B.G. Jenks

Neuropeptide Y: localization in the central nervous system and neuroendocrine functions

Summary— Neuropeptide Y (NPY) is a 36‐amino acid peptide first isolated and characterized from porcine brain extracts. A number of immunocytochemical investigations have been conducted to determine the localization of NPY‐containing neurons in various animal species including both vertebrates and invertebrates. These studies have established the widespread distribution of NPY in the brain and in sympathetic neurons. In the rat brain, a high density of immunoreactive cell bodies and fibers is observed in the cortex, caudate putamen and hippocampus. In the diencephalon, NPY‐containing perikarya are mainly located in the arcuate nucleus of the hypothalamus; numerous fibers innervate the paraventricular and suprachiasmatic nuclei of the hypothalamus, as well as the paraventricular nucleus of the thalamus and the periaqueductal gray. At the electron microscope level, using the pre‐ and post‐embedding immunoperoxidase techniques, NPY‐like immunoreactivity has been observed in neuronal cell body dendrites and axonal processes. In nerve terminals of the hypothalamus, the product of the immunoreaction is associated with large dense core vesicles. In lower vertebrates, including amphibians and fish, neurons originating from the diencephalic (or telencephalic) region innervate the intermediate lobe of the pituitary where a dense network of immunoreactive fibers has been detected. At the ultrastructural level, positive endings have been observed in direct contact with pituitary melanotrophs of frog and dogfish. These anatomical data suggest that NPY can act both as a neurotransmitter (or neuromodulator) and as a hypophysiotropic neurohormone.

An npy-like peptide may function as msh-release inhibiting factor in xenopus-laevis

This study demonstrates the presence of a rich plexus of neuropeptide Y (NPY) immunoreactive fibers in the hypothalamus and in the intermediate lobe of the pituitary of Xenopus laevis. During superfusion of neurointermediate lobe tissue, synthetic NPY induces a rapid, powerful and dose-dependent inhibition of in vitro release of MSH, endorphin and other proopiomelanocortin (POMC) derived peptides. Therefore, NPY undoubtedly is one of the growing number of neuropeptides that are likely involved in control of the amphibian MSH cells. Although a number of stimulatory neuropeptides have been found, this is the first neuropeptide to apparently function through an inhibitory mechanism. In that a 2-hr treatment with NPY did not influence POMC biosynthesis, nor processing of this prohormone to smaller peptides, we conclude that the primary action of NPY is a direct effect on the secretory process of the MSH cell.

Regulation of msh release from the neurointermediate lobe of xenopus-laevis by crf-like peptides

Immunocytochemical studies showed the presence of a fiber system containing a CRF-like peptide in the median eminence and in the neural lobe of the pituitary gland of Xenopus laevis. During in vitro superfusion of neurointermediate lobe tissue, CRF, sauvagine and urotensin I induced a rapid and dose-dependent stimulation of secretion of MSH and endorphin. Tissue of white-background adapted animals displayed a remarkably higher sensitivity to CRF and sauvagine than tissue from animals that were adapted to a black background. During superfusion of isolated melanotrope cells in suspension, it was shown that CRF and sauvagine exerted their effect directly on the melanotrope cell. We therefore conclude that there is morphological and biochemical evidence to consider a CRF-like peptide as a physiological MSH-releasing factor.

Assessment of trh as a potential msh release stimulating factor in xenopus-laevis

This study considers the possible involvement of the tripeptide TRH (thyrotropin releasing hormone) in the physiological regulation of melanophore stimulating hormone (MSH) secretion from the pars intermedia of the toad, Xenopus laevis. TRH was shown to stimulate release of MSH from superfused neurointermediate lobes obtained from white-background adapted animals, but had no effect on secretion from lobes of black-background adapted animals. Immunohistochemical analysis revealed a rich TRH-containing neuronal network terminating in the neural lobe of the Xenopus pituitary. Plasma levels of TRH, determined with a specific radioimmunoassay, proved to be extremely high and no significant difference in this level could be found between white- and black-adapted animals. Plasma TRH probably originates from the skin, and our results show that its concentration is within the effective concentration range established for this peptide in stimulating MSH release from the pars intermedia. Therefore, while both our superfusion and immunohistochemical results argue favourably for a function of TRH in the regulation of MSH secretion, we conclude that, in any regulatory role, it would likely have to function within the pars intermedia at concentrations exceeding the high plasma values. While TRH could be involved in short-term activation of the secretory process in white-background adapted animals or in animals undergoing the initial stages of black background adaptation, our results indicate that this peptide may have no function in the maintenance of secretion from the pars intermedia of animals fully adapted to black background.

Characteristics of receptors for dopamine in the pars-intermedia of the amphibian xenopus-laevis

While mammalian dopamine receptors have been extensively characterized, very little attention has been given to these receptors in lower vertebrates. Dopamine is thought to be a physiologically important melanotropin release-inhibiting factor in amphibians. By administering selective dopamine receptor agonists and antagonists to superfused neurointermediate lobes of Xenopus laevis and monitoring their effect on the release of melanophore-stimulating hormone (MSH), we have examined the characteristics of the receptors involved in the dopamine-induced inhibition of MSH secretion. The results show that the receptor system involved has characteristics of both classical D-2 receptors and alpha-adrenergic receptors. This is concluded from: the agonistic effect of both the D-2 receptor agonists LY-171555 and apomorphine; the antagonism of this effect, not only by the D-2 receptor antagonists sulpiride, domperidon and haloperidol, but also by the alpha-adrenergic receptor antagonist phentolamine, and the observation that the effect of adrenaline, which is extremely potent in inhibiting MSH release, could be fully blocked by D-2 receptor antagonists. Both the fact that the dopamine-induced inhibition of MSH secretion could not be blocked by haloperidol, domperidon and phentolamine, and that adrenaline-induced inhibition was hardly blocked by phentolamine, led us to suggest the possible presence of multiple receptors or receptor sites. We find no evidence for the involvement of dopamine D-1 receptors in the regulation of MSH release. The effects of dopamine agonists and antagonists were also studied in vivo by monitoring changes in pigment dispersion of dermal melanophores. The results are consistent with our in vitro findings and indicate, moreover, that in the living animals there must also be a non-catecholaminergic system involved in the inhibition of MSH release from the pars intermedia.

Biosynthesis, processing and release of pro-opiomelanocortin related peptides in the intermediate lobe of the pituitary gland of the frog (Rana ridibunda).

The biosynthesis of pro-opiomelanocortin (POMC) and related peptides by the intermediate lobe of the pituitary gland was studied in the frog Rana ridibunda using the pulse-chase technique. Analysis of radioactive proteins by dodecyl sulfate polyacrylamide gel electrophoresis showed that during pulse incubations a 36,000 dalton (36K) glycosylated prohormone was synthesized. It disappeared slowly during chase incubations, giving rise to another glycosylated protein (Mr 18K), identified as the N-terminal fragment of POMC. This latter protein was secreted to the incubation medium. High performance liquid chromatography analysis of peptides synthesized during chase incubations revealed the biosynthesis of two peptides related to gamma-MSH, three peptides related to alpha-MSH, one endorphin-related and one CLIP-related peptides. These newly synthesized peptides were slowly secreted to the incubation medium. Among the alpha-MSH related peptides, only the des-N alpha-acetyl alpha-MSH form of the peptide was found to be present within the cells, in contrast to the incubation medium where the presence of des-N alpha-acetyl alpha-MSH and a modified alpha-MSH was demonstrated.

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 gamma-aminobutyric 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.

Regulation of melanotropin release from the pars intermedia of the amphibian Xenopus laevis: Evaluation of the involvement of serotonergic, cholinergic, or adrenergic receptor mechanisms

Melanophore-stimulating hormone (MSH) release from the pars intermedia of the pituitary gland is probably regulated by multiple factors of hypothalamic origin. We have examined a number of potential regulatory factors for their effects on MSH release from the amphibian Xenopus laevis. Serotonin and acetylcholine have no effect on MSH release. Both adrenaline and noradrenaline inhibit release of MSH in a dose-dependent manner. Studies with specific receptor agonists and antagonists reveal that these neurotransmitters exert their in vitro effects primarily through a dopamine D-2 receptor, although an alpha-adrenergic receptor could not be excluded. We further conclude that the pars intermedia of X. laevis lacks a beta-adrenergic receptor for the regulation of MSH secretion from the pars intermedia. In mammals, this receptor activates the adenylate cyclase system. Our studies reveal that despite the lack of beta-adrenergic receptors, cyclic-AMP is likely an intracellular factor involved in the stimulation of MSH release.

Regulation of biosynthesis and release of pars-intermedia peptides in rana-ridibunda - dopamine affects both acetylation and release of alpha-msh

Dopamine is likely an important physiological melanotropin release-inhibiting factor in amphibians. This study concerns the effects of dopamine on the biosynthesis and release of peptides from the pars intermedia of the frog Rana ridibunda. Our results show that this secretagogue has no immediate effects on either the rate of biosynthesis of pro-opiomelanocortin nor on the direction of processing of this prohormone. Pulse-chase experiments revealed that dopamine inhibited the release of all newly synthesized POMC-related peptides in a dose-dependent manner. For each dopamine concentration tested, the degree of inhibition exerted on the release of the various newly synthesized peptides by a given concentration of secretagogue was approximately the same, with the exception of that found for the alpha-MSH related peptides. Analysis of the release of these melanotropins was complex because dopamine not only inhibited their release but also, either directly or indirectly, inhibited the acetylation reaction which converts des-N alpha-acetyl alpha-MSH to alpha-MSH. Dopamine was shown to be less potent in inhibiting the release of des-N alpha-acetyl alpha-MSH than inhibiting release of the acetylated form of the peptide. In amphibians a preferential release of the less-bioactive non-acetylated form of MSH under inhibitory conditions induced by dopamine may be of physiological importance.

Electrophysiological evidence for the existence of GABAA receptors in cultured frog melanotrophs

The neurotransmitter GABA exerts a biphasic effect on alpha-melanocyte-stimulating hormone (alpha-MSH) secretion from pars intermedia cells: GABA induces a rapid and transient stimulation followed by a sustained inhibition of alpha-MSH release. In the present study, we have investigated the effect of GABA on the electrophysiological properties of frog melanotrophs in primary culture using the patch-clamp technique in the whole cell configuration. In all cells tested, GABA stimulated an inward current and induced depolarization. A transient period of intense firing was consistently observed at the onset of GABA administration. During the depolarization phase, the membrane potential reached a plateau corresponding to the Cl- equilibrium potential. When repeated hyperpolarizing pulses were applied, an increase of membrane conductance was observed throughout the response evoked by GABA. The effect of GABA was abolished by the chloride channel blocker picrotoxin, and by antagonists of GABAA receptors (bicuculline and SR 95531). The depolarizing action of GABA was mimicked by muscimol, an agonist of GABAA receptors. Taken together, our results indicate that the rapid and transient stimulation of alpha-MSH release induced by GABA can be accounted for by activation of a chloride conductance which causes membrane depolarization. These data support the notion that the transient stimulation of alpha-MSH secretion induced by GABA can be accounted for by membrane depolarization which provokes activation of voltage-operated calcium channels. Since no evidence was found for GABA-induced hyperpolarization, the intracellular mechanisms leading to the strong inhibitory effect of GABA on alpha-MSH secretion remain to be elucidated.