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
G. Bhat et al., 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, Neuroendocrinology, 64(2), 1996, pp. 93-102
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.