Low temperature stimulates alpha-melanophore-stimulating hormone secretion and inhibits background adaptation in Xenopus laevis

It is well-known that alpha-melanophore-stimulating hormone (alpha-MSH) release from the amphibian pars intermedia (PI) depends on the light condition of the animal's background, permitting the animal to adapt the colour of its skin to background light intensity. In the present study, we carried out nine experiments on the effect of low temperature on this skin adaptation process in the toad Xenopus laevis, using the skin melanophore index (MI) bioassay and a radioimmunoassay to measure skin colour adaptation and alpha-MSH secretion, respectively. We show that temperatures below 8 degrees C stimulate alpha-MSH secretion and skin darkening, with a maximum at 5 degrees C, independent of the illumination state of the background. No significant stimulatory effect of low temperature on the MI and alpha-MSH plasma contents was noted when the experiment was repeated with toads from which the neurointermediate lobe (NIL) had been surgically extirpated. This indicates that low temperature stimulates alpha-MSH release from melanotrope cells located in the PI. An in vitro superfusion study with the NIL demonstrated that low temperature does not act directly on the PI. A possible role of the central nervous system in cold-induced alpha-MSH release from the PI was tested by studying the hypothalamic expression of c-Fos (as an indicator for neuronal activity) and the coexistence of c-Fos with the regulators of melanotrope cell activity, neuropeptide Y (NPY) and thyrotrophin-releasing hormone (TRH), using double fluorescence immunocytochemistry. Upon lowering temperature from 22 degrees C to 5 degrees C, in white-adapted animals c-Fos expression decreased in NPY-producing suprachiasmatic-melanotrope-inhibiting neurones (SMIN) in the ventrolateral area of the suprachiasmatic nucleus (SC) but increased in TRH-containing neurones of the magnocellular nucleus. TRH is known to stimulate melanotrope alpha-MSH release. We conclude that temperatures around 5 degrees C inactivate the SMIN in the SC and activate TRH-neurones in the magnocellular nucleus, resulting in enhanced alpha-MSH secretion from the PI, darkening the skin of white-adapted X. laevis.     

Demonstration of coexisting catecholamine (dopamine), amino acid (GABA), and peptide (NPY) involved in inhibition of melanotrope cell activity in Xenopus laevis: a quantitative ultrastructural, freeze-substitution immunocytochemical study.

This quantitative ultrastructural immunocytochemical study demonstrates the coexistence of a catecholamine [dopamine (DA)], an amino acid (GABA), and a neuropeptide [neuropeptide Y (NPY)] in axon varicosities innervating the pars intermedia of Xenopus laevis. The varicosities are assumed to control the pars intermedia melanotrope cells, which regulate skin color during the physiological process of background adaptation. Varicosity profiles appear to abut melanotrope cells and folliculostellate cells, star-shaped cells that intimately contact the melanotropes. All varicosity profiles contain two morphological types of vesicle. Monolabeling studies on routinely fixed and freeze-substituted tissues showed that the small, electron-lucent vesicles store GABA, whereas DA and NPY occur in larger, electron-dense ones. Double and triple labeling experiments, in which the degree of immunoreactivity was quantified per varicosity profile and per vesicle, led to the conclusion that (1) DA, GABA, and NPY coexist within almost all varicosity profiles and (2) DA and NPY are costored within electron-dense vesicles. Varicosity profiles that about melanotrope cells show a much higher ratio between the numbers of electron-lucent and electron-dense vesicles than varicosities contacting folliculostellate cells (15.8 and 3.3, respectively). This differential distribution is in line with the previous demonstration that, in contrast to GABA, NPY does not act directly on the melanotrope cells but indirectly, by controlling the activity of the folliculostellate cells.     

Identification of suprachiasmatic melanotrope-inhibiting neurons in Xenopus laevis: A confocal laser-scanning microscopy study

The amphibian Xenopus laevis is able to adjust its skin color to the light intensity of the environment. Paling of the skin is achieved by inhibiting the release of α-melanophore-stimulating hormone (α-MSH) from the melanotrope cells in the pars intermedia of the pituitary gland. The release of α-MSH is inhibited by γ-aminobutyric acid (GABA), neuropeptide Y (NPY), and dopamine (DA). To locate and identify neurons that might be responsible for the inhibitory input, double and triple immunocytochemistry, retrograde tracing from the pars intermedia with the carbocyanine membrane probe 1,1′dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine, 4-chlorobenzene-sulfonate (Fast DiI), and confocal laser-scanning microscopy were combined. Glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), and NPY were found to coexist in an axonal network innervating the pars intermedia. The suprachiasmatic nucleus (SC) contained different populations of neurons that were single, double, or triple labelled for GAD, NPY, and TH. In the lateral SC, NPY⁺ neurons were observed. TH-immunoreactive (TH-IR) neurons occurred in the medial, dorsolateral, lateral, and ventrolateral SC. Neurons that were double labelled for NPY and TH and triple labelled for Fast DiI, NPY, and TH were present in the ventrolateral SC. This same area contained neurons that were triple labelled for GAD, NPY, and TH. It is concluded that the triple-labelled and probably the double-labelled ventrolateral SC neurons (suprachiasmatic melanotrope-inhibiting neurons) innervate the pituitary pars intermedia and are responsible for the NPY-, DA-, and GABA-mediated inhibition of melanotrope cell activity in Xenopus laevis. J. Comp. Neurol. 397:60–68, 1998. © 1998 Wiley-Liss, Inc.     

Functional organization of the suprachiasmatic nucleus of Xenopus laevis in relation to background adaptation

The process of background adaptation in the toad Xenopus laevis is controlled by neurons in the suprachiasmatic nucleus (SC) that inhibit the release of alpha-melanophore-stimulating hormone from the neuroendocrine melanotrope cells in the pituitary gland. We have identified the structural and functional organization of different neuropeptide Y (NPY)-containing cell groups in the Xenopus SC in relation to background adaptation. A ventrolateral, a dorsomedial, and a caudal group were distinguished, differing in location as well as in number, size, and shape of their cells. They also show different degrees of NPY immunoreactivity in response to different background adaptation conditions. In situ hybridization using a Xenopus mRNA probe for the exocytosis protein DOC2 revealed that melanotrope cells of black-adapted animals have a much higher expression of DOC2-mRNA than white-adapted ones. This establishes that the degree of DOC2-mRNA expression is a good parameter to measure cellular secretory activity in Xenopus. We show that in the ventrolateral SC group, more NPY-positive neurons express DOC2-mRNA in white- than in black-adapted animals. In contrast, NPY-positive neurons in the dorsomedial group have a high secretory activity under the black-adaptation condition. We propose that in black-adapted animals, NPY-positive neurons in the ventrolateral group, known to inhibit the melanotrope cells in white-adapted animals synaptically, are inhibited by NPY-containing interneurons in the dorsmedial group. NPY-positive neurons in the caudal group have similar secretory dynamics as the dorsomedial NPY neurons, indicating that they also play a role in background adaptation, distinct from that exerted by the ventrolateral and dorsomedial group.     

Plasticity of melanotrope cell regulations in Xenopus laevis

This review focuses on the plasticity of the regulation of a particular neuroendocrine transducer cell, the melanotrope cell in the pituitary pars intermedia of the amphibian Xenopus laevis. This cell type is a suitable model to study the relationship between various external regulatory inputs and the secretion of an adaptive endocrine message, in this case the release of α-melanophore-stimulating hormone, which activates skin melanophores to darken when the animal is placed on a dark background. Information about the environmental conditions is processed by various brain centres, in the hypothalamus and elsewhere, that eventually control the activity of the melanotrope cell regarding hormone production and secretion. The review discusses the roles of these hypothalamic and extrahypothalamic nuclei, their neurochemical messengers acting on the melanotrope, and the external stimuli they mediate to control melanotrope cell functioning.     

Serotonergic innervation of the pituitary pars intermedia of xenopus laevis

At this point three brain centres are thought to be involved in the regulation of the melanotrope cells of the pituitary pars intermedia of Xenopus laevis: the magnocellular nucleus, the suprachiasmatic nucleus and the locus coeruleus. This study aims to investigate the existence of a fourth, serotonergic, centre controlling the melanotrope cells. In-vitro superfusion studies show that serotonin has a dose-dependent stimulatory effect on peptide release (1.6 x basal level at 10(-6) M serotonin) from single melanotrope cells. Retrograde neuronal tract tracing experiments, with the membrane probe FAST Dil applied to the pars intermedia, reveals retrogradely labelled neurones in the magnocellular nucleus, the suprachiasmatic nucleus, the locus coeruleus and the raphe nucleus. Of these brain centres, after immunocytochemistry only the raphe nucleus revealed serotonin-immunoreactive cell bodies. In addition, serotonin-immunoreactive cell bodies were found in the nucleus of the paraventricular organ, the posteroventral tegmental nucleus and the reticular istmic nucleus. In the pituitary, the pars nervosa, pars intermedia and pars distalis all reveal serotonin-immunoreactive nerve fibres. With immunocytochemical double-labelling for tyrosine hydroxylase and serotonin no colocalization of serotonin and tyrosine hydroxylase was observed in cell bodies in the brain, and in the pituitary hardly any colocalization was found in the nerve fibres. However, after in-vitro loading of neurointermediate lobes with serotonin, tyrosine hydroxylase and serotonin appear to coexist in a fibre network in the pars intermedia. On the basis of these data we propose that the melanotrope cells in the Xenopus pars intermedia are innervated by a 5-HT network originating in the raphe nucleus; this network represents the first identified stimulatory input to the pars intermedia of this species.     

Alpha-melanophore-stimulating hormone in the brain,cranial placode derivatives,and retina of Xenopus laevis during development in relation to background adaptation

The amphibian Xenopus laevis can adapt the color of its skin to the light intensity of the background. A key peptide in this adaptation process is alpha-melanophore-stimulating hormone (alpha-MSH), which is derived from proopiomelanocortin (POMC) and released by the endocrine melanotrope cells in the pituitary pars intermedia. In this study, the presence of alpha-MSH in the brain, cranial placode derivatives, and retina of developing Xenopus laevis was investigated using immunocytochemistry, to test the hypothesis that POMC peptide-producing neurons and endocrine cells have a common embryonic origin and a common function, i.e., controlling each other's activities and/or being involved in the process of physiological adaptation. The presence of alpha-MSH-positive cells in the suprachiasmatic nucleus, ventral hypothalamic nucleus, epiphysis, and endocrine melanotrope and corticotrope cells, which are all involved in regulation of adaptation processes, has been detected from stage 37/38 onward. This is consistent with the presumed common origin of these cells, the anterior neural ridge (ANR) of the neural-plate-stage embryo. The olfactory epithelium and the otic and epibranchial ganglia also contain alpha-MSH, indicating that these placodal derivatives originate from a common placodal domain continuous with the ANR. Furthermore, we demonstrate the presence of alpha-MSH in a subpopulation of retinal ganglion cells (RGCs), which is possibly also derived from the ANR. Immunoreactivity for alpha-MSH in RGCs that are located in the dorsal part of the retina is dependent on the background light intensity, suggesting that these cells are involved in the regulation of background adaptation. Taken together, the results support the hypothesis that POMC peptide-producing cells have a common embryonic origin and are involved in adaptation processes.     

Activity-dependent dynamics of coexisting brain-derived neurotrophic factor, pro-opiomelanocortin and alpha-melanophore-stimulating hormone in melanotrope cells of Xenopus laevis

Brain-derived neurotrophic factor (BDNF) is involved as an autocrine factor in the regulation of the secretory activity of the neuroendocrine pituitary melanotrope cells of Xenopus laevis. We studied the subcellular distribution of BDNF in Xenopus melanotropes using a combination of high-pressure freezing, cryosubstitution and immunoelectron microscopy. Presence of BDNF, pro-opiomelanocortin (POMC) and alpha-melanophore-stimulating hormone (alphaMSH) within melanotrope secretory granules was studied by triple-labelling immunoelectron microscopy. In addition, intracellular processing of BDNF was investigated by quantifying the number of immunogold particles in different stages of secretory granule maturation, in animals adapted to black or white background light conditions. The high-pressure freezing technique provides excellent preservation of both cellular ultrastructure and antigenicity. BDNF coexists with POMC and alphaMSH within secretory granules. BDNF-immunoreactivity increases along the secretory granule maturation axis (i.e. from electron-dense, via moderately electron-dense, to electron-lucent secretory granules). Immature, low immunoreactive, electron-dense secretory granules are assumed to contain mainly or even exclusively proBDNF. Strongly immunoreactive electron-lucent secretory granules represent the mature granule stage in which proBDNF has been processed to mature BDNF. Furthermore, in moderately electron-dense secretory granules, immunoreactivity is markedly (+79%) higher in black-adapted than in white-adapted animals, indicating that stimulation of melanotrope cell activity by the black background condition speeds up processing of BDNF from its precursor in this granule stage. It is concluded that, in the Xenopus melanotrope, BDNF biosynthesis and processing occur along the secretory granule maturation axis, together with that of POMC-derived alphaMSH, and that the environmental light condition not only controls the biosynthesis and secretion of BDNF and of POMC end-products, but also regulates the rate of their intragranular processing.     

Co-localization of tyrosine hydroxylase, GABA and neuropeptide Y within axon terminals innervating the intermediate lobe of the frog Rana ridibunda.

Possible co-existence of gamma-aminobutyric acid (GABA), catecholamines, and neuropeptide Y (NPY) in the same nerve terminals of the frog intermediate lobe was investigated by immunocytochemistry at the electron microscopic level. Co-localization of GABA and tyrosine hydroxylase (TH) was studied by using a double immunogold labeling procedure. Co-localization of glutamate decarboxylase (GAD) and NPY was studied by combining, respectively, the peroxidase-antiperoxidase method and a radioimmunocytochemical labeling procedure. Catecholamines and GABA were systematically co-localized in nerve endings of the pars intermedia. Most of the NPY-immunoreactive fibers also contained GAD-like immunoreactivity. However, a few NPY-positive nerve terminals were not immunoreactive for GAD. These data provide evidence for co-existence of a regulatory peptide (NPY) and several neurotransmitters (i.e., GABA and catecholamines) within the same axon terminals in the intermediate lobe. Since GABA, dopamine, and NPY have all been shown to inhibit the activity of frog melanotrope cells, the present findings suggest that these neuroendocrine factors may interact either at the pre-synaptic or post-synaptic level.     

GABA and Dopamine Act Directly on Melanotropes of Xenopus to inhibit MSH secretion

The release of melanophore stimulating hormone (MSH) from the pars intermedia of the amphibian Xenopus laevis is regulated by multiple factors of hypothalamic origin. The aim of this study was to determine if potential secretagogues function through a direct action on the melanotrope cell. For this purpose an in vitro superfusion system containing isolated melanotropes (cell suspension) was utilized. The viability of the cells in suspension was tested by examining their ability to synthesize, process and release pro-opiomelanocortin (POMC) related peptides. All biosynthetic functions appeared normal, with the exception that the isolated melanotropes are unable to N-terminally acetylate MSH. Release of immunoreactive-MSH from these cells was shown to be Ca²⁺-dependent and high K⁺ stimulated release. Both the neurotransmitters dopamine and γ-aminobuiyric acid (GABA), which are thought to be physiologically important MSH-release inhibiting factors, were shown to inhibit MSH release from isolated melanotropes. Dopamine appeared to function through a dopamine D2 type receptor mechanism while for GABA, both a GABAa and GABAb receptor mechanism are involved.     

Distribution of the mRNAs encoding the thyrotropin-releasing hormone (TRH) precursor and three TRH receptors in the brain and pituitary of Xenopus laevis: effect of background color adaptation on TRH and TRH receptor gene expression

In amphibians, thyrotropin-releasing hormone (TRH) is a potent stimulator of alpha-melanotropin (alpha-MSH) secretion, so TRH plays a major role in the neuroendocrine regulation of skin-color adaptation. We have recently cloned a third type of TRH receptor in Xenopus laevis (xTRHR3) that has not yet been characterized in any other vertebrate species. In the present study, we have examined the distribution of the mRNAs encoding proTRH and the three receptor subtypes (xTRHR1, xTRHR2, and xTRHR3) in the frog CNS and pituitary, and we have investigated the effect of background color adaptation on the expression of these mRNAs. A good correlation was generally observed between the expression patterns of proTRH and xTRHR mRNAs. xTRHRs, including the novel receptor subtype xTRHR3, 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 expressed selectively in the distal lobe, and xTRHR3 was found exclusively in the intermediate lobe. Adaptation of frog skin to background illumination had no effect on the expression of proTRH and xTRHRs in the brain. In contrast, adaptation of the animals to a white background provoked an 18-fold increase in xTRHR3 mRNA concentration in the intermediate lobe of the pituitary. These data demonstrate that, in amphibians, the effect of TRH on alpha-MSH secretion is mediated through the novel receptor subtype xTRHR3.     

Localization and physiological regulation of the exocytosis protein SNAP-25 in the brain and pituitary gland of Xenopus laevis

In mammals, the synaptosomal-associated protein of 25 kDa, SNAP-25, is generally thought to play a role in synaptic exocytosis of neuronal messengers. Using a polyclonal antiserum against rat SNAP-25, we have shown the presence of a SNAP-25-like protein in the brain of the South-African clawed toad Xenopus laevis by Western blotting and immunocytochemistry. Xenopus SNAP-25 is ubiquitously present throughout the brain, where its distribution in various identified neuronal perikarya and axon tracts is described. Western blot analysis and immunocytochemistry also demonstrated the presence of SNAP-25 in the neural, intermediate and distal lobes of the pituitary gland. Intensity line plots of confocal laser scanning microscope images of isolated melanotropes indicated that SNAP-25 is produced and processed in the rough endoplasmatic reticulum and Golgi apparatus, and is associated with the plasma membrane. Immunoelectron microscopy substantiated the idea that SNAP-25 is present in the plasma membrane but also showed a close association of SNAP-25 with the bounding membrane of peptide-containing secretory granules in both the neurohemal axon terminals in the neural lobe and the endocrine melanotropes in the intermediate lobe. Quantitative Western blotting revealed that adapting Xenopus to a dark background has a clear stimulatory effect on the expression of SNAP-25 in the neural lobe and in the melanotrope cells. This background light intensity-dependent stimulation of SNAP-25 expression was confirmed by the demonstration of increased immunofluorescence recorded by confocal laser scanning microscopy of individual melanotropes of black background-adapted toads. On the basis of this study on Xenopus laevis, we conclude that SNAP-25 (i) plays a substantial role in the secretion of a wide variety of neuronal messengers; (ii) functions in the central nervous system but also in neurohormonal and endocrine systems; (iii) acts at the plasma membrane but possibly also at the membrane of synaptic vesicles and peptide-containing secretory granules; (iv) acts not only locally (as in synapses), but at various sites of the plasma membrane (as in the endocrine melanotrope cell); and (v) can be upregulated in its expression by physiological stimuli that increase the extent of the molecular machinery involved in exocytosis.     

Characterization of pro-ACTH/endorphin-derived peptides in rat hypothalamus

The proteolytic processing pattern of pro-ACTH/endorphin in rat hypothalamus is similar to the pattern in the pars intermedia; peptides the size of beta-endorphin, gamma-lipotropin (gamma-LPH), corticotropin-like intermediate lobe peptide (CLIP), alpha-melanotropin (gamma-MSH), joining peptide, and glycosylated gamma 3-MSH all represent predominant end products. Equimolar amounts of beta-endorphin-, alpha-MSH-, CLIP-, gamma-LPH-, and joining peptide-related immunoreactivity are found in hypothalamic extracts (approximately 3 pmol per hypothalamus). Although the proteolytic processing pattern in the hypothalamus is similar to that in the pars intermedia, a tissue-specific posttranslational processing pattern was detected. Ion-exchange analysis of beta-endorphin-sized immunoreactive material from hypothalamic extracts resolves three major forms, corresponding to beta-endorphin(1-31), beta-endorphin(1-27), and beta-endorphin(1-26). The alpha-N-acetylated forms of endorphin represent less than 10% of the total beta-endorphin immunoreactivity. Analyses of hypothalamic alpha-MSH-sized molecules with acetyl- and amide-directed alpha-MSH antisera suggest that hypothalamic alpha-MSH is fully amidated, but largely not alpha-N-acetylated. Fractionation by reverse-phase high-performance liquid chromatography (HPLC) confirms that greater than 85% of the alpha-MSH immunoreactivity corresponds to ACTH(1-13)NH2 or its sulfoxide, and less than 10% corresponds to alpha-MSH [alpha-N-acetyl-ACTH(1-13)NH2] or its sulfoxide. Isoelectric focusing demonstrates that 83-93% of hypothalamic CLIP is phosphorylated. Isoelectric focusing suggests that the majority of the hypothalamic gamma-LPH-sized immunoreactive material is indistinguishable from gamma-LPH synthesized by pituitary melanotropes. The minor extent of alpha-N-acetylation of alpha-MSH and beta-endorphin, the limited carboxyl-terminal proteolysis of beta-endorphin, and the extensive phosphorylation of CLIP represent major differences between the posttranslational processing patterns of pro-ACTH/endorphin in the hypothalamus and pars intermedia.     

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

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

Sauvagine regulates Ca2+ oscillations and electrical membrane activity of melanotrope cells of Xenopus laevis

Ca2+ oscillations regulate secretion of the hormone alpha-melanphore-stimulating hormone (alpha-MSH) by the neuroendocrine pituitary melanotrope cells of the amphibian Xenopus laevis. These Ca2+ oscillations are built up by discrete increments in the intracellular Ca2+ concentration, the Ca2+ steps, which are generated by electrical membrane bursting firing activity. It has been demonstrated that the patterns of Ca2+ oscillations and kinetics of the Ca2+ steps can be modulated by changing the degree of intracellular Ca2+ buffering. We hypothesized that neurotransmitters known to regulate alpha-MSH secretion also modulate the pattern of Ca2+ oscillations and related electrical membrane activity. In this study, we tested this hypothesis for the secretagogue sauvagine. Using high temporal-resolution Ca2+ imaging, we show that sauvagine modulated the pattern of Ca2+ signalling by increasing the frequency of Ca2+ oscillations and inducing a broadening of the oscillations through its effect on various Ca2+ step parameters. Second, we demonstrate that sauvagine caused a small but significant decrease in K+ currents measured in the whole-cell voltage-clamp, whereas Ca2+ currents remained unchanged. Third, in the cell-attached patch-clamp mode, a stimulatory effect of sauvagine on action current firing was observed. Moreover, sauvagine changed the shape of individual action currents. These results support the hypothesis that the secretagogue sauvagine stimulates the frequency of Ca2+ oscillations in Xenopus melanotropes by altering Ca2+ step parameters, an action that likely is evoked by an inhibition of K+ currents.     

Enhanced inhibitory feeding response to alpha-melanocyte stimulating hormone in the diet-induced obese rat

A dysregulation in the hypothalamic neuropeptide systems involved in the control of appetite has previously been shown in models of diet-induced obesity. In the present study, male Sprague-Dawley rats were rendered obese by a highly palatable cafeteria-style diet over 20 weeks, while control rats had access to standard laboratory chow. Feeding responses to alpha-melanocyte stimulating hormone (alpha-MSH), an anorectic peptide and neuropeptide Y (NPY), a potent orexigenic agent were investigated in diet-induced obese and control animals. In addition, endogenous hypothalamic peptide levels were determined in these animals. Intracerebroventricular injections of either 4 nmol alpha-MSH or saline vehicle were given 10 min prior to the onset of the dark phase. Diet-induced obese rats had significantly enhanced nocturnal inhibitory feeding responses to alpha-MSH (P<0.05). The orexigenic feeding response induced by 1 nmol NPY was similar for both groups. At sacrifice, both alpha-MSH and NPY peptide content were selectively reduced in the paraventricular nucleus (PVN) of these animals (P<0.05). Although diet-induced obesity had no effect on responses to NPY, the significantly greater inhibition of nocturnal feeding by alpha-MSH and reduction in PVN alpha-MSH peptide level, suggests melanocortinergic signalling may be reduced in obesity which may account for the hyperphagia of these animals when presented with a palatable diet.     

The melanocortin agonist Melanotan-II reduces the orexigenic and adipogenic effects of neuropeptide Y (NPY) but does not affect the NPY-driven suppressive effects on the gonadotropic and somatotropic axes in the male rat

Neuropeptide Y (NPY) is a strong orexigenic neurotransmitter also known to modulate several neuroendocrine axes. alpha-Melanocyte-stimulating hormone (MSH) is an essential anorectic neuropeptide, acting on hypothalamic MC3/4 receptor subtypes. When given as an intracerebroventricular bolus injection, Melanotan-II (MT-II), a non selective MC receptor agonist, inhibits feeding, suppresses the NPY orexigenic action, and reduces basal insulinaemia. We evaluated the effects of a 7-day central infusion of MT-II (15 nmol/day) given either alone or in association with NPY (5 nmol/day) in male Sprague-Dawley rats. MT-II produced almost full anorexia for 1-2 days but then feeding gradually returned to normal despite continued MT-II infusion. When coinfused with NPY, MT-II also produced the same initial anorectic episode but then maintained feeding to upper normal levels, thus cancelling the hyperphagia driven by NPY. Whereas NPY infusion produced a doubling of fat pad weight, MT-II reduced adiposity by a factor of two compared to pair-fed rats, and vastly curtailed the NPY-driven increase in fat pad weight. MT-II infusion also significantly curtailed the NPY-induced rise in insulin and leptin secretions. NPY infusion significantly inhibited hypothalamic pro-opiomelanocortin mRNA expression, most likely cancelling the alpha-MSH anorectic activity. As expected from previous studies, chronic NPY infusion strongly inhibited both the gonadotropic and somatotropic axes, and coinfusion of MT-II did not reverse these NPY-driven effects, in sharp contrast with that seen for the metabolic data. MT-II infusion alone had little effect on these axes. In conclusion, chronic MT-II infusion generated a severe but transient reduction in feeding, suggesting an escape phenomenon, and clearly reduced fat pad size. When coinfused with NPY, MT-II was able to cancel most of the NPY effects on feeding, but not those on the neuroendocrine axes. It appears therefore that, as expected, NPY and alpha-MSH closely interact in the control of feeding, whereas the neural pathways by which NPY affects growth and reproduction are distinct and not sensitive to MC peptide modulation.     

Inhibitory effects of NPY on ganglionic transmission in myenteric neurones of the guinea-pig descending colon

Intracellular recordings were made from myenteric neurones of the guinea-pig descending colon. Neuropeptide Y (NPY) and related pancreatic polypeptides were applied by superfusion and the effects upon the amplitude of fast excitatory synaptic potentials (ESPs) and the ratio of paired fast ESPs evoked by stimulation of internodal fibre tracts were noted. NPY produced a concentration-dependent inhibition in fast ESP amplitude in the majority of neurones (17/21) with a calculated IC50 value of 7 nM; in some neurones this inhibition was mediated via the local release of noradrenaline. Peptide YY (PYY) (eight out of 11 neurones; IC50 = 1 nM), NPY(3-36) (three out of three neurones) and [Leu31, Pro34]NPY (four out of five neurones) also decreased the amplitude of fast ESPs. The effects of two or more pancreatic polypeptides or analogues on fast synaptic transmission were compared directly in six neurones; the apparent relative potency of agonists suggested the involvement of Y2-receptors and at least one other Y-receptor type. In the absence of any direct postsynaptic effects of pancreatic polypeptides on the active or passive properties of myenteric neurones, or on their sensitivity to ionophoretically applied acetylcholine, inhibition of fast ganglionic transmission was presumed to be presynaptic in origin. It is concluded that, in addition to their previously described depressant actions on neuro-effector transmission to colonic smooth muscle, pancreatic polypeptides can exert powerful inhibitory effects on myenteric neurones of the descending colon.