In hepatocytes the regulation of NOS-2 activity at physiological L-arginine levels suggests a close link to the urea cycle.
High-output synthesis of nitric oxide (NO) by the inducible isoform of NO-synthases (NOS-2) plays an important role in hepatic pathophysiological processes and may contribute to both organ protection and organ destruction during inflammatory reactions. As they compete for the same substrate, L-arginine, an interdependence of NOS-2 and arginase-1 has been repeatedly observed in cells where arginase-1 is cytokine-inducible. However, in hepatocytes, arginases are constitutively expressed and thus, their impact on hepatic NOS-2-derived NO synthesis as well as the influence of L-arginine influx via cationic amino acid transporters during inflammatory reactions are still under debate. Freshly isolated rat hepatocytes were cultured for 24h in the presence of various L-arginine concentrations with or without cytokine addition and nitrite and urea accumulation in culture supernatants was measured. We find that both, cytokine-induced NOS-2 and arginase activities strongly depend on extracellular L-arginine concentrations. When we competed for L-arginine influx via the cationic amino acid transporters by addition of L-lysine, we find a 60-70% inhibition of arginase activity without significant loss of NOS-2 activity. Addition of L-valine, as an arginase inhibitor, leads to a 25% increase in NO formation and an 80-90% decrease in arginase activity. Interestingly, product inhibition of arginase and competitive inhibition of CATs through the addition of L-ornithine leads to a highly significant increase in hepatocytic NOS-2 activity with a concomitant and complete abolishment of its dependence on extracellular L-arginine concentrations. In conclusion, hepatocytic NOS-2 activity shows a surprising pattern of dependence on exogenous L-arginine concentrations. Inhibition and competition experiments suggest a relatively tight link of NOS-2 and urea cycle activities. These data stress the hypothesis of a metabolon-like organization of the urea cycle together with NOS-2 in hepatocytes as excess L-ornithine will be metabolized to l-arginine and thereby increases NO production.
Lerzynski
Guido
G
Research Group Immunobiology, Heinrich-Heine-University Düsseldorf, P.O. Box 101007, 40001 Düsseldorf, Germany.
Suschek
Christoph V
CV
Kolb-Bachofen
Victoria
V
D026923:Amino Acid Transport Systems, Basic / Q000378:metabolism; D000818:Animals; D001119:Arginase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D007249:Inflammation / Q000473:pathology; D008239:Lysine / Q000494:pharmacology; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D009952:Ornithine / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D014508:Urea / Q000378:metabolism*; D014633:Valine / Q000494:pharmacology
D026923:Amino Acid Transport Systems, Basic; D016207:Cytokines; D004791:Enzyme Inhibitors; D009569:Nitric Oxide; D014508:Urea; D001120:Arginine; D009952:Ornithine; D052247:Nitric Oxide Synthase Type II; D001119:Arginase; D014633:Valine; D008239:Lysine