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

Abstract

Recent studies suggest that nitric oxide (NO) may function as a neurotransmitter in the hypothalamus. In order to provide further evidence supporting this contention, we examined: (1) whether the hypothalamus displays significant NO synthase (NOS) activity and whether the activity is inhibited by an NOS inhibitor, (2) whether the different NOS isoforms [brain (b)-NOS, endothelial (e)-NOS and macrophage (m)-NOS] are expressed in the various nuclei of the hypothalamus of the random cycling adult female rat, (3) whether the NO donor molecule, sodium nitroprusside (SNP), regulates the heme-containing enzyme, guanylate cyclase in the preoptic area and medial basal hypothalamus of the random cycling adult female rat as well as the ovariectomized steroid (estradiol-17 beta)-treated rat. The results of the study showed that the preoptic area (POA) and medial basal hypothalamus (MBH) of the adult female rat displays significant NOS activity which can be dose-dependently inhibited 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 antibodies to the specific NOS isoform proteins revealed that b-NOS represented the major form of NOS in the hypothalamus based on density and distribution 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 nucleus/median eminence. The pattern and density of b-NOS staining closely mirrored our previously reported pattern of NADPH-diaphorase staining 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 hypothalamus, we observed no specific staining for m-NOS in the hypothalamus. e-NOS immunostaining, on the other hand, was present in the hypothalamus, but to a much lesser extent than b-NOS. Light e-NOS staining was observed in the OVLT, MPOA, supraoptic nucleus and arcuate nucleus/median eminence. That NO can regulate guanylate cyclase as a potential mediator of its effects was demonstrated using SNP which dose-dependently elevated cGMP levels in the POA and MBH of random cycling rats and estrogen-primed ovariectomized rats. The effect of SNP was due to its NO donor ability as it was blocked by the NO scavenger molecule, hemoglobin. 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 demonstrates 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 the female hypothalamus as evidenced by the potent ability of SNP to elevate cGMP levels in the POA and MBH.