Attenuation of morphine tolerance and dependence by alpha-melanocyte stimulating hormone(alpha-MSH).

Repeated administration of morphine resulted in significant reduction of its analgesic potency. If 0.1 mg/kg α-MSH was coadministered, the tolerance development was attenuated, 1 mg/kg MIF (MSH release inhibiting factor), given simultaneously with morphine, did not affect tolerance. Injecting, however, MIF 1 hour prior to the daily opiate treatment resulted in accelerated development of tolerance supposedly by lowering the plasma α-MSH level at the time of morphine administration. Of the morphine abstinence symptoms the naloxone-induced jumping in morphine pretreated mice could not be modified either by α-MSH coadministration or by MIF pretreament, but the withdrawal body weight loss was found to be diminished by the former and increased by the latter peptide. The possible role of α-MSH in preventing the development of tolerance to the analgesic effect of endogenous opioid peptides is discussed.     

The influence of beta-LPH fragments on alpha-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 $\text{in bitro}$, 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 induces an increase of the plasma levels of immunoreactive alpha-MSH, beta-endorphin, ACTH and of corticosterone

Infusion of I-isoproterenol in anesthetised rats induced a dose-dependent increase of the plasma levels of immunoreactive α-MSH (α-MSHi), β-endorphin (β-ENDi), ACTH (ACTHi) and of corticosterone (B). Steady state levels of all of these substances were reached within 20 min after the start of the infusion. The ED₅₀ values of I-isoproterenol for the increase of plasma α-MSHi, β-ENDi, ACTHi and B were similar (42, 86, 84 and 48 ng/kg. min, respectively). Infusion of I-epinephrine also induced a dose-dependent increase of plasma α-MSHi, β-ENDi, ACTHi and B with similar ED₅₀ values (89, III, 110 and 46 ng/kg. min, respectively). The I-epinephrine-induced increase of plasma α-MSHi, β-ENDi, ACTHi and B was blocked by I-propranolol but not by d-propranolol.Pretreatment of rats with dexamethasone (2.0 mg/kg s.c., 16 hr) completely prevented the I-epinephrine-induced increase of plasma ACTHi and B, without affecting the increase of plasma α-MSHi and β-ENDi.We conclude that catecholamines can stimulate the secretion of peptides from the intermediate lobe (e.g. α-MSH, β-END) as well as from the anterior lobe (e.g. ACTH) of the pituitary gland via interaction with one or more β-adrenergic receptor mechanisms.     

Involvement of noradrenergic transmission in the PVN on CREB activation, TORC1 levels, and pituitary-adrenal axis activity during morphine withdrawal

Experimental and clinical findings have shown that administration of adrenoceptor antagonists alleviated different aspects of drug withdrawal and dependence. The present study tested the hypothesis that changes in CREB activation and phosphorylated TORC1 levels in the hypothalamic paraventricular nucleus (PVN) after naloxone-precipitated morphine withdrawal as well as the HPA axis activity arises from α(1)- and/or β-adrenoceptor activation. The effects of morphine dependence and withdrawal on CREB phosphorylation (pCREB), phosphorylated TORC1 (pTORC1), and HPA axis response were measured by Western-blot, immunohistochemistry and radioimmunoassay in rats pretreated with prazosin (α(1)-adrenoceptor antagonist) or propranolol (β-adrenoceptor antagonist). In addition, the effects of morphine withdrawal on MHPG (the main NA metabolite at the central nervous system) and NA content and turnover were evaluated by HPLC. We found an increase in MHPG and NA turnover in morphine-withdrawn rats, which were accompanied by increased pCREB immunoreactivity and plasma corticosterone concentrations. Levels of the inactive form of TORC1 (pTORC1) were decreased during withdrawal. Prazosin but not propranolol blocked the rise in pCREB level and the decrease in pTORC1 immunoreactivity. In addition, the HPA axis response to morphine withdrawal was attenuated in prazosin-pretreated rats. Present results suggest that, during acute morphine withdrawal, NA may control the HPA axis activity through CREB activation at the PVN level. We concluded that the combined increase in CREB phosphorylation and decrease in pTORC1 levels might represent, in part, two of the mechanisms of CREB activation at the PVN during morphine withdrawal.     

Effect of nicotine on in vivo secretion of melanocorticotropic hormones in the rat

Corticosterone, ACTH, β-endorphin and α-MSH were measured in rat plasma by radioimmunoassay before and 2,5,15,30 minutes after an intraperitoneal injection of nicotine (500 μg/Kg b.w.). Nicotine induced an increase of plasma corticosterone (p < 0.05 at t + 15 min), ACTH and β-endorphin (p < 0.01 at t + 5 min) and a decrease of α-MSH (p < 0.005 at t + 15 min). Dose response experiments showed an increase of corticosterone, ACTH, β-endorphin 15 min after 250 μg/Kg b.w. nicotine I.P., no effect being observed after injection of 100 μg/Kg b.w. The decrease of α-MSH was observed 15 min after 100, 250 or 500 μg/Kg b.w. nicotine I.P. Our results suggest that the increase of corticosterone is mediated through ACTH release.     

The effect of phencyclidine on dopamine metabolism in the mouse brain

The administration of phencyclidine (PCP) to mice resulted in no change in brain levels of tyrosine, dopamine (DA), norepinephrine (NE), or homovanillic acid (HVA). Although PCP reduced plasma tyrosine levels, no effect of PCP on the utilization of DA of NE after blockade of synthesis with α-methyl-p-tyrosine (AMPT) was observed. In addition, PCP did not affect the probenecid-induced accumulation of HVA. However, PCP was observed to potentiate the haloperidol-induced increase in HVA concentration, and the haloperidol-induced decline in DA levels after AMPT. The former effect was blocked by baclofen, suggesting that PCP mobilizes DA for impulse-dependent release. This effect could not be attributed to an antagonism of presynaptic DA receptors. These effects are similar to those of the “non-amphetamine” stimulant class of drugs.     

Analysis of the UV-Induced Melanogenesis and Growth Arrest of Human Melanocytes

Cultured human melanocytes derived from different skin types responded to frequent treatment with ultraviolet (UV) light with increased melanin synthesis, decreased proliferation, and morphologic signs of aging. These effects were augmented by increased frequency of irradiation with 15.5 mJ/cm² UV light. Stimulation of melanogenesis by UV light involved an increase in tyrosinase activity, without any change in the amounts of either tyrosinase or tyrosinase-related protein (TRP)-1, and a decrease in the amount of TRP-2, as determined by Western blot analysis. These results are different from the mechanisms by which other melanogenic agents, such as cholera toxin and isobutyl methylxanthine, stimulated melanogenesis, whereby the amounts of tyrosinase, TRP-1 and TRP-2 were increased. The decrease in the amount of TRP-2 might be significant in that it might alter the properties of the newly synthesized melanin. The UV irradiation protocol that was followed blocked melanocytes in G₂-M phase of the cell cycle without compromising cellular viability. Following three rounds of UV irradiation, melanocytes could recover from the growth arrest and resume proliferation. Treatment with 0.1 μM α-melanocyte stimulating hormone (α-MSH) postirradiation enhanced the melanogenic effect of UV light and stimulated the melanocytes to proliferate. The effects of α-MSH on the UV induced responses and their implications on photocarcinogenesis are being further investigated. Analyzing the mechanisms by which UV light exposure affects normal melanocytes might lead to a better understanding of how these cells undergo malignant transformation, and why individuals with different skin types differ in their susceptibility to skin cancers.     

Tolerance to effects of clonidine and morphine on sulfobromophthalein disposition in mice

Chronic treatment of mice with clonidine or morphine caused tolerance to the analgesic and thermoregulatory effects of these drugs. After chronic morphine, mice also became tolerant to the analgesic and thermoregulatory effects of clonidine. Cross tolerance to the hypothermic effect of morphine was demonstrated after chronic clonidine administration, but no diminution of morphine-induced analgesia could be shown. Morphine and clonidine acutely increased the retention of sulfobromophthalein (BSP) in plasma and liver. Chronic dosing with morphine or clonidine caused partial tolerance and cross-tolerance to the rise in hepatic BSP caused by an acute challenge with either agonist. However, both drugs elevated plasma BSP levels similarly in tolerant and non-tolerant mice. Thus, regimens which readily induced tolerance to the analgesic and hypothermic effects of morphine or clonidine were only partially effective in modifying the acute hepatobiliary effects of these drugs.     

The role of adrenal corticosteroids in induction of micronuclei by morphine.

It has previously been shown that morphine can increase the frequency of micronucleated splenocytes when administered to mice, but not when cells are exposed to the opiate in vitro. Morphine treatment is also known to increase circulating levels of glucocorticosteroids, which have been reported to produce genetic damage in vivo and in vitro. In order to determine whether adrenal hormones might mediate the genotoxic effects of morphine, adrenalectomized and sham-operated mice were treated with morphine sulfate. In sham-operated animals administration of morphine produced a dose-related increase in the frequency of micronucleated cells, whereas adrenalectomy abolished the effect. When plasma from morphine-treated mice was used to supplement growth medium of untreated splenocytes, the frequency of micronucleated cells increased, an effect partially blocked by the steroid antagonist RU 486. The N-methylmorphine, which does not stimulate the release of corticosterone from adrenal glands, induced micronuclei formation in splenocytes, and administration of metyrapone, an inhibitor of corticosterone biosynthesis, blocked the morphine-induced increase in corticosterone secretion, but had no effect on the frequency of micronuclei formation. These results indicate that basal levels of glucocorticosteroids are required for induction of micronuclei by morphine in murine splenocytes, but activation of the hypothalamo-pituitary-adrenal (HPA) axis by morphine does not contribute to the observed response.     

Vasopressin and oxytocin reduce plasma prolactin levels of conscious rats in basal and stress conditions. Study of the characteristics of the receptor involved

Subcutaneous administration of arginine vasopressin (AVP) to conscious rats induced a dose-dependent increase of plasma ACTH and beta-endorphin levels and decrease of plasma prolactin (PRL) levels 30 min later. AVP similarly reduced PRL increase induced by exposure to a novel environment stress. Oxytocin (OT) was also active but 5-fold less potent than AVP. The study of several analogs with specific agonistic and antagonistic activity on the oxytocic, vasopressor and antidiuretic receptors of OT and AVP suggests that the receptor involved in this effect does not fit into this classification.     

Effects of beta-endorphin fragments on plasma levels of vasopressin

Plasma vasopressin levels are significantly decreased after intracerebroventricular (icv) administration of β-endorphin (βE), but not of des-tyrosine βE (DTβE). The βE induced decrease of vasopressin levels, which occurs in normal as well as in water deprivated rats, can partially be blocked by naltrexone. γ-Endorphin (γE), α-endorphin (αE), DTγE and DTαE did not affect basal levels of vasopressin, but γE and DTγE further increased the elevated vasopressin levels in water deprivated rats. Naltrexone antagonized this increase following γE administration, but not that induced by DTγE. The results suggest that the effects of βE and its fragments on plasma vasopressin levels are mediated by multiple opiate and non-opiate receptor systems.     

The metabolic effects of oxytocin are mediated by a uterine type of receptor and are inhibited by oxytocin antagonist and by arginine vasopressin in the dog.

Infusion of oxytocin (OT) into normal dogs, in doses which produced plasma levels of OT in the physiological range, has been shown to increase plasma levels of glucose, insulin and glucagon and increase rates of glucose production and uptake. This study sought to determine whether there was a correlation between these metabolic effects and the oxytocic potency of four less potent oxytocic analogues when infused into normal dogs. The rank order of oxytocic potency of all 4 correlated well with the rise in plasma glucose levels, and in 3 of the 4 with the rise in plasma insulin levels. An antagonist of the oxytocic effect of OT suppressed the usual OT-induced rise in plasma glucose, insulin and glucagon as well as the increased glucose production and uptake. Arginine vasopressin (AVP) infusion, which by itself did not produce any metabolic effects, blocked completely the effects of OT infusion to raise plasma glucose and insulin levels and increase glucose production and uptake. The data suggest that the metabolic effects of OT in the dog are mediated by OT receptors that are similar to those producing the oxytocic effects. Whether the inhibition by AVP of the metabolic and hormonal effects of OT occurs at the receptor or post receptor level or via other mechanisms remains to be determined.     

Hemodynamic actions and mechanisms of systemically administered α-MSH analogs in mice

α-Melanocyte-stimulating hormone (α-MSH) regulates important physiological functions including energy homeostasis and inflammation. Potent analogs of α-MSH, [Nle⁴, d-Phe⁷]-α-MSH (NDP-α-MSH) and melanotan-II (MT-II), are widely used in pharmacological studies, but the hemodynamic effects associated with their systemic administration have not been thoroughly examined. Therefore, we investigated the hemodynamic actions of these compounds in anesthetized and conscious C57Bl/6N mice using peripheral routes of administration. NDP-α-MSH and MT-II induced mild changes in blood pressure and heart rate in anesthetized mice compared to the effects observed in conscious mice, suggesting that anesthesia distorts the hemodynamic actions of α-MSH analogs. In conscious mice, NDP-α-MSH and MT-II increased blood pressure and heart rate in a dose-dependent manner, but the tachycardic effect was more prominent than the pressor effect. Pretreatment with the melanocortin (MC) 3/4 receptor antagonist SHU9119 abolished these hemodynamic effects. Furthermore, the blockade of β₁-adrenoceptors with metoprolol prevented the pressor effect and partly the tachycardic action of α-MSH analogs, while the ganglionic blocker hexamethonium abrogated completely the difference in heart rate between vehicle and α-MSH treatments. These findings suggest that the pressor effect is primarily caused by augmentation of cardiac sympathetic activity, but the tachycardic effect seems to involve withdrawal of vagal tone in addition to sympathetic activation. In conclusion, the present results indicate that systemic administration of α-MSH analogs elevates blood pressure and heart rate via activation of MC_3/4 receptor pathways. These effects and the consequent increase in cardiac workload should be taken into account when using α-MSH analogs via peripheral routes of administration.     

Responses of melanocytes and melanomacrophages of Eupemphix nattereri (Anura: Leiuperidae) to Nle4, D-Phe7-α-melanocyte stimulating hormone and lipopolysaccharides

Melanocytes are found in various organs of ectothermic animals, playing a protective role against bacteria and free radicals. It is known that pigment cells from hematopoietic organs have immune functions. However, the role of visceral melanocytes is not well understood. Cutaneous melanocytes are responsive to α-melanocyte stimulating hormone (α-MSH), which is associated with the dispersion of melanin granules within melanocytes. α-MSH has also been reported to inhibit most forms of inflammatory responses by decreasing the pro-inflammatory cytokines and neutrophil migration. The present study evaluated the influence of an α-MSH analog (Nle⁴, D-Phe⁷-α-MSH) and lipopolysaccharides (LPS) from Escherichia coli on the liver and testicular tissues of the anuran Eupemphix nattereri. The tested hypotheses were: (i) the pigmented area will increase following hormone and LPS administration, (ii) pre-treatment with α-MSH will decrease the number of mast cells, and (iii) the hormone will have protective effects against LPS-induced responses. We found that hormone administration did not change hepatic pigmentation, but increased testicular pigmentation. Testicular pigmentation quickly increased after LPS administration, whereas there was a late response in the liver. The response of enhanced pigmentation was delayed and the number of mast cells decreased in animals previously treated with the α-MSH analog when compared to the LPS group. Hemosiderin and lipofuscin were found in melanomacrophages, but not in testicular melanocytes. Although both the liver and the testes of E. nattereri have pigmented cells, these are distinct in morphology, embryonic origin, and pigmentary substances. These differences may be responsible for the different responses of these cells to the α-MSH analog and LPS administration.     

Brain-derived adrenomedullin controls blood volume through the regulation of arginine vasopressin production and release

Central nervous system-derived adrenomedullin (AM) has been shown to be a physiological regulator of thirst. Administration of AM into the lateral ventricle of the brain attenuated water intake, whereas a decrease in endogenous AM, induced by an AM-specific ribozyme, led to exaggerated water intake. We hypothesized that central AM may control fluid homeostasis, in part by regulating plasma arginine vasopressin (AVP) levels. To test this hypothesis, AM or a ribozyme specific to AM was administered intracerebroventricularly, and alterations in plasma AVP concentrations were examined under basal and stimulated (hypovolemic) conditions. Additionally, we examined changes in blood volume, kidney function, and plasma electrolyte and protein levels, as well as changes in plasma aldosterone concentrations. Intracerebroventricular administration of AM increased plasma AVP levels, whereas AM ribozyme treatment led to decreased plasma AVP levels under stimulated conditions. During hypovolemic challenges, AM ribozyme treatment led to an increased loss of plasma volume compared with control animals. Although overall plasma osmolality did not differ between treatment groups during hypovolemia, aldosterone levels were significantly higher and, consequently, plasma potassium concentrations were lower in AM ribozyme-treated rats than in controls. These data suggest that brain-derived AM is a physiological regulator of vasopressin secretion and, thereby, fluid homeostasis.     

Alpha-MSH and coat color changes in the mouse

The effect of -MSH on coat color was examined in viable yellow mice (C3H/He-A*^vy). These mice normally grow a coat of darkly pigmented hair at puberty. This darkening effect was also evident in hair that grew in a region that had been plucked at 13 days of age. Administration of -MSH increased the darkness of this hair and the hair which grew naturally in an unplucked area. However, the natural coat darkening that occurred at puberty was not associated with an increase in plasma immunoreactive -MSH levels. Moreover, although bromocryptine, a dopamine agonist that inhibits -MSH release from the pituitary reduced the darkness of the coat that grew after plucking the reduction in coat darkening was unrelated to changes in plasma -MSH. Nevertheless, this effect of bromocryptine was reversed when -MSH was administered together with the drug. Apomorphine had no effect on coat darkening and produced only a slight decrease in plasma -MSH. Melatonin reduced coat darkening slightly but, like apomorphine, had little effect on plasma -MSH concentrations. Although -MSH may have a physiological role in coat darkening in the C3H/He-A*^vy mouse at puberty the response seems to be unrelated to an increase in circulating -MSH. Thus, other factors, such as changes in melanocyte sensitivity to -MSH or inhibitory mechanisms that prevent coat darkening during prepubertal and adult life may be involved in regulation of coat color in the viable yellow mouse.     

Attenuation of morphine analgesia by α-MSH,MIF-I,melatonin and naloxone in the rat

In two experiments the effects of the pituitary peptide α-MSH, the hypothalamic tripeptide MIF-I (P-L-G-NH₂) and the pineal hormone melatonin were investigated on the attenuation of morphine analgesia measured by a tail flick test. In Experiment 1, α-MSH had minimal effect on morphine analgesia, whereas, MIF-I and melatonin clearly delayed the onset of morphine analgesia, and melatonin also shortened the duration of analgesia. Experiment 2 was designed to investigate the possible synergistic effect of MIF-I and melatonin. The combined treatment of MIF-I and melatonin significantly delayed the onset of morphine analgesia, and melatonin alone shortened the duration of analgesia. The relationshps among the pituitary, hypothalamus and the pineal for the modulation of pain and response to morphine were discussed.     

α-MSH inhibits TNF-α-induced maturation of human dendritic cells in vitro through the up-regulation of ANXA1

α-Melanocyte-stimulating hormone (α-MSH), an anti-inflammatory and immunomodulatory neuropeptide, has been shown to be effective in the experimental treatment of autoimmune diseases and allograft rejection. However, its regulatory mechanism is still unclear. Mature dendritic cells (DCs) are pivotal initiators of immune response and inflammation. We hypothesized that the regulatory role of α-MSH in DC maturation would contribute to the effects of α-MSH in immune-response-mediated disease models. It was found that α-MSH inhibited tumor necrosis factor-alpha (TNF-α)-induced maturation of human peripheral-monocyte-derived DCs (MoDCs), both phenotypically and functionally. This occurred through the down-regulation of the expression of co-stimulatory molecules CD83 and CD86, the production of IL-12, the promotion of IL-10 secretion, and the MoDC phagocytic activity, suggesting that the inhibition of DC maturation by α-MSH could contribute to the anti-inflammatory effect of this neuropeptide. Furthermore, increased expression of annexin A1 (ANXA1) was found to be responsible for the α-MSH inhibiting effect on TNF-α-induced MoDC maturation, which could be abolished by the treatment of MoDCs with specific, small interfering RNAs targeting ANXA1 (ANXA1-siRNA), suggesting that α-MSH-induced ANXA1 mediates the inhibition. Therefore, α-MSH inhibits TNF-α-induced maturation of human DCs through α-MSH-up-regulated ANXA1, suggesting that inhibition of the maturation of DCs by α-MSH could mediate the anti-inflammatory effect of the neuropeptide. Furthermore, ANXA1 could be identified as a new therapeutic drug target based on the role of DCs in immune-mediated inflammatory diseases.     

Effects of corticotropin-releasing hormone on proopiomelanocortin derivatives and monocytic HLA-DR expression in patients with septic shock

Little is known about interactions between immune and neuro-endocrine systems in patients with septic shock. We therefore evaluated whether the corticotropin-releasing hormone (CRH) and/or proopiomelanocortin (POMC) derivatives [ACTH, β-endorphin (β-END), β-lipotropin (β-LPH), α-melanocyte stimulating hormone (α-MSH) or N-acetyl-β-END (Nac-β-END)] have any influences on monocyte deactivation as a major factor of immunosuppression under septic shock conditions. Sixteen patients with septic shock were enrolled in a double-blind, cross-over and placebo controlled clinical study; 0.5 μg/(kg_bodyweight h) CRH (or placebo) were intravenously administered for 24 h. Using flow cytometry we investigated the immunosuppression in patients as far as related to the loss of leukocyte surface antigen-DR expression on circulating monocytes (mHLA-DR). ACTH, β-END immunoreacive material (IRM), β-LPH IRM, α-MSH and Nac-β-END IRM as well as TNF-α and mHLA-DR expression were determined before, during and after treatment with CRH (or placebo). A significant correlation between plasma concentration of α-MSH and mHLA-DR expression and an inverse correlation between mHLA-DR expression and TNF-α plasma level were found. Additionally, a significant increase of mHLA-DR expression was observed 16 h after starting the CRH infusion; 8 h later, the mHLA-DR expression had decreased again. Our results indicate that the up-regulation of mHLA-DR expression after CRH infusion is not dependent on the release of POMC derivatives. From the correlation between plasma concentration of α-MSH and mHLA-DR expression, we conclude that in patients with septic shock the down-regulation of mHAL-DR expression is accompanied by the loss of monocytic release of α-MSH into the cardiovascular compartment.     

Alpha-MSH injected into the substantia nigra or intraventricularly alters behavior and the striatal dopaminergic activity

Rats were injected with 1 μg of alpha-melanocyte stimulating hormone (α-MSH) into the third ventricle and locally in the ventral tegmental area and in different regions of the substantia nigra. The modifications produced on grooming behavior and locomotion as well as on the dopamine content of the nucleus accumbens and the caudate putamen, were studied. Both intraventricular peptide administration and microinjections into the ventral tegmental area induced excessive grooming and a significant increase of the locomotor activity. The dopamine content of the nucleus accumbens and caudate putamen was markedly reduced. Injections of the peptide into the substantia nigra pars compacta failed to induce excessive grooming but did provoke a slight increase in locomotor activity and a smaller change in caudate dopamine content than that observed by injections in the ventral tegmental area or in the third ventricle. Dopamine levels in the nucleus accumbens were not changed. Finally, the injections of α-MSH into the lateral substantia nigra did not produce either biochemical or behavioral changes.The results suggests that α-MSH can modify, directly or indirectly, the striatal dopaminergic activity and that the behavioral alterations observed such as excessive grooming, could be mediated by the activation of the dopamine cells from the ventral tegmental area, that in turn may provoke a significative release of dopamine at the caudate putamen nucleus as well as in nucleus accumbens.