Schizophrenia susceptibility loci on chromosomes 13q32 and 8p21.

Schizophrenia is a common disorder characterized by psychotic symptoms; diagnostic criteria have been established. Family, twin and adoption studies suggest that both genetic and environmental factors influence susceptibility (heritability is approximately 71%; ref. 2), however, little is known about the aetiology of schizophrenia. Clinical and family studies suggest aetiological heterogeneity. Previously, we reported that regions on chromosomes 22, 3 and 8 may be associated with susceptibility to schizophrenia, and collaborations provided some support for regions on chromosomes 8 and 22 (refs 9-13). We present here a genome-wide scan for schizophrenia susceptibility loci (SSL) using 452 microsatellite markers on 54 multiplex pedigrees. Non-parametric linkage (NPL) analysis provided significant evidence for an SSL on chromosome 13q32 (NPL score=4.18; P=0.00002), and suggestive evidence for another SSL on chromosome 8p21-22 (NPL=3.64; P=0.0001). Parametric linkage analysis provided additional support for these SSL. Linkage evidence at chromosome 8 is weaker than that at chromosome 13, so it is more probable that chromosome 8 may be a false positive linkage. Additional putative SSL were noted on chromosomes 14q13 (NPL=2.57; P=0.005), 7q11 (NPL=2.50, P=0.007) and 22q11 (NPL=2.42, P=0.009). Verification of suggestive SSL on chromosomes 13q and 8p was attempted in a follow-up sample of 51 multiplex pedigrees. This analysis confirmed the SSL in 13q14-q33 (NPL=2.36, P=0.007) and supported the SSL in 8p22-p21 (NPL=1.95, P=0.023).

Effect of vasotocin on cytosolic free calcium concentrations in frog adrenocortical cells in primary culture.

In a previous report we have shown that vasotocin (AVT), the amphibian counterpart of vasopressin, is a potent stimulator of corticosterone and aldosterone secretion by frog adrenocortical cells. We have also observed that the stimulatory effect of AVT on corticosteroid secretion is mediated through activation of receptors positively coupled to phospholipase-C. In the present study we examined the effect of AVT on cytosolic Ca2+ concentrations ([Ca2+]i). Since the interrenal (adrenal) gland of the frog is composed of a mixed population of chromaffin and adrenocortical cells, cytochemical identification of cultured cells was performed by immunofluorescence, using antibodies to AVT or 11 beta-hydroxylase as markers of chromaffin cells or steroid-producing cells, respectively. Cultured interrenal cells were loaded with the fluorescent Ca2+ indicator indo-1, and variations in [Ca2+]i were studied using dual emission wavelength microfluorimetry. Exposure of adrenocortical cells to AVT induced elevation of [Ca2+]i. Prolonged infusion of AVT caused an immediate increase in [Ca2+]i, followed by a sustained response of adrenocortical cells. Repeated pulses of AVT resulted in a gradual decline in the [Ca2+]i increase, suggesting the existence of a desensitization phenomenon. The effect of AVT on calcium mobilization was totally blocked when the cells were incubated in the presence of the V2 antagonist [d(CH2)5,D-Phe2,Ile4,Ala9-NH2]AVP. In calcium-free medium, the AVT-evoked increase in [Ca2+]i was suppressed. In contrast, when Ca2+ was replaced by Mn2+ in the incubation medium, the early response of the cells (transient peak of [Ca2+]i) was preserved, while the plateau phase disappeared. Incubation of the cells with the dihydropyridine Ca2+ channel blocker nifedipine did not affect the AVT-induced [Ca2+]i rise. These results indicate that AVT exerts a dual action on [Ca2+]i in frog adrenocortical cells. The initial rise of [Ca2+]i can be ascribed to immediate mobilization of intracellular Ca2+ stores, probably mediated by inositol trisphosphategated channels, whereas the sustained increase in [Ca2+]i results from nifedipine-insensitive plasma membrane Ca2+ channels.

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.

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.

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.

Evidence for the involvement of chromaffin cells in the stimulatory effect of tachykinins on corticosteroid secretion by the frog adrenal gland.

The adrenal gland of the frog is innervated by a network of fibers containing two tachykinins (ranakinin and [Leu3,Ile7]neurokinin A), which both stimulate corticosteroid secretion from frog adrenal tissue. The aim of the present study was to determine the mode of action of tachykinins on the frog adrenal gland. Double immunolabeling of tissue sections with a monoclonal antibody to tyrosine hydroxylase and an antiserum to substance P showed that tachykinin-containing fibers are preferentially apposed onto chromaffin cells. Immunocytochemical labeling at the electron microscope level revealed that tachykinin-immunoreactive fibers establish close contacts only with adrenochromaffin cells. Ranakinin stimulated corticosterone and aldosterone secretion from perifused adrenal slices, but had no stimulative effect on dispersed adrenal cells. Cytoautoradiographic labeling of frog adrenal cells in primary culture with [3H]substance P revealed the existence of specific binding sites located exclusively on chromaffin cells. Microfluorimetric measurement of cytosolic calcium concentrations ([Ca2+]i) in cultured adrenal cells showed that ranakinin induced a dose-dependent increase in [Ca2+]i in chromaffin cells (ED50 = 2 x 10(-7) M). In contrast, ranakinin did not affect [Ca2+]i in adrenocortical cells. The present results indicate that in the frog adrenal gland, tachykinin-containing fibers make preferential contacts with chromaffin cells, and tachykinins directly activate chromaffin cells. These data suggest that the stimulative effect of tachykinins on corticosteroid secretion is mediated via presynaptic activation of adrenochromaffin cells.

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.

The endozepine ODN stimulates polyphosphoinositide metabolism in rat astrocytes.

Astrocytes synthesize a series of peptides called endozepines which act as endogenous ligands of benzodiazepine receptors. The present study demonstrates that the endozepine ODN causes a dose-dependent increase in inositol trisphosphate and a parallel decrease in phosphatidylinositol bisphosphate in cultured rat astrocytes. Pre-incubation of astrocytes with the phospholipase C inhibitor U 73122 or with pertussis toxin totally blocked polyphosphoinositide metabolism. These data show that, in rat astrocytes, ODN stimulates a phospholipase C coupled to a pertussis toxin-sensitive G protein.

Dual effects of thyrotrophin-releasing hormone (TRH) on K+ conductance in frog pituitary melanotrophs. TRH-induced alpha-melanocyte-stimulating hormone release is not mediated through voltage-sensitive K+ channels.

Modulation of the activity of K+ channels by TRH and the possible involvement of this modulation in TRH-induced release of alpha-MSH were studied in cultured frog melanotrophs, using patch-clamp and perifusion techniques. Pars intermedia cells were enzymatically dispersed and cultured in Leibovitz medium. In order to test the viability of cultured cells, the amount of alpha-MSH released into the medium was measured by radioimmunoassay every day for 1 week of culture. The total amount of alpha-MSH released during the first 4 days of culture was 8.6 times higher than the intracellular content of alpha-MSH on day 1. Melanotrophs were identified by an indirect immunofluorescence technique using a specific antiserum to alpha-MSH. Recordings obtained in whole-cell, cell-attached and excised patch-clamp configurations showed that TRH induced a transient polarization concomitant with an increase in the probability of opening of Ca2+-activated K+ channels. This transient response was followed by a depolarization accompanied by an enhanced frequency of action potential discharge. TRH also induced a decrease in voltage-dependent K+ conductance. Application of tetraethylammonium, a K+ channel blocker, depolarized the cells and increased the basal secretory level without noticeable changes in TRH-evoked alpha-MSH release. These results demonstrate that the neuropeptide TRH both stimulates Ca2+-sensitive K+ channels and inhibits voltage-dependent K+ current in pituitary melanotrophs. Our data indicate that TRH-induced secretion of alpha-MSH is not a direct consequence of the lowering of K+ conductance. It thus appears that basal and TRH-induced alpha-MSH release occur through distinct pathways; the spontaneous release of alpha-MSH is probably linked to membrane potential, while modulation of the electrical activity is not directly involved in TRH-induced activation of the secretory process.

Mechanism of action of gamma-aminobutyric acid on frog melanotrophs.

We have previously demonstrated that gamma-aminobutyric acid (GABA) is a potent regulator of secretory and electrical activity in melanotrophs of the frog pituitary. The aim of the present study was to investigate the intracellular events which mediate the response of melanotrophs to GABA. We first observed that GABA (1-100 microM) inhibited both basal and forskolin-stimulated cyclic AMP (cAMP) formation. The inhibitory effect of GABA on cAMP levels was mimicked by the GABAB receptor agonist baclofen (100 microM) and totally abolished by a 4-h pretreatment with pertussis toxin (0.1 microgram/ml). In contrast, the specific GABAA agonist 3-aminopropane sulphonic acid (3APS) did not affect cAMP production. Both GABA and 3APS (100 microM each) induced a biphasic effect on alpha-MSH release from perifused frog neurointermediate lobes, i.e. a transient stimulation followed by an inhibition of alpha-MSH secretion. Administration of forskolin (10 microM) prolonged the stimulatory phase and attenuated the inhibitory phase evoked by GABA and 3APS, indicating that cAMP modulates the response of melanotrophs to GABAA agonists. Ejection of 3APS (1 microM) in the vicinity of cultured melanotrophs caused a massive increase in intracellular calcium concentration ([Ca2+]i). The stimulatory effect of 3APS on [Ca2+]i was abolished when the cells were incubated in a chloride-free medium. The formation of inositol trisphosphate was not affected by 3APS, suggesting that the increase in [Ca2+]i cannot be ascribed to mobilization of intracellular calcium stores. omega-Conotoxin did not alter the secretory response of frog neurointermediate lobes to 3APS, while nifedipine blocked the stimulation of alpha-MSH secretion induced by 3APS. In conclusion, the present data indicate that, in frog pituitary melanotrophs, (i) the stimulatory phase evoked by GABAA agonists can be accounted for by an influx of calcium through L-type calcium channels, (ii) the inhibitory effect evoked by GABAB agonists can be ascribed to inhibition of adenylate cyclase activity and (iii) cAMP attenuates the inhibitory phase evoked by GABAA agonists. Taken together, these data suggest that activation of GABAB receptors may modulate GABAA receptor function.

Role of calcium in thyrotrophin-releasing hormone-stimulated release of melanocyte-stimulating hormone from frog neurointermediate lobe.

The effect of modifications of extracellular calcium concentrations on alpha-MSH release has been studied using perifused frog neurointermediate lobes. Increasing concentrations of calcium (from 2 to 10 mmol/l) gave rise to a dose-related stimulation of alpha-MSH secretion, whereas reduction of Ca2+ from 2 to 1.5 mmol/l partially inhibited alpha-MSH release. The direct effect of extracellular Ca2+ on alpha-MSH secretion was confirmed by the dose-dependent stimulation of alpha-MSH release induced by the calcium ionophore A23187. Perifusion with a calcium-free medium or blockade of Ca2+ channels by 4 mmol Co2+/l both resulted in an inhibition of spontaneous and TRH-induced alpha-MSH release. Conversely, administration of verapamil or methoxyverapamil (10 mumol/l each) did not alter basal secretion and had no effect on the response of the glands to TRH. Nifedipine (10 mumol/l), which was able to block KCl (20 mmol/l)-evoked alpha-MSH release, induced a slight inhibition of basal alpha-MSH secretion, indicating that extracellular Ca2+ levels may regulate alpha-MSH release in part by Ca2+ influx through voltage-dependent Ca2+ channels. In contrast TRH-induced alpha-MSH release was not affected by nifedipine or dantrolene (10 mumol/l), and BAY-K-8644 (1 mumol/l) did not significantly modify the response of neurointermediate lobes to TRH. Taken together, these results suggest that TRH-induced alpha-MSH secretion is associated with calcium influx across the plasma membrane and that calcium entry caused by TRH may occur through nifedipine/verapamil-insensitive Ca2+ channels.

Three distinct thyrotropin-releasing hormone-immunoreactive axonal systems project in the median eminence-pituitary complex of the frog Rana ridibunda. Immunocytochemical evidence for co-localization of thyrotropin-releasing hormone and mesotocin in fibers innervating pars intermedia cells.

The localization of thyrotropin-releasing hormone-immunoreactive structures was investigated in the hypothalamo-hypophyseal complex of the frog, Rana ridibunda, by light and electron microscopy using the conventional indirect immunoperoxidase technique and the immuno-gold technique, respectively. The localization of mesotocin-, vasotocin- and neurophysin-immunoreactive elements was compared to that of thyrotropin-releasing hormone either by comparing homologous fields on serial sections or by staining the same section with two different antibodies. Thyrotropin-releasing hormone-immunoreactive perikarya occurred mainly in the anterobasal periventricular area and dorsal extension of the preoptic nucleus, and in the lateral zone of the infundibular nucleus. In the anterobasal preoptic nucleus, the distribution of thyrotropin-releasing hormone-immunoreactive perikarya partially overlapped that of vasotocin- and mesotocin-containing neurons; however, co-localization of thyrotropin-releasing hormone with either nonapeptide could not be detected there. In contrast, in the caudal extension of the preoptic nucleus, thyrotropin-releasing hormone- and mesotocin-like immunoreactivities were frequently co-localized in the same neurons. In the external zone of the median eminence, abundant networks of thyrotropin-releasing hormone- and vasotocin-immunoreactive nerve fibers were found in the vicinity of portal capillaries, while mesotocin-immunoreactive axons were only found in the internal zone. Using the immuno-gold technique at the electron microscopic level, three distinct thyrotropin-releasing hormone-immunoreactive systems were identified in the median eminence-neurointermediate lobe complex. (1) In the external zone of the median eminence, a conspicuous population of pericapillary endings contained 100-nm dense core vesicles immunoreactive solely for thyrotropin-releasing hormone. (2) In the neural lobe of the pituitary, thyrotropin-releasing hormone immunoreactivity occurred on secretory vesicles in a subpopulation of the mesotocinergic axons containing 160-nm secretory granules; co-localization with vasotocin was never seen. (3) In the intermediate lobe, thyrotropin-releasing hormone- and mesotocin (or neurophysin I)-immunoreactivities were systematically found in the same 120-nm dense core vesicles; these thyrotropin-releasing hormone-/mesotocin-immunoreactive axon terminals frequently made synaptic contacts with melanotropic cells. The possible modulatory effect of mesotocin on thyrotropin-releasing hormone-induced alpha-melanocyte-stimulating hormone secretion was investigated using perifused frog neurointermediate lobes. Administration of graded doses of mesotocin (from 10(-10) to 10(-5) M) did not affect the spontaneous release of alpha-melanocyte-stimulating hormone. In addition, mesotocin (10(-7) and 10(-6) M) did not modify thyrotropin-releasing hormone-evoked alpha-melanocyte-stimulating hormone release.(ABSTRACT TRUNCATED AT 400 WORDS)

Central-type benzodiazepines and the octadecaneuropeptide modulate the effects of GABA on the release of alpha-melanocyte-stimulating hormone from frog neurointermediate lobe in vitro.

The involvement of the GABA-benzodiazepine receptor complex in the regulation of melanotropin secretion has been investigated using perfused frog neurointermediate lobes. The GABAA agonist 3-amino-1 propane sulfonic acid mimicked the biphasic effect of GABA on alpha-melanocyte-stimulating hormone secretion: a brief stimulation followed by an inhibition of melanotropin secretion. The GABAA antagonist SR 95531 (10(-4) M) inhibited both stimulation and inhibition of alpha-melanocyte-stimulating hormone release induced by GABA (10(-4) M). Since the inhibitory effect of baclofen (10(-4) M) was partially antagonized by SR 95531 (10(-4) M), it appears that the GABAergic control of alpha-melanocyte-stimulating hormone release is mainly achieved through activation of GABAA receptors. GABA-induced stimulation of alpha-melanocyte-stimulating hormone release was inhibited by tetrodotoxin (10(-5) M), an Na+ -channel blocker, or nifedipine (10(-5) M), a voltage-dependent Ca2+ -channel blocker, suggesting that Na+ and Ca2+ ions are involved in the stimulatory phase of GABA action. Only central-type benzodiazepine binding site agonists such as clonazepam (10(-4) M) modified alpha-melanocyte-stimulating hormone release. In fact, clonazepam (10(-7) to 10(-5) M) led to a dose-dependent potentiation of both GABA-induced stimulation and inhibition of alpha-melanocyte-stimulating hormone release. This potentiating effect was antagonized by the GABAA antagonist SR 95531 (10(-4) M) or by the central-type benzodiazepine binding site antagonist flumazenil (10(-4) M), whereas picrotoxin (10(-4) M) abolished only the stimulatory phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dopamine on adenylate cyclase activity, polyphosphoinositide metabolism and cytosolic calcium concentrations in frog pituitary melanotrophs.

It has previously been shown that dopamine plays a pivotal role in the regulation of alpha-melanocyte-stimulating hormone (alpha-MSH) secretion from the intermediate lobe of the pituitary. In the present study, we have investigated the various intracellular mechanisms that are associated with the action of dopamine on frog pituitary melanotrophs. Dopamine reduced forskolin-stimulated cyclic adenosine monophosphate (cAMP) production and the inhibitory effect of dopamine was blocked by the dopaminergic D2 receptor antagonist sulpiride. The D2 receptor agonist apomorphine inhibited incorporation of [3H]inositol into membrane phospholipids. Dopamine also inhibited the formation of inositol trisphosphate and provoked accumulation of phosphatidylinositol bisphosphate. The inhibitory effect of dopamine on inositol trisphosphate production was mimicked by D2 receptor agonists and blocked by sulpiride. Using a double-wavelength microfluorimetric approach, we found that dopamine caused a rapid and transient decrease in K(+)-evoked stimulation of intracellular calcium concentration. The time-courses of the responses of the various intracellular messengers indicate that blockage of voltage-dependent calcium channels is the primary event associated with activation of dopamine D2 receptors, while inhibition of polyphosphoinositide breakdown, related to blockage of voltage-dependent calcium channels, and reduction of cAMP production are secondary events which may contribute to the sustained inhibitory effect of dopamine on alpha-MSH release.

Effect of serotonin on alpha-melanocyte-stimulating hormone secretion from perifused frog neurointermediate lobe: evidence for the presence of serotonin-containing cells in the frog pars intermedia.

We have examined the presence of 5-hydroxytryptamine (serotonin; 5-HT) in the intermediate lobe of the frog pituitary and investigated the effect of exogenous 5-HT on alpha-melanocyte-stimulating hormone (alpha-MSH) release from the perifused neurointermediate lobe (NIL). Using a specific antiserum against 5-HT, the indirect immunofluorescence technique revealed the presence of 5-HT-like immunoreactivity (5-HT-LI) in discrete cells, generally gathered in small clusters among parenchymal cells, and in numerous neurites surrounding melanotrophic cells. At the electron microscopic level, using a silver-gold intensification procedure, 5-HT-LI was localized in dense-core secretory vesicles within specific pituitary cells which appear to be different from pituitary melanotrophs. Dense accumulation of gold particles was also observed in nerve fibres running between parenchymal cells. A combination of high-performance liquid chromatography analysis and electrochemical detection showed the presence of both 5-HT and its metabolite 5-hydroxyindol acetic acid (5-HIAA) in frog NIL extracts (534 +/- 40 and 1245 +/- 65 (S.E.M.) pg/mg wet tissue respectively). Administration of graded doses of 5-HT (from 1 to 30 mumol/l) to perifused frog NIL induced a dose-dependent inhibition of alpha-MSH release. Repeated pulses of 5-HT (10 mumol/l each) induced a reproducible inhibition of alpha-MSH without any desensitization phenomena. The inhibitory effect of 5-HT was partially blocked by the serotonergic antagonists methysergide and ICS-205-930 (10 mumol/l each). Concomitant administration of methysergide and ICS-205-930 (10 mumol/l each) totally abolished 5-HT-evoked inhibition of alpha-MSH. Fenfluramine, a releaser of 5-HT, induced a slight but significant reduction of alpha-MSH secretion. While 5-HT caused a marked inhibition of alpha-MSH release from intact NIL, 5-HT was devoid of effect on acutely dispersed pars intermedia cells suggesting that 5-HT does not exert a direct action on pituitary melanotrophs. We have examined the effect of specific dopaminergic, GABAergic and alpha-adrenergic antagonists on 5-HT-induced alpha-MSH inhibition. We observed that sulpiride and SR 95531 (10 mumol/l each) did not affect the response of NIL to 5-HT while yohimbine (10 mumol/l) suppressed the inhibitory action of 5-HT. Taken together, our results indicate that discrete cells of the frog pars intermedia contain the neurotransmitter 5-HT which may act locally to inhibit alpha-MSH release.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium waves in frog melanotrophs are generated by intracellular inactivation of TTX-sensitive membrane Na+ channel.

Two models of plasma membrane oscillators may explain the regulation of calcium homeostasis in frog melanotrophs. In the majority (70%) of cells a high frequency and small amplitude fluctuations characterize the spontaneous calcium level. In the 30% of remaining cells a low frequency and high amplitude oscillations were observed. Utilization of EGTA, U73122 and ryanodine suggested that calcium homeostasis in frog melanotrophs is dependent on extra- but not on intracellular calcium pools. EGTA was able to block calcium oscillations and to decrease basal calcium level in non-oscillatory cells. omega-Conotoxin, N-type calcium channels antagonist, stopped calcium oscillations but not modified calcium level in non-oscillatory cells. Nifedipine, antagonist of L-type calcium channels, had no effect either on calcium waves formation or on basal level of calcium in non-oscillatory cells. omega-Conotoxin and nifedipine were able to decrease the spontaneous alpha-MSH release from whole NILs while only omega-conotoxin had inhibitory effect on hormonal output from dispersed melanotrophs. Nickel (Ni2+) provoked dose-dependent effect. At 2 mM concentration Ni2+ blocked either calcium oscillations or alpha-MSH release. In contrast, a 0.5 mM concentration had stimulatory effect on both the phenomenons. Similarly, mibefradil (antagonist of T-type calcium channel), was able to induce an increase in [Ca2+](i) after modification of calcium fluctuations in non-oscillatory cells. Utilization of veratridine and TTX, agonist and antagonist of Na channels, respectively, indicated that mobilization of extracellular sodium, by TTX-sensitive and TTX-resistant Na channels, stimulates a hormonal output resulting from increase of [Ca2+](i). In the presence of TTX, veratridine was able to generate a calcium oscillations, which were also observed after inactivation of TTX-sensitive channel. Bepridil (antagonist of Na-Na exchange of the Na+/Ca2+ exchanger) and Na-free medium had powerful effect on increase of [Ca2+](i). The same observations obtained after administration of ouabain, antagonist of Na+/K+ dependent ATPase, confirmed dependence of calcium homeostasis on sodium distribution. Furthermore, dibutyryl-cAMP induced calcium oscillations suggesting implication of intracellular phosphorylation in the generation of calcium waves. Taken together, our results suggest that each type of calcium homeostasis is controlled by different mechanisms. Calcium fluctuations may be ascribed to the high frequency activity of T-type calcium channel, TTX-sensitive and TTX-resistant sodium channels. Calcium oscillations may be generated by the destabilization of the steady-state Na+/Ca2+ gradient provoked by intracellular inactivation of TTX-sensitive Na channel. This ionic unbalance would increase Ca-Ca exchange of Na+/Ca2+ exchanger, which by local depolarization promotes opening of N-type calcium channel responsible for calcium wave. In both types of homeostasis, the calcium and sodium overload is avoided by opening of K+ voltage- and Ca-dependent channels, and by increase in activities of Na+/K+ ATPase and forward mode of Na+/Ca2+ exchanger.

Biphasic effect of thyrotropin-releasing factor (TRH) on alpha-melanotropin secretion from frog intermediate lobe in vitro.

The kinetics of alpha-MSH secretion induced by prolonged TRH infusion were studied using perfused frog neurointermediate lobe (NIL). During a 2 h administration of TRH (10(-8) M), the secretion rate of alpha-MSH displayed two phases. During the first phase, secretion of alpha-MSH increased rapidly reaching a maximum within 20 min and then, despite continued TRH infusion, this secretion slowly declined. The second phase was characterized as plateau of elevated release (relative to basal secretion); within this second phase there was often a small peak of released alpha-MSH occurring at about 100 min. Exposure of NIL to another TRH (10(-8) M) pulse 90 min later induced a normal stimulation of alpha-MSH secretion, thus demonstrating the viability of tissue in perifusion. Continuous infusion of cycloheximide (10(-5) M) during a 5 h period totally inhibited the biosynthetic activity of NIL but did not influence TRH-induced alpha-MSH secretion. In particular, cycloheximide had no effect on the second phase of the response to prolonged infusion of TRH. Similarly, during continuous infusion of the monovalent carboxylic ionophore monensin (10(-6) M), the biphasic response to prolonged infusion of TRH (10(-8) M) was still observed. Administration of a short pulse of TRH (10(-7) M) during the declining part of the first phase or during the second phase of prolonged TRH (10(-8) M) infusion induced a significant enhancement of alpha-MSH stimulation. From these results we conclude that prolonged TRH infusion causes alpha-MSH release in a biphasic manner; attenuation of the secretory response to continuous TRH administration does not result from exhaustion of the releasable pool of alpha-MSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of extracellular and intracellular calcium sources in TRH-induced alpha-MSH secretion from frog melanotrope cells.

The stimulatory effect of thyrotropin-releasing hormone (TRH) on alpha-melanocyte stimulating hormone (MSH) secretion from the frog pars intermedia is mediated through the phospholipase C (PLC) pathway but requires extracellular Ca2+. The aim of the present study was to investigate the respective contribution of extracellular and intracellular Ca2+ in the action of TRH on cytosolic calcium concentration ([Ca2+]i) and alpha-MSH release. In normal conditions, TRH (10(-7) M; 5 s) evoked two types of Ca2+ responses: in 63% of the cells, TRH caused a sustained and biphasic increase in [Ca2+]i while in 37% of the cells, TRH only induced a transient response. In the presence of EGTA or Ni2+, the stimulatory effect of TRH on [Ca2+]i and alpha-MSH secretion was totally suppressed. Nifedipine (10(-6) M) reduced by approximately 50% the amplitude of the two types of Ca2+ responses whereas omega-conotoxin GVIA (10(-7) M) suppressed the plateau-phase of the sustained response indicating that the activation of L-type Ca2+-channels (LCC) is required for initiation of the Ca2+ response while N-type Ca2+-channels (NCC) are involved in the second phase of the response. Paradoxically, neither nifedipine nor omega-conotoxin GVIA had any effect on TRH-induced alpha-MSH secretion. The PLC inhibitor U-73122 (10(-6) M) significantly reduced the transient increase in [Ca2+]i and totally suppressed the sustained phase of the Ca2+ response but had no effect on TRH-induced alpha-MSH secretion. The stimulatory effect of TRH on PLC activity was not effected by nifedipine and omega-conotoxin GVIA but was abolished in Ca2+-free medium. Ryanodine had no effect on the TRH-induced stimulation of [Ca2+]i and alpha-MSH secretion. Concomitant administration of nifedipine/omega-conotoxin GVIA or U-73122/omega-conotoxin GVIA markedly reduced the response to TRH but did not affect TRH-evoked alpha-MSH release. In contrast, concomitant administration of U-73122 and nifedipine significantly reduced the effect of TRH on both [Ca2+]i and alpha-MSH release. Taken together, these data indicate that, in melanotrope cells, activation of TRH receptors induces an initial Ca2+ influx through nifedipine- and omega-conotoxin-insensitive, Ni2+-sensitive Ca2+-channels which subsequently activates LCC and causes Ca2+ mobilization from intracellular pools by enhancing PLC activity. Activation of the PLC causes Ca2+ entry through NCC which is responsible for the plateau-phase of sustained Ca2+ response. Although nifedipine and U-73122, separately used, were devoid of effect on secretory response, Ca2+ entry through LCC and mobilization of intracellular Ca2+ are both involved in TRH-evoked alpha-MSH release because only one source of Ca2+ is sufficient for inducing maximal hormone release. In contrast, the Ca2+ influx through NCC does not contribute to TRH-induced alpha-MSH secretion.

The endogenous benzodiazepine receptor ligand ODN increases cytosolic calcium in cultured rat astrocytes.

We have investigated the production of diazepam-binding inhibitor (DBI)-related peptides by astrocytes in primary culture and we have determined the effect of the octadecaneuropeptide DBI[33-50] (ODN) on the intracellular calcium concentration ([Ca2+]i) in astrocytes. Immunocytochemical labeling with antibodies against ODN showed that cultured astrocytes retain their ability to synthesize DBI in vitro. Cultured astrocytes were also found to release substantial amounts of ODN-immunoreactive material, and a brief exposure of astrocytes to a depolarizing potassium concentration resulted in a 5-fold increase in the rate of release of the ODN-like peptide. Microfluorimetric measurement of [Ca2+]i with the fluorescent probe indo-1 showed that nanomolar concentrations of ODN induced a marked increase in [Ca2+]i. The stimulatory effect of ODN on [Ca2+]i was not affected by calcium channel blockers or by incubation in Ca(2+)-free medium. In contrast, thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase activity, totally abolished the ODN-induced increase in [Ca2+]i. Repeated pulses of ODN caused attenuation of the response, indicating the existence of a desensitization phenomenon. Preincubation of astrocytes with pertussis toxin totally blocked the effect of ODN on [Ca2+]i. The present study indicates that ODN-related peptides are synthesized and released by glial cells. Our results also show that synthetic ODN induces calcium mobilization from an intracellular store through stimulation of pertussis toxin-sensitive G protein. Taken together, these data suggest that endozepines act as paracrine and/or autocrine factors controlling the activity of astroglial cells.

Adenosine inhibits alpha-melanocyte-stimulating hormone release from frog pituitary melanotrophs. Evidence for the involvement of a(1) adenosine receptors negatively coupled to adenylate cyclase.

Adenosine is recognized as an important modulator of cell activity. In particular, adenosine regulates the secretion of adrenocorticotropin from anterior pituitary cells. However, the possible role of adenosine on the pars intermedia has never been investigated. In the present study, we have examined the effect of adenosine on α-melanotropin (α-MSH) secretion from the intermediate lobe of the pituitary of the frog (Rana ridibunda), using the perifusion technique. When whole neurointermediate lobes were exposed to graded doses of adenosine (10(-9) to 10(-4) M), a dose-dependent inhibition of a-MSH release was observed. Repeated pulses of adenosine (5 ± 10(-5) M) induced a reproducible inhibition of α-MSH secretion without any desensitization phenomenon. The effect of adenosine was mimicked by the non-selective agonist 5'-N-ethylcarboxamide-adenosine and the highly specific adenosine A, receptor agonist N(6) -[R-phenylisopropyl]-adenosine (R-PIA). In contrast the selective adenosine A(2) receptor agonist, CGS 21680, induced a slight stimulation of α-MSH release. Adenosine-induced inhibition of α-MSH secretion was blocked by the non-selective adenosine antagonist, 8-(p-sulfophenyl)-theophyline. Adenosine and R-PIA also inhibited α-MSH secretion from acutely dispersed pars intermedia cells. Adenosine did not block thyrotropin-releasing hormone-induced α-MSH release from perifused neurointermediate lobes. In contrast, adenosine inhibited both acetylcholine-evoked and muscarine-evoked α-MSH secretion. Finally, R-PIA induced a significant inhibition of basal and forskolin-stimulated cyclic AMP levels in whole neurointermediate lobes. The present results demonstrate that adenosine exerts a direct inhibitory effect on α-MSH release from melanotrope cells through activation of the A(1) receptor subtype, negatively coupled to adenylate cyclase. These data suggest that adenosine may play a physiological role in the regulation of hormone release from the intermediate lobe of the pituitary.