Subcellular localization of immunoreactive α-melanocyte stimulating hormone in human brain

The regional and subcellular distribution of immunoreactive α-melanocyte stimulating hormone (α-MSH₁) in the post mortem adult human brain was investigated. α-MSH_i was highly concentrated in medial basal hypothalamic tissue (1.02 ng/mg protein). Lower levels of α-MSH_i were present in the optic chiasm and mammillary bodies, 0.08 and 0.11 ng/mg protein, respectively. The concentrations of α-MSH_i in cerebellum and frontal cerebral cortex were 1/1,000th that of the medial basal hypothalamus. When medial basal hypothalamic homogenates were subjected to discontinuous or continuous sucrose density gradients, α-MSH_i was found to be associated primarily with subcellular particles which resembled isolated nerve terminals, i.e., synaptosomes. Low to undetectable amounts of α-MSH_i were found in the cytosol or the myelin/microsome fraction of the gradients. The results of these studies are consistent with the view that α-MSH is a neuronal peptide in the human brain.     

Cardiovascular effects of intrahypothalamic injections of α-melanocyte stimulating hormone

Injection of α-melanocyte stimulating hormone (α-MSH, 0.6–12 nmol in 100–300 nl) into the rostral dorsomedial hypothalamic nucleus of the halothane anesthetized rat resulted in a 12% increase in heart rate (41 ± 4bpm) which was accompanied by a slight increase in blood pressure (5 ± 1mm Hg). The response was characterized by a gradual onset, with a peak increase at 7 ± 1 min and a duration of 51 ± 6 min. Tachyphylaxis to the response was apparent for at least 180 min following initial exposure to the peptide. In contrast to the increase in heart rate observed following α-MSH injection into the dorsomedial nucleus, injections into the medial preoptic, anterior, paraventricular or posterior hypothalamic nuclei had no significant effects on blood pressure and heart rate. These data suggest a possible role for brain α-MSH in the central control of heart rate at a site within the dorsomedial nucleus of the hypothalamus.     

Differential projections of two immunoreactive α-melanocyte stimulating hormone (α-MSH) neuronal systems in the rat brain

Immunocytochemical localization of α-MSH was performed in the brain of rats of which the arcuate nucleus has been destroyed by treatment with monosodium glutamate in the neonatal period. In these animals, α-MSH cell bodies normally found in the arcuate nucleus were almost completely absent. The reactive fibers found in the ACTH-β-LPH pathway were also markedly decreased. On the other hand, α-MSH cell bodies located in the dorsolateral hypothalamus as well as fibers located outside the ACTH-β-LPH pathway were not decreased. These results strongly suggest that α-MSH cell bodies in dorsolateral hypothalamus have projections completely different from those located in the arcuate nucleus.     

Lateral hypothalamic innervation of the cerebral cortex: Immunoreactive staining for a peptide resembling but immunochemically distinct from pituitary/arcuate α-melanocyte stimulating hormone

The combination of retrograde transport of fluorescent dyes and indirect immunofluorescence has been used to study the putative neurotransmitter specificity of the tuberal lateral hypothalamic projection to the cerebral cortex. Injections of either fast blue or diamidino yellow dye into the cerebral cortex or hippocampus retrogradely labeled large, multipolar neurons scattered through the lateral hypothalamic area and zona incerta at the level of the ventromedial nucleus of the hypothalamus. Approximately 80% of these neurons stained immunohistochemically with an antiserum against α-melanocyte stimulating hormone (α-MSH). A second population of smaller, predominantly bipolar α-MSH-like immunoreactive neurons was seen in the arcuate nucleus and retrochiasmatic area, but none of these projected to the cerebral cortex. Immunohistochemical staining for ACTH (18–24), another proopiomelanocortin series peptide, or with an antiserum against α-MSH (4–10) demonstrated only the second of these cell groups. Our results indicate that the tuberal lateral hypothalamic projection to the cerebral cortex contains a substance similar but not identical to α-MSH, and that this material is probably not derived from the same proopiomelanocortin precursor as true α-MSH.     

α-Melanocyte stimulating hormone discloses a stimulatory effect of β-endorphin on somatostatin release

The influence of alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin (beta-END) on the secretion of somatostatin (SRIF) from the median eminence (ME) was studied using an in vitro incubation system. The MEs from adult male rats were first preincubated at 37 degrees C for 30 min with constant shaking in 0.4 ml of Krebs-Ringer bicarbonate-glucose buffer (pH 7.4) containing bacitracin in an atmosphere of 95% O2/5% CO2. Medium was discarded and replaced by medium containing different doses of alpha-MSH, beta-END, or a fixed dose of alpha-MSH (10(-7) M or 10(-9) M) plus beta-END at various concentrations. By themselves alpha-MSH and beta-END did not alter basal SRIF release, but in the presence of alpha-MSH (10(-7) M) beta-END stimulated somatostatin release. This effect was significant at concentrations of beta-END of 10(-8) M and higher. The permissive effect of alpha-MSH was observed at a concentration as low as 10(-9) M, but in this case the stimulatory effect of beta-END became evident only at higher doses tested (10(-7) M). It is suggested that alpha-MSH and beta-END participate in the modulation of SRIF release. By themselves beta-END and alpha-MSH did not affect basal release of SRIF but in the presence of alpha-MSH, beta-END had a stimulatory effect on SRIF release. The mechanism for this interaction is unknown. The results are consistent with the possibility that beta-END neurons have stimulatory and inhibitory effects on SRIF release and that alpha-MSH, by blocking the inhibitory components, discloses the stimulatory effect of beta-END on SRIF release.     

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 effect of α-melanocyte stimulating hormone (α-MSH) on the metabolism of blogenlc amines in the rat brain

The effect of the chronic administration of α-MSH on the incorporation of tritiated tyrosine into noradrenalin and dopamine and of tritiated tryptophan into serotonin was studied in different regions of the rat brain. α-MSH increased the incorporation of tritiated tryptophan into serotonin in the cortex and slightly decreased that of tyrosine into the dopamine in the hypothalamus. As the brain concentration of serotonin was unchanged in the animals treated with α-MSH, it is suggested that some of the changes in behavior, which other investigators have found following the administration of peptides containing the same sequence of amino acids to that found in MSH, could be associated with an increased turnover of cortical serotonin.     

Extrahypophyseal distribution of α-melanocyte stimulating hormone (α-MSH)-like immunoreactivity in postmortem brains from normal subjects and Alzheimer-type dementia patients

Using a sensitive double-antibody solid-phase enzyme immunoassay method α-melanocyte stimulating hormone-like immunoreactivity (α-MSH-LI) was measured in 21 regions of postmortem brains from 8 normal subjects and 5 patients with Alzheimer-type dementia (ATD). In the brains from the normal subjects, the highest concentration of α-MSH-LI was found in the hypothalamus. Relatively high concentration were also measured in the locus coeruleus, substantia innominata, substantia nigra, amygdala and medial nucleus of thalamus. α-MSH-LI in other regions was approximately1/100 of the hypothalamic content. This data is consistent with the existence of α-MSH in extrahypophyseal regions and indicates its regional distribution in the human brain. In the Alzheimer brains, although the temporal cortex and hippocampus had normal concentrations of α-MSH-LI, the cingulate cortex, caudate and substantia nigra showed significantly lower concentrations of α-MSH-LI than those of the control brains. This data suggests that further studies of α-MSH content in a larger number of ATD brains would be useful.     

Bilateral α-melanocyte stimulating hormonergic fiber system from zona incerta to cerebral cortex: combined retrograde axonal transport and immunohistochemical study

We employed a highly sensitive combination method of retrograde tracing and immunohistochemistry to identify an α-melanocyte-stimulating hormone (α-MSH)-containing fiber pathway from zona incerta to cerebral cortex. Biotin-horseradish peroxidase injected into the parietal cortex of the rat labeled a number of neurons in the zona incerta bilaterally, and stimultaneous staining with an α-MSH antiserum revealed that a part of these neurons are α-MSHergic.     

Immunohistochemical localization of γ-aminobutyric acid in the rat pituitary gland and related hypothalamic regions

The distribution on γ-aminobutyric acid (GABA) containing neurons in the rat pituitary gland and related hypothalamic areas was immunohistochemically investigaed using antibodies raised against GABA conjugated to bovine serum albumin by glutaraldehyde. A dense network of GABA-like immunoreactive fine varicose nerve fibers was observed within the posterior and intermediate lobes of the pituitary gland, surrounding endocrine cells and capillaries, but not in the anterior lobe. In the pituitary stalk, the dense varicose fibers ran along the anterior wall of the posterior lobe into the posterior and intermediate lobes. A small number of GABA-like immunoreactive cell bodies were evident in the intermediate lobe. GABA-like immunoreactive fibers occurred at low to high density in most parts of the hypothalamus. GABA-like immunoreactive neurons were observed in some regions related to the pituitary gland (such as periventricular nucleus, paraventricular nucleus, arcuate nucleus and accessory magnocellular nucleus). These results provide morphological evidence for the presence of GABAergic neurons in the rat hypothalamo-pituitary system.     

Copper-induced release of immunoreactive α-melanotropin from isolated hypothalamic granules

Previously, we demonstrated that copper chelates stimulate the release of luteinizing hormone releasing hormone (LHRH) from isolated hypothalamic granules. To assess the generality of the copper-stimulated release process, we determined the effects of copper on the release of immunoreactive α-melanotropin (α-MSH₁) from isolated granules. When granules were incubated with various copper complexes, CuATP stimulated α-MSH₁ release by 54 ± 6% (mean ± S.E.), Cu tartarate by 56 ± 4%, CuBSA by 32 ± 5% and Cu histidine by 29 ± 2%. CuATP-stimulated α-MSH₁ release from granules incubated under N₂ was 57% of that incubated under air. Furthermore, the reducing agent dithiothreitol (DTT) inhibited CuATP-stimulated α-MSH₁ release (p < 0.01), whereas oxidized DTT did not do so. Pretreatment of granules with the thiol-blocking reagents iodoacetic acid or 5, 5'-dithiobis-(2-nitrobenzoic acid) inhibited CuATP-stimulated α-MSH₁ release by 52 ± 3 and 38 ± 4%, respectively. Thus, chelated copper, rather than ionic copper, is the active form of the metal and the action of copper involves the oxidation of thiols. These data are similar to those previously observed for the copper-stimulated release of LHRH. Hence, the effects of copper on the permeability of granule membranes may be a generalized phenomenon which underlies susceptibility of storage granules to the reduction-oxidation status of the cellular milieu.     

The distribution and projection of γ-melanocyte stimulating hormone in the rat brain: An immunohistochemical analysis

The distribution and projection of immunoreactive γ-melanocyte-stimulating hormone (γ-MSHI) in the rat brain was examined by indirect immunofluorescence using an antiserum against synthetic rat γ-MSH. The present study confirmed the presence of γ-MSHI neurons in the arcuate nucleus and further demonstrated that the n. communissuralis is a new γ-MSHI neurons-containing site. We also found a γ-MSHI fiber network in the hypothalamus, thalamus, amygdala, central gray matter of the midbrain and upper pons, and further demonstrated a much more extensive distribution of these fibers particularly in the medulla oblongata, an area previously thought not to contain γ-MSHI structures. The present observation on the normal distribution of γ-MSHI suggested the existence of two different systems: one is the arcuatofugal γ-MSH system and the other n. commissuralis γ-MSH system. Using experimental manipulations, we clearly established that γ-MSHI fibers in the forebrain, diencephalon, midbrain and upper pons originate from γ-MSHI neurons in the arcuate nucleus and those in the medulla oblongata from the n. commissuralis.     

Activation of supraoptic magnocellular neurons by gamma 2-melanocyte stimulating hormone (gamma 2-MSH)

The effects of intracarotid infusions of the peptide gamma₂-melanocyte stimulating hormone (γ₂-MSH) on electrophysiologically and immunohistochemically identified supraoptic nucleus (SON) units were investigated Over a wide dose range this agent always excited SON units, while control infusions of vehicle had no effect. Because neural responses invariably preceded blood pressure elevation, it appears that γ₂-MSH excitation of the magnocellular system was due to a direct effect on the central nervous system and was not a result of systemic cardiovascular responses. These results suggest a forebrain γ₂-MSH sensitive site in the activation of SON magnocellular neurons.     

Influence of centrally administered α- and γ2-melanocyte-stimulating hormone on hypothalamic blood flow autoregulation in the rat

The effect of intracerebroventricularly (i.c.v.) administered α-melanocyte-stimulating hormone (MSH) and γ₂-MSH on hypothalamic blood flow autoregulation was studied in anesthetized rats at different levels of standardized arterial hypotension. Autoregulation was impaired upon i.c.v. administration of 5 γ g/kg γ₂-MSH while α-MSH caused no change.. Since this effect of γ₂-MSH wa identical to that produced by i.c.v. naloxone in the same model, γ₂-MSH may be a functional antagonist of central opioid mechanisms participating in the control of cerebral blood flow autoregulation.     

Plasma and cerebrospinal fluid α-MSH levels in the rat after hypophysectomy and stimulation of pituitary α-MSH secretion

Immunoreactive α-MSH was measured in cerebrospinal fluid (CSF) and plasma of rats. While treatment with haloperidol increased α-MSH levels in the plasma concentration of α-MSH in the CSF showed little change. Hypophysectomy also had little effect on the concentration of α-MSH in the CSF despite the fall in plasma α-MSH levels. This lack of correlation between α-MSH levels in the CSF and plasma suggests that the systemic circulation does not deliver α-MSH to the CSF. The apparently normal levels of α-MSH in the hypothalamus after hypophysectomy suggests that this tissue is able to synthesize α-MSH and it is possible that the hypothalamus is a source of the α-MSH in the CSF.     

Activation of supraoptic magnocellular neurons by gamma2-melanocyte stimulating hormone (γ2-MSH)

The effects of intracarotid infusions of the peptide gamma₂-melanocyte stimulating hormone (γ₂-MSH) on electrophysiologically and immunohistochemically identified supraoptic nucleus (SON) units were investigated Over a wide dose range this agent always excited SON units, while control infusions of vehicle had no effect. Because neural responses invariably preceded blood pressure elevation, it appears that γ₂-MSH excitation of the magnocellular system was due to a direct effect on the central nervous system and was not a result of systemic cardiovascular responses. These results suggest a forebrain γ₂-MSH sensitive site in the activation of SON magnocellular neurons.     

Release of hypothalamic corticotropin-releasing hormone and arginine-vasopressin by interleukin 1β and αMSH: studies in rats with different susceptibility to inflammatory disease

The susceptibility of Lewis rats is related to blunted hypothalamic-pituitary-adrenal (HPA) axis responsiveness to a variety of inflammatory and neuroendocrine stimuli. In contrast resistance to inflammatory disease of histocompatible Fischer rats is associated with their intact HPA axis responses to the same stimuli. We have examined the contribution of IL-1β to in vitro corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) release from hypothalamic explants derived from LEW/N and F344/N rats. The same animal model has been used to investigate the regulatory effect of αMSH, an immunosuppressive neurohormone, on IL-1β stimulated CRH and AVP secretion. CRH basal release in both strains was similar. However, LEW/N hypothalamic AVP basal secretion was significantly elevated. CRH relative response of LEW/N hypothalamic explants to IL-1β stimulation was lower compared to Fischer, which is consistent with their hyporesponsiveness to inflammatory mediators. AVP secretion however, was significantly decreased in hypothalamic explants from both strains after 40 min exposure to IL-1β. αMSH suppressed basal CRH and AVP release in both LEW/N and F344/N rats and prevented IL-1β stimulated CRH secretion in these strains. AVP was further diminished in F344/N explants following incubation with αMSH+IL-1β, while LEW/N level was significantly elevated. However, AVP levels remained significantly below baseline in explants from both strains after final incubation with IL-1β. Although our findings indicate a modulatory action of αMSH in HPA axis regulation in vitro, the physiological importance of this phenomenon in Lewis and Fischer rats requires further investigation.     

The influence of heparin and indol on the catalytic properties of α- and β/δ -thrombins

Heparin was shown to form an equimolar complex with α- and β/δ -forms of thrombin. The formation of the complex resulted in inhibition of the TAME esterase activity of thrombin ( by 40% form α- and 17% for β/δ-form ) and in stimulation of its BAME esterase activity ( by 50% for α- and 64% for β/δ-form ). Heparin caused the 70% inhibition of the activity of both forms of the enzyme towards the synthetic amid substrate Bz-Phe-Val-Arg-pNA; at the same time it had little if any effect on the enzyme activity towards Tos-Gly-Pro-Arg-pNA and stimulated the α- and β/δ-thrombins activities towards H-D-Phe-Pip-Arg-pNA by 16% and 57% respectively. In the case of both ester and amid substrates heparin exerted its effect on kcat, but had no effect on Km(app).Indol was shown to activate the TAME hydrolysis catalyzed by α- and β/δ-thrombins. The identity of the binding site for indol and for the additional TAME molecule in the effect of substrate activation was demonstrated. Heparin did not prevent the effects of indol and substrate activation of the thrombin-catalyzed hydrolysis of ester substrates. Moreover the kinetic parameters of indol activation are similar for the free enzyme and its complex with heparin indicating the different localization of the binding sites for indol and heparin in the molecule of thrombin.     

Lack of effect of interleukins 1α and 1β, during in vitro perifusion,on anterior pituitary release of adrenocorticotropic hormone and β endorphin in the male rat

It has been demonstrated that interleukin 1 (IL1) injection provokes a great variety of biological effects, notably an activation of the corticotropic axis, increasing plasma adrenocorticotropic hormone (ACTH) and corticosterone. However, the primary site of action of IL1 is still controversial. In the present study, we first verified the in vivo capability of human interleukins 1α (hIL1α) and 1β (hIL1β) to release ACTH and β endorphin (β EP) in the normal male rat, before investigating, through an anterior pituitary (AP) perifusion system, the hIL1α and hIL1β effects on basal and corticotropin-releasing factor (CRF)-induced ACTH and β EP secretions. This system enabled the examination of a dynamic profile of hormones secretion, avoiding the possibility of feedback mechanisms, as is the case with the use of regular but very often longtime incubations. The results showed that in a perifusion system, with a short duration treatment (below 2 hr) compatible with the kinetics of action observed in vivo, basal and CRF-induced ACTH and β EP release were not modified in the presence of a broad range of concentrations (from 10^?12 to 10^?9 M) of hIL1α or hIL1β. Taken together, these results clearly show that in an in vitro situation close to physiological conditions, the primary site of action of hIL1α and hIL1β on ACTH and β EP release is not located at the AP level in the male rat. © 1993 Wiley-Liss, Inc.