The effect of nitric oxide on cytokine-induced release of PGE2 by human cultured astroglial cells

1 The role of the L-arginine-nitric oxide (NO) pathway on the formation of prostaglandin E-2 (PGE(2) by human cultured astroglial cells incubated with interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) was investigated. 2 Incubation of T 67 astroglial cell line with IL-beta (10 ng ml(-1)) and TNF-alpha (500 u ml(-1)) produced a significant (P < 0.05) increase of both nitrite (the breakdown product of NO), cyclic GMP and PGE(2) levels in cell supernatants. N-omega-nitro-L-arginine methyl ester (L-NAME; 20-300 mu M), an inhibitor of NO synthase (NOS), inhibited the increase of cyclic GMP and nitrite levels found in supernatants of cytokine-treated astroglial cells and reduced the release of PGE(2). The latter effect showed that the enhanced arachidonic acid (AA) metabolism subsequent to stimulation of astroglial cells with IL-1 beta and TNF-alpha was, at least in part, induced by NO. This occurred also when sodium nitroprusside (SNP; 120 mu M), an NO donor, was incubated with astroglial cells, an effect antagonized by oxyhaemoglobin (OxyHb; 10 mu M). 3 The inhibition elicited by L-NAME on PGE(2)-release by cytokine-treated astroglial cells was reversed by adding AA (40 mu M), showing that the effect of NO on cytokine-dependent PGE(2) release occurred at the cyclo-oxygenase (COX) level. Furthermore, the release of PGE(2) in cytokine-treated astroglial cells was inhibited by indomethacin (10 mu M), a COX inhibitor as well as by preincubating cells with dexamethasone (20 mu M), an inhibitor of inducible enzymes, showing that the inducible isoform of COX (COX-2) was involved. 4 On the other hand, pretreating astroglial cells with methylene blue (MB; 10 mu M), an inhibitor of NO biological activity acting at the guanylate cyclase level, failed to affect PGE(2) release in cytokine-treated astroglial cells, leading to the conclusion that cyclic GMP changes related to NO formation are not involved in the generation of AA metabolites. 5 The present experiments demonstrated that the release of PGE(2) by astroglial cells pretreated with IL-1 beta and TNF-alpha is due to enhanced COX-2 activity via activation of the L-arginine-NO pathway, and this may be relevant to the understanding of the pathophysiological mechanisms underlying neuroimmune disorders.