Neuropeptide Y inhibits the biosynthesis of sulfated neurosteroids in the hypothalamus through activation of Y(1) receptors

We have recently shown that hydroxysteroid sulfotransferase (HST), the enzyme responsible for the biosynthesis of pregnenolone sulfate (Delta(5)PS) and dehydroepiandrosterone sulfate (DHEAS), is expressed in neurons located in the anterior preoptic area and the dorsal magnocellular nucleus of the frog diencephalon. As these two nuclei are richly innervated by NPY-immunoreactive fibers, we investigated the possible implication of NPY in the control of Delta(5)PS and DHEAS biosynthesis. Double labeling of frog brain sections revealed that 42% of the HST-immunoreactive perikarya in the diencephalon were contacted by NPY-containing fibers. In situ hybridization studies showed that Y(1) and Y(5) receptor mRNAs are expressed in the anterior preoptic area and the dorsal magnocellular nucleus. Pulse-chase experiments with (35)S-labeled 3'-phosphoadenosine 5'-phosphosulfate as a sulfate donor demonstrated that frog NPY (fNPY) inhibited the conversion of [(3)H]Delta(5)P and [(3)H]dehydroepiandrosterone ([(3)H]DHEA) into [(3)H,(35)S]Delta(5)PS and [(3)H,(35)S]DHEAS by diencephalic explants. The inhibitory effect of fNPY on Delta(5)PS and DHEAS formation was mimicked by (pPYY) and [Leu(31),Pro(34)]pNPY, which is an agonist for non-Y(2) receptors in mammals, and was completely suppressed by the Y(1) receptor antagonist BIBP3226. Conversely, the Y(2) receptor agonist pNPY-(13-36) and the Y(5) receptor agonist [D-Trp(32)]pNPY did not significantly modify the biosynthesis of [(3)H,(35)S]Delta(5)PS and [(3)H,(35)S]DHEAS. The present study provides the first evidence for the innervation of neurosteroid-producing neurons by NPY fibers. Our data also demonstrate that NPY, acting via Y(1) receptors, exerts an inhibitory effect on the biosynthesis of sulfated neurosteroids.     

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.     

Neuropeptide Y inhibits thyrotropin-releasing hormone-induced stimulation of melanotropin release from the intermediate lobe of the frog pituitary

Previous studies have shown that the release of melanotropin from frog neurointermediate lobes is under the control of two neuropeptides: thyrotropin-releasing hormone (TRH) stimulates, while neuropeptide Y (NPY) inhibits alpha-melanocyte-stimulating hormone (alpha-MSH) secretion from intact neurointermediate lobes in vitro. The aim of the present study was to investigate possible interactions between the two regulatory peptides at the pituitary level. Whole neurointermediate lobes or acutely dispersed pars intermedia cells from Rana ridibunda were perifused in vitro for 2 to 7.5 hr and the concentrations of alpha-MSH released into the effluent perifusate were monitored by radioimmunoassay. Administration of TRH (10(-7) M) or NPY (10(-7) M) to dispersed cells induced, respectively, marked stimulation or inhibition of alpha-MSH release. The effects of the two neuropeptides were similar to those observed using intact neurointermediate lobes, suggesting that TRH and NPY act directly on melanotropic cells. Perifused whole neurointermediate lobes were exposed to NPY (10(-8) to 3 x 10(-7) M) for 120 min and a single dose of TRH (10(-8) M) was administered during the prolonged infusion of NPY. Using this procedure, we observed a dose-dependent inhibition of TRH-evoked alpha-MSH release. These data support the concept that TRH and NPY act through a common intracellular pathway to regulate alpha-MSH release.     

Thyrotrophin-releasing hormone stimulates polyphosphoinositide metabolism in the frog neurointermediate lobe

Previous studies have demonstrated that TRH is a potent stimulator of alpha-MSH secretion from frog pituitary melanotrophs. In order to determine the intracellular events responsible for TRH-evoked alpha-MSH release, we have investigated the effect of TRH on polyphosphoinositide breakdown in frog pars intermedia. Neurointermediate lobes were labelled to isotopic equilibrium with myo-[3H]inositol. TRH stimulated the rate of incorporation of [3H]inositol into the phospholipid fraction. The effect of TRH was concentration-dependent; half-maximal stimulation of alpha-MSH release and inositol incorporation occurred at 12 and 28 nmol TRH/l respectively. In prelabelled neurointermediate lobes, lithium (10 mmol/l) enhanced the radioactivity in inositol monophosphate, bisphosphate (IP2) and trisphosphate (IP3). LiCl (10 mmol/l) induced a 38% inhibition of alpha-MSH release from perifused neurointermediate lobes but did not impair TRH-induced alpha-MSH secretion. In the presence of LiCl, TRH (1 mumol/l) induced a transient increase of the radioactivity in IP3, which was evident by 30 s and maximal by 1 min (+100%). TRH treatment also increased the radioactivity in IP2, which reached a plateau after 5 min (+100%). The increase in radioactivity in IP3 induced by TRH was closely paralleled by a rapid loss of [3H]phosphatidylinositol bisphosphate (PIP2), which was maximal by 1 min (-70%). These results indicate that, in frog pars intermedia, TRH-evoked alpha-MSH secretion is coupled to breakdown of PIP2. The data suggest that, in amphibian melanotrophs, as previously shown in GH3 tumour cells and in rat pituitary mammotrophs, TRH causes rapid stimulation of polyphosphoinositide-hydrolysing phospholipase C.     

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.     

In vitro study of frog (Rana ridibunda pallas) neurointermediate lobe secretion by use of a simplified perifusion system: III. Effect of neuropeptides on α-MSH secretion

It has been previously demonstrated that thyrotropin-releasing hormone (TRH) stimulates in vitro the release of α-melanocyte-stimulating hormone (α-MSH) in frog. In the present study, the effects of various neuropeptides on spontaneous and/or TRH-induced α-MSH secretion were investigated, using a well-defined perifusion system technique. Vasoactive intestinal peptide, (VIP) a neurohormone which stimulates TRH target cells in mammals, was totally devoid of effect on frog melanotrophs although VIP-like material could be detected in neurointermediate lobe extracts. Somatostatin-like immunoreactive material was found in high concentrations in the frog neurointermediate lobe complex, but synthetic somatostatin (from 10^?10 to 10^?6M) did not modify the spontaneous release of α-MSH. At doses of 10^?8 and 10^?6M, synthetic somatostatin did not modify TRH-induced α-MSH secretion. Morphine (10^?5M) and opioid peptides (10^?10 to 10^?6M) had no effect on spontaneous α-MSH secretion. In addition, methionine enkephalin (10^?5M) did not modify the stimulatory effect of TRH on α-MSH secretion. From these results we conclude that, among the neuropeptides which modulate prolactin secretion in mammals, only TRH is involved in α-MSH secretion in the frog.     

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)     

Lack of effect of TRH on alpha-MSH release from the neurointermediate lobe of the lizard Lacerta vivipara.

Thyrotropin-releasing hormone (TRH) is a potent stimulator of melanotropin (alpha-MSH) release from pituitary melanotrophs in pig, frog, and fish. Concurrently, it has recently been shown that injection of TRH induces skin darkening in the lizard Anolis carolinensis (Licht and Denver, 1988). In the present study, we have thus investigated in vitro the possible effect of TRH on alpha-MSH release from the lizard (Lacerta vivipara) neurointermediate lobe, by means of the perifusion technique. Using our radioimmunoassay procedure, we found that serial dilutions of L. vivipara NIL extracts and synthetic alpha-MSH gave parallel binding curves. Administration of graded doses of TRH (10(-8)-10(-6) M) did not cause any modification of alpha-MSH release. In contrast, infusion of a depolarizing concentration of K+ induced a robust stimulation of alpha-MSH secretion. These results indicate that, in the lizard L. vivipara, the neuropeptide TRH does not stimulate pituitary melanotrophs.     

Comparative effects of corticotropin-releasing factor, arginine vasopressin, and related neuropeptides on the secretion of ACTH and alpha-MSH by frog anterior pituitary cells and neurointermediate lobes in vitro

The ability of corticoliberin (CRF), urotensin I, sauvagine, arginine-vasopressin (AVP), and mesotocin to stimulate ACTH release by frog anterior pituitary cells and alpha-melanotropin (MSH) by frog neurointermediate lobe was studied in vitro using a perifusion technique. CRF and AVP were found to be potent stimulators of ACTH secretion, whereas urotensin I and sauvagine were totally inactive. In opposition to recent findings in the rat. CRF did not modify alpha-MSH secretion by the frog neurointermediate lobe. Mesotocin, which is present in the parenchymal cells of the frog pars intermedia, had no effect on alpha-MSH release in vitro. No potentiation of CRF-induced ACTH release was observed when anterior pituitary cells were incubated with a combination of AVP and CRF. Together with the recent elucidation of a CRF-like molecule in the frog diencephalon, these results suggest that, in Amphibia, CRF and AVP exert their stimulatory action specifically on distal lobe corticotrophs.     

Innervation of the pars intermedia and control of alpha-melanotropin secretion in the newt

In this study we have investigated the presence of nerve fibers containing dopamine, thyrotropin-releasing hormone (TRH) and neuropeptide Y (NPY) in the pars intermedia of the crested newt and we have examined the possible effect of these neurohormones on the release of alpha-melanotropin (alpha-MSH) by neurointermediate lobes in vitro. By means of immunohistochemistry, we observed the presence of tyrosine hydroxylase (TH)-immunoreactive fibers in the pars intermedia of the crested newt. Using a specific antiserum to dopamine, these fibers appeared to be mainly dopaminergic in nature. Unlike anurans, urodele amphibians do not exhibit TRH or NPY-like immunoreactivity in the pars intermedia. A perifusion system technique for newt pituitaries was developed to investigate the effect of dopamine, TRH and NPY on alpha-MSH secretion. Administration of increasing concentrations of dopamine (from 10(-9) to 10(-5)M) induced a dose-related inhibition of alpha-MSH release. This inhibitory effect was mimicked by the dopamine agonist apomorphine (10(-6)M). In contrast, the secretory activity of the newt pars intermedia was not affected by administration of synthetic TRH or NPY (up to 10(-7) and 10(-6)M, respectively). These results indicate that the neurotransmitter dopamine likely plays a pivotal role in the regulation of melanotropin secretion in urodele amphibians.     

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.     

Role of calcium in TRH-induced secretion of alpha-MSH in the frog

In amphibians, alpha-MSH secretion is stimulated by thyrotropin-releasing hormone (TRH). In the present study we show that extracellular calcium influences directly the spontaneous secretion of alpha-MSH and that it participates in the mechanism of action of TRH on frog melanotrophs.     

Pituitary proopiomelanocortin-derived peptides and hypothalamus-pituitary-interrenal axis activity in gilthead sea bream (Sparus aurata) during prolonged crowding stress: differential regulation of adrenocorticotropin hormone and alpha-melanocyte-stimulating hormone release by corticotropin-releasing hormone and thyrotropin-releasing hormone

Plasma levels of cortisol, growth hormone (GH), adrenocorticotropin hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), N-acetyl-beta-endorphin, in vitro ACTH-stimulated cortisol secretion, and in vitro corticotropin-releasing hormone (CRH)- and thyrotropin-releasing hormone (TRH)-stimulated ACTH and alpha-MSH secretion were investigated in gilthead sea bream exposed to high stocking density (30 kg m(-3)) for 23 days. Within 3 days after the onset of crowding, plasma levels of cortisol, ACTH, alpha-MSH, and N-acetyl-beta-endorphin were above control values. After 7 days, plasma parameters had returned to control levels, but at 23 days, cortisol, alpha-MSH, and N-acetyl-beta-endorphin levels were again elevated over controls, indicating a long-term activation of the melanotrope cells. In contrast, crowding stress elicited a prolonged reduction in plasma GH levels concomitant with the increased hypothalamus-pituitary-interrenal axis (HPI) activation. Crowding stress enhanced cortisol secretory activity of the unstimulated interrenal cells. However, interrenal tissue from crowded fish in vitro displayed an attenuated response to ACTH stimulation compared with tissue from control fish, indicating a desensitization of these cells to ACTH during crowding. The involvement of pituitary proopiomelanocortin-derived peptides in the HPI axis of sea bream is indicated by the observed modulation of the CRH and TRH responsiveness of the corticotropes and melanotropes in crowded fish. At day 1, when there were crowding-induced plasma increases in ACTH and alpha-MSH, there was an attenuated CRH-stimulated but not TRH-stimulated, ACTH release. However, at that time, CRH- and TRH-induced responses of alpha-MSH secretion, and the unstimulated secretory activity of the MSH cells, were enhanced in crowded sea bream. These data provide evidence for stimulatory roles of multiple hypothalamic (CRH and TRH) and pituitary (ACTH and alpha-MSH) peptides in the activation of the hypothalamus-pituitary-interrenal axis under crowding conditions in sea bream.     

Effect of Tyr-Pro-Leu-Gly-NH2 on melanotropin-stimulating hormone release in the frog and rat

The tetrapeptide Tyr-Pro-Leu-Gly-NH2 (Tyr-MIFI) has been recently characterized in the hypothalamus and pineal of the rat. Since the concentration of Tyr-MIFI in the brain is increased by pinealectomy and is higher when the animals are in the dark, the possibility that Tyr-MIFI could be a physiological regulator of melanotropin secretion has been investigated. For this study a well-defined perifusion model has been applied, using whole neurointermediate lobes from male frogs (Rana ridibunda Pallas) or male Wistar rats. The amount of alpha-MSH released in the effluent perifusate was measured by means of a sensitive and specific radioimmunoassay method. For concentrations ranging from 10(-10) to 10(-6) M, Tyr-MIFI did not significantly alter the spontaneous release of alpha-MSH in the frog nor did it alter the release of alpha-MSH in the rat. Since it has been recently demonstrated that the tripeptide pGlu-His-Pro-NH2 (mammalian TRH) is a specific MSH-releasing factor in the frog, the possibility that Tyr-MIFI could modulate the response of the intermediate lobe of the frog to TRH has also been investigated. No alteration of TRH-induced alpha-MSH release was observed in the presence of a 100-fold excess of Tyr-MIFI. In addition, Tyr-MIFI was found to be unable to lighten the skin of the frog (Rana pipiens) when applied directly to the pituitary of the darkened animals. Thus these results definitively rule out the possibility that Tyr-MIFI is the melanotropin-release inhibiting factor in the frog or rat.     

Plasticity in the melanotrope neuroendocrine interface of Xenopus laevis

Melanotrope cells of the amphibian pituitary pars intermedia produce alpha-melanophore-stimulating hormone (alpha-MSH), a peptide which causes skin darkening during adaptation to a dark background. The secretory activity of the melanotrope of the South African clawed toad Xenopus laevis is regulated by multiple factors, both classical neurotransmitters and neuropeptides from the brain. This review concerns the plasticity displayed in this intermediate lobe neuroendocrine interface during physiological adaptation to the environment. The plasticity includes dramatic morphological plasticity in both pre- and post-synaptic elements of the interface. Inhibitory neurons in the suprachiasmatic nucleus, designated suprachiasmatic melanotrope-inhibiting neurons (SMINs), possess more and larger synapses on the melanotrope cells in white than in black-background adapted animals; in the latter animals the melanotropes are larger and produce more proopiomelanocortin (POMC), the precursor of alpha-MSH. On a white background, pre-synaptic SMIN plasticity is reflected by a higher expression of inhibitory neuropeptide Y (NPY) and is closely associated with postsynaptic melanotrope plasticity, namely a higher expression of the NPY Y1 receptor. Interestingly, melanotrope cells in such animals also display higher expression of the receptors for thyrotropin-releasing hormone (TRH) and urocortin 1, two neuropeptides that stimulate alpha-MSH secretion. Possibly, in white-adapted animals melanotropes are sensitized to neuropeptide stimulation so that, when the toad moves to a black background, they can immediately initiate alpha-MSH secretion to achieve rapid adaptation to the new background condition. The melanotrope cell also produces brain-derived neurotrophic factor (BDNF), which is co-sequestered with alpha-MSH in secretory granules within the cells. The neurotrophin seems to control melanotrope cell plasticity in an autocrine way and we speculate that it may also control presynaptic SMIN plasticity.     

D2 Dopamine receptor subtype mediates the inhibitory effect of dopamine on TRH-induced prolactin release from the bullfrog pituitary

Dopamine receptors in mammals are known to consist of two D1-like receptors (D1 and D5) and three D2-like receptors (D2, D3 and D4). The aim of this study was to determine the dopamine receptor subtype that mediates the inhibitory action of dopamine on the release of prolactin (PRL) from the amphibian pituitary. Distal lobes of the bullfrog (Rana catesbeiana) were perifused and the amount of PRL released in the effluent medium was measured by means of a homologous enzyme-immunoassay. TRH stimulated the release of PRL from perifused pituitaries. Dopamine suppressed TRH-induced elevation of PRL release. Quinpirole (a D2 receptor agonist) also suppressed the stimulatory effect of TRH on the release of PRL, whereas SKF-38393 (a D1 receptor agonist) exhibited no such an effect. The inhibitory action of dopamine on TRH-induced PRL release from the pituitary was nullified by the addition of L-741,626 (a selective D2 receptor antagonist) to the medium, but not by the addition of SCH-23390 (a selective D1 receptor antagonist). These data indicate that the inhibitory effect of dopamine on TRH-evoked PRL release from the bullfrog pituitary gland is mediated through D2 dopamine receptors.     

In vitro study of frog (Rana ridibunda pallas) neurointermediate lobe secretion by use of a simplified perifusion system: II. Lack of action of thyroxine on TRH-induced α-MSH secretion

Thyrotropin-releasing hormone (TRH) stimulates α-melanocyte-stimulating hormone (α-MSH) secretion in amphibia as well as thyrotropin-stimulating hormone (TSH) and prolactin secretions in mammals. Since thyroid hormones regulate the stimulatory effect of TRH on TSH and prolactin, the possible role of thyroxine (T₄) in the control of α-MSH secretion in amphibia, has been investigated. Neurointermediate lobes of Rana ridibunda were perifused in amphibian culture medium for 7 hr and the amounts of α-MSH released into the effluent perfusate were measured by radioimmunoassay. In vivo treatment with T₄ (0.5 mg/kg twice a day for 9 days) did not modify the in vitro response of the neurointermediate lobes to TRH (10^?9 to 10^?7M). In addition, prolonged infusion of T₄in vitro did not alter spontaneous and TRH-induced α-MSH release. In spite of the inhibitory effect of T₄ on TRH-induced TSH and prolactin secretions in mammals, the present data show that, in frogs, thyroid hormone does not modulate the stimulation of α-MSH secretion induced by TRH.     

Dopamine inhibition of gonadotropin and alpha-melanocyte-stimulating hormone release in vitro from the pituitary of the goldfish (Carassius auratus)

The purpose of the present investigation was to examine the receptor specificity of dopamine inhibition of gonadotropin (GtH) and alpha-melanocyte-stimulating hormone (alpha-MSH) release from the goldfish (Carassius auratus) pituitary in vitro. Pars distalis (PD) and neurointermediate lobe (NIL) fragments of the goldfish pituitary were superfused in vitro under various experimental paradigms; eluate from PD and NIL fragments was analyzed for (GtH) and (alpha-MSH), respectively. Spontaneous GtH release from PD fragments was relatively constant over 6 hr; continuous superfusion with dopamine reversibly inhibited spontaneous GtH release with an estimated ED50 of 10(-4.4) M. Domperidone, a specific D-2 receptor antagonist, reversed the inhibitory action of dopamine and increased spontaneous GtH release. Acute treatment of PD fragments with salmon GnRH (sGnRH) stimulated GtH release; dopamine inhibited GtH release from similarly treated fragments with an ED50 of 10(-7.5) M. The spontaneous release of alpha-MSH from NIL fragments was relatively constant over 6 hr; continuous superfusion with dopamine reversibly inhibited this release with an ED50 of 10(-7.2) M. Acute treatment of NIL fragments with thyrotropin-releasing hormone (TRH) caused acute dose-related increases in alpha-MSH release with an ED50 of 10(-8.2) M; dopamine reversibly inhibited alpha-MSH release from similarly treated fragments with an ED50 of 10(-7.7) M. Both stereoisomers of apomorphine, a dopamine agonist, inhibited GtH release from PD fragments treated with sGnRH; in contrast, alpha-MSH release from NIL fragments treated with TRH was stereospecifically inhibited by (-)-apomorphine, but not by (+)-apomorphine. Domperidone reversed (ED50 = 10(-6.6) M) dopamine (10(-6.3) M) inhibition of GtH release from PD fragments treated with sGnRH. In NIL fragments, the inhibitory action of dopamine (10(-6.3) M) was reversed by domperidone (ED50 = 10(-5.5) M), which restored the acute alpha-MSH release response to TRH. These results suggest the involvement of a low-affinity dopamine/neuroleptic receptor in dopamine inhibition of GtH and alpha-MSH release from the pituitary of the goldfish.     

Neuropeptide Y impairs insulin-stimulated translocation of glucose transporter 4 in 3T3-L1 adipocytes through the Y1 receptor

Neuropeptide Y (NPY) is expressed in adipose tissue and is involved in adipocyte metabolism. Although NPY impacts on glucose utilization in vivo, the underlying cellular mechanism is yet to be fully elucidated.In this study we investigated the effect of NPY on the insulin-stimulated translocation of glucose transporter 4 (GLUT4) from intracellular stores to the cell surface in vitro. Using cellular fractionation and immunofluorescence we analyzed the cellular localization and content of GLUT4 in 3T3-L1 adipocytes. Additionally we investigated the effect of NPY on insulin action in adipocyte cultures by assessing the phosphorylation of Akt and [³H]-deoxyglucose uptake.Our data suggest that in 3T3-L1 adipocytes NPY inhibits insulin-stimulated glucose uptake in a GLUT4-dependent manner. The insulin induced translocation of GLUT4 was attenuated by the Y1 receptor agonist [Phe(7),Pro(34)] pNPY, demonstrating an essential role of the Y1 receptor in GLUT4 translocation. Additionally, we observed an NPY dose-dependent impairment of Akt phosphorylation.This study provides evidence that NPY impairs the insulin sensitivity of adipocytes and suggests that the Y1 receptor could be a potential therapeutic target for type 2 diabetes.