Inhibition of Stimulated Ascorbic Acid and Luteinizing Hormone-Releasing Hormone Release by Nitric Oxide Synthase or Guanyl Cyclase Inhibitors

Abstract

Ascorbic acid (AA), an antioxidant, is present in high concentrations in the hypothalamus. Previously, we have shown that AA inhibited stimulated release of luteinizing hormone-releasing hormone (LHRH) from medial basal hypothalami in vitro. We have also demonstrated that cell membrane depolarization by high [K⁺] media-induced AA release that is blocked by N^G-monomethyl-l-arginine, a competitive inhibitor of nitric oxide synthase (NOS), indicating that the release process is mediated by NO. The release of LHRH is also mediated by NO. We hypothesized that AA is a co-transmitter released with classical transmitters from synaptic vesicles that acts to reduce chemically the NO formed, thereby providing feed-forward inhibitory control over LHRH release. Because NO acts by activating guanylyl cyclase (GC) resulting in production of cGMP, in the present investigation we studied the effects of an NOS inhibitor LY 83583 and GC inhibitor, O.D.Q. to further characterize the role of NO in high [K⁺]-induced AA and LHRH release. Medial basal hypothalami were incubated in 0.5 ml of Krebs-Ringer Bicarbonate buffer or medium containing increased potassium [K⁺ = 56 mM] for 1 hr or combinations of high [K⁺] + LY 83583 or O.D.Q. for 1 hr. AA and LHRH released into the incubation medium were measured by high-pressure liquid chromatography and radioimmunoassay, respectively. Cell membrane depolarization with high [K⁺] produced a significant increase in both AA and LHRH release. A combination of high [K⁺] + LY 83583 or high [K⁺] + O.D.Q. decreased basal AA and completely blocked high [K⁺]-induced AA and LHRH release. As in the case of high [K⁺], LHRH release induced by the excitatory amino acid N-methyl-d-aspartic acid (NMDA) was blocked by both the inhibitors. NMDA alone failed to alter AA release, but the combined presence of NMDA and the inhibitors totally blocked AA release. Because LY 83583 and O.D.Q. were shown to inhibit NOS and soluble GC, respectively, the data demonstrate that basal and high [K⁺]-induced AA and high [K⁺] and NMDA-stimulated LHRH release were mediated by NO by its activation of GC and consequent generation of cGMP.