Nitric Oxide (NO) Stimulates Gonadotropin Secretion in vitro through a Calcium-Dependent, cGMP-Independent Mechanism

Abstract

In the last few years, nitric oxide (NO) has emerged as an important intra- and intracellular messenger involved in the control of hypothalamic-pituitary function. The present experiments were undertaken in order to evaluate the pituitary component in the modulatory action of NO on gonadotropin secretion, as well as the second messenger pathway(s) involved. In a first step, we assessed LH and FSH secretion by hemipituitaries incubated in the presence of increasing concentrations of sodium nitroprusside (SNP), a potent NO donor, and cyclic guanosin monophosphate (cGMP), the second messenger for a wide range of NO actions. In addition, given that SNP induces the release of NO and cyanide ions, the response to SNP was tested in the presence of hemoglobin (an NO scavenger) or rhodanese + sodium thiosulfate (inactivators of cyanides) in order to ensure that the effects of SNP on gonadotropin secretion were mediated by the release of NO. SNP (10^–4–10^–3  M) stimulated gonadotropin secretion in our incubation system, whereas cGMP, at all doses tested, was ineffective. Similar results were obtained using dispersed pituitary cells. The stimulatory action of SNP is attributable to its ability to induce NO release since it was blocked by hemoglobin, but preserved after incubation with rhodanese + sodium thiosulfate. In further experiments, we aimed to identify the mechanism(s) underlying SNP-induced gonadotropin secretion. First, to evaluate the involvement of calcium (Ca²⁺), the effects of SNP were analyzed in a calcium-free medium, after depletion of Ca²⁺ stores by caffeine, in the presence of the Ca²⁺ chelator ethylene glycol bis ( p-aminoethyl ether) N,N-tetra-acetic acid (EGTA), and after incubation with the Ca²⁺ channel blockers verapamil and nifedipine. Second, to confirm that cGMP is not involved in the stimulatory action of SNP, the effects of the latter on gonadotropin secretion were tested in the presence of the antagonists of the guanylyl cyclases oxadiazoloquinoxaline and LY 83,583. Our results showed that the stimulatory action of SNP on gonadotropin release is blunted in Ca²⁺-free medium and after incubation with EGTA, verapamil, nifedipine, and caffeine. On the contrary, the effect of SNP remained unaltered after antagonization of guanylyl cyclases. We conclude that NO, acting at the pituitary level, stimulates gonadotropin secretion through a calcium-dependent, cGMP-independent mechanism.