GnRH-dependent up-regulation of nitric oxide synthase I level in pituitary gonadotrophs mediates cGMP elevation during rat proestrus

Abstract We have recently provided evidence that the concentration and activity of the enzyme nitric oxide synthase type I (NOS I) was stimulated in gonadotrophs by GnRH, suggesting a role for the NOS I/NO pathway in the GnRH control of cell functions. To further establish the GnRH regulation of pituitary NOS I under physiological circumstances, we have examined the expression of NOS I during the 4-day estrus cycle in rats. Western blot analysis demonstrated that NOS I was present in the anterior pituitary at low levels during diestrus I (DI) and diestrus II, then was subject to a significant increase on the afternoon of proestrus to reach a maximal 3-fold increase between 19:00 and 20:00 h, after which NOS I decreased to return, during estrus, to levels within the range detected in diestrus. No such variation was apparent in the posterior pituitary lobe. NADPH–diaphorase histochemistry combined with immuno-identification of the cells revealed that active NOS I was expressed in both the gonadotrophs and the folliculo-stellate cells throughout the whole cycle but only the gonadotrophs showed an up-regulation during proestrus. A high temporal correlation was observed in the profiles of NOS I, pituitary cGMP and serum luteinizing hormone (LH) suggesting an implication of GnRH. Consistently, the administration of a potent GnRH antagonist to rats totally abolished the rise in pituitary NOS I and cGMP, in addition to suppressing, as expected, the surge in the secretion of LH. A role of NOS I as a mediator in the GnRH-induced augmentation in cGMP was further established in vitro by incubating anterior pituitaries in the presence of the NOS inhibitor, L-NMMA (1 mM). Altogether, these data demonstrate that the level and activity of NOS I is up-regulated in gonadotrophs during proestrus, in a manner consistent with a major implication of GnRH over a period during which its release from the hypothalamus, as well as gonadotroph responsiveness, are at maximum. This effect is accompanied by a NOS/NO-mediated rise in cGMP. In the absence of obvious effect on gonadotropin release, the roles of NO and cGMP in the regulation of gonadotroph functions, especially during proestrus, remain to be established.