Activation of neuronal nitric oxide release inhibits spontaneous firing in adult gonadotropin-releasing hormone neurons: a possible local synchronizing signal

The activation of nitric oxide (NO) signaling pathways in hypothalamic neurons plays a key role in the control of GnRH secretion that is central to reproductive function. It is unknown whether NO directly modulates the firing behavior of GnRH neurons in the preoptic region of the mature brain. Using patch-clamp recordings from GnRH neurons expressing green fluorescent protein in adult mice brain slices, we demonstrate that the NO precursor, L-arginine (Arg), or the NO donor, diethylamine/NO, induced a robust and reversible reduction in the spontaneous firing activity of GnRH neurons, including bursting activity. The effects of L-Arg were prevented by the NO synthase inhibitor N omega-nitro-L-Arg methyl ester hydrochloride. Histochemical studies revealing a close anatomical relationship between neurons producing NO and GnRH perikarya, together with the loss of the L-Arg-mediated inhibition of GnRH neuronal activity via the selective blockade of neuronal NO synthase, suggested that the primary source of local NO production in the mouse preoptic region was neuronal. Synaptic transmission uncoupling did not alter the effect of NO, suggesting that NO affects the firing pattern of GnRH neurons by acting at a postsynaptic site. We also show that the NO-mediated changes in membrane properties in the GnRH neurons require soluble guanylyl cyclase activity and may involve potassium conductance. By revealing that NO is a direct modulator of GnRH neuronal activity, our results introduce the intriguing possibility that this gaseous neurotransmitter may be used by the sexual brain to modulate burst firing patterns. It may set into phase the bursting activity of GnRH neurons at key stages of reproductive physiology.     

Estradiol and progesterone modulate the nitric oxide/cyclic gmp pathway in the hypothalamus of female rats and in GT1-1 cells

Considerable evidence suggests that the nitric oxide (NO)/cGMP signaling pathway plays an important role in the expression of reproductive behavior and in gonadotropin-releasing hormone (GnRH) release from the hypothalamus The effects of the NO/cGMP pathway on GnRH release and gene expression have also been examined in GT1 cells. However, it is still controversial whether NO/cGMP signaling facilitates or inhibits GnRH release in these cells. The current study examined the effects of estradiol and progesterone on neuronal NO synthase (nNOS), soluble guanylyl cyclase (sGC), and NO-dependent cGMP production in the preoptic area (POA) and hypothalamus (HYP) as well as in GT1-1 cells. Ovariectomized female rats received vehicle, estradiol benzoate (48 h) and/or progesterone (3–4 h) before preparation of brain slices. GT1-1 cells were incubated with vehicle, estradiol (48 h), progesterone (3–4 h), or with both hormones. The combination of estradiol and progesterone increased the expression of nNOS protein in the POA and HYP. Hormones had little effect on the abundance of sGC. Estradiol and progesterone together greatly enhanced NO-stimulated sGC activity in HYP-POA slices. In GT1-1 cells, NO-stimulated sGC activity was significantly increased by estradiol and progesterone, alone or in combination, but sGC expression was not altered by hormones.     

Evidence that neuronal nitric oxide synthase but not heme oxygenase increases in the hypothalamus on proestrus afternoon

Nitric oxide (NO) has been implicated in the control of the proestrus luteinizing hormone (LH) surge in the rat but to date no studies have attempted to measure neuronal nitric oxide synthase (nNOS) or NO production on proestrus in the hypothalamus in order to determine if endogenous NO is increased on proestrus afternoon to activate gonadotropin-releasing hormone (GnRH) neurons. To address this deficit in our knowledge, we measured nNOS mRNA and protein levels as well as NOS activity levels in rat preoptic area (POA) and medial basal hypothalamus (MBH) fragments at 12.00, 14.00, 16.00, and 18.00 h of proestrus. Serum LH levels were also assessed to determine whether NOS changes correlate to the LH surge. To determine the specificity of observed changes we also measured mRNA levels for the enzyme heme oxygenase-2, which is responsible for production of another putative gaseous transmitter, carbon monoxide. In all studies a metestrus 12.00 h control group was included since steroid and LH levels would be basal at this time as compared to proestrus. The results revealed that nNOS mRNA and protein levels, as well as NOS activity did not change significantly in the MBH on proestrus. In contrast, nNOS mRNA levels were significantly elevated in the POA at proestrus 12.00 and 14.00 h, as compared to metestrus 12.00 h. Likewise, at the protein and activity level, nNOS protein levels in the POA were significantly elevated on proestrus at 14.00 and 16.00 h, with NOS activity significantly increased at 16.00 h on proestrus. The elevation of nNOS protein and activity levels in the POA occurred at the time of initiation of the LH surge. The elevation of nNOS was specific as mRNA levels for the CO-synthetic enzyme heme oxygenase-2 did not change significantly on proestrus in the POA or MBH. As a whole, the current studies provide new evidence that nNOS is elevated in the POA on proestrus, and thus could play a role in the activation of GnRH neurons to produce the preovulatory LH surge.     

Somatostatin inhibition of GnRH neuronal activity and the morphological relationship between GnRH and somatostatin neurons in rats

In rodents, GnRH neurons are diffusely distributed from the medial septum through to the medial preoptic area and control gonadal functions through the pituitary. The activity of GnRH neurons is regulated by a variety of bioactive substances, including the inhibitory peptide somatostatin. In the present study, we focused on somatostatin because intracerebroventricular injection of somatostatin inhibits the LH surge in rats and reduces LH secretion in ewes. Somatostatin also decreases GnRH release from rat hypothalamic slices. In mice, somatostatin is also thought to suppress GnRH neuronal activity through contact on the soma of GnRH neurons. However, similar data are missing in rats. Moreover, rat GnRH neurons receive only a few synaptic inputs. In this study, we assessed the morphological relationship between GnRH and somatostatin neurons. Confocal microscopy on the sections from the medial septum through medial preoptic area revealed about 35 close contacts per rat between the GnRH and somatostatin neuronal fibers in the organum vasculosum of the lamina terminalis region. No contact of somatostatin fibers on the GnRH neuronal somata was observed. Multicell RT-PCR for somatostatin receptor mRNA in rat GnRH neurons was also performed, which revealed moderate expression of somatostatin receptor subtypes 1-5. In addition, patch clamp experiments were carried out in acute slice preparations. Somatostatin suppressed neuronal firing in cells recorded in a cell-attached configuration and also induced whole-cell outward currents in GnRH neurons. These findings suggest that somatostatin directly inhibits the activity of rat GnRH neurons through volume transmission in the organum vasculosum of the lamina terminalis region.     

Differential effects of hypothalamic IGF-I on gonadotropin releasing hormone neuronal activation during steroid-induced LH surges in young and middle-aged female rats

Interactions between brain IGF-I receptors and estrogen receptors regulate female reproductive physiology and behavior. The present study investigated potential mechanisms by which IGF-I receptors in the neuroendocrine hypothalamus regulate GnRH neuronal activation and LH release in young and middle-aged female rats under estradiol (E2) positive feedback conditions. We infused vehicle, IGF-I, or JB-1, a selective antagonist of IGF-I receptors, into the third ventricle of ovariectomized female rats primed with E2 and progesterone or vehicle. In young females, blockade of IGF-I receptors attenuated the steroid hormone-induced LH surge, reduced the percent of GnRH neurons expressing c-fos on the day of the LH surge, and decreased the total number of neurons expressing c-fos in the preoptic area. Middle-aged females had fewer GnRH neurons expressing c-fos during the LH surge than young females, and the LH surge amplitude was attenuated. Infusion of an IGF-I dose previously shown to increase LH surge amplitude did not increase the percent of GnRH neurons expressing c-fos in middle-aged females. Brain IGF-I receptor blockade did not modify E2 induction of progestin receptor-immunoreactive neurons in the preoptic area, arcuate, or ventromedial hypothalamus of young rats. These findings indicate that brain IGF-I receptors are required for E2 activation of GnRH neurons in young rats and for robust GnRH release from axon terminals in middle-aged females. IGF-I likely exerts its effects by actions on E2-sensitive neurons that are upstream of GnRH neurons and terminals.     

Changes in renal hemodynamics and structure in the aging kidney; sexual dimorphism and the nitric oxide system

With advancing age the kidney shows both functional declines (falls in GFR) and development of structural damage. In most individuals this occurs slowly and does not lead to severe renal impairment unless additional insults are superimposed. There is a pronounced sexual dimorphism with females protected, due both to beneficial effects of the estrogens and damaging effects of androgens, some of which act directly on the glomerular mesangial cell to regulate growth and extracellular matrix production. Nitric oxide is a major factor in regulation of vascular tone and growth and becomes deficient with advancing age, as endothelial dysfunction develops. Although the abundance of the substrate, L-arginine, is well maintained during aging, there are increases in the concentration of circulating endogenous nitric oxide synthase (nNOS) inhibitors, which will contribute, to the endothelial dysfunction. There is a clear sexual dimorphism in the NO system, with pre-menopausal females producing more NO than men. Within the kidney, declines in the abundance and activity of the neuronal form of the nitric oxide synthase (nNOS) correlate with development of disease. In the male rat where injury and dysfunction occurs, nNOS abundance declines markedly, whereas in the protected female, renal nNOS abundance is maintained. Taken together, it is likely that age-dependent declines in NO generation contribute to age-dependent kidney damage.     

In human brain ornithine transcarbamylase (OTC) immunoreactivity is strongly expressed in a small number of nitrergic neurons

There is recent evidence for ornithine transcarbamylase (OTC) expression in adult human brain. We decided to immunocytochemically map OTC throughout brain, and to further characterize OTC-immunopositive neurons. By using double immunolabeling technique for OTC and neuronal nitric oxide synthase (nNOS) OTC protein expression was revealed in a small subset of nitrergic (nNOS) neurons. Since citrulline (the reaction product of OTC) enhances the bioavailability of L-arginine, the substrate of nNOS, it is conceivable that OTC activity supports NO production in nitrergic neurons.

     

Growth hormone-releasing hormone and gonadotropin-releasing hormone stimulate nitric oxide production in 17beta-estradiol-primed rat anterior pituitary cells

It was reported that neuronal nitric oxide synthase (nNOS) was expressed only in gonadotrophs and folliculo-stellate cells in the anterior lobe of the pituitary gland. However, recent studies have demonstrated the occurrence of nNOS in the somatotrophs and lactotrophs. In the present study, we investigated effects of growth hormone-releasing hormone (GHRH), gonadotropin-releasing hormone (GnRH), and 17β-estradiol on nitric oxide (NO) release in cultured rat anterior pituitary cells in vitro. The NO ₂ ⁻ level in the incubation medium of the rat anterior pituitary cells was dependent on the cell density. Pretreatment with 10 μM 17β-estradiol resulted in an increase in medium NO ₂ ⁻ level. GHRH and GnRH failed to change medium NO ₂ ⁻ levels, but they elicited increases in medium NO ₂ ⁻ levels in estrogen-treated cells. The GHRH-induced increase in NO ₂ ⁻ level was inhibited by Nχ-nitro-l-arginine methyl ester, a NOS inhibitor. These findings suggest that GnRH and GHRH could activate nNOS in the gonadotrophs and the somatotrophs, respectively.     

A comparative study of the effects of nitric oxide and carbon monoxide on the in vivo release of gonadotropin-releasing hormone and neuropeptide Y from rat hypothalamus during the estradiol-induced luteinizing hormone surge: estimation by push-pull perfusion

Recent evidence suggests that nitric oxide (NO), a free radical gas, plays an important role in regulating the function of a variety of neuroendocrine systems. With respect to the hypothalamo-pituitary-gonadal axis, a stimulatory effect of NO on the release of gonadotropin-releasing hormone (GnRH) from rat hypothalamus has been demonstrated in vitro. However, no previous study has reported NO-stimulated secretion of GnRH from in vivo hypothalamus, and also the precise cellular site of action of NO within the GnRH neuronal system remains to be elucidated. In the present study, utilizing the push-pull perfusion technique of rat hypothalamus, we examined the effect of L-arginine (L-Arg), an NO donor, on the release of GnRH, neuropeptide Y and cyclic GMP (c-GMP), which is a pivotal second messenger molecule of the NO system. For comparison, we also examined the effect of carbon monoxide (CO), another putative gaseous neurotransmitter, using hematin, a CO donor. During the period of 11.00-18.00 h, we collected blood and hypothalamic perfusates from ovariectomized adult rats that had been implanted with an estradiol capsule 2 days before. During the entire period of observation, L-Arg (1.0 or 10 mM), hematin (10 or 100 microM) or artificial cerebrospinal fluid alone (as the control) was infused into the medial preoptic area (MPOA) where there are cell bodies of GnRH neurons, or the median eminence-arcuate nucleus complex (ME-ARC) where axon terminals of GnRH neurons are localized. Although 10 mM of L-Arg significantly stimulated GnRH and c-GMP, but not neuropeptide Y, levels in both the MPOA and ME-ARC, GnRH and c-GMP in the ME-ARC were already increased by 1.0 mM of L-Arg. By contrast, both concentrations of hematin were without effect at either site of the hypothalamus. This study is the first to demonstrate that NO is capable of stimulating GnRH release from rat hypothalamus in vivo. Our data also suggests that both cell bodies and axon terminals of GnRH neurons may be sites of action of NO. Our data do not support a previous study by other investigators that reported a stimulatory effect of CO on the GnRH release.     

Projections of arcuate nucleus and rostral periventricular kisspeptin neurons in the adult female mouse brain

The important role of kisspeptin neurons in the regulation of GnRH neuron activity is now well accepted. However, the ways in which kisspeptin neurons located in the arcuate nucleus (ARN) and rostral periventricular area of the third ventricle (RP3V) control GnRH neurons are poorly understood. The present study used anterograde and retrograde tracing techniques to establish the neuronal projection patterns of kisspeptin cell populations in the female mouse brain. Anterograde tracing studies revealed that kisspeptin neurons in the ARN innervated a wide number of hypothalamic and associated limbic region nuclei, whereas RP3V kisspeptin neurons projected to a smaller number of mostly medially located hypothalamic nuclei. Retrograde tracing confirmed a major projection of RP3V kisspeptin neurons to the ARN and showed that kisspeptin neurons located in the rostral half of the ARN projected to the rostral preoptic area. Peripheral administration of Fluorogold was found to label the majority of GnRH neurons but no kisspeptin neurons. Together, these studies highlight the complexity of the brain kisspeptin neuronal system and indicate that both ARN and RP3V kisspeptin neurons participate in a variety of limbic functions. In relation to the GnRH neuronal network, these investigations demonstrate that, alongside the RP3V kisspeptin cells, rostral ARN kisspeptin neurons may also project to GnRH neuron cell bodies. However, no kisspeptin neurons innervate GnRH nerve terminals in the external layer of the median eminence. These studies provide a neuroanatomical framework for the further elucidation of the functions of the ARN and RP3V kisspeptin neuron populations.     

Evidence for a spontaneous nitric oxide release from the rat median eminence: influence on gonadotropin-releasing hormone release

The involvement of nitric oxide (NO) as a gaseous neurotransmitter in the hypothalamic control of pituitary LH secretion has been demonstrated. NO, as a diffusible signaling gas, has the ability to control and synchronize the activity of the neighboring cells. NO is secreted at the median eminence (ME), the common termination field for the antehypophysiotropic neurons, under the stimulation of other signaling substances. At the ME, NO stimulates GnRH release from neuroendocrine terminals. The present studies were undertaken to determine whether NO is secreted spontaneously from ME fragments ex vivo and whether its secretion is correlated to GnRH release. To accomplish this, female rats were killed at different time points of the day and/or of the estrous cycle. The spontaneous NO release was monitored in real time, with an amperometric probe, during 4 periods of 30 min, from individual ME fragments (for each time point, n = 4). GnRH levels were measured in parallel for each incubation-period by RIA. The results revealed that NO was released in a pulsatile manner from female ME fragments and, unambiguously, that the amplitude of NO secretion varied markedly across the estrous cycle. Indeed, though the NO pulse period (32 +/- 1 min, n = 36) and duration (21 +/- 2 min, n = 36) did not vary significantly across the estrous cycle, the amplitude of this secretion pulse was significantly higher on proestrus (Pro; 39 +/- 3 nM, n = 20), compared with diestrus (16 +/- 1 nM, n = 8) or estrus (23 +/- 3 nM, n = 8, P < 0.05). The GnRH levels in the incubation medium were positively correlated to NO secretion across the estrous cycle (r = 0.86, P < 0.003, n = 9), confirming that NO and GnRH release are coupled. Furthermore, 5 x 10(-7) M L-N(5)-(1-iminoethyl)ornithine (L-NIO), a NO synthase inhibitor, succeeded in inhibiting the strong NO-GnRH secretory coupling and GnRH release on PRO: Because at this concentration, L-NIO selectively inhibits endothelial NO synthase, the results further demonstrate that the major source of NO involved in GnRH release at the ME is endothelial in origin. Additionally, the induction of a massive NO/GnRH release in 15-day ovariectomized rat treated with estradiol benzoate strongly suggested that estradiol is participating in the stimulation of NO release activity between diestrus II and PRO: The present study is the first demonstrating that ME can spontaneously release NO and that NO's rhythm of secretion varies markedly across the estrous cycle. This pulsatile/cyclic ME NO release may constitute the synchronizing link to anatomically scattered GnRH neurons.     

Vascular-brain signaling in hypertension: Role of angiotensin II and nitric oxide

Paracrine signaling by nitric oxide (NO) released from microvasculature within the brain affects multiple neuronal functions. Reviewed here is a role in central cardiovascular control. Within the nucleus tractus solitarii (NTS), a major regulatory region for arterial pressure, angiotensin II stimulates NO generation from endothelial nitric oxide synthase (eNOS). This enhances γ-aminobutyric acid release to depress baroreflex function. In the spontaneously hypertensive rat (SHR), eNOS mRNA in the NTS is elevated compared to normotensive rats. Chronic inhibition of eNOS activity in the NTS of SHR reduced arterial pressure and increased baroreflex gain. Thus, eNOS-generated NO in the NTS plays a major role in control of baroreflex gain and arterial pressure. Indeed, its activity contributes to hypertension in the SHR. We propose that eNOS-generated NO in the SHR may be a compensatory mechanism for any potential threat to an adequate blood supply to the brain (eg, from genetically small arteries supplying the brainstem).     

NELL2, a neuron-specific EGF-like protein, is selectively expressed in glutamatergic neurons and contributes to the glutamatergic control of GnRH neurons at puberty

NELL2, a protein containing EGF-like repeats, is almost exclusively expressed in the nervous system. In the mammalian brain, NELL2 expression is mostly neuronal. NELL2 was previously found to be a secreted protein that functions during embryonic development as a neuronal differentiation and survival factor. We now show that the Nell2 gene is selectively expressed in the two major subtypes of glutamatergic neurons described in the postnatal brain: those containing the vesicular glutamate transporter 1 and those expressing vesicular glutamate transporter 2. No Nell2 mRNA is detected in GABAergic neurons. Likewise, GnRH neurons are devoid of NELL2. During prepubertal development of the female rat, Nell2 mRNA abundance increases selectively in the medial basal hypothalamus, reaching maximal values at the end of the juvenile period, to decline at the time of puberty to intermediate levels. Similar, but less pronounced changes are observed in the preoptic area, but they are absent in the cerebral cortex. A well-established glutamatergic function in the neuroendocrine brain is to enhance release of GnRH, the neurohormone controlling sexual development and the time of puberty. In vivo disruption of NELL2 synthesis via intraventricular administration of antisense oligodeoxynucleotides reduced GnRH release from the medial basal hypothalamus and delayed the initiation of female puberty. These results identify NELL2 as a new component of glutamatergic neurons and provide evidence for its physiological involvement in a major, glutamate-dependent, process of neuroendocrine regulation.     

Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity.

Using antibodies that react selectively with peptide sequences unique to endothelial nitric oxide synthase (eNOS), we demonstrate localizations to neuronal populations in the brain. In some brain regions, such as the cerebellum and olfactory bulb, eNOS and neuronal NOS (nNOS) occur in the same cell populations, though in differing proportions. In the hippocampus, localizations of the two enzymes are strikingly different, with eNOS more concentrated in hippocampal pyramidal cells than in any other brain area, whereas nNOS is restricted to occasional interneurons. In many brain regions NADPH diaphorase staining reflects NOS catalytic activity. Hippocampal pyramidal cells do not stain for diaphorase with conventional paraformaldehyde fixation but stain robustly with glutaraldehyde fixatives, presumably reflecting eNOS catalytic activity. eNOS in hippocampal pyramidal cells may generate the NO that has been postulated as a retrograde messenger of long-term potentiation.     

Huntingtin-associated protein (HAP1): discrete neuronal localizations in the brain resemble those of neuronal nitric oxide synthase.

Huntington disease stems from a mutation of the protein huntingtin and is characterized by selective loss of discrete neuronal populations in the brain. Despite a massive loss of neurons in the corpus striatum, NO-generating neurons are intact. We recently identified a brain-specific protein that associates with huntingtin and is designated huntingtin-associated protein (HAP1). We now describe selective neuronal localizations of HAP1. In situ hybridization studies reveal a resemblance of HAP1 and neuronal nitric oxide synthase (nNOS) mRNA localizations with dramatic enrichment of both in the pedunculopontine nuclei, the accessory olfactory bulb, and the supraoptic nucleus of the hypothalamus. Both nNOS and HAP1 are enriched in subcellular fractions containing synaptic vesicles. Immunocytochemical studies indicate colocalizations of HAP1 and nNOS in some neurons. The possible relationship of HAP1 and nNOS in the brain is reminiscent of the relationship of dystrophin and nNOS in skeletal muscle and suggests a role of NO in Huntington disease, analogous to its postulated role in Duchenne muscular dystrophy.     

Forebrain gonadotropin-releasing hormone neuronal development: insights from transgenic medaka and the relevance to X-linked Kallmann syndrome

Neurons that synthesize and release GnRH are essential for the central regulation of reproduction. Evidence suggests that forebrain GnRH neurons originate in the olfactory placode and migrate to their final destinations, although this is still a matter of controversy. X-linked Kallmann syndrome (X-KS), characterized by failed gonadal function secondary to deficient gonadotropin secretion, is caused by a mutation in KAL1, which is suggested to regulate the migration of forebrain GnRH neurons. Because rodents lack Kal1 in their genome and have GnRH neurons scattered throughout their forebrain, the development of forebrain GnRH neurons and the pathogenesis of X-KS have been difficult to study. In the present study, we generated transgenic medaka that expressed green fluorescent protein under the control of the gnrh1 and gnrh3 promoters for analyzing forebrain GnRH neuronal development. Our data revealed the presence of the following four gnrh1 neuronal populations: an olfactory region-derived ventral preoptic population, a dorsal preoptic population that migrates from the dorsal telencephalon, a medial ventral telencephalic population that migrates from the anterior telencephalon, and a nonmigratory ventral hypothalamic population. We found that all forebrain gnrh3 neurons, extending from the terminal nerve ganglion to the anterior mesencephalon, arise from the olfactory region and that trigeminal ganglion neurons express gnrh3. Maternal gnrh3 expression was also observed in oocytes and early embryos. We subsequently identified a KAL1 ortholog and its paralogous form in the medaka. Consistent with the X-KS phenotype, antisense knockdown of the medaka KAL1 ortholog resulted in the disruption of forebrain GnRH neuronal migration. Thus, these transgenic medaka provide a useful model system for studying GnRH neuronal development and disorders of GnRH deficiency.     

Thyroid hormone and estrogen regulate brain region-specific messenger ribonucleic acids encoding three gonadotropin-releasing hormone genes in sexually immature male fish, Oreochromis niloticus

The present study was undertaken to determine whether T3, estrogen, and 11-ketotestosterone could alter a specific population of GnRH-containing neurons, as indicated by a change in messenger RNA (mRNA) levels in sexually immature male tilapia, Oreochromis niloticus. Two weeks after castration, fish were assigned to four treatment groups. One group served as the control (sesame oil); a single ip injection of (T3; 5 microg/g), estradiol benzoate (EB; 5 microg/g), or 11-ketotestosterone (KT; 5 microg/g) was administered to the remaining three groups. Twenty-four hours after the injection, brains were collected and processed for in situ hybridization histochemistry using 35S-labeled 30-mer antisense oligonucleotide probes complementary to the GnRH-coding region of chicken II, salmon, and seabream GnRH. Computerized image analysis was performed to quantify mRNA concentrations, neuronal numbers, and neuronal size of the terminal nerve-nucleus olfactoretinalis, preoptic, and midbrain GnRH neurons. KT had no effect on any of the above neuronal parameters examined for salmon or seabream GnRH. Neither T3, EB, nor KT was effective to induce changes in midbrain chicken GnRH II mRNA concentrations, neuronal numbers, and neuronal size, indicating that an as yet unknown regulatory mechanism may operate midbrain GnRH neurons. T3 specifically suppressed the concentration of terminal nerve salmon GnRH mRNA, and EB significantly increased preoptic seabream GnRH neuronal numbers. These results are consistent with the hypothesis that thyroid hormone, by suppressing terminal nerve GnRH expression, promotes inhibition of sexual maturation. Furthermore, the failure of KT, a nonaromatizable androgen, to influence preoptic GnRH neurons emphasizes that an estrogenic pathway, at the onset of sexual maturation, is responsible for the recruitment of additional preoptic GnRH neurons that are fundamental to reproduction and behavior.     

Increased neuronal nitric oxide synthase-derived NO production in the failing human heart

Experimental data suggest that nitric oxide (NO) generated from neuronal NO synthase (nNOS) modulates the myocardial inotropic state. To assess the contribution of NO, derived from endothelial and neuronal isoforms, to the pathophysiology of congestive heart failure in human beings, we compared expression, localisation, and specific activity of NOS isoforms in myocardium from patients with dilated cardiomyopathy with those in controls who had died from head trauma or intracranial bleeds. Diseased hearts had a significant increase in nNOS mRNA and protein expression, and activity associated with the translocation of nNOS to the sarcolemma through interactions with caveolin 3. Enhanced nNOS activity counteracted a decrease in eNOS expression and activity. Our results provide evidence of increased nNOS-derived NO in the failing human heart. Such altered regulation may be important in the pathophysiology of cardiac dysfunction in human congestive heart failure.