α-MSH Release Research Articles

α-MSH and Pro-Leu-Gly-NH 2 (PLG; MIF-1): Influence on dopamine (DA) uptake in crude synaptosomal preparations from rat mediobasal hypothalamus (MBH) and caudate putamen (CP)

The uptake of tritiated dopamine [ 3H] (DMI insensitive DA uptake) by synaptosomal fractions isolated from rat mediobasal hypothalamus (MBH) and caudate putamen (CP) was measured in the presence of different concentrations of α-melanocyte stimulating hormone (α-MSH) and Pro-Leu-Gly-NH 2 (PLG; MIF-1) which is an inhibitor of α-MSH release. Compared to control, [ 3H]DA uptake increased significantly when the synaptosomal fraction of CP was incubated with 0.1 and 1 μM of α-MSH and also when the rat was previously injected with α-MSH. A simultaneous reduction of endogenous dopamine content was observed. Kinetic studies suggest that the enhanced uptake induced by α-MSH 1 μM is the consequence of the rise in Vmax, without changes in the apparent km. The uptake of [ 3H]DA in hypothalamic (MBH) preparations on the other hand, was not modified by the presence of α-MSH. PLG did not have any significant effect on [ 3H]DA uptake either in the CP or in the MBH. α-MSH may act as a modulator of the dopaminergic nigrostriatal system and the results obtained incubating CP synaptosomes in its presence demonstrate a possible direct modulator action by α-MSH on the terminal area of the substantia nigra neurons.

Dual population of GABAA and GABAB receptors in rat pars intermedia demonstrated by release of alpha MSH caused by barium ions.

We have studied the effects of selective GABAA and GABAB agonists on alpha-melanophore stimulating hormone (alpha MSH) release from intact rat neurointermediate lobes (NIL) in vitro. Agonist effects were tested against either basal alpha MSH output or BaCl2 (5 mM)-evoked release. GABA (50 microM) produced a biphasic effect on basal release, with an enhancement followed by inhibition of release. The enhancement but not the inhibition was blocked by bicuculline methiodide (100 microM). Baclofen (10 microM), a specific GABAB agonist, reduced the basal and Ba2+-evoked hormonal release in a stereospecific manner. (-)-Baclofen (5 microM) was active whereas the (+)-isomer was inactive at the same concentration. Isoguvacine (50 microM) a specific GABAA agonist, potentiated the Ba2+-evoked release of alpha MSH. GABA (50 microM) mimicked this effect, and its action was antagonized by bicuculline methiodide (200 microM). The results suggest that both GABAA and GABAB receptors are present on the endocrine cells of the intermediate lobe.

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 −6 M) did not modify the spontaneous release of α-MSH. At doses of 10 −8 and 10 −6 M, synthetic somatostatin did not modify TRH-induced α-MSH secretion. Morphine (10 −5 M) and opioid peptides (10 −10 to 10 −6 M) had no effect on spontaneous α-MSH secretion. In addition, methionine enkephalin (10 −5 M) 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.

Effect of Tyr-Pro-Leu-Gly-NH 2 on melanotropin-stimulating hormone release in the frog and rat

The tetrapeptide Tyr-Pro-Leu-Gly-NH 2 (Tyr-MIF I) has been recently characterized in the hypothalamus and pineal of the rat. Since the concentration of Tyr-MIF I in the brain is increased by pinealectomy and is higher when the animals are in the dark, the possibility that Tyr-MIF I 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 α-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-MIF I did not significantly alter the spontaneous release of α-MSH in the frog nor did it alter the release of α-MSH in the rat. Since it has been recently demonstrated that the tripeptide pGlu-His-Pro-NH 2 (mammalian TRH) is a specific MSH-releasing factor in the frog, the possibility that Tyr-MIF I could modulate the response of the intermediate lobe of the frog to TRH has also been investigated. No alteration of TRH-induced α-MSH release was observed in the presence of a 100-fold excess of Tyr-MIF I. In addition, Tyr-MIF I 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-MIF I is the melanotropin-release inhibiting factor in the frog or rat.

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 (T 4) 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 T 4 (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 −7 M). In addition, prolonged infusion of T 4 in vitro did not alter spontaneous and TRH-induced α-MSH release. In spite of the inhibitory effect of T 4 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.

Multiple hormonal control of pars intermedia cell activity.

In rat pars intermedia cells, the rate of alpha-melanocyte-stimulating hormone (alpha-MSH) secretion was so far known to result from a balance between the stimulatory effect of beta-adrenergic agonists and the inhibitory influence of dopaminergic substances. Recently, we have identified a second stimulatory substance, namely corticotropin-releasing factor (CRF). CRF is a potent stimulator of pars intermedia adenylate cyclase activity, cAMP accumulation and alpha-MSH release. A requirement for calcium ions was observed on basal as well as on CRF-induced alpha-MSH secretion. The beta-adrenergic and CRF effects on adenylate cyclase activity, as well as the dopamine inhibition of adenylate cyclase activity, are potentiated by guanine nucleotides (GTP). Stimulation of the beta-adrenergic receptor with isoproterenol causes a rapid loss in cAMP responsiveness, which can be completely blocked by beta-adrenergic antagonists and partially prevented by dopamine. These findings suggest that CRF should now be considered, in addition to beta-adrenergic agents, as a stimulator of the activity of pars intermedia cells and that cAMP is also involved as mediator of its action. Changes of receptor sensitivity, as well as interaction of the two stimulatory receptors with the inhibitory dopaminergic receptor, are involved in the fine control of pars intermedia cell activity. All three receptors appear to exert their action through a common pathway, namely changes of adenylate cyclase activity.

Effect of 41-CRF antiserum on the secretion of ACTH, B-Endorphin and α-MSH in the rat

In order to elucidate the physiological role of the 41 amino-acid residue corticotropin-releasing factor (41-CRF) on the secretion of ACTH, B-Endorphin and α-MSH, plasma levels of these peptides were measured by radioimmunoassay in intact and adrenalectomized rats, two hours after the injection of either 41-CRF antiserum (CRF-AS) or normal rabbit serum for controls. The administration of CRF-AS strikingly lowered the plasma ACTH levels in both intact and adrenalectomized rats. A statistically significant reduction of plasma levels of B-Endorphin was also observed in the same rats. However, the effect of CRF-AS on B-Endorphin release was less pronounced than the effect on ACTH release. No changes in plasma α-MSH levels were observed after passive immunization with CRF-AS. We conclude that, in the rat, 41-CRF plays a physiological role in the regulation of ACTH and B-Endorphin secretion, but is not involved in the regulation of α-MSH release from the pituitary gland.

Pro-opiomelanocortin peptides in the human fetal pituitary

Levels of immunoreactive pro-opiomelanocortin (POMC) peptides (N- and C-terminal ACTH, N- and C-terminal LPH and α-MSH) have been measured in pituitary extracts from human fetuses of 12–22 weeks gestation. The levels of ACTH were 30–200 times higher than α-MSH in all fetuses studied. Sephadex G-75 and G-25 chromatography of 8 extracts showed peaks of 34 kilodaltons (K) POMC, 22K ACTH, β-LPH, γ-LPH, β-endorphin, approximately 8K ACTH, 1–39 ACTH, α-MSH and CLIP. The 8K and 22K forms of ACTH are both partly glycosylated. In vitro culture of pituitaries from 2 fetuses (22 and 26 weeks gestation) gave a detectable basal output of ACTH but not of α-MSH. Stimulation of these pituitary cells with human fetal and rat hypothalamic extracts and with synthetic ovine CRF-41 produced a significant increase in ACTH release, and either small or undetectable amounts of α-MSH. These results demonstrate the presence of POMC-related peptides in early gestation human fetal pituitaries and suggest that ACTH, and not α-MSH, is the major corticotrophic hormone at this stage of gestation.

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

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

Inhibition by L-Prolyl-L-Leucyl-Glycinamide (PLG) of Alpha-melanocyte stimulating hormone release from hypothalamic slices

Release of α-MSH from male rat hypothalamic slices was studied using a sensitive bioassay (1–2 pg). Addition of 60 mM KC1 to superfusion medium resulted in a twofold increase in α-MSH release compared to spontaneous release. Both spontaneous and potassium- induced release were inhibited in a dose-response manner by the tripeptide Pro-Leu-Gly-NH 2 (PLG, or MIF-1); 0.04 μg to 1 μg PLG inhibited the α-MSH release but the lowest dose demonstrated a greater inhibitory effect; high concentrations of PLG, on the other hand, did not modify either spontaneous or potassium-evoked α-MSH release from the slices. Contrarily, DA did not modify either spontaneous or potassium- induced α-MSH release at any of the doses tested. These findings demonstrate that the inhibitory behavior of PLG and DA in the central nervous system (CNS) differs from their behavior towards α-MSH release in the pituitary. This suggests differences in the regulation of α-MSH release from the pituitary and the CNS.

CRF stimulates α-MSH secretion and cyclic AMP accumulation in rat pars intermedia cells

Ovine corticotropin-releasing factor (CRF) stimulates α-MSH release and cyclic AMP accumulation in rat pars intermedia cells in culture at ED 50 values of 1 and 6 nM, respectively. The stimulatory effect of CRF on both parameters is inhibited by the dopaminergic agonist 2-bromo-α-ergocryptine (CB-154). The present data show that CRF is a potent stimulator of peptide secretion in the intermediate lobe of the pituitary gland and suggest a role of this pituitary lobe in the response to stress. In addition, the present data clearly indicate a role of cyclic AMP as mediator of the action of CRF in pars cells.

α-MSH levels in cerebrospinal fluid and blood of rats during behavioral manipulations

It was shown previously that α-MSH levels in peripheral blood of rats subjected to passive avoidance training did not correlate with the behavioral performance of the animals. We have investigated whether α-MSH levels in cerebrospinal fluid (CSF) change during passive avoidance behavior. It appeared that throughout adaptation, acquisition and retention of this avoidance behavior, α-MSH levels in the CSF did not change significantly. In an additional experiment in which the effects of an electric footshock versus a psychological stimulus were tested, α-MSH levels in CSF also remained unchanged. Since CSF α-MSH levels appear to be relatively stable under these behavioral conditions, it seems unlikely that the CSF functions as a direct and specific route for the afferent transport of the behaviorally active neuropeptide α-MSH to its sites of action in the brain. However, the psychological stimulus, which consisted of the fear of receiving an unavoidable electric footshock, did induce a significant enhancement of α-MSH levels in peripheral blood, suggesting that psychological stress may be involved in the release of α-MSH into the peripheral circulation. These results support the idea of a differentiated system of secretion of α-MSH into CSF and peripheral blood.

In vitro study of frog ( Rana ridibunda pallas) neurointermediate lobe secretion by use of a simplified perifusion system : I. Effect of TRH analogs upon α-MSH release

We have recently shown that the tripeptide l-pyroglutamyl- lhistidyl- l-proline-amide (mammalian TRH) stimulates α-MSH secretion in amphibia. Using a perifusion system technique, we have compared the stereochemical requirements for hormone-receptor interaction of frog melanotrophs with mammalian thyrotrophs and mammatrophs. Of all the analogs tested, only the TRH analog l- N-(2-oxopiperidine-6-yl-carbonyl)-histidyl-thiazolidine-4-carboxamide (MK-771) was equipotent with TRH. All analogs which were known to be TRH agonists in mammals (e.g., [Pic] 3-TRH, [Pro-hydrazide] 3-TRH) were also relatively active on α-MSH release. Seven analogs were totally inactive on both mammalian pars distalis and frog pars intermedia. The discrepancies concerned only two TRH analogs in which the histidine moiety has been altered [Tyr] 2-TRH and [Lys] 2-TRH). The biological potencies of these analogs were 17 and 8% on α-MSH release whereas both molecules were devoid of activity in mammals.

N alpha-acetylation is linked to alpha-MSH release from pars intermedia of the amphibian pituitary gland.

Most amphibians can adapt their colour to that of their background by a mechanism which is controlled by melanophore stimulating hormone (MSH), a product of the pars intermedia of the pituitary gland1. Although the melanotropic peptides of amphibians have never been sequenced, immunological evidence indicates that at least some of them are structurally related to α-MSH2–4. In our studies of the biosynthesis and structure of melanotropic peptides in the pars intermedia of Xenopus laevis, we have found evidence, reported here, for des-Nα-acetyl-α-MSH as the immediate precursor of α-MSH, with the acetylation taking place just before or during release from the pituitary.

The influence of β-LPH fragments on α-MSH release: The involvement of a dopaminergic system

β-Endorphin was able to enhance plasma α-MSH levels in rats after intracerebroventricular injection. This effect could be inhibited by naloxone or by removing tyrosine from position 61 of the peptide. Neither α- and γ-endorphin nor their des-tyrosine analogs appeared to be able to modify plasma α-MSH levels. The stimulating effect of β-endorphin on plasma α-MSH levels could be completely blocked by a simultaneous injection of apomorphine, in an amount in which apomorphine itself had no effect on α-MSH levels in plasma. A single injection of haloperidol increased plasma α-MSH levels in a dose related manner. A dose of haloperidol, which caused an apomorphine antagonizable increase in plasma α-MSH, did not modify β-endorphin elevated α-MSH levels. A high concentration of haloperidol was able to stimulate the basal release of α-MSH from isolated pituitaries in bitro , whereas β-endorphin appeared to be inactive in this respect. These observations indicate a central opiate receptor-mediated influence of β-endorphin on α-MSH release and the possible involvement of a dopaminergic system, mediating the β-endorphin effect.

Beta-Adrenergic stimulation of the release of ACTH- and LPH-related peptides from the pars intermedia of the rat pituitary gland.

The intermediate lobe of the rat pituitary gland produces a series of peptides related to ACTH and LPH. The spontaneous and isoproterenol-stimulated release of such peptides was studied during in vitro superfusion of rat neurointermediate lobes with Krebs-Ringer medium. Products released into the superfusion medium were quantified by direct measurement or after chromatography on Sephadex G-50. ACTH bioactivity was determined by use of adrenal cortical cell suspension assay. In addition, NH2-terminal ACTH, CO2H-terminal ACTH, alpha-MSH and beta-endorphin radioimmunoassays were used. The results show that 1. neurointermediate lobes of rats secret spontaneously various ACTH- and LPH-related peptides in amounts proportional to the amounts in which these peptides are found in extracts of the neurointermediate lobe; 2. the beta-adrenergic agonist, isoproterenol, stimulated the spontaneous release of various peptides, including alpha-MSH, ACTH, CLIP, glycosylated CLIP, and beta-endorphin-like peptides; 3. isoproterenol induced a dose-dependent (10(-9)-10(-7) M), parallel increase in the release of alpha-MSH and ACTH following similar time courses and showing identical EC50 values (about 10(-8)M). Although the spontaneous release of alpha-MSH and ACTH from rat neurointermediate lobes is not strictly coupled under the conditions used in this study, isoproterenol seems to affect the spontaneous release of these peptides to the same relative extent.

Parallel stimulation of ACTH, β-LPH + β-endorphin and α-MSH release by α-adrenergic agents in rat anterior pituitary cells in culture

Characteristics of the α-adrenergic stimulation of ACTH, β-endorphin + β-LPH and α-MSH release were studied in rat anterior pituitary cells in primary culture. Parallel changes of ACTH, β-endorphin + β-LPH and α-MSH release were found under all stimulatory and inhibitory conditions by natural and synthetic catecholamine agonists and antagonists. (−)Epinephrine and (−)norepinephrine lead to a 8–10-fold stimulation of peptide release at ED 50 values of 20 and 90 nM, respectively. The stereoselectivity of the α-adrenergic stimulatory action on peptide release is indicated by a 100-fold higher activity of (−)- than (+)norepinephrine while (−)-epinephrine is 10 times more potent than the corresponding (+) stereoisomer. The involvement of a typical α-adrenergic mechanism in the control of release of ACTH, β-endorphin and related peptides in rat anterior pituitary gland is indicated by the following order of potency of a series of catecholaminergic agents (ED 50 values): (−)epinephrine (20 nM) > (−)norepinephrine (90 nm) > phenylephrine (400 nM) > isoproterenol (6000 nM). The stimulatory effect of (−)epinephrine or phenylephrine is completely reversed by low concentrations of the α-adrenergic antagonist phentolamine while the β-adrenergic antagonist propranolol has no effect up to 10 μM. Beside providing an easily accessible pure population of post-synaptic α-adrenergic receptors having potential applications as a model for other less accessible α-adrenergic brain systems, the present data suggest the possibility of the direct involvement of a catecholamine in the physiological control of ACTH secretion in the rat anterior pituitary gland.

Behavioral profile of ψ-MSH: Relationship with acta and β-endorphin action

In view of the close structural similarity of the pro-opiocortin fragment γ-MSH and of ACTH/MSH type peptides, the behavioral profile of γ-MSH was explored. Attention was first focussed on behavioral procedures in which ACTH/MSH related neuropeptides have been found effective. Using different procedures to test avoidance behavior, it was found that γ-MSH and ACTH-like neuropeptides had opposite effects. In this respect the activity of γ-MSH resembles that of opiate antagonists rather than that of β-endorphin. Accordingly, ACTH 1–24-induced excessive grooming which is blocked by opiate antagonists, is attenuated by γ-MSH. In addition, γ-MSH injected into the periaqueductal gray matter of the brainstem of opiate naive rats elicited symptoms reminiscent of those seen after opiate withdrawal. γ-MSH attenuated more or less several effects of intracerebroventricularly administered β-endorphin (e.g. antinociception, hypothermia, α-MSH release) and decreased acquisition of heroin self-administration. Although γ-MSH at rather high doses displaced naloxone from its specific binding sites in brain homogenates, it did not interfere with β-endorphin-induced effects on in vitro muscle preparations (guinea pig ileum, rat rectum). Interestingly, γ-MSH induced relaxation of the rat rectum in vitro. It is postulated that γ-MSH may attenuate β-endorphin-induced effects by acting via γ-MSH receptor sites (functional antagonism), although a pharmacological antagonism cannot be excluded as yet.

Stimulatory effect of prostaglandin E 1 on thyroliberin-induced α-melanotropin release from perifused neuro-intermediate lobes of frog pituitary gland

A possible direct effect of prostaglandins on α-melanotropin (α-MSH) release at the level of the intermediate lobe of the frog pituitary was investigated in vitro using a perifusion system technique. The effect of prostaglandins was studied on both spontaneous and TRH-stimulated α-MSH secretion. No significant effect of PGE 1, PGE 2, PGF 1α or PGF 2α on basal release of α-MSH could be detected. Indomethacin did not alter the α-MSH release induced by TRH. Conversely a significant increase in TRH-induced α-MSH secretion was observed in the presence of 1 x 10 −6M PGE 1. This magnifying effect was directly related to the concentration of TRH for doses ranging from 1 x 10 −8M to 1 x 10 −6M.

Ion-induced release of LHRH, TRH and alpha-MSH from hypothalamic synaptosomes and its inhibition by vinblastine.

Homogenates of rat hypothalamic tissue were fractionated by means of discontinuous sucrose density gradient centrifugation. Immunoreactive luteinizing hormone releasing hormone (LHRH), thyrotropin releasing hormone (TRH), and α-melanocyte stimulating hormone (α-MSH) were found to be concentrated in the synaptosome-enriched fraction. This fraction was suspended in 0.32 M sucrose and the release of the three peptides was investigated. After incubation, the synaptosomes were re-isolated by ultrafiltration, and the concentration of each peptide in the ultrafiltrate was determined by radioimmunoassay. When the synaptosomal fraction was incubated at 30° C in 0.32 M sucrose containing either 60 mM K+-2 mM Ca2+ or 140 mM Na+ alone a release of LHRH, TRH, and α-MSH occurred. Of the total content 30–50% of LHRH but only about 10% of TRH and α-MSH was releasable. When the synaptosome preparation was preincubated for 30 min at 30°C with 10−4 M vinblastine. K+- as well as Na+-induced release of LHRH, THR, and α-MSH was inhibited, and the stimulatory effect of each cation was almost totally blocked by preincubation with 5×10−4 and 10−3 M vinblastine. The inhibitory action of vinblastine (5×10−4 M) did not affect the oxidation of glucose to CO2 by the synaptosomes. The results of the present investigation demonstrate that synaptosome-enriched fractions of hypothalamic origin are more stable with respect to LHRH, TRH, and α-MSH release during incubation in isotonic sucrose than they are in ionic solutions, and that the peptides are released by a vinblastine-sensitive mechanism.