Intracameral administration of α-MSH increases intraocular pressure in rabbits

A growing body of evidence suggests that neural peptides may induce important modulations on vegative and motor functions of the eye. The present study was designed to evaluate the effect of intracameral (I.C.) administration of α-melanocyte-stimulating hormone (α-MSH) and several other ocular peptides on intraocular pressure (IOP) in rabbits. α-MSH (5 μg) produced a significant and prolonged unilateral increase of IOP. This efeect of I.C. α-MSH was dose-dependent (ED₅₀ = 2.5 μg). Structure-activity studies revealed that equimolar doses of β-MSH and γ-MSH, unlike α-MSH, were totally ineffective. In addition, the structurally unrelated peptides β-endorphin, thyrotropin-releasing hormone (TRH) and gonadotropin-releasing hormone (Gn-RH) did not affect IOP, when tested in a dose equimolar to 5 μg of α-MSH.These results confirm and extend previous observations, suggesting that α-MSH may be an important factor involved in regulation of IOP.     

α-Melanocyte-stimulating hormone (α-MSH) release from perifused rat hypothalamic slices

A perifusion system was developed to investigate the control of α-melanocyte-stimulating hormone (α-MSH) release from rat brain. Hypothalamic slices were perifused with Krebs-Ringer bicarbonate (KRB) medium supplemented with glucose, bacitracin and bovine serum albumine. Fractions were set apart every 3 min and α-MSH levels were measured by means of a specific and sensitive radioimmunoassay method. Hypothalamic tissue in normal KRB medium released α-MSH at a constant rate corresponding to 0.1% of the total hypothalamic content per 3 min. The basal release was not altered by Ca²⁺ omission in the medium or addition of the sodium channel blocker tetrodotoxine (TTX). Depolarizing agents such as potassium (50 mM) and veratridine (50 μM), which is known to increase Na⁺ conductance, significantly stimulated α-MSH release in a Ca²⁺-dependent manner. When Na⁺-channels were blocked by TTX (0.5 μM) the stimulatory effect of veratridine was completely abolished whereas the K⁺-evoked release was unaffected. These findings suggest that: (1) voltage-dependent sodium channels are present on α-MSH hypothalamic neurons; (2) depolarization by K⁺ induces a marked stimulation of α-MSH release; (3) K⁺- and veratridine-evoked releases are calcium-dependent. Altogether, these data provide evidence for a neurotransmitter or neuromodulator role for α-MSH in rat hypothalamus.     

Molecular mechanisms involved in interleukin 1-beta (IL-1β)-induced memory impairment. Modulation by alpha-melanocyte-stimulating hormone (α-MSH)

Pro-inflammatory cytokines can affect cognitive processes such as learning and memory. Particularly, interleukin-1β (IL-1β) influences the consolidation of hippocampus-dependent memories. We previously reported that administration of IL-1β in dorsal hippocampus impaired contextual fear memory consolidation. Different mechanisms have been implicated in the action of IL-1β on long-term potentiation (LTP), but the processes by which this inhibition occurs in vivo remain to be elucidated. We herein report that intrahippocampal injection of IL-1β induced a significant increase in p38 phosphorylation after contextual fear conditioning. Also, treatment with SB203580, an inhibitor of p38, reversed impairment induced by IL-1β on conditioned fear behavior, indicating that this MAPK would be involved in the effect of the cytokine. We also showed that IL-1β administration produced a decrease in glutamate release from dorsal hippocampus synaptosomes and that treatment with SB203580 partially reversed this effect. Our results indicated that IL-1β-induced impairment in memory consolidation could be mediated by a decrease in glutamate release. This hypothesis is sustained by the fact that treatment with d-cycloserine (DCS), a partial agonist of the NMDA receptor, reversed the effect of IL-1β on contextual fear memory. Furthermore, we demonstrated that IL-1β produced a temporal delay in ERK phosphorylation and that DCS administration reversed this effect. We also observed that intrahippocampal injection of IL-1β decreased BDNF expression after contextual fear conditioning. We previously demonstrated that α-MSH reversed the detrimental effect of IL-1β on memory consolidation. The present results demonstrate that α-MSH administration did not modify the decrease in glutamate release induced by IL-1β. However, intrahippocampal injection of α-MSH prevented the effect on ERK phosphorylation and BDNF expression induced by IL-1β after contextual fear conditioning. Therefore, in the present study we determine possible molecular mechanisms involved in the impairment induced by IL-1β on fear memory consolidation. We also established how this effect could be modulated by α-MSH.     

Characterization of α-melanocyte-stimulating hormone (α-MSH)-like peptides in discrete regions of the rat brain. In vitro release of α-MSH from perifused hypothalamus and amygdala

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is synthesized by discrete populations of hypothalamic neurons which project in different brain regions including the cerebral cortex, hippocampus and amygdala nuclei. The purpose of the present study was to identify the α-MSH-immunoreactive species contained in these different structures and to compare the ionic mechanisms underlaying α-MSH release at the proximal and distal levels, i.e. within the hypothalamus and amygdala nuclei, respectively. The molecular forms of α-MSH-related peptides stored in discrete areas of the brain were characterized by combining high-performance liquid chromatography (HPLC) separation and radioimmunoassay detection. In mediobasal and dorsolateral hypothalamic extracts, HPLC analysis confirmed the existence of a major immunoreactive peak which co-eluted with the syntheticdes-Nα-acetyl α-MSH standard. In contrast, 3 distinct forms of immunoreactive α-MSH, which exhibited the same retention times as synthetic des-, mono- and di-acetyl α-MSH, were resolved in amygdala nuclei, hippocampus, cortex and medulla oblongata extracts. The proportions of acetylated α-MSH (authentic α-MSH plus diacetyl α-MSH) contained in these extrahypothalamic structures were, respectively, 78, 80, 60 and 92% of the total α-MSH immunoreactivity. In order to compare the ionic mechanisms underlaying α-MSH release from hypothalamic and extrahypothalamic tissues, we have investigated in vitro the secretion of α-MSH by perifused slices of hypothalamus and amygdala nuclei. High potassium concentrations induced a marked increase of α-MSH release from both tissue preparations. However, a higher concentration of KCl was required to obtain maximal stimulation of amygdala nuclei (90 mM) than hypothalamic tissue (50 mM). The effect of depolarizing concentrations of KCl was totally suppressed in the absence of Ca²⁺, indicating that high-K⁺ induced the opening of voltage-operated Ca²⁺ (VOC) channels. Veratridine (50 μM), a depolarizing agent which activates Na⁺ conductances, caused a robust stimulation of α-MSH release from hypothalamic slices but had virtually no effect on amygdala nuclei. ω-Conotoxin (1 μM), a peptide toxin which blocks L- and N-type VOC channels, caused a slight reduction of K⁺-evoked α-MSH release from hypothalamic slices but induced a dramatic decrease of α-MSH release from amygdala nuclei. These data suggest that acetylation of α-MSH to generate the biologically active forms of the peptide is a slow process which occurs gradually during axonal transport. Our results also indicate that release of α-MSH at the hypothalamic level mainly results from activation of T-type VOC channels whereas, in the amygdala nuclei, L- and (or) N-type VOC channels are involved in the regulation of α-MSH secretion.     

ACTH- and α-MSH-induced grooming, stretching, yawning and penile erection in male rats: Site of action in the brain and role of melanocortin receptors

The effect of adrenocorticotropin (ACTH)(1-24) and α-melanocyte stimulating hormone (α-MSH) on grooming, stretching, yawning and penile erection was studied after injection into different brain areas. Both peptides induce the above responses when injected into the hypothalamic periventricular region of the third ventricle. This region includes the paraventricular nucleus, the dorsomedial nucleus, the ventromedial nucleus and the anterior hypothalamic area. The minimal effective dose of both peptides was 0.5 μg and the maximal effect was seen with 2 μg, the highest dose tested. Irrespective of the injection site, grooming started 5–7 min after injection of either peptide, while stretching, yawning and penile erection started only after 15–35 min and lasted for 90–120 min. In contrast both peptides were ineffective when injected into the preoptic area, the caudate nucleus or the CA1 field of the hippocampus. Grooming, stretching and yawning, but not penile erection, were prevented by cyclic[AcCys¹¹, D-Nal¹⁴, Cys¹⁸, AspNH₂²²]-β-MSH (11-22) (HS014), a selective melanocortin 4 receptor antagonist, injected into the same periventricular area 10 min before of ACTH(1-24) or α-MSH. The results show that ACTH(1-24) and α-MSH act in the hypothalamic periventricular region to induce the above responses and that grooming, stretching and yawning, but not penile erection, are mediated by melanocortin 4 receptors.     

Effects of massive doses of α-MSH on thermoregulation in the rabbit

α-MSH-related compounds may prove to be clinically useful antipyretics since the parent peptide is extremely potent in reducing fever, it is effective when given orally, and it neither stimulates corticosteroid activity nor has marked melanotropic effects in man. To determine whether or in what doses α-MSH might cause harmful side-effects, we injected doses greatly exceeding those required to reduce fever into a lateral cerebral ventricle of afebrile rabbits. One hundred to seven hundred and fifty μg α-MSH caused large and prolonged reductions in body temperature and the dose-response relation was bell-shaped for both magnitude and duration. These doses caused no apparent injury to the animals. One mg α-MSH elicited hyperthermic responses that were variable in magnitude and duration. Animals that had previously received large doses of α-MSH ( 100 μg) did not develop hyperthermia, even when given 2 mg, indicating an acquired tolerance to this hyperthermic action of α-MSH. All animals, tolerant or previously uninjected, showed symptoms with doses 1 mg α-MSH that included: increased salivation, agitation, ataxia, respiratory distress, and death (in 30% of the animals); those that recovered from these large doses resumed outwardly healthy appearance and behavior. Although α-MSH is toxic when given centrally in large doses, the 5000-fold difference between antipyretic and toxic doses indicates a wide safety margin should this peptide be used clinically as an antipyretic drug.     

Biological and immunological characterization of α-melanocyte-stimulating hormone (α-MSH) in two neuronal systems of the rat brain

Recent immunocytochemical studies have demonstrated the existence of two different neuronal systems containing α-MSH-like material in the brain: one originating from the arcuate nucleus and the other one from the dorsolateral hypothalamus. The aim of the present study was to further characterize α-MSH in these two discrete regions of the rat diencephalon. Intracerebroventricular administration of colchicine resulted in a marked decrease in the number of ACTH and β-endorphin nerve fibers and a significant reduction in ACTH and β-endorphin content in the dorsolateral hypothalamus. Conversely, colchicine treatment did not alter α-MSH, ACTH or β-endorphin content in the arcuate nucleus and did not significantly affect α-MSH concentration in the dorsal region. Reverse-phase high-performance liquid chromatography showed that the major α-MSH-like compound localized in the dorsal hypothalamus co-migrated exactly with synthetic α-MSH, whereas the arcuate nucleus contained 5 peptides cross-reacting with α-MSH antibodies, 4 of them being different from standard α-MSH. Significant amounts of biologically active melanotropin, which migrated on Sephadex G-25 columns like synthetic α-MSH, were also detected in both the arcuate nucleus and dorsolateral hypothalamus. Taken together, these data demonstrate that the α-MSH cell bodies located in the dorsolateral hypothalamus specifically produce authentic α-MSH, whereas the α-MSH cell bodies in the arcuate nucleus also contain ACTH, β-endorphin and several peptides immunologically related but not identical to α-MSH.     

Localization and identification of α-melanocyte-stimulating hormone (α-MSH) in the frog brain

The distribution of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. α-MSH-like containing perikarya were localized in the infundibular region, mainly in the ventral hypothalamic nucleus. A rich plexus of immunoreactive fibers directed towards the ventral telencephalic region was detected. Reverse-phase high-performance liquid chromatography and radioimmunoassay were used to characterize α-MSH-like peptides in the frog brain. Chromatographic separation revealed that immunoreactive α-MSH coeluted with synthetic des-Nα-acetyl α-MSH, authentic α-MSH and their sulfoxide derivatives. The heterogeneity of α-MSH-like material in the frog brain was in marked contrast with the figure observed in the intermediate lobe of the pituitary gland where only des-Nα-acetyl α-MSH is present. These findings support the existence of discrete α-MSH immunoreactive neurons in the frog brain containing both desacetyl and authentic α-MSH.     

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.     

The antihyperalgesic activity of a selective P2X7 receptor antagonist, A-839977, is lost in IL-1αβ knockout mice

The pro-inflammatory cytokine interleukin-1β (IL-1β) has been implicated in both inflammatory processes and nociceptive neurotransmission. Activation of P2X7 receptors is the mechanism by which ATP stimulates the rapid maturation and release of IL-1β from macrophages and microglial cells. Recently, selective P2X7 receptor antagonists have been shown to reduce inflammatory and neuropathic pain in animal models. However, the mechanisms underlying these analgesic effects are unknown. The present studies characterize the pharmacology and antinociceptive effects of a structurally novel P2X7 antagonist. A-839977 potently (IC₅₀ = 20–150 nM) blocked BzATP-evoked calcium influx at recombinant human, rat and mouse P2X7 receptors. A-839977 also potently blocked agonist-evoked YO-PRO uptake and IL-1β release from differentiated human THP-1 cells. Systemic administration of A-839977 dose-dependently reduced thermal hyperalgesia produced by intraplantar administration of complete Freund's adjuvant (CFA) (ED₅₀ = 100 μmol/kg, i.p.) in rats. A-839977 also produced robust antihyperalgesia in the CFA model of inflammatory pain in wild-type mice (ED₅₀ = 40 μmol/kg, i.p.), but the antihyperalgesic effects of A-839977 were completely absent in IL-1αβ knockout mice. These data demonstrate that selective blockade of P2X7 receptors in vivo produces significant antinociception in animal models of inflammatory pain and suggest that the antihyperalgesic effects of P2X7 receptor blockade in an inflammatory pain model in mice are mediated by blocking the release of IL-1β.     

Release of immunoreactive α-MSH by synaptosome-enriched fractions of homogenates of hypothalami

Immunoreactive α-melanocyte-stimulating hormone (α-MSH) was found to be concentrated in a synaptosome-enriched fraction prepared by differential centrifugation of rat hypothalamic homogenates. The release of the hormone from this preparation was investigated. After incubation, the synaptosomes were isolated by ultrafiltration and α-MSH in the ultrafiltrate was determined by radioimmunoassay. Particle-bound α-MSH, recovered by extraction with acid ethanol, and α-MSH released from the synaptosome preparation, were immunologically similar to synthetic α-MSH and had an accompanying melanotropic activity. Less than 10% of the particle-bound α-MSH was released during incubation in 0.32 M sucrose. However, in the presence of 2 mM Ca²⁺, α-MSH release increased with increasing concentrations (30–150 mM) of K⁺. The stimulatory effect of 60 mM K⁺ was complete within 2 min and was potentiated by increasing Ca²⁺ concentrations over the range of 0 to 2 mM. K⁺-induced release of α-MSH was independent of temperature from 1 to 30°C, and neither glucose (10 mM) nor dopamine (10⁻¹⁰-10⁻² M) had any effect on the release of the peptide. It is concluded that a synaptosome-enriched fraction from the hypothalamus contains a releasable pool of immunoreactive α-MSH that is mobilized by depolarizing concentrations of K⁺ in a Ca²⁺-dependent manner.     

ARC POMC mRNA and PVN α-MSH are lower in obese relative to lean Zucker rats

Effects of obesity on gene expression for opioid peptides and neuropeptide-Y (NPY) in the arcuate nucleus (ARC), and on opioid peptides and α-melanocyte stimulating hormone (α-MSH) in the paraventricular nucleus (PVN) were examined in obese Zucker rats (18 weeks old). Obese Zucker rats are insulin-resistant, diabetic and hyperleptinemic as indicated by high serum glucose, insulin and leptin levels. ARC proOpiomelanocortin (POMC) mRNA levels were significantly lower in the obese relative to lean Zucker rats and ARC proNeuropeptide Y (proNPY) mRNA levels were higher (P<0.05). There were no differences in proDynorphin and proEnkephalin mRNA levels in the ARC (P>0.05). Obese Zucker rats had lower α-MSH and dynorphin A_1–17 peptide levels in the paraventricular nucleus (PVN) (P<0.05), but did not have lower PVN β-endorphin peptide levels (P>0.05). The decrease in POMC in the ARC and decrease in α-MSH in the PVN seen in the obese Zucker rat in the present study suggest that reduced activity of the melanocortin system in the ARC to PVN pathway may contribute to the related hyperphagia. Reduced activity of the melanocortin system in the ARC to PVN pathway may be due to a disturbance of leptin signaling coupling to POMC.     

α-Melanocyte-stimulating hormone infused ICV fails to affect body temperature or endotoxin fever in the cat

Permanent cannulae for intracerebroventricular (ICV) infusion were implanted bilaterally in cats following stereotaxic procedures. After colonie temperature was recorded for a one-hour baseline, a 300 μl ICV infusion was given of CSF control vehicle, 1 : 100 dilution of W3110 E. coli endotoxin (10⁸ organisms/ml) or α-melanocyte-stimulating hormone (α-MSH) in one of seven doses ranging from 50.0 ng to 50.0 μg. Whereas ICV E. coli always induced an intense and prolonged fever of rapid onset, α-MSH infused similarly was essentially without effect on the deep body temperature of the normothermic cat. When each of the doses of α-MSH was infused ICV, either during the rising phase of an E. coli fever or after the febrile response had reached its asymptote, the core temperature of the cat was unaffected. Similarly, a mixture of E. coli combined with α-MSH given ICV failed to alter the characteristics of the rapidly developing fever produced in the cat by this endotoxin. On the other hand, either excess Ca⁺⁺ ions (50 mM) given ICV or the antipyretic drug, Dipyrone, administered systemicatly during the course of an endotoxin fever effectively attenuated the animal's elevated body temperature. These results demonstrate that α-MSH is apparently neither involved in the central mechanisms underlying normal thermoregulation, nor does it act as an endogenous antipyretic in the cat as has been postulated in another species.     

Infusion of 3α,5α-THP to the pontine reticular formation attenuates PTZ-induced seizures

Whether progesterone (P₄) and its metabolite, 5α-pregnan-3α-ol-20-one (3α,5α-THP) have anti-seizure effects through actions in the pontine reticular formation (PRF) was investigated. Concentrations of P₄ and 3α,5α-THP in the PRF were greater in proestrous and hormone-primed rats, that are typically more resistant to seizure-induction, than diestrous and males rats. Ovx, Long-Evans rats with unilateral microinjections into the PRF of 3α,5α-THP (5 μg/0.2 μl), but not P₄ (11 μg/0.2 μl) or vehicle (β-cyclodextrin), had a greater latency and lower incidence of tonic-clonic seizures induced by pentylenetetrazol (PTZ; 70 mg/kg, IP) administration. Infusions that missed the PRF were not effective. These data suggest 3α,5α-THP has anti-seizure effects in part through actions in the PRF.     

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.     

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.     

(±)-α-Methyl-4-carboxyphenylglycine, a metabotropic glutamate receptor blocker, impairs retention of an inhibitory avoidance task in rats when infused into the basolateral nucleus of the amygdala

The amygdala is important for memory processes of emotionally motivated learning and the amygdala glutamatergic system may play a key role in this process. In this study we assessed the effect of the infusion of (±)-α-methyl-4-carboxyphenylglycine (MCPG), a metabotropic glutamate receptor (mGluR) antagonist, into the basolateral complex of the amygdala (BLA) on the learning and retention of an emotionally motivated task. Rats received either vehicle or three different doses of MCPG (0.2, or 1.0, or 5.0 μg/0.2μl/side, respectively) bilaterally into the BLA, 5 min before they were trained in a continuous multiple-trial inhibitory avoidance (CMIA) task. Response latencies during the training were recorded. Retention was assessed 8 days later. MCPG in the doses given did not significantly affect the acquisition of the CMIA task. However, MCPG at a dose of 5.0 μg/0.2 μl/side impaired the long-term retention test performance. Additionally, a nociception test indicated that dose of MCPG infused into the BLA did not affect the footshock sensitivity. Our results indicate that MCPG, when infused into the BLA of rats prior to the training, impaired long-term memory of aversive training without affecting acquisition.