Endogenous production of nitric oxide and effects of nitric oxide and superoxide on melanotrope functioning in the pituitary pars intermedia of Xenopus laevis

Previous studies have focused on the immunohistochemical detection of a nit ric oxide (NO)-cyclic 3',5'-monophosphate (cGMP) pathway in the brain and p ituitary of the aquatic toad Xenopus laevis. We here investigate the endoge nous production and possible involvement of NO signaling in the regulation of melanotrope cell activity in the pituitary pars intermedia of this amphi bian, Using immunohistochemical staining of cultured cells with a polyclona l antiserum against inducible NO synthase (iNOS), immunoreactivity was obse rved both in melanotropes and in stellate-shaped cells. Part of these stell ate-shaped cells is characterized as folliculo-stellate cells by their capa city of beta-Ala-Lys-N-e-AMCA uptake, Using chemiluminescence detection we demonstrate the presence of NO and reaction products like nitrite (NO2-) or peroxynitrite (ONOO-) in the incubation medium of cultured melanotropes. B acterial lipopolysaccharide (LPS) stimulates the generation of NO and react ion products, the effect of which was blocked by S-methyl-L-thiocitrulline hydrochloride, a potent general NOS inhibitor. With [H-3]lysine incorporati on and a superfusion technique, it is shown that peptide release from melan otropes is stimulated by administration of superoxide dismutase (SOD), whic h was added to the superfusion medium to prevent scavenging of NO by supero xide anions, Pretreating the cells with the general NOS inhibitor L-nitroar ginine methyl ester for 48 h attenuated the SOD-induced stimulation, but di d not affect the stimulation by sodium nitroprusside (SNP) or 3-morpholinyl -sydnoneimine chloride (SIN-1), whereas hemoglobin blocked the combined eff ect of SOD plus NO donors. The soluble guanylate cyclase inhibitor 1H-[1,2, 4]oxadiazolo[4,3 alpha]-quinoxaline-1-one did not inhibit but even signific antly potentiated the effect of NO donors on peptide release without affect ing the SOB-induced stimulation of peptide release. In addition to the prev iously described neuronal NOS (nNOS) immunoreactivity in nerve fibers in th e pars intermedia of Xenopus, the present data reveal. iNOS and nNOS as pot ential sources of endogenous NO production in cultured cells of the pars in termedia. Our study shows that also in nonmammalian vertebrates endogenous NO production may be physiologically relevant under conditions where protec tion against oxidative damage is needed. The endocrine cells of the pars in termedia themselves, as well as the folliculo-stellate cells, under such co nditions may dispose of a protective mechanism against oxidative stress, Th e sensitivity of the endogenous NO production to LPS suggests that NO may a lso play a role during systemic inflammation. (C) 2000 Academic Press.