought to determine whether the cyclic AMP-induced effects were associated with increases in the polymerized actin pool (F-actin). Both cholera toxin and forskolin led to apparent endothelial cell spreading and quantitative increases in endothelial cell F-actin fluorescence. In conclusion, increased endothelial cell cyclic adenine nucleotide activity was an important determinant of endothelial barrier function in vitro. The barrier enhancement was associated with increased endothelial apposition and increases in F-actin, suggesting that influences on cytoskeletal assembly may be involved in this process.

Fawn hooded rats (FHR), a strain of rat with a hereditary bleeding tendency due to a genetic defect in platelet aggregation, have recently been found to develop pulmonary hypertension. However, whether the pulmonary hypertension in FHR has a genetic basis or simply reflects the influence of extrinsic factors known to increase pulmonary artery pressure in other rat strains has not been fully evaluated. To further examine the structural and hemodynamic changes of pulmonary hypertension in FHR, and to investigate the extent to which alveolar hypoxia may have promoted these abnormalities, hemodynamic and morphometric measurements were made in FHR (4 to 24 wk) and compared with age-matched Sprague-Dawley (SDR) control rats. Increases in mean pulmonary artery pressure, total pulmonary resistance, and right ventricular enlargement were present in both male and female FHR and were evident at an early age (4 wk). Morphometric analysis of barium gelatin-infused lungs revealed marked pulmonary vascular remodelling in FHR characterized as extension of muscle into more peripheral pulmonary vessels, medial hypertrophy of proximal vessels, and reduced number of barium-filled arteries. The increases in pulmonary artery pressure in FHR were not due to the influence of more severe hypoxia, hypoventilation, or polycythemia, as blood gas tension and hematocrit were similar in FHR and SDR. Moreover, we found that pulmonary hypertension could be transmitted to backcross and second filial generation offspring arising from selective matings between FHR and control Wistar Kyoto rats, confirming the heritable basis for pulmonary hypertension in the FHR.

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Pulmonary edema after ascent to altitude is well recognized but its pathogenesis is poorly understood. To determine whether altitude exposure increases lung vascular permeability, we exposed rats to a simulated altitude of -14,500 feet (barometric pressure [Pbi 450 Torr) and measured the pulmonary transvascular escape of radiolabeled '25l-albumin corrected for lung blood content with 51Cr-tagged red blood cells (protein leak index = PLI). Exposures of 24 and 48 h caused significant increases in PLI (2.30±0.08 and 2.40±0.06) compared with normoxic controls (1.76±0.06), but brief hypoxic exposures of 1-13 h produced no increase in PLI, despite comparable increases in pulmonary artery pressure. There were associated increases in gravimetric estimates of lung water in the altitude-exposed groups and perivascular edema cuffs on histologic examination. Normobaric hypoxia (48 h; fractional inspired oxygen concentration [FI021 = 15%) also increased lung transvascular protein escape and lung water.Dexamethasone has been used to prevent and treat altitude-induced illnesses, but its mechanism of action is unclear. Dexamethasone (0.5 or 0.05 mg/kg per 12 h) started 12 h before and continued during 48 h of altitude exposure prevented the hypoxia-induced increases in transvascular protein escape and lung water. Hemodynamic measurements (mean pulmonary artery pressure and cardiac output) were unaffected by dexamethasone, suggesting that its effect was not due to a reduction in pulmonary artery pressure or flow. The role of endogenous glucocorticoid activity was assessed in adrenalectomized rats that showed augmented hypoxia-induced increases in transvascular protein escape, which were prevented by exogenous glucocorticoid replacement. In summary, subacute hypoxic exposures increased pulmonary transvascular protein escape and lung water in rats. Dexamethasone prevented these changes independent of reductions of mean pulmonary artery pressure or flow, whereas adrenalectomy increased pulmonary vascular permeability and edema at altitude. Increases in vascular permeability in hypoxia could contribute to the development of high-altitude pulmonary edema and endogenous glucocorticoids may have an important influence on pulmonary vascular permeability in hypoxia. Introduction Rapid ascent to high altitude may be associated with the development of pulmonary edema (HAPE)' (1,2 Despite this, attempts to demonstrate hypoxia-induced changes in permeability in the laboratory have produced conflicting results. Bland et al. (5) and Landolt et al. (6) failed to detect increases in lung lymph flow in adult sheep during hypoxia, even with the added stress of increased blood flow. In contrast, other studies have demonstrated increases in lymph flow in newborn lambs (7) and dogs (8-10) after hypoxia. Furthermore, studies in isolated perfused dog lungs have revealed increased filtration and reflection coefficients consistent with increased vascular permeability after brief exposures to anoxic gas (1 1, 12). Differences in both species and ...

Endothelin 1 (ET-1), a peptide produced by endothelial cells, causes transient dilation of some systemic vascular beds. To test whether low concentrations of ET-1 could also dilate the pulmonary vascular bed, we examined its effects in isolated blood- and salt solution-perfused rat lungs and in conscious catheterized rats. In blood-perfused lungs undergoing hypoxic (3% O2) vasoconstriction, repeated additions of 0.5 nM ET-1 to the perfusate elicited transient partial vasodilations. The higher concentration of 5 nM caused a larger transient vasodilation followed by vasoconstriction. In nine conscious rats exposed to 8% O2, intravenous ET-1 (0.2 nmol/kg) reversed the hypoxic pressor response by 63 +/- 8% without affecting cardiac output. In eight salt solution-perfused lungs vasoconstricted with 25 mM KCl, 0.5 nM ET-1 caused a maximum vasodilation of 35 +/- 3% with a half-life of 10.7 +/- 1.1 min. The vasodilation was not inhibited by blockers of cyclooxygenase (3.1 microM meclofenamate), platelet-activating factor receptors (10 microM Web 2086), histamine H1 receptors (50 microM chlorpheniramine), or endothelium-derived relaxing factor activity (10 microM hemoglobin and 50 microM methylene blue). However, it was reduced by approximately 50% with the K+ channel blockers, tetraethylammonium chloride (10 mM) and glybenclamide (10 microM), and the inhibitor of Na(+)-K+ pumping, ouabain (0.1 mM). These results indicate that ET-1 is a potent dilator of the pulmonary vascular bed of the rat and that the mechanism of dilation may involve activation of ATP-sensitive K+ channels and membrane hyperpolarization.