Gastric electrical stimulation modulates hypothalamic corticotropin-releasing factor-producing neurons during post-operative ileus in rat.

High-frequency/low-energy gastric electrical stimulation (GES) is an efficient therapy to treat gastric emptying-related disorders but its mechanism of action remains poorly understood. We aimed to assess the effects of high-frequency/low-energy GES on corticotropin-releasing factor (CRF)-producing neurons in the paraventricular nucleus of the hypothalamus (PVN), which are involved in gastric ileus induced by laparotomy. Two electrodes were implanted in the rat gastric antrum during laparotomy, then stimulation (amplitude: 2 mA; pulse duration 330 micros; frequency: 2 Hz; 1 min ON/2 min OFF) or sham stimulation (control group) were applied. Using immunohistochemistry, the number of c-Fos protein-expressing neurons (c-Fos protein-immunoreactive cells, Fos-IR) was quantified in the PVN after 1 h of stimulation. The number of neurons expressing simultaneously c-Fos protein and CRF mRNA was measured by means of immunocytochemistry combined with in situ hybridization. Finally, c-Fos and CRF mRNA levels in the hypothalamus were determined by in situ hybridization or quantitative reverse transcriptase-polymerase chain reaction. Fos-IR in the PVN was significantly decreased 1 h after GES (P<0.05) but was not affected by sub-diaphragmatic vagotomy. The number of neurons containing c-Fos protein and CRF mRNA was lower in the GES group compared with the control group (P<0.05). In addition, c-Fos and CRF mRNA levels in the PVN were significantly decreased by GES (P

Adenosine A2A receptor gene disruption provokes marked changes in melanocortin content and pro-opiomelanocortin gene expression.

A2A receptor knockout (A2AR-/-) mice are more anxious and aggressive, and exhibit reduced exploratory activity than their wild-type littermates (A2AR+/+). Because alpha-melanocyte-stimulating hormone (alpha-MSH) influences anxiety, aggressiveness and motor activity, we investigated the effect of A2AR gene disruption on alpha-MSH content in discrete brain regions and pro-opiomelanocortin (POMC) expression in the hypothalamus and pituitary. No modification in alpha-MSH content was observed in the hypothalamus and medulla oblongata where POMC-expressing perikarya are located. In the arcuate nucleus of the hypothalamus, POMC mRNA levels were not affected by A2AR disruption. Conversely, in A2AR-/- mice, a significant increase in alpha-MSH content was observed in the amygdala and cerebral cortex, two regions that are innervated by POMC terminals. In the pars intermedia of the pituitary, A2AR disruption provoked a significant reduction of POMC mRNA expression associated with a decrease in alpha-MSH content. By contrast, in the anterior lobe of the pituitary, a substantial increase in POMC mRNA and adrenocorticotropin hormone concentrations was observed, and plasma corticosterone concentration was significantly higher in A2AR-/- mice, revealing hyperactivity of their pituitary-adrenocortical axis. Together, these results suggest that adenosine, acting through A2A receptors, may modulate the release of alpha-MSH in the cerebral cortex and amygdala. The data also indicate that A2A receptors are involved in the control of POMC gene expression and biosynthesis of POMC-derived peptides in pituitary melanotrophs and corticotrophs.

Galanin modulates the activity of proopiomelanocortin neurons in the isolated mediobasal hypothalamus of the male rat.

It has become apparent that galanin as well as proopiomelanocortin-derived peptides, such as beta-endorphin, play an important role in the hypothalamic circuitry that regulates neuroendocrine functions and appetite behavior. We have recently shown that GalR1 and GalR2 galanin receptor mRNAs are expressed in proopiomelanocortin neurons of the arcuate nucleus, suggesting a direct modulatory action of galanin on the proopiomelanocortin neuronal system. In the present study, we investigated the effect of galanin on beta-endorphin release and proopiomelanocortin mRNA expression from male rat mediobasal hypothalamic fragments incubated ex vivo. Galanin induced a decrease of spontaneous beta-endorphin release within the first 30-60 min of incubation and this effect was blocked by the galanin receptor antagonist galantide. Co-incubation of galanin with FK-506 (tacrolimus), a calcineurin inhibitor, suppressed the inhibitory effect of galanin on beta-endorphin release, suggesting that calcineurin is involved in the galanin-evoked decrease in beta-endorphin release. Measurement of beta-endorphin levels in the tissues at the end of the incubation period (120 min) revealed that galanin caused a two-fold increase of beta-endorphin peptide concentration in the mediobasal hypothalamic tissues. Concurrently, galanin induced an increase in the mean density of silver grains overlying proopiomelanocortin neurons after 60 min of incubation, an effect antagonized by galantide. Finally, reverse transcription-polymerase chain reaction analysis revealed that the mRNAs for the three galanin receptor subtypes (i.e. GalR1, GalR2, and GalR3) were expressed in the incubated mediobasal hypothalamic fragments. Taken as a whole, our results indicate that galanin plays a modulatory role on proopiomelanocortin neurons and this interrelation contributes to the elucidation of the neural circuitry that controls, among others, gonadotropin-releasing hormone function.

Molecular cloning, characterization of cDNA, and distribution of mRNA encoding the frog prohormone convertase PC1.

Prohormone convertases (PCs) are calcium-dependent serine endoproteases of the subtilisin/kexin family that play a key role in the posttranslational processing of precursors for biologically active peptides. In this study, we have characterized the cDNA encoding PC1 in the European green frog Rana ridibunda. A frog brain cDNA library was screened by using a heterologous probe at low stringency, and a 2.3-kb cDNA clone encoding PC1 was isolated. This cDNA encodes a 736-residue protein with a 26-amino-acid signal peptide. Comparative structural analysis revealed that frog PC1 exhibits a high degree of amino acid identity with its mammalian counterparts, in particular in the subtilisin-like catalytic domain. Northern blot analysis resolved two major transcripts of 3.0 kb and 5.0 kb that were expressed differentially in the brain and pituitary. In situ hybridization studies showed that, in the frog brain, the highest densities of PC1 mRNA are present in the amygdala, the hypothalamus, and the anterior preoptic area. High concentrations of PC1 mRNA also were found in the pars distalis and pars intermedia of the pituitary, whereas the pars nervosa was devoid of hybridization signal. The wide distribution of PC1 mRNA in the brain and pituitary suggests that, in frog, PC1 is involved in the processing of a number of hormone and neuropeptide precursors.

Interrelations between hypothalamic somatostatin and proopiomelanocortin neurons.

Somatostatin receptors were visualized by [125I]-Tyr0-DTrp8-somatostatin radioautography on 35% of arcuate neurons containing proopiomelanocortin (POMC) mRNA, as identified by in situ hybridization using a [35S] labelled riboprobe on 5 microm-thick consecutive sections. Furthermore, double immunohistochemical staining revealed contacts of beta-endorphin or alpha-MSH containing fibres with a majority of somatostatin perikarya in the anterior hypothalamic periventricular nucleus. Taken together, these data indicate that hypothalamic somatostatin and POMC neurons are interconnected. The results are discussed in term of intrahypothalamic control of GH secretion.

Effect of centrally administered neuropeptide Y on hypothalamic and hypophyseal proopiomelanocortin-derived peptides in the rat.

In a previous study, we have shown that neuropeptide Y inhibits the release of alpha-melanocyte-stimulating hormone from the rat hypothalamus in vitro. The aim of the present study was to investigate the possible effect of neuropeptide Y on the regulation of proopiomelanocortin-derived peptides in vivo. Rats received acute or chronic administration of neuropeptide Y in the lateral ventricle and the amount of alpha-melanocyte-stimulating hormone was measured in the hypothalamus and in the neurointermediate lobe of the pituitary. In the same experiments, the amounts of corticotropin-releasing factor and corticotropin were quantified in the hypothalamus and anterior pituitary, respectively. Acute treatment with synthetic neuropeptide Y (0.1 to 10 micrograms/rat) did not modify the amount of alpha-melanocyte-stimulating hormone in the hypothalamus. In contrast, chronic infusion of neuropeptide Y (1.25 micrograms/h) over a seven day period significantly decreased the hypothalamic content of alpha-melanocyte-stimulating hormone, suggesting that neuropeptide Y regulates the synthesis and/or the processing of proopiomelanocortin. Concurrently, we found that both acute and chronic infusion of neuropeptide Y induced a significant reduction in corticotropin-releasing factor in the hypothalamus as well as a significant decrease in alpha-melanocyte-stimulating hormone and corticotropin in the neurointermediate and anterior lobes, respectively. Quantitative in situ hybridization histochemistry showed that chronic administration of neuropeptide Y also caused a reduction of proopiomelanocortin messenger RNA levels both in the intermediate and anterior lobes of the pituitary. Administration of neuropeptide Y (10(-6) M) on perifused rat hypothalamic slices caused a significant increase in corticotropin-releasing factor release.(ABSTRACT TRUNCATED AT 250 WORDS)

Visualization of gamma-aminobutyric acid A receptors on proopiomelanocortin-producing neurons in the rat hypothalamus.

It has recently been shown that gamma-aminobutyric acid (GABA) and central-type benzodiazepine receptor agonists inhibit the expression of the POMC gene and the release of POMC-derived peptides from hypothalamic neurons. To determine whether the inhibitory effect of GABA could be accounted for by a direct action on POMC neurons, we investigated the localization of the beta 1-subunit of the GABAA-benzodiazepine-receptor complex in the arcuate nucleus. Using a monoclonal antibody raised against a synthetic fragment of the beta 1-subunit, we demonstrate the presence of GABAA receptor on POMC neurons. The proportion of POMC neurons that exhibit immunoreactivity for the beta 1-subunit of the GABAA receptor was not significantly different in the posterior portion (73.0-76.0%) and anterior portion (61.3-62.7%) of the arcuate nucleus. The data also revealed that in the arcuate nucleus, a majority of neurons that were immunostained by the antibody to the beta 1-subunit were not POMC positive. The present results support the concept that GABAA and central-type benzodiazepine receptor agonists exert a direct inhibitory action on POMC neurons. The data also indicate the existence of subsets of POMC neurons within the arcuate nucleus.

Neuropeptide Y inhibits alpha-MSH release from rat hypothalamic slices through a pertussis toxin-sensitive G protein.

The arcuate nucleus of the hypothalamus contains various types of peptidergic neurons. In particular, two distinct populations of neurosecretory neurons containing neuropeptide Y (NPY)- and alpha-melanocyte-stimulating hormone (alpha-MSH)-like immunoreactivity have been identified in the arcuate nucleus. Double-labeling immunocytochemical data have recently shown that NPY-containing fibers make synaptic contacts with proopiomelanocortin (POMC) immunoreactive neurons. We have thus investigated the possible effect of NPY on the release of alpha-MSH from rat hypothalamic slices in vitro, using the perifusion technique. NPY significantly inhibited KCl-stimulated alpha-MSH release in a dose-dependent manner. The inhibitory effect of NPY was mimicked by the Y2 agonist, NPY-(13-36), while the Y1 agonist, [Leu31,Pro34]NPY, was devoid of effect. Pretreatment of hypothalamic slices with pertussis toxin (PTX) blocked the inhibitory effect of NPY, suggesting that the action of NPY on POMC neurons is mediated through a PTX-sensitive G protein. These results support the notion that NPY may play a physiological role in the regulation of alpha-MSH release from hypothalamic neurons.

Central-type benzodiazepines inhibit release of alpha-melanocyte-stimulating hormone from the rat hypothalamus.

In a previous work, we have shown that GABA inhibits the release of alpha-melanocyte-stimulating hormone (alpha-melanotropin) from hypothalamic neurons through activation of GABAA receptors [Delbende et al. (1989) Brain Res. 497, 86-93]. Since GABA-gated channel activity can be allosterically modulated by a variety of compounds including benzodiazepines, we have investigated the effect of benzodiazepines in the control of alpha-melanotropin release by the rat basal hypothalamus. This study was conducted in vitro using perifused rat hypothalamic slices and the amount of alpha-melanotropin release was monitored with a sensitive and highly specific radioimmunoassay. Infusion of clonazepam (50 microM), a selective agonist for central-type benzodiazepine binding sites, induced an inhibition of KCl (50 mM)-evoked alpha-melanotropin release. The inhibitory effect of clonazepam was rapid and reversible. Administration of Ro 15-1788 (100 microM), a specific antagonist for central-type benzodiazepine receptors or SR 95531, a GABAA receptor antagonist, completely reversed the inhibitory effect of clonazepam. In addition, Ro 15-1788 and SR 95531 both enhanced the amplitude of the response observed during prolonged KCl infusion on alpha-melanotropin neurons, suggesting the existence of a tonic inhibitory effect of endogenous GABA and/or benzodiazepines in the release of alpha-melanotropin by hypothalamic neurons. To investigate further the effect of benzodiazepines in the regulation of alpha-melanotropin neurons, rats were treated in vivo with clonazepam (5 mg/kg) or the non-selective benzodiazepine receptor agonist diazepam (3 mg/kg). Both compounds caused a significant increase in the content of alpha-melanotropin and beta-endorphin in the rat hypothalamus within 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ions and ionic channel activators or blockers on release of alpha-MSH from perifused rat hypothalamic slices.

The involvement of sodium and chloride ions in the process of alpha-melanocyte-stimulating hormone (a-MSH) release from hypothalamic neurons was investigated using perifused rat hypothalamic slices. Three different stimuli were found to increase a-MSH release from hypothalamic slices: high K+ concentration (50 mM), veratridine (50 microM), and the Na+/K(+)-ATPase inhibitor ouabain (1 mM). Spontaneous or K(+)-evoked a-MSH release was insensitive to the specific Na+ channel blocker tetrodotoxin (TTX; 1.5 microM) and to the blocker of K+ channels tetraethylammonium (TEA; 30 mM) or 4-aminopyridine (4-AP; 4 mM). In contrast, blockage of ouabain-sensitive Na+/K(+)-ATPase increased the resting level of a-MSH and caused a dramatic potentiation of K(+)-evoked a-MSH release. The Na+ channel activator veratridine (50 microM) triggered a-MSH release. This stimulatory effect was blocked by TTX and prolonged by TEA application, indicating the occurrence of voltage-sensitive Na+ and K+ channels on a-MSH neurons. Replacement of Na+ by impermeant choline ions from 95 to 60 mM did not alter K(+)-evoked a-MSH release. Conversely, dramatic reduction of the external Na+ concentration to 16 mM caused a robust increase of a-MSH secretion from hypothalamic neurons, likely through activation of the Na+/Ca2+ exchange system. These data indicate that the depolarizing effect of K+ results from direct activation of voltage-operated Ca2+ channels. The lack of effect of TEA on basal a-MSH release prompted us to investigate the possible involvement of chloride ions in the regulation of the spontaneous activity of a-MSH neurons. Substitution of Cl- for impermeant acetate ions did not affect basal or K(+)-evoked a-MSH release.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of alpha-melanocyte-stimulating hormone (alpha-MSH)-like peptides in discrete regions of the rat brain. In vitro release of alpha-MSH from perifused hypothalamus and amygdala.

The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) is synthesized by discrete populations of hypothalamic neurons which project in different brain regions including the cerebral cortex, hippocampus and amygdala nuclei. The purpose of the present study was to identify the alpha-MSH-immunoreactive species contained in these different structures and to compare the ionic mechanisms underlaying alpha-MSH release at the proximal and distal levels, i.e. within the hypothalamus and amygdala nuclei, respectively. The molecular forms of alpha-MSH-related peptides stored in discrete areas of the brain were characterized by combining high-performance liquid chromatography (HPLC) separation and radioimmunoassay detection. In mediobasal and dorsolateral hypothalamic extracts, HPLC analysis confirmed the existence of a major immunoreactive peak which co-eluted with the synthetic des-N alpha-acetyl alpha-MSH standard. In contrast, 3 distinct forms of immunoreactive alpha-MSH, which exhibited the same retention times as synthetic des-, mono- and di-acetyl alpha-MSH, were resolved in amygdala nuclei, hippocampus, cortex and medulla oblongata extracts. The proportions of acetylated alpha-MSH (authentic alpha-MSH plus diacetyl alpha-MSH) contained in these extrahypothalamic structures were, respectively, 78, 80, 60 and 92% of the total alpha-MSH immunoreactivity. In order to compare the ionic mechanisms underlaying alpha-MSH release from hypothalamic and extrahypothalamic tissues, we have investigated in vitro the secretion of alpha-MSH by perifused slices of hypothalamus and amygdala nuclei. High potassium concentrations induced a marked increase of alpha-MSH release from both tissue preparations. However, a higher concentration of KCl was required to obtain maximal stimulation of amygdala nuclei (90 mM) than hypothalamic tissue (50 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of platelet-activating factor on hypothalamic and hypophyseal pro-opiomelanocortin-related peptides and hypothalamo-pituitary-adrenal axis in the rat.

To examine the effect of platelet-activating factor (PAF-acether) on pro-opiomelanocortin (POMC)-related peptides and on the hypothalamo-pituitary-adrenal axis, we administered PAF-acether and BN 52021, a selective PAF-acether antagonist, to freely moving rats. Minipumps loaded with either PAF-acether (30 micrograms/kg) or the vehicle alone were connected to the jugular vein for 7 days and positioned under the back skin of rats. A group of animals treated with PAF-acether also received 15 mg/kg of BN 52021 orally twice a day. In vivo treatment with PAF-acether alone or in association with BN 52021 did not affect the hypothalamic concentrations of corticotropin-releasing factor (CRF), alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin. Using a perifusion system for rat hypothalamic slices, we did not observe any effect of PAF-acether on spontaneous or potassium-induced release of alpha-MSH in vitro. In addition, treatment of rats with PAF-acether alone or in association with BN 52021 did not modify the alpha-MSH or beta-endorphin concentration in the neurointermediate lobe of the pituitary. In contrast, in vivo administration of PAF-acether caused a significant reduction of ACTH concentration in the anterior lobe of the pituitary and a marked decrease in the corticosterone level in plasma and adrenal glands. The inhibitory effect of PAF-acether was reversed by concomitant administration of BN 52021. The ineffectiveness of PAF-acether to modulate in vitro ACTH release from perifused anterior pituitary fragments ruled out a direct effect of PAF-acether on corticotrophs. These findings support the view that PAF-acether exerts a specific inhibitory effect on the hypothalamo-pituitary-adrenal axis.

gamma-Aminobutyric acid inhibits the release of alpha-melanocyte-stimulating hormone from rat hypothalamic slices.

The effect of gamma-aminobutyric acid (GABA) on release of alpha-melanocyte-stimulating hormone (alpha-MSH) from hypothalamic neurons was investigated in vitro using the perifusion technique. Rat hypothalamic slices were continuously superfused with Krebs-Ringer medium and the release of alpha-MSH in the effluent perifusate was monitored by means of a sensitive and specific radioimmunoassay method. Infusion of 50 mM K+ for 15 min induced a transient increase of alpha-MSH release (5- to 8-fold above the spontaneous level). Infusion of the same dose of K+ for 75 min caused a brief discharge of alpha-MSH during the first 30 min followed by sustained release of the neuropeptide. The effect of GABA was investigated 27 min after the onset of KCl infusion. Application of GABA (5 x 10(-5) M) resulted in a significant and reversible inhibition of K+-induced alpha-MSH release. The GABAA agonist, muscimol (10(-4) M), produced a prolonged inhibition of K+-evoked alpha-MSH release, while the GABAB agonist, baclofen (10(-4) M), was devoid of effect on hypothalamic alpha-MSH release. Bicuculline (10(-4) M), a specific GABAA antagonist, had no effect when added alone to the medium but totally reversed the inhibitory effect of GABA on K+-induced alpha-MSH release. Taken together, these data suggest that exogenous GABA exerts an inhibitory control on alpha-MSH neurons. Our data also show that the effect of GABA on alpha-MSH release by hypothalamic neurons is mediated through GABAA-type receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of voltage-operated calcium channels in alpha-melanocyte-stimulating hormone (alpha-MSH) release from perifused rat hypothalamic slices.

The contribution of voltage-operated calcium (VOC) channels in the mechanism of release of alpha-melanocyte-stimulating hormone (alpha-MSH) from hypothalamic neurons was investigated using perifused rat hypothalamic slices. The stimulatory effect of potassium (50 mM) on alpha-MSH release was completely blocked by cadmium (1 mM) a calcium competitor which indifferently blocks T-, L-and N-type VOC channels. To determine the nature of calcium conductances involved in K+-evoked alpha-MSH release, we have investigated the effect of a VOC channel agonist and 3 antagonists on the secretion of the neuropeptide. Administration of synthetic omega-conotoxin fraction GVIA (1 microM), a peptide toxin which blocks both N- and L-type VOC channels, reduced by 33% K+-induced alpha-MSH release. In contrast, the 1,4-dihydropyridine (DHP) antagonist nifedipine, at concentrations up to 100 microM, did not affect the response of hypothalamic alpha-MSH neurons to depolarizing concentrations of KCl. In addition, the secretion of alpha-MSH induced by high K+ concentrations was not reduced by nifedipine (10 microM) in the presence of diltiazem (1 microM), a benzothiazepine derivative which increases the affinity of the DHP antagonist for L-type VOC channels. The DHP agonist BAY K 8644 (0.1-10 microM) did not modify the early phase of the response of alpha-MSH neurons to K+-induced depolarization. In contrast BAY K 8644 (1 or 10 microM) significantly prolonged the duration of K+-induced alpha-MSH release. This sustained release of alpha-MSH induced by BAY K 8644 (10 microM) was totally suppressed by nifedipine (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of alpha-MSH-related peptides released from rat hypothalamic neurons in vitro.

Reverse-phase high-performance liquid chromatography analysis, coupled with a sensitive radioimmunoassay for alpha-melanocyte-stimulating hormone (alpha-MSH), was used to characterize the alpha-MSH-related peptides stored in the rat hypothalamus or released from perifused hypothalamic slices. Four peaks of alpha-MSH-like immunoreactivity (alpha-MSH-LI) co-eluting with synthetic des-N alpha-acetyl alpha-MSH, alpha-MSH and their respective sulfoxide derivatives were resolved and quantified. In hypothalamic extract, deacetyl alpha-MSH which was the predominant peptide represented 94.4% of total alpha-MSH-LI content, while the relative amount of alpha-MSH was only 5.6%. Analysis of alpha-MSH-related peptides contained in effluent perifusates showed that deacetyl alpha-MSH and its oxidized form were the major peptides released from neurons in basal conditions or under KCl-induced depolarization (50 mM KCl for 75 min). However, the proportion of acetylated peptide was 3-4 times higher in the perifusion medium than in hypothalamic extracts. Our data indicate that acetylation of des-N alpha-acetyl alpha-MSH may occur during the process of exocytosis. Since acetylation of alpha-MSH markedly increases the behavioural potency of the peptide, these results suggest that regulation of the acetyltransferase activity could be a key mechanism to modulate the bioactivity of alpha-MSH-related peptides in the brain.

Hypothalamic alpha-melanocyte-stimulating hormone (alpha-MSH) is not under dopaminergic control.

A possible dopaminergic regulation of hypothalamic proopiomelanocortin (POMC)-containing neurons has been investigated in rats by means of in vivo and in vitro approaches. Acute or 3-weeks chronic in vivo treatments with the dopaminergic agonists apomorphine (1 mg/kg: s.c.) and 2-Br-alpha-ergocriptine (2.5 mg/kg; s.c.) or the dopaminergic antagonist haloperidol (0.15-3 mg/kg; i.p.) had no significant effect on the concentration of alpha-melanocyte-stimulating hormone (alpha-MSH) in two hypothalamic regions: arcuate nucleus (AN) and dorsolateral area (DLH). In the same way, chronic administration of the dopaminergic agonists or antagonist did not induce any change in hypothalamic contents of beta-endorphin, another peptide derived from POMC. Reverse-phase high-performance liquid chromatographic analysis revealed that acetic acid extracts of AN and DLH both contained two major forms of alpha-MSH-like peptides: deacetylated alpha-MSH and authentic alpha-MSH. The ratio between these two forms was not altered after acute haloperidol treatment (3 mg/kg, i.p.). The possible effect of dopamine on the release of hypothalamic alpha-MSH was studied in vitro using perifused rat hypothalamic slices. Infusion of dopamine (10(-7)-10(-5)M) or its antagonist haloperidol (10(-5)M) had no effect on spontaneous alpha-MSH release from hypothalamic tissue. In addition, none of these drugs had any effect on potassium (50 mM)-induced alpha-MSH release. It is concluded that dopaminergic neurons are not involved in the regulation of synthesis, post-translational processing (acetylation) or release of hypothalamic alpha-MSH.

alpha-Melanocyte-stimulating hormone (alpha-MSH) release from perifused rat hypothalamic slices.

A perifusion system was developed to investigate the control of alpha-melanocyte-stimulating hormone (alpha-MSH) release from rat brain. Hypothalamic slices were perifused with Krebs-Ringer bicarbonate (KRB) medium supplemented with glucose, bacitracin and bovine serum albumin. Fractions were set apart every 3 min and alpha-MSH levels were measured by means of a specific and sensitive radioimmunoassay method. Hypothalamic tissue in normal KRB medium released alpha-MSH at a constant rate corresponding to 0.1% of the total hypothalamic content per 3 min. The basal release was not altered by Ca2+ omission in the medium or addition of the sodium channel blocker tetrodotoxin (TTX). Depolarizing agents such as potassium (50 mM) and veratridine (50 microM), which is known to increase Na+ conductance, significantly stimulated alpha-MSH release in a Ca2+-dependent manner. When Na+-channels were blocked by TTX (0.5 microM) the stimulatory effect of veratridine was completely abolished whereas the K+-evoked release was unaffected. These findings suggest that: voltage-dependent sodium channels are present on alpha-MSH hypothalamic neurons; depolarization by K+ induces a marked stimulation of alpha-MSH release; K+- and veratridine-evoke releases are calcium-dependent. Altogether, these data provide evidence for a neurotransmitter or neuromodulator role for alpha-MSH in rat hypothalamus.

Localization and identification of alpha-melanocyte-stimulating hormone (alpha-MSH) in the frog brain.

The distribution of alpha-melanocyte-stimulating hormone (alpha-MSH) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. alpha-MSH-like containing perikarya were localized in the infundibular region, mainly in the ventral hypothalamic nucleus. A rich plexus of immunoreactive fibers directed towards the ventral telencephalic region was detected. Reverse-phase high-performance liquid chromatography and radioimmunoassay were used to characterize alpha-MSH-like peptides in the frog brain. Chromatographic separation revealed that immunoreactive alpha-MSH coeluted with synthetic des-N alpha-acetyl alpha-MSH, authentic alpha-MSH and their sulfoxide derivatives. The heterogeneity of alpha-MSH-like material in the frog brain was in marked contrast with the figure observed in the intermediate lobe of the pituitary gland where only des-N alpha-acetyl alpha-MSH is present. These findings support the existence of discrete alpha-MSH immunoreactive neurons in the frog brain containing both desacetyl and authentic alpha-MSH.