Medial Basal Hypothalamic Explants Research Articles

Hypothalamic cocaine- and amphetamine-regulated transcript (CART) and agouti-related protein (AgRP) neurons coexpress the NOP1 receptor and nociceptin alters CART and AgRP release.

Nociceptin or orphanin FQ (N/OFQ) and its receptor NOP1 are expressed in hypothalamic nuclei involved in energy homeostasis. N/OFQ administered by intracerebroventricular or arcuate nucleus (ARC) injection increases food intake in satiated rats. The mechanisms by which N/OFQ increases food intake are unknown. We hypothesized that N/OFQ may regulate hypothalamic neurons containing peptides involved in the control of food intake such as cocaine- and amphetamine-regulated transcript (CART), alphaMSH, neuropeptide Y (NPY), and agouti-related protein (AgRP). We investigated the ability of N/OFQ to alter the release of CART, alphaMSH, NPY, and AgRP using ex vivo medial basal hypothalamic explants. Incubation of hypothalamic explants with N/OFQ (1, 10, 100 nM) resulted in significant changes in CART and AgRP release. One hundred nanomoles N/OFQ caused a 33% decrease in release of CART (55-102) immunoreactivity (IR) and increased release of AgRP-IR to 163% but produced no change in either alphaMSH-IR or NPY-IR. Double immunocytochemistry/in situ hybridization demonstrated that CART-IR and NOP1 mRNA are colocalized throughout the hypothalamus, in particular in the paraventricular nucleus, lateral hypothalamus, zona incerta, and ARC, providing an anatomical basis for N/OFQ action on CART release. Dual in situ hybridization demonstrated that AgRP neurons in the ARC also express the NOP1 receptor. Our data suggest that nociceptin via the NOP1 receptor may increase food intake by decreasing the release of the anorectic peptide CART and increasing the release of the orexigenic peptide AgRP.

Alcohol inhibits luteinizing hormone-releasing hormone release by activating the endocannabinoid system

We hypothesized that ethanol (EtOH) might act through the endocannabinoid system to inhibit luteinizing hormone-releasing hormone (LHRH) release. Therefore, we examined the mechanism by which EtOH and anandamide (AEA), an endogenous cannabinoid, inhibit LHRH release from incubated medial basal hypothalamic explants. In previous work, we demonstrated that EtOH inhibits the N-methyl-D-aspartic acid-stimulated release of LHRH by increasing the release of two neurotransmitters: beta-endorphin and gamma-aminobutyric acid (GABA). In the present work, bicuculline, a GABAergic antagonist, completely prevented the inhibition of AEA (10(-9)M) on N-methyl-D-aspartic acid-induced LHRH release, but naltrexone, a micro-opioid receptor antagonist, had no effect. AEA also significantly increased GABA release but had no effect on beta-endorphin release. Therefore, AEA could inhibit LHRH release by increasing GABA but not beta-endorphin release. Because EtOH and AEA acted similarly to inhibit LHRH release, we investigated whether both substances would affect the adenylate cyclase activity acting through the same GTP-coupled receptors, the cannabinoid receptors 1 (CB1-rs). AEA and EtOH (10(-1)M) reduced the forskolin-stimulated accumulation of cAMP, but AM251, a specific antagonist of CB1-r, significantly blocked that inhibition. Additionally we investigated whether CB1-r is involved in the inhibition of LHRH by EtOH and AEA. AEA and EtOH reduced forskolin-stimulated LHRH release, but AM251 significantly blocked that inhibition. Also, we demonstrated that EtOH did not act by increasing AEA synthase activity to inhibit LHRH release in our experimental conditions. Therefore, our results indicate that EtOH inhibits the release of LHRH acting through the endocannabinoid system.

Ascorbic acid stimulates gonadotropin release by autocrine action by means of NO.

Because high concentrations of ascorbic acid (AA) are found in the adenohypophysis, we hypothesized that it might have an acute effect on the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the gland, particularly because we have reported that AA rapidly inhibits stimulated LH-releasing hormone (LHRH) release from medial basal hypothalamic explants. Incubation of anterior pituitary halves from adult male rats with graded concentrations of AA for 1 h induced highly significant release of both FSH and LH with a minimal effective concentration of 10(-5) M. Release remained on a plateau from 10(-5) to 10(-2) M. When both AA and an effective concentration of LHRH were incubated together, there was no additive response to LHRH and the response was the same as to either compound alone. The FSH and LH release in response to AA was blocked by incubation with N(G)-monomethyl-l-arginine (NMMA) (300 microM), a competitive inhibitor of NO synthase. NMMA also inhibited LHRH-induced LH and FSH release and gonadotropin release in the presence of both LHRH and AA, whereas sodium nitroprusside, a releaser of NO, stimulated LH and FSH release. Membrane depolarization caused by incubation in high potassium (K(+) = 28 or 56 mM) medium stimulated release of FSH, LH, and AA that was blocked by NMMA. We hypothesize that AA is released with FSH and LH from secretory granules. AA is transported back into gonadotropes by the AA transporter and increases intracellular [Ca(2+)]-activating NO synthase that evokes exocytosis of gonadotropins and AA by cGMP.

The effects of ciliary neurotrophic factor on the hypothalamo-pituitary gonadal axis in vitro in female rats.

Ciliary neurotrophic factor (CNTF) is a member of the neuropoietic family of cytokines. CNTF exerts its actions through activation of a receptor complex, which shows similarity of sequence, second messenger systems and distribution to the leptin receptor. Leptin has been demonstrated to exert profound effects on the hypothalamo-pituitary gonadal axis. This study examines the in vitro effects of CNTF on hypothalamic luteinizing hormone releasing hormone release (LHRH) and pituitary luteinizing hormone (LH) release compared to those of leptin in the female. We report that CNTF stimulates LHRH release from medial basal hypothalamic explants harvested from proestrous female rats and this effect is of similar magnitude to that seen with leptin. In contrast, CNTF suppresses LHRH-stimulated LH release from dispersed anterior pituitary cells harvested from proestrous female rats but has no effect on basal LH release. Leptin stimulates basal LH release but has no effect on LHRH-stimulated LH release. The suppressive effect of CNTF on LHRH-stimulated LH release has been confirmed in perifused anterior hemipituitaries. These results suggest a differential effect of CNTF on the hypothalamo-pituitary gonadal axis and a possible role in the modulation of pituitary gonadal function.

Inhibitory pathways and the inhibition of luteinizing hormone-releasing hormone release by alcohol.

In this research we examined the mechanisms by which ethanol (EtOH) inhibits luteinizing hormone-releasing hormone (LHRH) release from incubated medial basal hypothalamic explants. EtOH (100 mM) stimulated the release of two inhibitory neurotransmitters: gamma-aminobutyric acid (GABA) and beta-endorphin. EtOH also inhibited NO production, indicative of a suppression of nitric oxide synthase (NOS) activity. This inhibition was reversed by naltroxone (10(-8) M), a micro-opioid receptor blocker, indicating that the inhibition of NOS by EtOH is mediated by beta-endorphin. EtOH also blocked N-methyl-d-aspartic acid-induced LHRH release, but the blockade could not be reversed by either the GABA receptor blocker, bicuculline (10(-5) M), naltroxone (10(-8) M), or both inhibitors added together. However, increasing the concentration of naltrexone (10(-6) M) but not bicuculline (10(-4) M) reversed the inhibition. When we lowered the concentration of EtOH (50 mM), the EtOH-induced blockade of LHRH release could be reversed by either bicuculline (10(-5) M), naltroxone (10(-8) M), or the combination of the two blockers. Therefore, GABA is partially responsible for the blockade of N-methyl-d-aspartic acid-induced LHRH release. The block by GABA was exerted by inhibiting the activation of cyclooxygenase by NO, because it was reversed by prostaglandin E(2), the product of activation of cyclooxygenase. Because the inhibition caused by the higher concentration of EtOH could not be reduced by bicuculline (10(-4) M) but was blocked by naltroxone (10(-6) M), the action of alcohol can be accounted for by stimulation of beta-endorphin neurons that inhibit LHRH release by inhibition of activation of NOS and stimulation of GABA release.

Ascorbic acid acts as an inhibitory transmitter in the hypothalamus to inhibit stimulated luteinizing hormone-releasing hormone release by scavenging nitric oxide.

Because ascorbic acid (AA) is concentrated in synaptic vesicles containing glutamic acid, we hypothesized that AA might act as a neurotransmitter. Because AA is an antioxidant, it might therefore inhibit nitric oxidergic (NOergic) activation of luteinizing hormone-releasing hormone (LH-RH) release from medial basal hypothalamic explants by chemically reducing NO. Cell membrane depolarization induced by increased potassium concentration [K(+)] increased medium concentrations of both AA and LH-RH. An inhibitor of NO synthase (NOS), N(G)-monomethyl-l-arginine (NMMA), prevented the increase in medium concentrations of AA and LH-RH induced by high [K(+)], suggesting that NO mediates release of both AA and LH-RH. Calcium-free medium blocked not only the increase in AA in the medium but also the release of LH-RH. Sodium nitroprusside, which releases NO, stimulated LH-RH release and decreased the concentration of AA in the incubation medium, presumably because the NO released oxidized AA to dehydro-AA. AA (10(-5) to 10(-3) M) had no effect on basal LH-RH release but completely blocked high [K(+)]- and nitroprusside-induced LH-RH release. N-Methyl-d-aspartic acid (NMDA), which mimics the action of the excitatory amino acid neurotransmitter glutamic acid, releases LH-RH by releasing NO. AA (10(-5) to 10(-3) M) inhibited the LH-RH-releasing action of NMDA. AA may be an inhibitory neurotransmitter that blocks NOergic stimulation of LH-RH release by chemically reducing the NO released by the NOergic neurons.

Agouti Related Peptide (Agrp) Stimulates the Hypothalamo Pituitary Gonadal Axis in Vivo & in Vitro in Male Rats

Abstract. Agouti related peptide (Agrp), the endogenous antagonist of the melanocortin 3 and 4 receptors (MC3-R and MC4-R), is expressed at high levels in the arcuate nucleus. Agrp immunoreactive terminals are found in the medial preoptic area (MPO), an area that also contains luteinizing hormone releasing hormone (LHRH) neurons. This study was designed to examine the action of Agrp and αMSH on gonadotropin release in vivo following intracerebroventricular (ICV) injection and on LHRH and luteinizing hormone (LH) release in vitro in male rats. We report that ICV administration of Agrp (83–132) significantly increased plasma LH at 40 min post-injection (Agrp (83–132) 2 nmol 0.6 ± 0.1 vs. saline 0.4 ± 0.02 ng/ml, p < 0.05). Plasma follicle stimulating hormone (FSH) was also significantly increased by Agrp (83–132) at 40 min post-injection (Agrp (83–132) 2 nmol 15.6 ± 2.1 ng/ml vs. saline 9.6 ± 1.1 ng/ml, p < 0.05). Agrp (83–132) significantly stimulated the release of LHRH from medial basal hypothalamic explants (Agrp (83–132) 100 nM 170 ± 23% vs. control 100 ± 20%, p < 0.05). In contrast, Agrp (83–132) (0.1–100 nM) had no effect on either basal or LHRH-stimulated LH release from dispersed anterior pituitary cells (Agrp (83–132) 100 nM 10 ± 0.4 ng/ml vs. control 12 ± 1.3 ng/ml, p = ns; LHRH 10 nM + Agrp (83–132) 100 nM 28 ± 1.5 ng/ml vs. LHRH 10 nM 28 ± 1.2 ng/ml, p = ns). We have demonstrated that Agrp (83–132), which blocks melanocortin receptors, significantly increases plasma LH and FSH following ICV administration and stimulates LHRH release from hypothalamic explants but has no direct effect on LH release from dispersed anterior pituitary cells. These results suggest that the melanocortin system plays a role in the control of the hypothalamo-pituitary gonadal axis and may act as a link between the control of appetite and reproductive function.

Role of nitric oxide in the neuroendocrine responses to cytokines.

During infection, bacterial and viral products, such as bacterial lipopolysaccharide (LPS), cause the release of cytokines from immune cells. These cytokines can reach the brain by several routes. Furthermore, cytokines, such as interleukin-1 (IL-1), are induced in neurons within the brain by systemic injection of LPS. These cytokines determine the pattern of hypothalamic-pituitary secretion which characterizes infection. IL-2, by stimulation of cholinergic neurons, activates neural nitric oxide synthase (nNOS). The nitric oxide (NO) released diffuses into corticotropin-releasing hormone (CRH)-secreting neurons and releases CRH. IL-2 also acts in the pituitary to stimulate adrenocorticotropic hormone (ACTH) secretion. On the other hand, IL-1 alpha blocks the NO-induced release of luteinizing hormone-releasing hormone (LHRH) from LHRH neurons, thereby blocking pulsatile LH but not follicle-stimulating hormone (FSH) release and also inhibiting sex behavior that is induced by LHRH. IL-1 alpha and granulocyte macrophage colony-stimulating factor (GMCSF) block the response of the LHRH terminals to NO. The mechanism of action of GMCSF to inhibit LHRH release is as follows. It acts on its receptors on gamma-aminobutyric acid (GABA)ergic neurons to stimulate GABA release. GABA acts on GABAa receptors on the LHRH neuronal terminal to block NOergic stimulation of LHRH release. This concept is supported by blockade of GMCSF-induced suppression of LHRH release from medial basal hypothalamic explants by the GABAa receptor blocker, bicuculline. IL-1 alpha inhibits growth hormone (GH) release by inhibiting GH-releasing hormone (GHRH) release, which is mediated by NO, and stimulating somatostatin release, also mediated by NO. IL-1 alpha-induced stimulation of prolactin release is also mediated by intrahypothalamic action of NO, which inhibits release of the prolactin-inhibiting hormone dopamine. The actions of NO are brought about by its combined activation of guanylate cyclase-liberating cyclic guanosine monophosphate (cGMP) and activation of cyclooxygenase and lipoxygenase with liberation of prostaglandin E2 and leukotrienes, respectively. Thus, NO plays a key role in inducing the changes in release of hypothalamic peptides induced in infection by cytokines. Cytokines, such as IL-1 beta, also act in the anterior pituitary gland, at least in part via induction of inducible NOS. The NO produced inhibits release of anterior pituitary hormones.

Oxytocin stimulates the release of luteinizing hormone-releasing hormone from medial basal hypothalamic explants by releasing nitric oxide.

Oxytocin induces mating behavior in rats of both sexes. Previous experiments revealed that progesterone-induced sex behavior in ovariectomized, estrogen-primed rats was caused by release of NO from NOergic neurons that stimulated the release of luteinizing hormone-releasing hormone (LHRH). The LHRH activated brain-stem neurons that initiated the lordosis reflex. We hypothesized that oxytocin might similarly release NO in the medial basal hypothalamic region that would stimulate release of LHRH into the hypophyseal portal vessels to release luteinizing hormone. To investigate this hypothesis, medial basal hypothalamic explants were preincubated in Krebs-Ringer bicarbonate buffer for 30 min, followed by a 30-min incubation in fresh Krebs-Ringer bicarbonate buffer containing the compounds to be tested. Oxytocin stimulated LHRH release 3- to 4-fold at the lowest concentration tested (10(-10) M). Values remained at a plateau as the concentration was increased to 10(-7) M and then declined in a concentration-dependent manner, so that there was no stimulation with a concentration of 10(-5) M. Oxytocin (10(-7) M) stimulated release of prostaglandin E2 into the medium, a finding consistent with a role of NO in the response. That NO indeed mediated the action of oxytocin was supported by blockade of the action of oxytocin by the competitive inhibitor of NO synthase (NOS), N(G)-monomethyl-L-arginine (300 microM). Furthermore, oxytocin (10(-9) to 10(-7) M) activated NOS as measured at the end of the experiments. Oxytocin appeared to act to stimulate norepinephrine terminals in the medial basal hypothalamus, which activated NOS by alpha1-adrenergic receptors, because prazocine, an alpha1 receptor blocker, inhibited the LHRH-releasing action of oxytocin. Finally, incubation of neural lobe explants with sodium nitroprusside, a NO releasor, revealed that nitroprusside (300-600 microM, but not 900 microM) inhibited oxytocin release. Therefore, the NO released by oxytocin also diffuses into the oxytocin neuronal endings and inhibits oxytocin release, forming a negative feedback loop. The results indicate that oxytocin is important not only in induction of mating, but also in stimulating LHRH release with subsequent luteinizing hormone discharge that plays a crucial role in reproduction.

Granulocyte-macrophage colony stimulating factor suppresses LHRH release by inhibition of nitric oxide synthase and stimulation of gamma-aminobutyric acid release.

Several cytokines produced by immune cells act within the hypothalamus and/or on the pituitary to produce the pattern of pituitary hormone secretion that characterizes infection. Granulocyte-macrophage colony stimulating factor (GMCSF) was first described as a hematopoietic cytokine; however, its synthesis is also stimulated during infection, and it has been found in glia in the brain. Previous research indicates that interleukin-1 inhibits release of luteinizing hormone-releasing hormone (LHRH) both in vivo and in vitro. In the present study, we determined that GMCSF inhibited the release of LHRH in vitro and evaluated the mechanisms involved. After a 1-hour preincubation in Krebs-Ringer bicarbonate glucose buffer (KRB), medial basal hypothalamic explants were incubated in KRB together with recombinant murine GMCSF for 0.5 h in a Dubnoff metabolic shaker (50 cycles/min) in an atmosphere of 95% O2/5% CO2. LHRH release into the media was determined by radioimmunoassay. At concentrations of 10(-12) and 10(-11) M, GMCSF significantly inhibited LHRH release. There was a U-shaped dose-response curve and LHRH release was not inhibited at lower or higher cytokine concentrations. The inhibition was specific since it was completely blocked by GMCSF antiserum. Since sodium nitroprusside (NP; 300 microM), a releaser of nitric oxide (NO), stimulates LHRH, presumably by acting within the LHRH neurons, we examined the effect of GMCSF (10(-11) M) on NP-induced LHRH release. It completely suppressed NP-induced release of LHRH. Bicuculline (10(-5) M), a gamma-aminobutyric acid (GABA) receptor antagonist, partially reversed the inhibitory effects of GMCSF on LHRH release. This dose completely reversed the suppression of LHRH release induced by GABA. The present results indicate that the inhibitory effects of GMCSF on LHRH release are partially caused by blockade of NO-induced LHRH release by its activation of GMCSF receptors on GABAergic neurons. The stimulated release of GABA acts on the GABA-a receptors on the LHRH terminals to inhibit their response to NO. At the end of the experiment, NO synthase (NOS) activity was measured in the tissue homogenate by the citrulline method. NOS activity was highly significantly reduced by GMCSF (10(-11) M) indicating that part of its suppressive action on LHRH release is mediated by reduction in NOS activity in the medial basal hypothalamus.

Nitric oxide controls the hypothalamic-pituitary response to cytokines.

During infection, bacterial products, such as lipopolysaccharide (LPS), and viral products release cytokines from immune cells. These cytokines reach the brain by several routes. Furthermore, cytokines such as interleukin-1 (IL-1) are induced in central nervous system neurons by systemic injection of LPS. These cytokines determine the pattern of hypothalamic-pituitary secretion which occurs in infection. IL-2, by stimulation of cholinergic neurons, activates neural nitric oxide synthase (NOS). The nitric oxide (NO) released diffuses into corticotropin-releasing hormone (CRH)-secreting neurons and releases CRH. IL-2 also acts in the pituitary to stimulate adrenocorticotropic hormone secretion. On the other hand, IL-1 alpha blocks the NO-induced release of luteinizing-hormone-releasing hormone (LHRH) from neurons, thereby blocking pulsatile luteinizing hormone (LH), but not follicle-stimulating hormone release, and also inhibiting sexual behavior which is induced by LHRH. IL-1 alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) block the response of the LHRH terminals to NO. GM-CSF inhibits LHRH release by acting on its receptors on gamma-aminobutyric acid (GABA)ergic neurons to stimulate GABA release. GABA acts on GABA-A receptors on the LHRH neuronal terminal to block NOergic stimulation of LHRH release. This concept is supported by a blockade of GM-CSF-induced suppression of LHRH release from medial basal hypothalamic explants by the GABA-A receptor blocker, bicuculline. IL-1 alpha inhibits growth hormone (GH) release by inhibiting GH-releasing hormone release mediated by NO and stimulating somatostatin release, also mediated by NO. IL-1 alpha-induced stimulation of prolactin release is also mediated by intrahypothalamic action of NO which inhibits release of the prolactin-inhibiting hormone, dopamine. The actions of NO are brought about by its combined activation of guanylate cyclase liberating cyclic guanosine monophosphate and activation of cyclooxygenase and lipoxygenase, with liberation of prostaglandin E2 and leukotrienes, respectively. Thus, NO plays a key role in inducing the changes in the release of hypothalamic peptides induced in infection by cytokines. Cytokines, such as IL-1 beta, also act in the anterior pituitary gland, at least in part, via induction of inducible NOS. The NO produced alters the release of anterior pituitary hormones.

Endotoxin-induced suppression of the somatotropic axis is mediated by interleukin-1 beta and corticotropin-releasing factor in the juvenile rat.

This study extends the neuroendocrine role of central interleukin-1 beta (IL-1 beta) during the stress of lipopolysaccharide (LPS) challenge to include inhibition of the somatotropic [GH-releasing hormone (GHRH)-somatostatin (SRIF)-GH] axis in juvenile male rats and clarifies the role of CRF in the mediation of LPS/IL-1-induced changes in GHRH and SRIF neurosecretion. The results of the in vivo component of this study demonstrated that LPS treatment (2.5 mg/kg twice daily for 5 days) caused a significant attenuation of body weight gain for 2 days (2.4 +/- 1.7% vs. 10.3 +/- 1.8% BW/day in saline controls; P < 0.05) and failure of catch-up growth thereafter even though a small transient suppression of food intake returned to normal by the second of 4 days of treatment. Associated with the first day of growth attenuation was an acute suppression of all plasma GH parameters, including GH mass (area under the curve, 1.972 +/- 0.1837 vs. 6.402 +/- 1.7 micrograms/ml.6 h for saline controls; P < 0.05), in animals receiving an acute bolus of LPS, which was blocked by prior microinjection of IL receptor antagonist protein (IRAP) into the third ventricle. In contrast, GH parameters associated with the second day of LPS-suppressed body weight gain were increased (GH mass, 9.4 +/- 2.2 vs. 3.5 +/- 0.5 micrograms/ml.4 h in saline controls; P < 0.05). These increases were reversed after another 2 days of LPS treatment. In a series of in vitro experiments using medial basal hypothalamic (MBH) explants incubated with LPS [100 ng/ml alone or with 10(-7) M IRAP or 10(-6) M CRF antagonist (CRF-ANT)], GHRH release from MBH incubated with LPS was significantly greater than that in controls (231 +/- 79% vs. 71 +/- 34% of baseline release; P < 0.05), and this stimulation was antagonized by both IRAP and CRF-ANT. SRIF release was significantly increased by incubation with LPS (163 +/- 28% vs. 97 +/- 20% of the baseline for controls; P < 0.05) and blocked (to 88 +/- 14% of the baseline) by IRAP, but not by CRF-ANT. Finally, when MBH explants were incubated with IL-1 beta (10(-9) M), there was a significant inhibition of in vitro GHRH release (37.9 +/- 6.7% vs. 74.9 +/- 16.6% for controls), which was reversed by IRAP and CRF-ANT, and a significant stimulation of SRIF release (168.7 +/- 37.5% vs. 98.0 +/- 11.6% for controls), which was reversed by IRAP, but not CRF-ANT.(ABSTRACT TRUNCATED AT 400 WORDS)

Nitric oxide inhibits hypothalamic luteinizing hormone-releasing hormone release by releasing gamma-aminobutyric acid.

Nitric oxide synthase (NOS)-containing neurons, termed NOergic neurons, occur in various regions of the hypothalamus, including the median eminence-arcuate region, which plays an important role in controlling the release of luteinzing hormone-releasing hormone (LHRH). We examined the effect of NO on release of gamma-aminobutyric acid (GABA) from medial basal hypothalamic (MBH) explants incubated in vitro. Sodium nitroprusside (NP) (300 microM), a spontaneous releaser of NO, doubled the release of GABA. This release was significantly reduced by incubation of the tissue with hemoglobin, a scavenger of NO, whereas hemoglobin alone had no effect on the basal release of GABA. Elevation of the potassium concentration (40 mM) in the medium increased GABA release 15-fold; this release was further augmented by NP. Hemoglobin blocked the increase in GABA release induced by NP but had no effect on potassium-induced release, suggesting that the latter is not related to NO. As in the case of hemoglobin, NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, had no effect on basal release of GABA, which indicates again that NO is not significant to basal GABA release. However, NMMA markedly inhibited the release of GABA induced by high potassium, which indicates that NO plays a role in potassium-induced release of GABA. In conditions in which the release of GABA was substantially augmented, there was a reduction in GABA tissue stores as well, suggesting that synthesis of GABA in these conditions did not keep up with release of the amine. Although NO released GABA, there was no effect of the released GABA on NO production, for incubation of MBH explants with GABA had no effect on NO release as measured by [14C]citrulline production. To determine whether GABA had any effect on the release of LHRH from these MBH explants, GABA was incubated with the tissue and the effect on LHRH release was determined. GABA (10(-5) or 10(-6) M) induced a 70% decrease in the release of LHRH, indicating that in the male rat GABA inhibits the release of this hypothalamic peptide. This inhibition in LHRH release induced by GABA was blocked by NMMA (300 microM), which indicates that GABA converts the stimulatory effect of NO on LHRH release into an inhibitory one, presumably via GABA receptors, which activate chloride channels that hyperpolarize the cell. Previous results have indicated that norepinephrine stimulates release of NO from the NOergic neurons, which then stimulates the release of LHRH. The current results indicate that the NO released also induces release of GABA, which then inhibits further LHRH release. Thus, in vivo the norepinephrinergic-driven pulses of LHRH release may be terminated by GABA released from GABAergic neurons via NO.

Ethanol inhibits luteinizing hormone-releasing hormone (LHRH) secretion by blocking the response of LHRH neuronal terminals to nitric oxide.

It has previously been shown that alcohol can suppress reproduction in humans, monkeys, and small rodents by inhibiting release of luteinizing hormone (LH). The principal action is via suppression of the release of LH-releasing hormone (LHRH) both in vivo and in vitro. The present experiments were designed to determine the mechanism by which alcohol inhibits LHRH release. Previous research has indicated that the release of LHRH is controlled by nitric oxide (NO). The proposed pathway is via norepinephrine-induced release of NO from NOergic neurons, which then activates LHRH release. In the present experiments, we further evaluated the details of this mechanism in male rats by incubating medial basal hypothalamic (MBH) explants in vitro and examining the release of NO, prostaglandin E2 (PGE2), conversion of arachidonic acid to prostanoids, and production of cGMP. The results have provided further support for our theory of LHRH control. Norepinephrine increased the release of NO as measured by conversion of [14C]arginine to [14C]citrulline, and this increase was blocked by the alpha 1 receptor blocker prazosin. Furthermore, the release of LHRH induced by nitroprusside (NP), a donor of NO, is related to the activation of soluble guanylate cyclase by NO since NP increased cGMP release from MBHs and cGMP also released LHRH. Ethanol had no effect on the production of NO by MBH explants or the increased release of NO induced by norepinephrine. Therefore, it does not act at that step in the pathway. Ethanol also failed to affect the increase in cGMP induced by NP. On the other hand, as might be expected from previous experiments indicating that LHRH release was brought about by PGE2, NP increased the conversion of [14C]arachidonic acid to its metabolites, particularly PGE2. Ethanol completely blocked the release of LHRH induced by NP and the increase in PGE2 induced by NP. Therefore, the results support the theory that norepinephrine acts to stimulate NO release from NOergic neurons. This NO diffuses to the LHRH terminals where it activates guanylate cyclase, leading to an increase in cGMP. At the same time, it also activates cyclooxygenase. The increase in cGMP increases intracellular free calcium, activating phospholipase A2 to provide arachidonic acid, the substrate for conversion by the activated cyclooxygenase to PGE2, which then activates the release of LHRH. Since alcohol inhibits the conversion of labeled arachidonic acid to PGE2, it must act either directly to inhibit cyclooxygenase or perhaps it may act by blocking the increase in intracellular free calcium induced by cGMP, which is crucial for activation of of both phospholipase A2 and cyclooxygenase.

Blockade by interleukin-1-alpha of nitricoxidergic control of luteinizing hormone-releasing hormone release in vivo and in vitro.

Nitric oxide (NO) synthase (NOS), the enzyme that converts arginine into citrulline plus NO, the latter a highly active free radical, occurs in a large number of neurons in the brain, including certain neurons in the hypothalamus. Our previous experiments have shown that norepinephrine (NE)-induced prostaglandin E2 (PGE2) release from medial basal hypothalamic explants (MBH) is mediated by NO. Because release of luteinizing hormone (LH)-releasing hormone (LHRH) is also driven by NE and PGE2, we hypothesized that NO controls pulsatile release of LHRH in vivo, which in turn induces pulsatile LH release. Indeed, in vivo and in vitro experiments using an inhibitor of NOS (NG-monomethyl-L-arginine; NMMA) demonstrated that pulsatile LH release is mediated by NO; LHRH release in vitro is also mediated by this free radical. Cytokines that are released from cells of the immune system during infection also inhibit LHRH release. We compared the action of one such cytokine, interleukin-1 alpha (IL-1 alpha), on LHRH release with that of substances which inhibit or induce NO release. Microinjection of IL-1 alpha (0.06 pmol in 2 microliters) into the third cerebral ventricle (3V) of conscious, castrated male rats had an action similar to that of 3V microinjection of NMMA (1 mg in 5 microliters): it blocked pulsatile LH, but not follicle-stimulating hormone (FSH) release. The only difference between the responses to NMMA and IL-1 alpha was that the latency to onset was greater with IL-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)