ARGINASE ACTIVITY IN ENDOTHELIAL-CELLS - INHIBITION BY N-G-HYDROXY-L-ARGININE DURING HIGH-OUTPUT NO PRODUCTION

Rat aortic endothelial cells were found to contain both constitutive a nd 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 pr oduction. Unactivated cells contained abundant arginase activity accom panied by continuous urea formation. LPS induced the formation of both inducible NOS (iNOS) and arginase, and this was accompanied by increa sed production of NO, citrulline, and urea. Immunoprecipitation experi ments revealed the constitutive presence of arginase-I in both unactiv ated and LPS-activated cells and arginase-II induction by LPS. Arginas e-I and iNOS were verified by reverse transcriptase-polymerase chain r eaction. Induction of large amounts of iNOS by LPS plus several cytoki nes resulted in large quantities of NO, citrulline, and N-G-hydroxy-L- arginine (NOHA), but urea production was markedly diminished. Decrease d urea production was attributed to increased formation of NOHA, the p recursor to NO and citrulline and a potent inhibitor of arginase-I act ivity with an inhibitory constant of 10-12 mu M. Inhibition of iNOS ac tivity by N-G-methyl-L-arginine decreased NO and NOHA production and i ncreased urea production. This study reveals for the first time that s ubstantial arginase activity is present constitutively in rat aortic e ndothelial cells, a different isoform of arginase is induced by LPS, a nd intracellular arginase activity can be markedly inhibited during cy tokine 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-ou tput production of NO.