Evidence for Alpha-MSH Binding Sites on Human Scalp Hair Follicles: Preliminary Results

Alpha-MSH, considered an important pigmentation hormone, binds to melanocytes and is thought to stimulate melanogenesis through a cyclic-AMP-dependent mechanism. The binding of alpha-MSH to follicular melanocytes has been investigated in human hair of different colors, ranging from black to blond and senile white. Hairs were plucked, the follicles were cut off, and an alpha-MSH binding assay, using a radiolabeled alpha-MSH analogue, was performed on these bulbs. As controls of each assay, fragments of hairs of the same person were used. The results show a dose-response relationship and the assay seems to be specific for alpha-MSH, because other peptides such as ACTH, beta-LPH and beta-endorphins do not compete for binding sites as alpha-MSH does. These binding sites seem to be present only on melanin synthesizing melanocytes, since the controls and follicles of senile white hair, which do not contain active melanocytes, show negative results. All the assays were performed on raw material, i.e., whole plucked hair follicles. This is the first time that binding sites for alpha-MSH have been demonstrated on human scalp hair follicles. In addition, their presence was found to be associated with active melanin production; their absence was demonstrated on senile white hair follicles.     

Evidence for alpha-MSH binding sites on human scalp hair follicles: preliminary results.

Alpha-MSH, considered an important pigmentation hormone, binds to melanocytes and is thought to stimulate melanogenesis through a cyclic-AMP-dependent mechanism. The binding of alpha-MSH to follicular melanocytes has been investigated in human hair of different colors, ranging from black to blond and senile white. Hairs were plucked, the follicles were cut off, and an alpha-MSH binding assay, using a radiolabeled alpha-MSH analogue, was performed on these bulbs. As controls of each assay, fragments of hairs of the same person were used. The results show a dose-response relationship and the assay seems to be specific for alpha-MSH, because other peptides such as ACTH, beta-LPH and beta-endorphins do not compete for binding sites as alpha-MSH does. These binding sites seem to be present only on melanin synthesizing melanocytes, since the controls and follicles of senile white hair, which do not contain active melanocytes, show negative results. All the assays were performed on raw material, i.e., whole plucked hair follicles. This is the first time that binding sites for alpha-MSH have been demonstrated on human scalp hair follicles. In addition, their presence was found to be associated with active melanin production; their absence was demonstrated on senile white hair follicles.     

Binding and effect of atrial natriuretic factor on cyclic GMP formation and alpha-MSH secretion of intermediate pituitary cells

The present study shows for the first time that in proopiomelanocortin cells of the rat intermediate pituitary gland ANF binds to two receptor forms, with apparent molecular weights of 150K and 70K. Scatchard plots revealed specific and high affinity non-interacting sites, with a KD value of about 3 nM and a density of 7,000 sites/cell. The presence of these binding sites was further confirmed by autoradiographic studies. Activation of these receptors led to an increase in cellular content of cGMP, with half-maximal effect being elicited with about 5 nM ANF, while cAMP formation was unaltered. Alpha-MSH secretion of intermediate pituitary cells was unaffected by ANF, whether the cells were incubated in the absence or presence of corticotropin-releasing factor or bromocryptine. These data thus indicate the presence of multiple ANF receptor sites in the intermediate pituitary which are coupled to cell production of cGMP, but independent of alpha-MSH secretion.     

Dopaminergic control of aldosterone: modulation of the response of rat adrenal zona glomerulosa cells to alpha-Msh by pretreatment with bromocriptine or metoclopramide

Bromocriptine treatment in rats (3 mg/kg per day, 7 days) significantly reduced alpha-msh and aldosterone plasma levels 2 hrs after the final treatment in animals on low, normal and high sodium diets. Alpha-MSH dose response curves for corticosterone and 18-hydroxydeoxycorticosterone (18-OH-DOC) in subsequently incubated glomerulosa cells gave stimulation at lower concentrations of alpha-MSH (10(-10) moles per litre) than in cells from untreated animals (10(-9) moles per 1). Curves for aldosterone (ald) and 18-hydroxycorticosterone (18-OH-B) were also affected in cells from animals on a low sodium diet. Fasciculata-reticularis cell responses to ACTH were unaffected. Metoclopramide (4 mg/kg per day, 7 days) elevated plasma alpha-MSH, although ald was unaffected, but inhibited the glomerulosa cell response to alpha-MSH in vitro. Acute dopaminergic responses in plasma ald may be mediated through alpha-MSH in rats, but chronically alpha-MSH may down- regulate glomerulosa cell alpha-MSH receptors. It is unlikely that alpha-MSH mediates the adrenocortical response to sodium depletion.     

Modulation of separation distress by alpha-MSH

The effects of centrally administered alpha-MSH on separation-induced distress vocalizations (DVs) and squatting were evaluated in domestic chicks for dose-response, time course, and interactions with peripheral naloxone and both peripheral and central morphine. Some of the tests were conducted in both the presence and absence of social stimuli (mirrors or a conspecific). Doses of 0.04 microgram of alpha-MSH or greater eliminated the usual suppression of DVs produced by mirrors or conspecifics. This effect lasted 10-15 minutes and was followed by inhibition of DVs, accompanied by a dose-dependent vigilant squatting posture, that lasted about one hour. These effects showed no development of tolerance to repeated alpha-MSH injections over a six-day period, and no apparent interaction with the effects of peripherally injected naloxone or either peripherally or centrally injected morphine. It is suggested that, in keeping with its role in defensive camouflage in amphibians, alpha-MSH in chicks may activate a central state akin to fear to adaptively modulate DVs and defensive hiding.     

Antiinflammatory activity of a COOH-terminal fragment of the neuropeptide alpha-MSH

The endogenous neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH 1-13), previously found to have marked antipyretic activity, inhibits histamine-induced increases in vasopermeability. The primary antipyretic amino acid message sequence is believed to be the COOH-terminal trieptide, lysine-proline-valine. In recent preliminary research this tripeptide inhibited increases in vasopermeability, raising the possibility that this portion of the alpha-MSH molecule has general antiinflammatory activity. To test this idea, the effects of graded doses of alpha-MSH [11-13] on ear swelling induced by picryl chloride in mice were compared with the effects of saline and a large dose of corticosteroid. Alpha-MSH [11-13] inhibited swelling in a dose-related fashion. This result, together with previous findings, suggests that endogenous circulating alpha-MSH and its COOH-terminal fragments may contribute to modulation of physiological responses in host defense. If this is true, it may be possible to develop new peptide drugs or mimetics based on the tripeptide that are useful in treating inflammation.     

ACTH1-17 is a more potent agonist at the human MC1 receptor than alpha-MSH.

The melanocortin receptor MC1 is expressed on melanocytes and is an important control point for melanogenesis and other responses. Alpha-MSH, which is considered to be the major ligand at the human melanocortin (MC)1 receptor (hMC1R), is produced from proopiomelanocortin (POMC) in the pituitary and in the skin by melanocytes and keratinocytes. Other POMC peptides are also produced in the skin and their concentrations exceed those of alpha-MSH by several fold. One of the most abundant is ACTH1-17. We have shown that adrenocorticotrophic hormone (ACTH)1-17 is more potent than alpha-MSH in stimulating melanogenesis in human melanocytes and unlike alpha-MSH produces a biphasic dose response curve. In this study we have examined the ability of ACTH1-17 to function as a ligand at the hMC1R. Competitive binding assays with [125I]Nle4 DPhe7 alpha-MSH as labelled ligand were carried out in HEK 293 cells transfected with the hMC1R. ACTH1-17 showed high affinity for the hMC1R with a Ki value of 0.21 +/- 0.03 nM which was slightly higher than that of 0.13 +/- 0.005 nM for alpha-MSH. ACTH1-17 was, however, more potent than alpha-MSH in increasing cAMP and IP3 production in the transfected cells. Our results demonstrate that ACTH1-17 is a potent agonist at the hMC1R. It is therefore possible that ACTH1-17, which is found in the skin in greater concentrations than alpha-MSH, has an important role in the regulation of human melanocytes and other cell types that express the hMC1R.     

Intravenous alpha-MSH reduces fever in the squirrel monkey

alpha-MSH reduces fever in rabbits when administered IV, ICV, or by gavage; however, the applicability of this finding to higher species, specifically to primates, has not been determined. In this study, we chose the squirrel monkey as an appropriate primate model since it responds reliably to peripheral administration of bacterial endotoxins that cause fever in man. From pilot studies, doses of S. typhosa endotoxin necessary to produce maximum fever and doses of alpha-MSH which did not cause hypothermia were determined for each animal. In the main experiments endotoxin was given via an indwelling catheter in the saphenous vein, followed by alpha-MSH injections when the rectal temperature increased 0.3 degrees C. alpha-MSH (100-400 micrograms) reduced the area under the fever curve an average of 50.0%, but had no effect on afebrile temperature. Molar equivalent amounts of the antipyretic drug acetaminophen had little effect on fever. These findings support the idea, based on research on rabbits, that alpha-MSH has a role in central modulation of fever.     

The effects of structure-conformation modifications of melanotropin analogs on learning and memory: D-amino acid substituted linear and cyclic analogs

Alpha-MSH has a wide variety of putative biological activities in addition to its classical melanocyte dispersing activity. Since each of these activities appears to be mediated by a discrete receptor, this peptide is an excellent candidate for exploring conformational restrictions which determine the chemical-physical basis for hormone action on specific activities. Experiments One and Two evaluated several cyclic and linear analogs of alpha-MSH on retrieval of memory during the reactivation of memory for a passive avoidance response following hypothermia-induced amnesia. Three of the cyclic analogs appear to have enhanced the peptide's ability to serve as a reactivation agent. One of the linear Nle4,D-Phe7 analogs antagonized whereas three others enhanced reactivation. The D-Phe7 substitution in cyclic analogs did not affect reactivation. Another group of animals were trained on a step-through passive avoidance task and tested 25 days later. The cyclic analog enhanced memory whereas the D-Phe7 analog and alpha-MSH had no effect. Finally, two analogs were tested on a black-white discrimination. Although the cyclic analog had no effect on either acquisition or reversal of this learning, the Nle4,D-Phe7 analog significantly impaired reversal learning. The results from these preliminary studies suggest that structural modifications of alpha-MSH do alter its potency and pattern of actions in learning and memory situations.     

Differential feeding responses to central alpha-melanocyte stimulating hormone in genetically low and high body weight selected lines of chickens

This study was conducted to compare the effects of central alpha-MSH, a potent anorexigenic signal, in lines of chickens that have undergone long-term divergent selection for low (LWS) or high (HWS) body weight. Chicks from both lines were centrally injected with 0, 24, 120 or 600 pmol alpha-MSH and feed and water intake were concurrently measured thereafter for a total of 180 min. The LWS line responded to all doses of alpha-MSH with a similar potent decrease in feed intake at all observation times. The HWS line only responded to 600 pmol alpha-MSH with decreased feed intake. alpha-MSH did not influence water intake in either line. To determine if differential hypothalamic signaling was associated with the anorexigenic effect, c-Fos immunoreactivity was measured in appetite-related hypothalamic nuclei after 600 pmol central alpha-MSH injections. c-Fos immunoreactivity was increased in the dorsomedial hypothalamus, paraventricular nucleus (PVN) and ventromedial hypothalamus in both lines after alpha-MSH; however, the magnitude of increase was greater in LWS than in HWS chicks at the PVN (136% vs. 47% increase over controls, respectively). Based on behavior observations, the number of feeding and exploratory pecks is decreased with greater magnitude after alpha-MSH in the LWS line. Additionally, alpha-MSH was associated with increased deep rest in both lines, and may be a secondary effect to reduced ingestion. These data support that the LWS line has a lower threshold for the anorexigenic effect of central alpha-MSH while in the HWS line this threshold is higher, and that this difference may be associated with differential hypothalamic signaling. Genetic variation exists in the threshold of anorexigenic response for central alpha-MSH in LWS and HWS lines of chickens with possible implications to other species including humans.     

Anti-inflammatory activity of alpha-MSH(11-13) analogs: influences of alteration in stereochemistry.

D-Amino acid substitutions in the anti-inflammatory/antipyretic Ac-alpha-MSH(11-13)-NH2 tripeptide of Ac-alpha-MSH(1-13)-NH2 were made and the altered peptides were injected in mice treated with picryl chloride. Ear swelling, measured 3 and 6 h after application of the irritant, was reduced by IP injections of Ac-alpha-MSH(11-13)-NH2, in confirmation of previous observations. Ac-[D-Lys11]alpha-MSH(11-13)-NH2 effected similar anti-inflammatory activity but Ac-[D-Pro12]alpha-MSH(11-13)-NH2 was inactive. Ac-[D-Val13]alpha-MSH(11-13)-NH2 and Ac-[D-Lys11,D-Val13]alpha-MSH(11-13)-NH2 generally had greater anti-inflammatory activity than the parent tripeptide molecule; the dose-response relations exhibited the bell-shaped characteristics seen previously with MSH peptides. The results indicate that the L-Pro12 is essential for the anti-inflammatory activity of Ac-alpha-MSH(11-13)-NH2 whereas the L-Lys11 is not. D-Val13 substitution increased anti-inflammatory activity approximately four-fold over Ac-alpha-MSH(11-13)-NH2. These results provide new structure-activity relationships of the anti-inflammatory Ac-alpha-MSH(11-13)-NH2 molecule. The data support the developing idea that alpha-MSH and its COOH-terminal fragments modulate host responses, perhaps by antagonizing the actions of cytokines.     

Antipyretic activity of a potent alpha-MSH analog

[Nle4,D-Phe7]-alpha-MSH has exceptional potency in certain biological assays of alpha-MSH activity such as skin darkening in frogs. However, this analog was equipotent to alpha-MSH in induction of grooming in the rat and had opposite effects on the performance of a visual discrimination task. These results led to the suggestion that distinct differences may exist between the melanocyte and CNS receptors for alpha-MSH. We determined the antipyretic and hypothermic potencies of centrally and peripherally administered [Nle4,D-Phe7]-alpha-MSH, relative to those of alpha-MSH, in the rabbit. Central injections of 40 and 80 ng of [Nle4,D-Phe7]-alpha-MSH caused hypothermia in afebrile rabbits, whereas 20 and 10 ng, which had no effect on afebrile body temperature, caused greater than 40% reduction in leukocytic pyrogen-induced fever. These results indicate that this analog is approximately 10 times more potent in reducing fever than alpha-MSH, making it the most potent antipyretic substance yet described. In contrast, IV administration of 16 micrograms of the analog, an extremely large dose relative to established antipyretic doses of alpha-MSH, elicited weak, variable responses. Since this analog is said to be resistant to degradation by serum enzymes, the contrast between the effects of central and peripheral administration may reflect a limited ability of the analog to cross the blood brain barrier when given IV. Our results do not suggest any distinct differences between the melanocyte receptors for alpha-MSH and those involved with CNS control of temperature. The marked central potency of [Nle4,D-Phe7]-alpha-MSH could result from an increased duration of action and/or a greater affinity for central receptor sites relative to alpha-MSH.     

Antipyretic effect of centrally administered CRF

CRF injected into the third cerebral ventricle (0.5-2.5 micrograms) caused dose-related reductions in fever induced in rabbits by IV administration of leukocytic pyrogen. Control injections of CRF when the same animals were afebrile did not alter normal body temperature. Intravenous injections of 5 and 20 micrograms CRF, doses known to release ACTH and corticosteroids into the bloodstream in other species, did not reduce fever. CRF injected into the cerebral ventricles may be antipyretic per se, or it may reduce fever by virtue of central release of the antipyretic peptides ACTH and alpha-MSH.     

Repeated central administration of alpha-MSH does not alter the antipyretic effect of alpha-MSH in young and aged rabbits

alpha-Melanocyte-stimulating hormone is a potent antipyretic when administered centrally or peripherally; however, there are no previous data on development of tolerance to the antipyretic action of this neuropeptide. In previous research, aged rabbits were more sensitive to low doses of alpha-MSH than young rabbits. We tested the antipyretic action of alpha-MSH in young and old rabbits after twice daily injections of the peptide for 10 days. Neither aged nor younger rabbits showed altered responses to alpha-MSH. This lack of tolerance underscores the importance of alpha-MSH to physiological control and survival of the host.     

Melanocortin-5 receptor deficiency promotes defensive behavior in male mice

The endogenous melanocortin, alpha-melanocyte-stimulating hormone (alpha-MSH), is a neurohormone secreted by the neurointermediate lobe of the pituitary. Alpha-MSH promotes intermale aggression in mice by influencing pheromone secretion, but the role of specific melanocortin receptors has not been determined. We assessed mice made deficient in the gene for the melanocortin-5 receptor (MC5R) to determine its role in pheromone-regulated behavior. In heterotypic pairs assessed in the social interaction test (SIT), MC5R-deficient mice exhibited less aggressive behavior and more defensive behavior than their wild-type opponents. By contrast, when assessed in homotypic pairs and against stimulus animals in the SIT, MC5R-deficient and wild-type mice behaved similarly. Moreover, urine from MC5R deficient mice stimulated more aggression than did urine from wild-type mice. The results suggest that MC5R deficiency disinhibits an aggression-suppressing pheromonal signal.     

Role of melanocortin signaling in the regulation of the hypothalamic-pituitary-thyroid (HPT) axis

The melanocortin signaling system is orchestrated by two, independent groups of neurons in the hypothalamic arcuate nucleus with opposing functions that synthesize either alpha-melanocyte stimulating hormone (alpha-MSH) or agouti-related protein (AGRP). These neurons exert regulatory control over hypophysiotropic TRH neurons in the hypothalamic paraventricular nucleus (PVN) at least in part through direct, overlapping, monosynaptic projections to the PVN. Alpha-MSH has an activating effect on hypophysiotropic TRH neurons via the phosphorylation of CREB, and when administered exogenously, can completely reverse fasting-induced suppression of TRH mRNA in the PVN. AGRP has a potent inhibitory effect on the hypothalamic-pituitary-thyroid axis in normally fed animals, mediated through actions at melanocortin 4 receptors. Inhibition of the HPT axis by fasting may be explained by inhibition of melanocortin signaling as a result of a reduction in alpha-MSH and increase in AGRP. Neuropeptide Y may also modulate the effects of the melanocortin signaling system during fasting by potentiating the inhibitory actions of AGRP on hypophysiotropic TRH neurons to prevent the phosphorylation of CREB and through direct inhibitory effects on alpha-MSH-producing neurons in the arcuate nucleus.     

Alpha-melanocyte stimulating hormone increases plasma levels of glucagon and insulin in rabbits

Intravenous injections of 25 and 2.5 micrograms alpha-melanocyte stimulating hormone (alpha-MSH) increased plasma levels of glucagon, insulin and free fatty acids in fasted and fed rabbits. 45 micrograms beta-melanocyte stimulating hormone (beta-MSH) had similar effects, whereas 22 micrograms gamma-2-melanocyte stimulating hormone (gamma-MSH) was inactive. The alpha-MSH-induced increases in the plasma levels of glucagon, insulin and free fatty acids were not inhibited by alpha- or beta-adrenergic blocking drugs. The alpha-MSH-induced increases in the plasma levels of insulin were, however, augmented by phentolamine (an alpha-adrenergic receptor blocking drug). The plasma levels of glucose were increased by 25 micrograms alpha-MSH in fed rabbits, only, and were decreased by alpha-MSH during alpha-receptor blockade. The acute in vivo effects of alpha-MSH and beta-MSH on the plasma levels of glucagon, insulin and free fatty acids were rather similar to those previously reported for corticotropin (ACTH). It is possible that the 4-10 ACTH sequence, present in alpha-MSH, beta-MSH and ACTH, but not in gamma-MSH, is a message sequence for the observed effects. However, ORG 2766, a 4-9 ACTH analogue, was inactive. The mechanism by which alpha-MSH increased the plasma levels of glucagon and insulin in rabbits remains to be determined. It is possible, that the effects were mediated by both a central nervous action and a direct action on the endocrine pancreas.     

Neuropeptide alpha-MSH antagonizes IL-6- and TNF-induced fever.

The pro-opiomelanocortin-derived peptide melanocyte stimulating hormone (alpha-MSH) antagonizes the fever induced by several stimuli including endotoxin, endogenous pyrogen, and certain cytokines. To determine if alpha-MSH can antagonize the pyrogenic action of recombinant IL-6 and TNF directly within the central nervous system, the cytokines were injected with and without alpha-MSH (200 ng) into a lateral cerebral ventricle of rabbits and rectal temperature was monitored continuously. Central administration of both cytokines caused fever. However, when alpha-MSH was injected after cytokine administration, the fevers were markedly reduced. The results are consistent with previous observations on the antipyretic effect of alpha-MSH and they show that the peptide can act within the brain to antagonize pyrogenic actions of specific cytokines believed to be important in CNS mediation of fever.