Anesthetic drugs modulate feeding behavior and hypothalamic expression of the POMC polypeptide precursor and the NPY neuropeptide.

BACKGROUND: Several hypnotic drugs have been previously identified as modulators of food intake, but exact mechanisms remain unknown. Feeding behavior implicates several neuronal populations in the hypothalamic arcuate nucleus including orexigenic neuropeptide Y and anorexigenic pro-opiomelanocortin producing neurons. The aim of this study was to investigate in mice the impact of different hypnotic drugs on food consumption and neuropeptide Y or pro-opiomelanocortine mRNA expression level in the hypothalamic arcuate nucleus. METHODS: Saline control, isoflurane, thiopental, midazolam or propofol were administered to C57Bl/6 mice. Feeding behavior was evaluated during 6 h. In situ hybridization of neuropeptide Y and pro-opiomelanocortine mRNAs in the hypothalamus brain region was also performed. Data were analyzed by Kruskal Wallis test and analysis of variance (p < 0.05). RESULTS: Midazolam, thiopental and propofol induced feeding behavior. Midazolam and thiopental increased neuropeptide Y mRNA level (respectively by 106 and 125%, p < 0.001) compared with control. Propofol and midazolam decreased pro-opiomelanocortine mRNA level by 31% (p < 0,01) compared with control. Isoflurane increased pro-opiomelanocortine mRNA level by 40% compared with control. CONCLUSION: In our murine model, most hypnotics induced food consumption. The hypnotic-induced regulation of neuropeptide Y and pro-opiomelanocortine hypothalamic peptides is associated with this finding. Our data suggest that administration of some hypnotic drugs may affect hypothalamic peptide precursor and neuropeptide expression and concomittantly modulate food intake. Thus, this questions the choice of anesthetics for better care management of patients undergoing major surgery or at risk of undernutrition.

Hypothalamic glucose-sensing: role of Glia-to-neuron signaling.

The hypothalamus senses hormones and nutrients in order to regulate energy balance. In particular, detection of hypothalamic glucose levels has been shown to regulate both feeding behavior and peripheral glucose homeostasis, and impairment of this regulatory system is believed to be involved in the development of obesity and diabetes. Several data clearly demonstrate that glial cells are key elements in the perception of glucose, constituting with neurons a "glucose-sensing unit". Characterization of this interplay between glia and neurons represents an exciting challenge, and will undoubtedly contribute to identify new candidates for therapeutic intervention. The purpose of this review is to summarize the current data that stress the importance of glia in central glucose-sensing. The nature of the glia-to-neuron signaling is discussed, with a special focus on the endozepine ODN, a potent anorexigenic peptide that is highly expressed in hypothalamic glia.

Identification and characterization of two novel (neuro)endocrine long coiled-coil proteins.

We have identified a novel vertebrate-specific gene by applying a Differential Display method on two distinct subtypes of pituitary melanotropes showing divergent secretory phenotypes of hypo- and hypersecretion. A paralogue of this gene was also identified. The existence of a long coiled-coil domain and a C-terminal transmembrane domain in the sequences, together with the Golgi distribution of the proteins in transfected cells, suggest that they can be considered as new members of the golgin family of proteins. Both genes were primarily expressed in (neuro)endocrine tissues in vertebrates thus supporting a role for these proteins in the regulated secretory pathway.

Effect of the diazepam-binding inhibitor-derived peptide, octadecaneuropeptide, on food intake in goldfish.

An endogenous ligand of central-type benzodiazepine receptors (CBR), the endozepine octadecaneuropeptide (ODN), is a very potent inhibitor of food intake in rodents. Although endozepines have been localized and characterized in the trout hypothalamus, so far, the action of these neuropeptides on feeding behavior has never been investigated in fish. In the present study, we have examined the effect of i.c.v. administration of synthetic rat ODN, its C-terminal octapeptide (OP) and the head-to-tail cyclic analog cyclo(1-8)OP (cOP) on feeding behavior in the goldfish model. i.c.v. injection of graded doses of ODN (2.5-10 pmol/g body weight (BW)) induced a dose-dependent inhibition of food intake, a significant decrease in cumulative food intake during the 60-min period after feeding being observed at doses of 5 and 10 pmol/g BW. The inhibitory effect of a 10 pmol/g BW dose of ODN on food consumption (-39%) was mimicked by an equimolar dose of OP (-42%) and cOP (-53%). The food intake-suppressing activity of ODN (10 pmol/g BW) was not affected by pre-injection of the CBR antagonist flumazenil (200 pmol/g BW). In contrast, the anorexigenic effect of ODN (10 pmol/g BW) was totally suppressed by a selective antagonist of metabotropic endozepine receptors, cyclo(1-8)[dLeu(5)]OP. These data indicate that, in goldfish as in rodents, ODN is a potent inhibitor of food consumption, and that the anorexigenic effect of ODN is not mediated through CBR but through the metabotropic endozepine receptor.

Differential expression and processing of chromogranin A and secretogranin II in relation to the secretory status of endocrine cells.

Chromogranin A (CgA) and secretogranin II (SgII) are neuroendocrine secretory proteins that participate in regulation of the secretory pathway and also serve as precursors of biologically active peptides. To investigate whether there is a relationship between the expression, distribution, and processing of CgA and SgII and the degree of secretory activity, we employed two melanotrope subpopulations of the pituitary intermediate lobe that exhibit opposite secretory phenotypes. Thus, although one of the melanotrope subtypes shows high secretory activity, the other exhibits characteristics of a hormone storage phenotype. Our data show that SgII expression levels were higher in secretory melanotropes, whereas CgA expression showed similar rates in both cell subsets. The use of various antibodies revealed the presence of the unprocessed proteins as well as three CgA-derived peptides (67, 45, and 30 kDa) and six SgII-derived peptides (81, 66, 55, 37, 32, and 30 kDa) in both subpopulations. However, the smallest molecular forms of both granins predominated in secretory melanotropes, whereas the largest SgII- and CgA-immunoreactive peptides were more abundant in storage melanotropes, which is suggestive of a more extensive processing of granins in the secretory subset. Confocal microscopy studies showed that CgA immunoreactivity was higher in storage cells, but SgII immunoreactivity was higher in secretory melanotropes. Taken together, our results indicate that SgII and CgA are differentially regulated in melanotrope subpopulations. Thus, SgII expression is strongly related to the secretory activity of melanotrope cells, whereas CgA expression may not be related to secretory rate, but, rather, to hormone storage in this endocrine cell type.

Role of androgens and glucocorticoids in the regulation of diazepam-binding inhibitor mRNA levels in male mouse hypothalamus.

In peripheral organs, gonadal and adrenal steroids regulate diazepam-binding inhibitor (DBI) mRNA expression. In order to further investigate the involvement of peripheral steroid hormones in the modulation of brain DBI mRNA expression, we studied by semiquantitative in situ hybridization the effect of adrenalectomy (ADX) and castration (CX) and short-term replacement therapy on DBI mRNA levels in the male mouse hypothalamus. Cells expressing DBI mRNA were mostly observed in the arcuate nucleus, the median eminence and the ependyma bordering the third ventricle. In the median eminence and the ependyma bordering the third ventricule, the DBI gene expression was decreased in ADX rats and a single injection of corticosterone to ADX rats induced a significant increase in DBI gene expression at 3 and 12 h time intervals without completely restoring the basal DBI mRNA expression observed in intact mice. In the arcuate nucleus, ADX and corticosterone administration did not modify DBI mRNA expression. CX down-regulated DBI gene expression in the ependyma bordering the third ventricle. The administration of dihydrotestosterone (3-24 h) completely reversed the inhibitory effect of CX. In the median eminence and arcuate nucleus, neither CX or dihydrotestosterone administration modified DBI mRNA levels. These results suggest that the effects of glucocorticoids on the hypothalamo-pituitary-adrenocortical axis and androgens on the hypothalamo-pituitary-gonadal axis are mediated by DBI.

In vivo action of a new octadecaneuropeptide antagonist on neuropeptide Y and corticotropin-releasing hormone mRNA levels in rat.

It has been reported that several of the effects induced by an octadecaneuropeptide (ODN), derived from an 86-amino-acid polypeptide termed diazepam-binding inhibitor, could be mediated by activation of a metabotropic receptor. In order to investigate the role and mechanism of action of ODN in the regulation of corticotropin-releasing factor (CRH) and neuropeptide Y (NPY) expression in the paraventricular nucleus and arcuate nucleus, respectively, we studied the effects of the acute intracerebroventricular administration of ODN (2 microg/rat) and the ODN antagonist to metabotropic receptor, cyclo(1-8)[Dleu5]OP (20 microg/rat), on the gene expression of the two neuropeptides in castrated male rat. ODN administration resulted in a 45% increase in CRH mRNA expression, an effect which was reversed by cyclo(1-8)[Dleu5]OP. When cyclo(1-8)[Dleu5]OP was administered alone, it induced a 19% decrease in CRH mRNA levels. ODN administration induced a 17% decrease in NPY mRNA expression while cyclo(1-8)[Dleu5]OP increased by 21% the hybridization signal. The administration of both ODN and ODN antagonist completely abolished the depressing effect of ODN on NPY mRNA. These data suggest that the effects of ODN on CRH and NPY mRNA might be mediated by interaction with metabotropic receptors. Moreover, since cyclo(1-8)[Dleu5]OP can by itself influence the expression of two peptide mRNAs, it might be suggested that ODN is exerting a tonic influence on NPY and CRH neurons.

Analysis of Rab18 and a new golgin in the secretory pathway.

Two new amphibian genes have been isolated and characterized from frog melanotropes, and the level of expression of these genes is related to the secretory status of the cells. Both genes, Rab18 and a novel member of the golgin family of proteins, are ubiquitously expressed in endocrine and nonendocrine tissues, and their corresponding proteins appear to show intracellular distributions associated with discrete vesicular and tubular structures, respectively, suggesting that they may play relevant roles in the regulation of the secretory pathway.

In situ hybridization localization of TRH precursor and TRH receptor mRNAs in the brain and pituitary of Xenopus laevis.

We examined the distribution of the mRNAs encoding proTRH and the three TRH receptor subtypes (xTRHR1, xTRHR2, and xTRHR3) in the Xenopus laevis CNS and pituitary. A positive correlation was generally observed between the expression patterns of proTRH and xTRHR mRNAs. xTRHRs were widely expressed in the telencephalon and diencephalon, where two or even three xTRHR mRNAs were often simultaneously observed within the same brain structures. In the pituitary, xTRHR2 was selectively expressed in the distal lobe, and xTRHR3 was found exclusively in the intermediate lobe of white background-adapted animals, indicating that, in amphibians, the effect of TRH on alpha-melanotropin (alpha-MSH) secretion from melanotrope cells is mediated through the novel receptor subtype xTRHR3.

Detection, characterization and biological activities of [bisphospho-thr3,9]ODN, an endogenous molecular form of ODN released by astrocytes.

Astrocytes synthesize and release endozepines, a family of regulatory neuropeptides, including diazepam-binding inhibitor (DBI) and its processing fragments such as the octadecaneuropeptide (ODN). At the molecular level, ODN interacts with two types of receptors, i.e. it acts as an inverse agonist of the central-type benzodiazepine receptor (CBR), and as an agonist of a G protein-coupled receptor (GPCR). ODN exerts a wide range of biological effects mediated through these two receptors and, in particular, it regulates astrocyte activity through an autocrine/paracrine mechanism involving the metabotropic receptor. More recently, it has been shown that Müller glial cells secrete phosphorylated DBI and that bisphosphorylated ODN ([bisphospho-Thr(3,9)]ODN, bpODN) has a stronger affinity for CBR than ODN. The aim of the present study was thus to investigate whether bpODN is released by mouse cortical astrocytes and to compare its potency to ODN. Using a radioimmunoassay and mass spectrometry analysis we have shown that bpODN as well as ODN were released in cultured astrocyte supernatants. Both bpODN and ODN increased astrocyte calcium event frequency but in a very different range of concentration. Indeed, ODN stimulatory effect decreased at concentrations over 10(-10)M whereas bpODN increased the calcium event frequency at similar doses. In vivo effects of bpODN and ODN were analyzed in two behavioral paradigms involving either the metabotropic receptor (anorexia) or the CBR (anxiety). As previously described, ODN (100ng, icv) induced a significant reduction of food intake. Similar effect was achieved with bpODN but at a 10 times higher dose (1000 ng, icv). Similarly, and contrasting with our hypothesis, bpODN was also 10 times less potent than ODN to induce anxiety-related behavior in the elevated zero maze test. Thus, the present data do not support that phosphorylation of ODN is involved in receptor selectivity but indicate that it rather weakens ODN activity.

Primary structure of frog pituitary adenylate cyclase-activating polypeptide (PACAP) and effects of ovine PACAP on frog pituitary.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a peptide of the glucagon-secretin-vasoactive intestinal polypeptide superfamily, was isolated in pure form from the brain of the European green frog, Rana ridibunda. The primary structure of the peptide indicates that evolutionary pressure to conserve the complete amino acid sequence has been very strong. Frog PACAP comprises 38 amino acid residues and contains only 1 substitution (isoleucine for valine at position 35) compared with human/ovine/rat PACAP. In the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, synthetic ovine PACAP-(1-38) produced a dose-dependent increase in the concentration of cAMP in isolated frog anterior pituitary fragments (ED50 = 2.1 +/- 0.6 x 10(-7) M; mean +/- SE; n = 6). Maximum stimulation (an approximately 8-fold increase in concentration over basal values) was produced by 10(-6) M peptide. The truncated form of PACAP [PACAP-(1-27)] also produced a dose-dependent increase in cAMP in frog anterior pituitary fragments, and the potency of the peptide (ED50 = 5.9 +/- 0.6 x 10(-8) M) was comparable to that of PACAP-(1-38). The data suggest, therefore, that the function as well as the structure of PACAP have been conserved during the evolution of amphibia to mammals.

Pituitary adenylate cyclase-activating polypeptide stimulates calcium mobilization in amphibian pituitary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide of the glucagon-secretin-vasoactive intestinal polypeptide superfamily. Although PACAP is a potent stimulator of adenylate cyclase activity in the adenohypophysis, the precise target cells for PACAP in the anterior pituitary remain unknown. The aim of the present study was to investigate whether PACAP could stimulate calcium mobilization in individual cells of the pituitary and to determine the type of cells that responded to PACAP. Enzymatically dispersed frog distal pituitary cells were plated on photoetched coverslips and cultured for 3-7 days. The cells were loaded with the fluorescent calcium indicator indo-1, and changes in intracellular calcium concentrations ([Ca2+]i) were monitored using dual wavelength microfluorimetry. The individual cells were localized with the aid of the alpha/numeric grid of the coverslips and identified retrospectively by immunofluorescence. Approximately 45% of GH and PRL cells and 25% of ACTH and TSH cells responded to PACAP (10(-5) M) ejection by an elevation of [Ca2+]i. Only 16% of gonadotropes were stimulated by PACAP. The time course of [Ca2+]i variations showed three different patterns: transient spikes, sustained stimulations, and oscillatory responses. In addition, heterogenous responses were observed within each cell type. These data provide evidence for the involvement of calcium mobilization in the mechanism of action of PACAP on pituitary cells. The results also indicate that in frogs, PACAP may stimulate the secretory activity of GH and PRL cells and, to a lesser extent, ACTH, TSH, and gonadotrope cells.

Dopamine inhibits inositol phosphate production, arachidonic acid formation, and corticosteroid release by frog adrenal gland through a pertussis toxin-sensitive G-protein.

We have previously shown that dopamine-evoked inhibition of corticosteroid production from adrenocortical cells is mediated through a decrease in prostaglandin biosynthesis. Since the catecholamine did not alter the stimulatory effect of arachidonic acid, it was proposed that dopamine may inhibit the formation of arachidonate from glycerophospholipids. To test this hypothesis, the effect of dopamine on phosphoinositol lipid metabolism was investigated in frog interrenal (adrenal) tissue. In [3H]myo-inositol-prelabeled frog interrenal slices, a short pulse of dopamine (50 microM) induced a biphasic effect on inositol phosphate production: a transient (1-min) increase, followed by a sustained inhibition. Concurrently, dopamine induced a transient reduction followed by a sustained increase in polyphosphoinositides. A 10-min pulse of the D2 dopamine receptor agonist apomorphine (50 microM) elicited a significant inhibition of basal levels of inositol phosphates (tris-, bis-, and mono-), and an increase in plasma membrane phosphoinositol lipid contents. The inhibitory effect of dopamine on inositol phosphate formation and corticosteroid release was abolished by a 24-h incubation of interrenal slices with pertussis toxin. In [3H]arachidonic acid-prelabeled interrenal slices, dopamine also decreased diacylglycerol (DG) and arachidonic acid (AA) concentrations. A delay of 1 min was observed between inhibition of DG and arachidonate, suggesting that AA is probably generated from DG. We conclude that in the adrenal cortex, activation of dopamine D2 receptors is coupled to a phosphoinositide-specific phospholipase-C mediated via a pertussis toxin-sensitive G-protein. Taken together, our data indicate that inhibition of inositol phosphate and AA formation is one of the mechanisms by which dopamine controls corticosteroid production by adrenocortical cells.

Regulation of the GABA(A) receptor by nitric oxide in frog pituitary melanotrophs.

Nitric oxide (NO) is implicated in the regulation of various endocrine functions, but the effect of NO on GABA(A) receptor transmission has never been reported in endocrine cells. In the present study, we have investigated the effects of various agents acting on the NO transduction pathway on GABA(A) receptor function in frog pituitary melanotrophs. Histochemical studies using the NADPH-diaphorase reaction and immunohistochemical labeling with antibodies against neuronal NO synthase (nNOS) revealed that nNOS is expressed in the intermediate lobe of the pituitary and in cultured melanotrophs. Whole-cell patch-clamp recordings showed that the specific substrate of NOS L-arginine (L-Arg, 10(-4) M) or the NO donor sodium nitroprusside (10(-5) M) provoked a long-lasting inhibition of the current evoked by GABA (5 x 10(-6) M). The NOS inhibitor L-nitroarginine (10(-5) M) produced a biphasic effect, i.e. a transient decrease followed by a delayed increase of the GABA-evoked current amplitude. Similarly, the specific nNOS inhibitor 7-nitroindazole and the specific inducible NOS (iNOS) inhibitor aminoguanidine (10(-5) M each) provoked a transient depression of the current followed by a sustained potentiation. Formation of cGMP in neurointermediate lobes was enhanced by L-Arg (10(-4) M) and by the calcium-releasing agent caffeine (10(-4) M), and inhibited by the calmodulin (CaM)/Ca2+ complex blocker W7 (10(-5) M). The GABA-evoked current was potentiated by the guanylyl cyclase inhibitor ODQ (10(-8)-10(-7) M) and inhibited by the protein kinase G (PKG) activator 8pCPT-cGMP (3 x 10(-7)-3 x 10(-5) M). The present data indicate that NO, produced by a CaM/Ca2+-dependent NOS in frog melanotrophs, exerts an autocrine inhibitory effect on the GABA-evoked current. The action of NO on the GABA(A) receptor function is mediated through activation of the cGMP/PKG pathway.

Localization of the endogenous benzodiazepine ligand octadecaneuropeptide in the rat testis.

Diazepam binding inhibitor (DBI) is the precursor of a family of peptides, including an octadecaneuropeptide (ODN), which share with DBI the ability to specifically displace benzodiazepines (BZD) from their receptors. BZD receptors have been found not only in the brain, but also in a variety of peripheral tissues, including the testis. To clarify the role of ODN in the testis, we have investigated the localization of ODN in the rat testis using two different cytochemical approaches: immunocytochemistry and in situ hybridization. Immunocytochemical localization was achieved using rabbit antibodies developed against rat ODN. At the light microscopic level, immunostaining was exclusively located in interstitial cells; the seminiferous tubules were totally unlabeled. In the developing rat, immunostaining in the interstitial cells was first detected in an 18-day-old fetus. The immunolabeling increased as a function of age to reach a plateau at 40 days of age. The ultrastructural localization of ODN was achieved by immunogold staining. The gold particles were exclusively found in the cytoplasm of Leydig cells. HPLC analysis performed in adult rat testicular extracts revealed that immunoreactive material was detected in a peak eluted later than synthetic rat ODN. The cellular distribution of ODN was also studied by in situ hybridization using a 35S-labeled single stranded RNA probe complementary to DBI mRNA. Hybridization signal obtained at the light microscopic level was only detected over interstitial cells. The data obtained clearly indicate that in the rat, Leydig cells synthesize ODN and accumulate ODN-like immunoreactivity. Since Leydig cells have been shown to contain BZD receptors, it might be hypothesized that ODN and/or other DBI-related peptides can play a role in Leydig cell regulation.

Acetylcholine stimulates alpha-melanocyte-stimulating hormone release from frog pituitary melanotrophs through activation of muscarinic and nicotinic receptors.

The release of alpha MSH from the pars intermedia of amphibians is regulated by multiple factors, including classical neurotransmitters and neuropeptides. In this study we have examined the possible involvement of acetylcholine (ACh) in the regulation of alpha MSH secretion from the pars intermedia of the frog (Rana ridibunda) using the perifusion technique. When intact neurointermediate lobes (NIL) were exposed to graded doses of ACh (3 X 10(-7) to 3 X 10(-4) M), a dose-dependent stimulation of alpha MSH release was observed. Repeated administration of ACh (10(-4) M) induced reproducible responses of NIL without any desensitization phenomenon. ACh was also capable of stimulating alpha MSH release from dispersed intermediate lobe cells, indicating that the neurotransmitter exerts its effect by acting directly on frog melanotrophs. Using the monoclonal antibody M-35 against calf muscarinic receptors we have visualized, by the immunofluorescence technique, the presence of muscarinic receptor-like immunoreactivity in the frog pars intermedia. The stimulatory action of ACh was mimicked by both nicotine and muscarine (10(-5) M each). Nicotine-induced stimulation of alpha MSH release was partially abolished by alpha-bungarotoxin (10(-6) M) and hexamethonium (10(-4) M). The stimulatory effect of muscarine was suppressed by atropine and the M1-muscarinic antagonist pirenzepine (10(-5) M), but not by the M2-muscarinic antagonist gallamine. We have investigated the effect of ACh during administration of specific nicotinic and muscarinic antagonists. While hexomethonium or atropine could block only part of the stimulatory effect of ACh, concomitant administration of these antagonists totally abolished the response of NIL to ACh. Finally, the stimulatory effect of ACh was not impaired during prolonged administration of the beta-adrenergic antagonist propranolol. These data show that ACh stimulates in vitro alpha MSH secretion by frog NIL. Our results also indicate that amphibian pars intermedia cells possess two types of cholinergic receptors, an M1-muscarinic receptor sensitive to pirenzepine and nicotinic receptors sensitive to hexamethonium and alpha-bungarotoxin.

Distribution and characterization of endogenous benzodiazepine receptor ligand (endozepine)-like peptides in the rat gastrointestinal tract.

Endozepine is the generic name for a family of peptides that are capable of displacing benzodiazepines and the 3-carboxylate ester of beta-carboline from their specific binding sites on synaptosomal membranes. The 104-amino acid polypeptide diazepam-binding inhibitor (DBI) and the octadecaneuropeptide (ODN) generated by tryptic digestion of DBI are two members of the endozepine family. In the present study we have used RIA, HPLC, in situ hybridization, and immunohistochemical techniques to identify and localize endozepine-like molecules in the rat gastrointestinal tract. Significant amounts of endozepine-like immunoreactivity (LI) were detected throughout the gut; the highest concentrations were found in the duodenum and antrum. HPLC analysis revealed that the immunoreactive material eluted as a major peak with a higher retention time than that of synthetic ODN. The distribution of the immunoreactive peptide(s) was studied using the peroxidase-antiperoxidase technique at the light microscope level. Endozepine-LI was localized only in the epithelial cell layer of the intestine in both goblet cells and enterocytes. In the stomach, endozepine-LI appeared to be restricted to deep layer of the epithelial cells. The diffuse neuroendocrine cells (amine precursor uptake and decarboxylation system) as well as myenteric and neuronal cells were devoid of immunoreactivity. A good correlation was observed between RIA and immunocytochemical data, in that the esophagus, which contained very low concentrations of endozepine-LI, also exhibited weak immunostaining of secretory cells. In situ hybridization using a 35S-labeled cRNA probe showed that endozepine mRNA was located in the mucosa. Taken together, these results show that in the rat, epithelial cells synthesize endozepine-LI material. Since epithelial cells also contain a high density of peripheral-type benzodiazepine-binding sites, our data indicate that endozepines may play a role in water, electrolyte, and/or mucus regulation in the rat gastrointestinal tract. The occurrence of high levels of endozepine-LI in the rat stomach also suggests that endozepines can be involved in the regulation of gastric acid secretion through modulation of local gamma-aminobutyric acid-ergic neurotransmission.

Pharmacological and functional characterization of muscarinic receptors in the frog pars intermedia.

The secretion of alphaMSH from the intermediate lobe of the frog pituitary is regulated by multiple factors, including classical neurotransmitters and neuropeptides. In particular, acetylcholine (ACh), acting via muscarinic receptors, stimulates alphaMSH release from frog neurointermediate lobes (NILs) in vitro. The aim of the present study was to characterize the type of receptor and the transduction pathways involved in the mechanism of action of ACh on frog melanotrope cells. The nonselective muscarinic receptor agonists muscarine and carbachol both stimulated alphaMSH release from perifused frog NILs, whereas the M1-selective muscarinic agonist McN-A-343 was virtually devoid of effect. Both the M1>M3 antagonist pirenzepine and the M3>M1 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide inhibited muscarine-induced alphaMSH release. Administration of a brief pulse of muscarine in the vicinity of cultured melanotrope cells provoked a 4-fold increase in the cytosolic calcium concentration ([Ca2+]i). Suppression of Ca2+ in the culture medium or addition of 3 mM Ni2+ abrogated the stimulatory effect of muscarine on [Ca2+]i and alphaMSH release. In contrast, omega-conotoxin GVIA and nifedipine did not significantly reduce the stimulatory effect of muscarine on [Ca2+]i and alphaMSH secretion. Exposure of NILs to muscarine provoked an increase in inositol phosphate formation, and this effect was dependent on extracellular Ca2+. The inhibitor of polyphosphoinositide turnover neomycin significantly attenuated the muscarine-evoked alphaMSH release. Similarly, pretreatment of frog NILs with phorbol ester markedly reduced the secretory response to muscarine. In contrast, the stimulatory effect of muscarine on alphaMSH release was not affected by the phospholipase A2 inhibitor dimethyl eicosadienoic acid or by the tyrosine kinase inhibitors lavendustin A, genistein, and tyrphostin 25. Muscarine at a high concentration (10(-4) M) only produced a 40% increase in cAMP formation. Preincubation of frog NILs with pertussis toxin did not significantly affect the muscarine-induced stimulation of alphaMSH release. These results indicate that frog melanotrope cells express a muscarinic receptor subtype pharmacologically related to the mammalian M3 receptor. Activation of this receptor causes calcium influx through Ni2+-sensitive Ca2+ channels and subsequent activation of the phopholipase C/protein kinase C transduction pathway.

Catecholaminergic control of alpha-melanocyte-stimulating hormone (alpha MSH) release by frog neurointermediate lobe in vitro: evidence for direct stimulation of alpha MSH release by thyrotropin-releasing hormone.

The role of dopaminergic and adrenergic innervation of the intermediate lobe of amphibian pituitary in the release of alpha MSH has been studied in vitro. Neurointermediate lobes of frog (Rana ridibunda Pallas) have been perifused in amphibian culture medium (ACM) for 5-7 h. alpha MSH released in the effluent perifusate was measured by means of a sensitive and specific RIA. No significant morphological alteration of neurointermediate lobe cells was observed during the perifusion experiment, even at the electron microscopic level. The existence of dopaminergic receptors, responsible for an inhibition of frog melanotrophs, was shown using the dopaminergic agonists apomorphine (10(-6) M) and bromo-2-ergocryptine (10(-8) and 10(-7) M), which initiated a marked reduction of alpha MSH secretion. The effect of apomorphine was obliterated by the dopaminergic antagonist haloperidol. Haloperidol itself induced a dose-related stimulation, and the monoamine oxidase inhibitor nialamide (4 x 10(-3) M) inhibited alpha MSH secretion. In addition, haloperidol led to a complete reversal of the inhibitory effect of nialamide on alpha MSH secretion. These results demonstrate the existence, in the parenchyme of the intermediate lobe, of dopaminergic nerve fibers that are functionally active. The beta-adrenergic agonist isoproterenol was responsible for a dose-related stimulation of alpha MSH secretion; the stimulatory effect was reversed by the beta-adrenergic antagonist propranolol. TRH is a potent stimulator of alpha MSH secretion in amphibians. Since haloperidol and propranolol did not abolish the stimulation of alpha MSH release induced by TRH, it appeared that TRH action was not mediated via an inhibition of dopamine release or via a stimulation of adrenergic nerve fibers.

Involvement of protein kinase C and protein tyrosine kinase in thyrotropin-releasing hormone-induced stimulation of alpha-melanocyte-stimulating hormone secretion in frog melanotrope cells.

We have previously shown that the stimulatory effect of TRH on alpha-MSH secretion from the frog pars intermedia is associated with Ca2+ influx through voltage-dependent Ca2+ channels, activation of a phospholipase C and mobilization of intracellular Ca2+ stores. The aim of the present study was to investigate the contribution of protein kinase C (PKC), adenylyl cyclase (AC), Ca2+/calmodulin-dependent protein kinase II (CAM KII), phospholipase A2, and protein tyrosine kinase (PTK) in TRH-induced alpha-MSH release. Incubation of frog neurointermediate lobes (NILs) with phorbol 12-myristate-13-acetate (24 h), which causes desensitization of PKC, or with the PKC inhibitor NPC-15437, reduced by approximately 50% of the effect of TRH on alpha-MSH release. In most melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i). Preincubation with phorbol 12-myristate-13-acetate or NPC-15437 suppressed the plateau phase of the Ca2+ response. Incubation of NILs with TRH (10(-6) M; 20 min) had no effect on cAMP production. In addition, the AC inhibitor SQ 22,536 did not affect the secretory response of NILs to TRH. These data indicate that the phospholipase C/PKC pathway, but not the AC/protein kinase A pathway, is involved in TRH-induced alpha-MSH release. The calmodulin inhibitor W-7 and the CAM KII inhibitor KN-93 did not significantly reduce the response to TRH. Similarly, the phospholipase A2 inhibitors quinacrine and 7-7'-DEA did not impair the effect of TRH on alpha-MSH secretion. The PTK inhibitors ST638 and Tyr-A23 had no effect on TRH-induced [Ca2+]i increase but inhibited in a dose-dependent manner TRH-evoked alpha-MSH release (ED50 = 1.22x10(-5) M and ED50 = 1.47x10(-5) M, respectively). Taken together, these data indicate that, in frog melanotrope cells, PKC and PTK are involved in TRH-induced alpha-MSH secretion. Activation of PKC is responsible for the sustained phase of the increase in [Ca2+]i, whereas activation of PTK does not affect Ca2+ mobilization.