Apoptosis may be important in the exacerbation of endothelial cell injury or limitation of endothelial cell proliferation. We have found that extracellular ATP (exATP) and adenosine cause endothelial apoptosis and that the development of apoptosis is linked to intracellular metabolism of adenosine [Dawicki, D. D., D. Chatterjee, J. Wyche, and S. Rounds. Am. J. Physiol. 273 ( Lung Cell Mol. Physiol. 17): L485–L494, 1997]. In the present study, we investigated the mechanism of this effect. We found that exATP, adenosine, and the S-adenosyl-l-homocysteine (SAH) hydrolase inhibitor MDL-28842 caused apoptosis and decreased the ratio of S-adenosyl-l-methionine to SAH compared with untreated control cells. Using release of soluble [3H]thymidine as a measure of DNA fragmentation, we found that the effect of adenosine on soluble DNA release was potentiated by coincubation with homocysteine. These results suggest that the mechanism of exATP- and adenosine-induced endothelial cell apoptosis involves inhibition of SAH hydrolase. exATP-induced apoptosis was enhanced by an inhibitor of adenosine deaminase, whereas exogenous adenosine-induced apoptosis was partially inhibited by an adenosine deaminase inhibitor. These results suggest that adenosine deaminase may also be involved in the mechanism of adenosine-induced endothelial cell apoptosis. Adenosine and MDL-28842 caused intracellular acidosis as assessed with the fluorescent probe 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. The cell-permeant base chloroquine prevented adenosine-induced acidosis but not apoptosis. Thus, although intracellular acidosis is associated with adenosine-induced apoptosis, it is not necessary for this effect. We speculate that exATP- and adenosine-induced endothelial cell apoptosis may be due to an inhibition of methyltransferase(s) activity. Purine-induced endothelial cell apoptosis may be important in limiting endothelial cell proliferation after vascular injury.

We determined the effects of oxidant stress by the use of tert-butyl hydroperoxide (t-BOOH) on Na+/H+ exchange in pulmonary artery endothelial cells. Monolayers were exposed to the hydroperoxide, followed by measurement of intracellular pH and the rate of recovery from acidosis by utilizing the pH-sensitive probe 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein. t-BOOH (0.4 mM) decreased the rate of acid recovery after a 2-h exposure without evidence of overt cytotoxicity (51Cr-release assay). Glutathione repletion (N-acetyl-L-cysteine) abolished the effect of the hydroperoxide. Lowering intracellular glutathione with buthionine sulfoximine decreased the acid recovery rate at a dose of t-BOOH (0.04 mM) that was not normally associated with a change in this parameter. Preincubation with vitamin E had no protective effect. Dithiothreitol abolished the effect of the hydroperoxide, suggesting oxidation of protein sulfhydryl groups as a mechanism for the altered kinetics of acid recovery. There was no difference in cell buffering capacity between control and treated monolayers. The findings suggest that the decrease in Na+/H+ antiport activity in this model of oxidant stress represents an early perturbation of membrane function and illustrate the role of the glutathione redox system in maintaining the functional integrity of the Na+/H+ antiport in these cells.

Little is known about the effects of prolonged hypoxic exposure on membrane ion transport activity. The Na+/H+antiport is an ion transport site that regulates intracellular pH in mammalian cells. We determined the effect of prolonged hypoxic exposure on human pulmonary arterial endothelial cell antiport activity, gene expression, and localization. Monolayers were incubated under hypoxic or normoxic conditions for 72 h. Antiport activity was determined as the rate of recovery from intracellular acidosis. Antiport isoform identification and gene expression were determined with RT-PCR and Northern and Western blots. Antiport localization and F-actin cytoskeleton organization were defined with immunofluorescent staining. Prolonged hypoxic exposure decreased antiport activity, with no change in cell viability compared with normoxic control cells. One antiport isoform [Na+/H+exchanger isoform (NHE) 1] that was localized to the basolateral cell surface was present in human pulmonary arterial endothelial cells. Hypoxic exposure had no effect on NHE1 mRNA transcript expression, but NHE1 protein expression was upregulated. Immunofluorescent staining demonstrated a significant alteration of the F-actin cytoskeleton after hypoxic exposure but no change in NHE1 localization. These results demonstrate that the decrease in NHE1 activity after prolonged hypoxic exposure is not related to altered gene expression. The change in NHE1 activity may have important consequences for vascular function.