| Arginase has two distinct isoforms and is the major enzyme for initiating arginine degradation in endothelial cells (EC). The objectives of this research were to investigate the role of arginase I and II in endothelial synthesis of ornithine, nitric oxide, polyamines, proline and glutamate, and cell proliferation. A sensitive, specific and rapid method for quantifying endothelial nitric oxide production was first developed, which involved chromatographic separation of nitrite and nitrate derivatives and fluorescence detection (detection limit, 10 pmol/ml). Overexpression of arginase I or II, achieved by gene transfection techniques, decreased cellular arginine content and nitric oxide synthesis, increased the production of ornithine, urea, polyamines, proline and glutamate, and stimulated EC proliferation. Metabolic studies using 10 or 50 μM putrescine and 5 mM DL-α-difluoromethylornithine (an inhibitor of ornithine decarboxylase) showed that an increase in putrescine and spermidine synthesis was necessary for the enhanced proliferation of the EC exhibiting elevated arginase expression. For defining the metabolic sources of the endothelial ornithine for polyamine synthesis, Nω-hydroxy-nor-L-arginine (a specific inhibitor of arginase) and gabaculine (an inhibitor of ornithine aminotransferase) were used. Nω-hydroxy-nor-L-arginine or gabaculine alone partially depleted intracellular ornithine, putrescine and spermidine. A combination of both inhibitors completely blocked ornithine production, resulting in no cellular ornithine and near absence of cellular putrescine and spermidine. In EC treated with Nω-hydroxy-nor-L-arginine, the increase in total cellular arginine content (14%) was much less than the inhibition of cellular arginase activity (98%), and the decrease in total cellular ornithine content (17%) was much less than the reduction in total cellular putrescine and spermidine content (65–74%). Collectively, these results demonstrate that (1) arginase I or II is normally a limiting or regulatory factor for endothelial arginine catabolism and cell proliferation, (2) the intracellular ornithine for endothelial polyamine synthesis was derived from both arginase and ornithine aminotransferase, and (3) there were intracellular compartmentations of arginine and ornithine for nitric oxide and polyamine synthesis in EC. The findings of this research provide a new metabolic basis for the use of arginine as a potentially novel nutritional strategy for preventing and treating cardiovascular disease. |
