EVIDENCE THAT GONADOTROPIN-RELEASING-HORMONE STIMULATES GENE-EXPRESSION AND LEVELS OF ACTIVE NITRIC-OXIDE SYNTHASE TYPE-I IN PITUITARY GONADOTROPHS, A PROCESS ALTERED BY DESENSITIZATION AND, INDIRECTLY, BY GONADAL-STEROIDS

To determine the site and mechanism of action of gonadal steroids on p ituitary nitric oxide synthase type I (NOS I), present in both gonadot rophs and folliculo-stellate cells, the effects of castration and ster oids were examined in male rats, in the presence of a GnRH antagonist (Antarelix). Western analysis showed a rapid and substantial increase with time, after orchidectomy, of NOS I protein, the concentration dou bling in 24 h and reaching a maximal 4- to B-fold increase after 3-7 d ays, followed by a progressive decline after 2 weeks. Testosterone or estradiol replacement, or administration of GnRH antagonist, totally a bolished the effects of castration, demonstrating a mediation of the s teroid effects via GnRH. In noncastrated rats, steroids and the GnRH a ntagonist also caused a reduction in the levels of NOS I (by 50-60%), consistent with inhibition of endogenous GnRH stimulation. In marked c ontrast, administration of a potent GnRH agonist (Triptorelin) to inta ct rats increased the levels of NOS I. A time-course study with a long -lasting formulation showed that rise in NOS I developed rapidly after a lag of approximately 5 h, with a 2-fold increase detectable after 8 h and a maximal 4.5-fold after 48 h. The level declined afterwards in a manner consistent with homologous desensitization that may occur in the continuous presence of GnRH; however, the profile was different a nd delayed compared with those of gonadotropin release. As observed fo r NOS I protein, NOS I messenger RNA concentration was increased by ca stration or GnRH agonist and reduced by steroids or GnRH antagonist. T aken together, these data demonstrate that steroids indirectly regulat e NOS I messenger RNA and protein levels, through the hypothalamic mod ulation of GnRH, which represents the primary regulator of NOS I. No e ffect of steroids on NOS I was seen in the posterior lobe. NADPH-diaph orase erase histochemistry coupled to immuno-identification of the cel ls revealed that the treatments affecting the concentration of NOS I c oncomitantly altered the activity but exclusively in gonadotrophs and not in folliculo-stellate cells (which do not respond to GnRH), reinfo rcing the idea that GnRH played a major regulatory role. Expression in gonadotrophs of a GnRH-dependent NOS I and the ensuing-production of nitric oxide represents a potentially novel signaling pathway for the neuropeptide in the anterior pituitary, consistent with the previously reported GnRH-induced cGMP production, the role of which remains to b e evaluated.