Catherine Blasquez

Regulation of α‐Melanocyte‐Stimulating Hormone Release from Hypothalamic Neuronsa

a-Melanocyte-stimulating hormone (a-MSH) is a tridecapeptide amide that was initially isolated and characterized from the intermediate lobe of the pituitary. a-MSH derives from a multifunctional precursor protein, called proopiomelanocortin (POMC), which can generate through proteolytic cleavage various regulatory peptides including corticotropin, melanotropins, lipotropins and endorphins . I The gene encoding POMC is expressed in corticotrope cells of the pars distalis, in melanotrope cells of the pars intermedia, and in two populations of neurons located respectively in the arcuate nucleus of the hypothalamus? and in the caudal portion of the nucleus s01itariu.s.~ POMC neurons of the arcuate nucleus project in a number of brain areas including several hypothalamic nuclei, the median eminence, the septum, the periventricular region of the thalamus, the amygdala, and the rh~mbencephalon.~ POMC-producing neurons of the nucleus sotitarius innervate the caudal rhombencephalon and the spinal cord.5 Processing of POMC occurs in a tissue-specific manner, depending on the enzymatic equipment that is available in each cell type. In corticotrope cells, POMC is cleaved at the level of three dibasic amino acid residues, thus generating a large terminal fragment (16 K peptide), the joining peptide, ACTH, and P-lipotropin. In melanotrope cells and in hypothalamic neurons, further processing occurs leading to the formation of several y-MSHs, a-MSH, corticotropinlike intermediate lobe peptide (CLIP), y-LPH, and P-end~rphin.~.’ A large body of evidence indicates that the neuropeptide a-MSH can be regarded as an authentic neurotransmitter and/or neuromoduiator. Intracerebral injection of a-MSH affects a number of behavioral processes8 such as learning, memory, attention, motivation, and grooming behavior. Furthermore, a-MSH exerts a trophic action on the central nervous system and induces a marked antipyretic effect. Electron microscopic studies revealed that a-MSH-immunoreactive material is sequestered in dense core secretory vesicle^.^ Concurrentiy, it

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)

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

Abstract Reverse-phase high-performance liquid chromatography analysis, coupled with a sensitive radioimmunoassay for α-melanocyte-stimulating hormone (α-MSH), was used to characterize the α-MSH-related peptides stored in the rat hypothalamus or released from perifused hypothalamic slices. Four peaks of α-MSH-like immunoreactivity (α-MSH-LI) co-eluting with synthetic des-Nα-acetyl α-MSH, α-MSH and their respective sulfoxide derivatives were resolved and quantified. In hypothalamic extract, deacetyl α-MSH which was the predominant peptide represented 94.4% of total α-MSH-LI content, while the relative amount of α-MSH was only 5.6%. Analysis of α-MSH-related peptides contained in effluent perifusates showed that deacetyl α-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-Nα-acetyl α-MSH may occur during the process of exocytosis. Since acetylation of α-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 α-MSH-related peptides in the brain.

Neuropeptide Y inhibits α-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.

γ-Aminobutyric acid inhibits the release of α-melanocyte-stimulating hormone from rat hypothalamic slices

Abstract The effect of γ-aminobutyric acid (GABA) on release of α-melanocyte-stimulating hormone (α-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 α-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 α-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 α-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 × 10 −5 M) resulted in a significant and reversible inhibition of K + -induced α-MSH release. The GABA A agonist, muscimol (10 −4 M), produced a prolonged inhibition of K + -evoked α-MSH release, while the GABA B agonist, baclofen (10 −4 M), was devoid of effect on hypothalamic α-MSH release. Bicuculline (10 −4 M), a specific GABA A antagonist, had no effect when added alone to the medium but totally reversed the inhibitory effect of GABA on K + -induced α-MSH release. Taken together these data suggest that exogenous GABA exerts an inhibitory control on α-MSH neurons. Our data also show that the effect of GABA on α-MSH release by hypothalamic neurons is mediated through GABA A -type receptors. To investigate further the involvement of GABA neuronal systems in the regulation of α-MSH release, rats were treated with the GABA transaminase inhibitor AOAA and the α-MSH content was assessed in both the intermediate lobe of the pituitary and in the hypothalamus. Three hours after intraperitoneal injection of aminooxyacetic acid hemihydrochloride (AOAA; 30 mg/kg), a significant increase of α-MSH concentration was observed in pars intermedia and hypothalamic extracts. These results, together with the presence of an abundant network of GABAergic nerve terminals in the basal hypothalamus, support the notion that the neurotransmitter GABA may play a physiological role in the regulation of α-MSH release by hypothalamic neurons.

Characterization of α-melanocyte-stimulating hormone (α-MSH)-like peptides in discrete regions of the rat brain. In vitro release of α-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)

Activation of the GABAA-benzodiazepine receptor complex inhibits proopiomelanocortin gene expression in the rat arcuate nucleus

Activation of the GABA(A)-benzodiazepine receptor complex has previously been shown to inhibit the release of alpha-melanocyte-stimulating hormone (alpha-MSH) from proopiomelanocortin (POMC) neurons of the hypothalamus. To examine whether long-term activation of the GABA(A) receptor may also modulate the expression of the POMC gene in hypothalamic neurons, we have investigated the effect of chronic treatment with the centraltype benzodiazepine receptor agonist clonazepam, alone or in combination with the GABA(A) receptor agonist muscimol, on POMC mRNA levels in four anatomical subdivisions of the arcuate nucleus of the rat hypothalamus, using quantitative in situ hybridization. Clonazepam treatment produced a significant decrease in POMC mRNA levels in all the regions of the arcuate nucleus with the exception of the most rostral one. Administration of both clonazepam and muscimol induced a marked reduction of mRNA levels in all the subdivisions of the arcuate nucleus. Chronic treatment with muscimol and clonazepam also induced a significant decrease in POMC mRNA level in the pars intermedia of the pituitary. These results, together with previous data, indicate that activation of the GABA(A)-benzodiazepine receptor complex inhibits the expression of the POMC gene as well as the release of POMC-derived mature peptides in both hypothalamic neurons and pituitary melanotrophs.

Effects of ions and ionic channel activators or blockers on release of α-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)

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.