Induction of citrulline–nitric oxide (NO) cycle enzymes and NO production in immunostimulated rat RPE-J cells

Abstract

Nitric oxide (NO) has been implicated in many physiological and pathological conditions in the eyes. 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. When rat RPE-J cells were treated with interferon-γ (IFNγ), tumor necrosis factor-α (TNFα) and lipopolysaccharide (LPS), and expression of the citrulline–NO cycle enzymes and related enzymes was analyzed, 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 TNFα, IFNγ 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. Our findings indicate that in activated RPE-J cells citrulline–arginine recycling is important for NO production.