Induction of Citrulline-Nitric Oxide (NO) Cycle Enzymes and NO Production in Immunostimulated Rat RPE-J Cells

Abstract

Abstract: : Purpose: The induction of inducible NO synthase (iNOS) and NO production have been noted in immunostimulated retinal pigment epithelial (RPE) cells. Cellular NO production depends on the availability of arginine, a substrate for NOS. Arginine can be regenerated from citrulline, another product of the NOS reaction, by argininosuccinate synthetase and argininosuccinate lyase, forming the citrulline-NO cycle. In this study, we aim to investigate the regulation of the citrulline-NO cycle enzymes and arginase isoforms in association with inducible nitric oxide synthase (iNOS) in immunostimulated RPE-J cells. Methods: When rat RPE-J cells were treated with interferon-g (IFNg), tumor necrosis factor-a (TNFa) and lipopolysaccharide (LPS), the expression of the citrulline-NO cycle enzymes and related enzymes was analyzed by RNA blot and immunoblot analysis. NO production was measured by NO2/NO3 assay kit. Results: iNOS and argininosuccinate synthetase were highly induced at both mRNA and protein levels. On the other hand, argininosuccinate lyase was not induced. Among other related enzymes and transporters, mRNA for cationic amino acid transporter (CAT)-1 was weakly induced, whereas those for CAT-2, arginase I and II, ornithine aminotransferase and ornithine decarboxylase remained little changed. NO was produced by cells after stimulation with TNFa, IFNg and LPS. The induction of iNOS mRNA and the production of NO by these immunostimulated cells was further enhanced by cAMP. NO was produced from citrulline as well as from arginine. Conclusion: Our findings indicate that in activated RPE-J cells citrulline-arginine recycling is important for NO production.

Keywords: nitric oxide • retinal pigment epithelium