Endothelial cell (EC) apoptosis is important in vascular injury, repair, and angiogenesis. Homocysteine and/or adenosine exposure of ECs causes apoptosis. Elevated homocysteine or adenosine occurs in disease states such as homocysteinuria and tissue necrosis, respectively. We examined the intracellular signaling mechanisms involved in this pathway of EC apoptosis. Inhibition of protein tyrosine phosphatase (PTPase) attenuated homocysteine- and/or adenosine-induced apoptosis and completely blocked apoptosis induced by the inhibition of S-adenosylhomocysteine hydrolase with MDL-28842. Consistent with this finding, the tyrosine kinase inhibitor genistein enhanced apoptosis in adenosine-treated ECs. Adenosine significantly elevated the PTPase activity in the ECs. Mitogen-activated protein kinase activities were examined to identify possible downstream targets for the upregulated PTPase(s). Extracellular signal-regulated kinase (ERK) 1 activity was slightly elevated in adenosine-treated ECs, whereas ERK2, c-Jun NH2-terminal kinase-1, or p38β activities differed little. The mitogen-activated protein kinase-1 inhibitor PD-98059 enhanced DNA fragmentation, suggesting that increased ERK1 activity is a result but not a cause of apoptosis in adenosine-treated ECs. Adenosine-treated ECs had diminished p38α activity compared with control cells; this effect was blunted on PTPase inhibition. These results indicate that PTPase(s) plays an integral role in the induction of EC apoptosis upon exposure to homocysteine and/or adenosine, possibly by the attenuation of p38α activity.

1998-08-01·American Journal of Physiology-Lung Cellular and Molecular Physiology

Mechanism of extracellular ATP- and adenosine-induced apoptosis of cultured pulmonary artery endothelial cells

Article

作者: Parks, Nancy ; Yee, Winnie Lin ; Dawicki, Doloretta D. ; Rounds, Sharon ; Cutaia, Michael V. ; Harrington, Elizabeth

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.

1997-08-01·American Journal of Physiology-Lung Cellular and Molecular Physiology

Extracellular ATP and adenosine cause apoptosis of pulmonary artery endothelial cells

Article

作者: Rounds, S. ; Wyche, J. ; Chatterjee, D. ; Dawicki, D. D.

ATP acts as an intracellular energy source and an extracellular signaling molecule. We report that extracellular ATP causes apoptosis in pulmonary artery endothelial cells, as assessed by morphological changes and internucleosomal DNA degradation. We investigated the mechanism of this effect using release of tritiated soluble DNA as a marker for apoptosis. We conclude that the metabolite adenosine is responsible for the apoptotic effect of ATP, since nucleotides that can be degraded to adenosine, as well as adenosine itself, cause DNA damage, whereas nonmetabolizable ATP analogs and uridine 5'-triphosphate are inactive. Furthermore, the ecto-5'-nucleotidase inhibitor alpha, beta-methylene-ADP blocks ATP-induced DNA fragmentation. The adenosine receptor agonist 5'-N-ethylcarboxamide adenosine does not cause DNA fragmentation, and adenosine receptor antagonists do not block adenosine-induced apoptosis. However, the nucleoside transport inhibitor dipyridamole prevents extracellular ATP-induced DNA cleavage. These findings indicate that ATP- and adenosine-mediated apoptosis are mediated via intracellular events rather than through cell surface receptor(s). The adenosine metabolites inosine, hypoxanthine, and xanthine do not cause apoptosis. The adenosine analogs 3-deazaadenosine and MDL-28842, which are not metabolized and are S-adenosylhomocysteine hydrolase inhibitors, also cause DNA fragmentation. Therefore, we speculate that extracellular ATP and adenosine cause apoptosis of pulmonary artery endothelial cells by altering methylation reactions that require S-adenosylmethionine as the methyl donor. We speculate that ATP released from cells undergoing cytolysis or degranulation may cause endothelial cell death. Endothelial cell apoptosis may be important in acute vascular injury or in limiting angiogenesis.

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