Arginase activity in endothelial cells: inhibition by NG-hydroxy-L-arginine during high-output NO production

Abstract

Rat aortic endothelial cells were found to contain both constitutive and lipopolysaccharide (LPS)-inducible arginase activity. Studies were performed to determine whether induction of nitric oxide synthase (NOS) by LPS and cytokines is accompanied by sufficient arginase induction to render arginine concentrations rate limiting for high-output NO production. Unactivated cells contained abundant arginase activity accompanied by continuous urea formation. LPS induced the formation of both inducible NOS (iNOS) and arginase, and this was accompanied by increased production of NO, citrulline, and urea. Immunoprecipitation experiments revealed the constitutive presence of arginase-I in both unactivated and LPS-activated cells and arginase-II induction by LPS. Arginase-I and iNOS were verified by reverse transcriptase-polymerase chain reaction. Induction of large amounts of iNOS by LPS plus several cytokines resulted in large quantities of NO, citrulline, and NG-hydroxy-L-arginine (NOHA), but urea production was markedly diminished. Decreased urea production was attributed to increased formation of NOHA, the precursor to NO and citrulline and a potent inhibitor of arginase-I activity with an inhibitory constant of 10-12 microM. Inhibition of iNOS activity by NG-methyl-L-arginine decreased NO and NOHA production and increased urea production. This study reveals for the first time that substantial arginase activity is present constitutively in rat aortic endothelial cells, a different isoform of arginase is induced by LPS, and intracellular arginase activity can be markedly inhibited during cytokine induction of iNOS because of NOHA formation. The inhibition of arginase activity that occurs by NOHA during marked iNOS induction may be a mechanism to ensure sufficient arginine availability for high-output production of NO.

Endothelial dysfunction in rat adjuvant‐induced arthritis: Up‐regulation of the vascular arginase pathway

“…Consistent with this hypothesis, increased vascular arginase activity was reported to contribute to decreased endotheliumdependent NO production in pathologic conditions such as hypertension (19,20), atherosclerosis (21), diabetes (22), and erectile dysfunction (23), or in aging (24). Moreover, even though the regulating factors of arginase expression/activity remain largely unknown, it was demonstrated that various proinflammatory cytokines can act as inducers of arginase expression in cultured endothelial cells (18,25,26).…”

“…Both arginase isoforms are expressed by endothelial and vascular smooth muscle cells (18). Because NOS and arginase use L-arginine as a common substrate, arginase may down-regulate NO biosynthesis by competing with NOS for L-arginine degradation.…”

Arginine metabolism: nitric oxide and beyond

“…In contrast, type II arginase, a mitochondrial enzyme, is expressed at lower levels in kidney, brain, small intestine, mammary gland and macrophages, but there is little or no expression in liver [138,162,198,199]. Rat aortic endothelial cells and murine macrophages express both type I and type II arginases [200][201][202], and it is likely that other cell types also express both isoenzymes. The different subcellular localization of the arginase isoenzymes may provide a mechanism for regulating the metabolic fate of arginine, as postulated for enterocytes [203].…”

“…For example, iNOS-expressing macrophages [228,229] and endothelial cells [200] can produce sufficient N G -hydroxyarginine to inhibit arginase activity. Because endothelial cells in intact animals are constantly perfused, whereas cultured cells are not, it is not clear whether the former would be exposed to sufficient N G -hydroxyarginine in i o to inhibit cellular arginase activity.…”

“…Ornithine\urea production is the predominant route of arginine catabolism in unactivated rat endothelial cells [200,290], but NO production predominates when endothelial cells are stimulated with the appropriate combination of cytokines [200]. NO\citrulline synthesis represents the vast majority of arginine metabolism in rat macrophages [223], but ornithine\urea production dominates in murine macrophages [187,254,291].…”