EVIDENCE THAT BRAIN NITRIC-OXIDE SYNTHASE IS THE MAJOR NITRIC-OXIDE SYNTHASE ISOFORM IN THE HYPOTHALAMUS OF THE ADULT FEMALE RAT AND THAT NITRIC-OXIDE POTENTLY REGULATES HYPOTHALAMIC CGMP LEVELS

Recent studies suggest that nitric oxide (NO) may function as a neurot ransmitter in the hypothalamus. In order to provide further evidence s upporting this contention, we examined: (1) whether the hypothalamus d isplays significant NO synthase (NOS) activity and whether the activit y is inhibited by an NOS inhibitor, (2) whether the different NOS isof orms [brain (b)-NOS, entothelial (e)-NOS and macrophage (m)-NOS] are e xpressed in the various nuclei of the hypothalamus of the random cycli ng adult female rat, (3) whether the NO donor molecule, sodium nitropr usside (SNP), regulates the heme-containing enzyme, guanylate cyclase in the preoptic area and medial basal hypothalamus of the random cycli ng adult female rat as well as the ovariectomized steroid (estradiol-1 7 beta)-treated rat. The results of the study showed that the preoptic area (POA) and medial basal hypothalamus (MBH) of the adult female ra t displays significant NOS activity which can be dose-dependently inhi bited by an NOS inhibitor. All three NOS isoform mRNA transcripts were present in the hypothalamus, with the order of expression being b-NOS >e-NOS>m-NOS. Immunohistochemical localization using monoclonal antibo dies to the specific NOS isoform proteins revealed that b-NOS represen ted the major form of NOS in the hypothalamus based on density and dis tribution of immunostaining. b-NOS immunostaining was especially dense in the organum vasculosum laminae terminalis (OVLT), medial preoptic area (MPOA), supraoptic nucleus, and moderately dense in the arcuate n ucleus/median eminence. The pattern and density of b-NOS staining clos ely mirrored our previously reported pattern of NADPH-diaphorase stain ing in the hypothalamus, and a polyclonal antibody to b-NOS yielded a similar staining pattern as that observed for the monoclonal antibody. In contrast to the dense staining observed for b-NOS in the hypothala mus, we observed no specific staining for m-NOS in the hypothalamus. e -NOS immunostaining, on the other hand, was present in the hypothalamu s, but to a much lesser extent than b-NOS. Light e-NOS staining was ob served in the OVLT, MPOA, supraoptic nucleus and arcuate nucleus/media n eminence. That NO can regulate guanylate cyclase as a potential medi ator of its effects was demonstrated using SNP which dose-dependently elevated cGMP levels in the POA and MBH of random cycling rats and est rogen-primed ovariectomized rats. The effect of SNP was due to its NO donor ability as it was blocked by the NO scavenger molecule, hemoglob in. Interestingly, hemoglobin alone caused a 50-60% reduction in basal cGMP levels, suggesting that endogenously produced NO regulates basal guanylate cyclase activity. Taken as a whole, the present study demon strates that b-NOS is the major NOS isoform in the hypothalamus and it also provides evidence that cGMP may be a mediator of NO effects in t he female hypothalamus as evidenced by the potent ability of SNP to el evate cGMP levels in the POA and MBH.