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Superfusion of rat hypothalamic slices with 10(-4) M N-methyl-D-aspartic acid (NMDA) resulted in increased release of alpha-melanocyte-stimulating hormone (alpha-MSH). Peptide release was blocked by 10(-6) M NG-nitro-L-arginine methyl ester (L-NAME) a specific competitive inhibitor of nitric oxide synthase but not by the inactive enantiomer D-NAME at 10(-6) M. The inhibition by L-NAME was reversed by the addition of 10(-5) mM L-arginine, an excess of enzyme substrate. Release of nitric oxide products into tissue superfusates was stimulated by a 50 mM concentration of potassium ions and by 10(-4) M NMDA. Potassium-stimulated release was blocked by L-NAME. Basal, potassium-stimulated and NMDA-stimulated release of nitric oxide products were significantly inhibited by the NMDA-receptor antagonist D(-)-2-amino-5-phosphopentanoic acid (AP5) at 10(-4) M and by the NMDA-channel blocker ketamine at 10(-4) M. We conclude that nitric oxide mediates the stimulatory action of glutamic acid on the release of alpha-MSH from the rat hypothalamus.

Release of alpha-melanocyte-stimulating hormone (alpha-MSH) and glutamic acid was quantified from superfused slices of rat hypothalamus. Application of L-glutamic acid 10(-4) M failed to evoke release of alpha-MSH but, in the presence of 10(-4) M dihydrokainic acid (DHK) an inhibitor of glutamate uptake systems, caused significant stimulation of release. DHK caused gradual and sustained increases in both alpha-MSH and glutamate release. That in alpha-MSH was blocked by 10(-4) M DL-2-amino-5-phosphopentanoic acid, a competitive N-methyl-D-aspartic acid (NMDA)-type glutamate receptor antagonist. We conclude that hypothalamic glutamate is subject to rapid uptake through mechanisms blocked by DHK and that alpha-MSH release is stimulated by endogenous and exogenous glutamate through NMDA-type receptors.

Release of immunoreactive alpha-melanocyte-stimulating hormone (alpha-MSH) from superfused slices of rat hypothalamus was stimulated by the gamma-aminobutyric acid (GABA) receptor antagonist, bicuculline, and inhibited by the benzodiasepine, chlordiazepoxide, an allosteric GABA receptor modulator, demonstrating the presence of tonic inhibition of alpha-MSH release by endogenous GABA in hypothalamic tissue. Chlordiazepoxide increased the effect of exogenous GABA which by inhibiting basal release of alpha-MSH demonstrated that the tonic inhibition was not maximal in the resting state.

N-methyl-D-aspartic acid (NMDA) 10(-4) M stimulated release of immunoreactive alpha-melanocyte-stimulating hormone (alpha-MSH) from superfused slices of rat hypothalamus through receptors which shared common features with other central NMDA-type glutamate receptors. The receptors possessed inhibitory sites for both Mg2+ and ketamine; basal and NMDA-stimulated alpha-MSH release was reduced by high (5 mM) Mg2+ ion concentrations and by 10(-4) M ketamine, whilst use of Mg(2+)-free media led to a prolongation of the NMDA-stimulated response. The receptors were also shown to possess an allosteric glycine site. The glycine site agonist D-serine 10(-4) M potentiated basal and NMDA-stimulated alpha-MSH release whilst the antagonist, 7-chlorokynurenic acid 10(-4) M, reduced NMDA-stimulated release, an effect which was partially reversed by 10(-4) M D-serine.

Increasing evidence supports a neurotransmitter or a neuromodulator action for peptides derived from proopiomelanocortin in the hypothalamus. Peptide release involves sodium, potassium and calcium ion channels and is dependent on the presence of extracellular calcium ions at the time of depolarisation of neuronal membranes. Dopaminergic and gamma-aminobutyric acid-containing neuronal systems inhibit POMC-derived peptide release from the hypothalamus through D2-dopamine and GABAA receptors, respectively. Serotoninergic mechanisms exert a biphasic effect on peptide release being directly stimulatory at low concentrations of serotonin and indirectly inhibitory at higher concentrations via interactions with the endogenous dopaminergic system. Cholinergic and glutamergic drugs stimulate peptide release through nicotinic and N-methyl-D-aspartate receptors, respectively. Finally, circulating steroids regulate the hypothalamic POMC system with testosterone stimulating POMC gene expression whilst oestradiol and glucocorticoids induce an inhibitory control.

Release of alpha-melanocyte-stimulating hormone (alpha-MSH) from frontal slices of rat hypothalamus superfused with oxygenated artificial cerebrospinal fluid (ACSF) was quantified by radioimmunoassay. Control depolarisations with 50 mM KCl-containing ACSF produced significant increases in alpha-MSH release which were partially blocked by 10(-6) M cinanserin, a serotonin (5-HT) receptor antagonist. Superfusion of the tissues with varying concentrations of 5-HT (10(-7) M to 10(-4) M) resulted in an inverted U-shaped dose-response curve, maximum alpha-MSH release being obtained with 10(-6) M 5-HT. Addition of 10(-6) M cinanserin shifted the 5-HT dose-response curve to the right whilst the presence of 10(-8) M flupenthixol, a dopamine receptor antagonist, resulted in a sigmoidal 5-HT dose-response curve. Superfusion with ACSF containing either 10(-7) M fluoxetine, a 5-HT re-uptake inhibitor, or 10(-7) M p-chloroamphetamine, an agent releasing 5-HT, induced significant increases in alpha-MSH release which were abolished in the presence of 10(-6) M cinanserin. These data demonstrate the presence of an endogenous 5-HT system that exerts a biphasic effect on alpha-MSH release. A stimulatory effect caused by lower 5-HT concentrations appears to be a direct action whilst an inhibitory effect at higher concentrations is mediated through an inhibitory endogenous dopaminergic system. A significant proportion of K+-stimulated peptide release is 5-HT-mediated.

Release of alpha-melanocyte-stimulating hormone (alpha-MSH) from slices of rat hypothalamus superfused with artificial cerebro-spinal fluid (ACSF) was quantified by radioimmunoassay. Addition of 10(-6) M quinpirole, a D2-dopamine receptor agonist, to the superfusion medium caused a significant (P less than 0.001) reduction in the amount of alpha-MSH released upon depolarisation with 50 mM potassium from 319 +/- 37% to 110 +/- 16% of basal release in normal ACSF (mean +/- S.E.M.). Basal peptide release in the presence of quinpirole was unaffected. Sulpiride, a D2-dopamine receptor antagonist, at a concentration of 10(-6) M, induced a significant (P less than 0.05) increase of both basal and potassium-stimulated alpha-MSH release to 203 +/- 21% and 447 +/- 88% of basal release in normal ACSF respectively. The latter increases were abolished when sulpiride and quinpirole were added in combination. SK&F 38393-A and SCH 23390, a D1-dopamine agonist and antagonist respectively, had no significant effect on either basal or potassium-stimulated alpha-MSH release. It is proposed that endogenous dopamine exerts an inhibitory control on alpha-MSH release from the rat hypothalamus via D2-dopamine receptors and that in isolated hypothalamic slices there is a tonic inhibition of peptide release due to the activity of this system.

The dopamine agonist apomorphine (10(-6) M) significantly reduced the 50 mM K+-stimulated, Ca2+-dependent release of immunoreactive alpha-melanocyte-stimulating hormone (alpha-MSH) from superfused slices of rat hypothalamus but did not alter baseline peptide release. Flupenthixol (10(-6) M), a dopamine receptor antagonist, significantly increased both basal and 50 mM K+-stimulated alpha-MSH release, possibly via blockade of inhibition caused by endogenous dopamine release. The latter increases were Ca2+-dependent and were inhibited by 10(-6) M apomorphine.

Release of alpha-melanocyte-stimulating hormone (alpha-MSH) from 250 micron frontal slices of rat hypothalamus superfused at 37 degrees C with oxygenated artificial cerebrospinal fluid (ACSF) was quantified in freeze-dried samples of ACSF by radioimmunoassay. Significant reproducible increases in alpha-MSH release were caused by 40-70 mM K+ in ACSF, maximum release being caused by 50 mM K+. Fifty mM K+-stimulated alpha-MSH release was abolished in the absence of Ca2+ from ACSF and by the presence of 10(-6) M tetrodotoxin. Tetrapentylammonium ions, 10(-4) M and 10(-3) M, stimulated dose-dependent increases in alpha-MSH release. The data support a putative neurotransmitter/neuromodulator role for alpha-MSH in the CNS.