Melanin concentrating hormone exhibits both MSH and MCH activities on individual melanophores

Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val (melanin concentrating hormone, MCH) and several fragment analogs (MCH1-14, MCH5-17, MCH5-14) were synthesized and their biological activities determined in a very sensitive fish skin bioassay. The potency ranking and minimum effective doses of the peptides were determined to be: MCH1-17 (10(-12)M) greater than less than MCH5-17 (10(-12)M) greater than MCH1-14 (10(-11)M) greater than MCH5-14 (2 X 10(-10)M). The melanosome aggregating activity of MCH could be completely reversed by a 100-fold higher concentration of pounds-MSH. MCH was self-antagonized in a dose-related manner by higher concentrations of the peptide as was the activity of the MCH1-14 fragment analog. The MCH activities of the MCH5-17 and MCH5-14 analogs were not compromised by even the highest concentrations of the peptides employed. The MSH-like activity of MCH appears to relate to the N-terminus of the peptide whereas MCH activity is more a function of the C-terminus of the hormone. Self-antagonism of MCH at high concentrations appears to relate to the N-terminal tetrapeptide, which is responsible for the intrinsic MSH-like activity of the hormone.     

Ionic requirements for melanin concentrating hormone (MCH) actions on teleost Poecilia reticulata melanophores

Melanin concentrating hormone (MCH) is a cyclic heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, synthesized in the hypothalamus and released by the neurohypophysis of teleost fish. This hormone is a potent lightening agent of fish skin. This lightening results from the stimulation of a centripetal melanosome (melanin granule) migration to a perinuclear position within integumental melanophores. MCH and related fragment analogues, MCH5-17 and MCH1-14 were used to investigate the ionic requirements for receptor activation by MCH on dermal melanophores of the fish Poecilia reticulata. In calcium-free saline, the sensitivity of the melanophores to MCH and MCH1-14 increased, whereas the sensitivity of the cells to MCH5-17 decreased. Verapamil diminished the sensitivity to MCH5-17, but did not affect melanophore responses to MCH or MCH1-14. The melanosome aggregating response to MCH was not affected in the presence of tetrodotoxin or in sodium- or potassium-free (choline-substituted) saline. These results suggest that neither TTX-sensitive sodium channels nor extracellular sodium or potassium ions play a role in MCH-induced melanosome aggregation. It is known that MCH and MCH1-14 also exhibit MSH-like melanosome dispersion within melanophores, skin darkening activity on fish melanophores whereas MCH5-17 lacks this characteristic. Since the darkening activity of MCH and MCH1-14 requires calcium, these analogues exhibited a diminished lightening (MCH-like) activity in the presence of the divalent cation. In the absence of the N-terminal tetrapeptide sequence (necessary for the expression of MSH-like activity), a role for calcium on melanosome aggregation became evident. These results demonstrate a bifunctional role of calcium on melanosome movements.     

Melanin concentrating hormone. III. Melanin concentrating hormone (MCH): the message sequence

Melanin concentrating hormone (MCH) is a heptadecapeptide synthesized by the hypothalamus and secreted by the neurohypophysis of the teleost pituitary gland. MCH stimulates melanosome aggregation within teleost melanocytes but also exhibits MSH-like (melanosome dispersing) activity on tetrapod (frog and lizard) melanocytes. We have synthesized a number of MCH analogues to determine the essential features of the primary structure necessary to stimulate either melanosome aggregation or dispersion in fish or tetrapod melanocytes, respectively. An analysis of the potencies and actions of these analogues on vertebrate melanocytes is provided and demonstrates that the two activities have different structural requirements.     

Melanin concentrating hormone (MCH): synthesis and bioactivity studies of MCH fragment analogues

Nineteen analogues of melanin concentrating hormone (MCH) were synthesized and tested for their skin-lightening activities in the in vitro eel skin (Synbranchus marmoratus) bioassay. All the analogues synthesized were fragments of the native sequence: Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val with sequential elimination of substituents from both the carboxy- and amino-termini. All the analogues that contained tryptophan in position 15 were found to be full agonists and equipotent to MCH. In the absence of Trp15, full agonist activity was maintained but potency was reduced ten-fold or more. The minimal fragment analogue possessing equipotency to the parent peptide, MCH, was the MCH(5-15) sequence. These observations coupled with results from work reported previously by our laboratories suggest the importance of the Trp15 residue for interaction with the MCH receptor in this assay system.     

alpha-MSH (melanocyte stimulating hormone) and MCH (melanin concentrating hormone) actions in Bufo ictericus ictericus melanophores

1. The darkening actions of MCH (melanin concentrating hormone), alpha-MSH and the synthetic analog [Nle4, D-Phe7]-alpha-MSH on the toad, Bufo ictericus ictericus, melanophores were studied regarding the role of calcium in the hormone receptor coupling, signal transduction and intracellular pigment translocation. 2. In the absence of external calcium, MCH and both melanotropins still elicit maximal skin darkening. 3. Verapamil, a calcium-channel blocker, completely abolishes the alpha-MSH-induced response and partially inhibits MCH-induced darkening, although the calcium carrier, ionophore A23187, was unable to promote any pigment translocation. 4. Since darkening responses promoted by cyclic nucleotides proceeded normally in the presence of verapamil and extracellular calcium was not necessary for melanotropin dispersing action, it is suggested that the blocking activity obtained with verapamil is probably due to an impairment of the Ca2+-dependent adenylate cyclase activity. 5. Reversal of melanotropin-induced darkening could be obtained with melatonin, in both normal and Ca2+-free Ringer, whereas MCH darkening is reversed by melatonin only in the absence of calcium. 6. The results seem to indicate that calcium is not required for hormone receptor binding and pigment migration, whereas it is specifically needed for signal transduction.     

Melanin concentrating hormone (MCH): structure-function aspects of its melanocyte stimulating hormone-like (MSH-like) activity

Melanin concentrating hormone (MCH) is a heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, synthesized in the brain and secreted from the pars nervosa of teleost fish. This hormone stimulates melanosome (melanin granule) aggregation within integumental melanocytes of fishes but, in contrast, stimulates melanosome dispersion within tetrapod (frog and lizard) melanocytes. We determined the message sequence of the primary structure of MCH which is responsible for its MSH-like component of activity. Removal of the N-terminal amino acid results in an almost total loss of MSH-like activity. The C-terminal amino acid is also essential for full MSH-like activity since the analogue, MCH(1-16), is about 100 times less active than MCH. Therefore, the entire heptadecapeptide sequence of MCH appears to contribute to the MSH-like activity of MCH. Ring-contracted analogues (e.g., [Ala5, Cys10]MCH) of MCH are almost devoid of any melanosome aggregating (MCH-like) activity but generally possess considerable or as great an MSH-like activity as MCH. Racemization of MCH by heat-alkali treatment drastically reduces the MCH-like activity of MCH, but does not enhance the MSH-like activity of the hormone.     

Melanin concentrating hormone. I. Influence of nerves and hormones on the control of trout melanophores

When melanophores on trout scales are cultured in vitro they show a transitory melanin concentration, which can be prevented by addition to the medium of the alpha adrenergic blocker, phentolamine. This indicates the release of endogenous nor-epinephrine from local nerve terminals. This initial phase of melanin aggregation is followed by redispersion and then by a second, more gradual melanin concentration over several days, which is not antagonized by phentolamine. A final melanophore index of between 2-2.5 is attained which may be the resting state of trout melanophores. Using short-term cultured melanophores which have passed the phase of endogenous nor-epinephrine release, it is shown that exogenous nor-epinephrine will interact synergistically with the melanin-concentrating hormone to achieve full melanin concentration. Evidence is discussed for believing that in the trout, such synergy is necessary to achieve maximum pallor in vivo.     

Melanin concentrating hormone (MCH) in the cerebrospinal fluid of ewes during spontaneous oestrous cycles and ram effect induced follicular phases

The melanin-concentrating hormone (MCH) is a neuropeptide synthesized by neurons of the lateral hypothalamus and incerto-hypothalamic area that project throughout the central nervous system. The aims of the present report were: (1) to determine if MCH levels in cerebrospinal fluid (CSF) of ewes vary between the mid-luteal and the oestrous phase of spontaneous oestrous cycles; and (2) to study if MCH levels in CSF of ewes vary acutely during the follicular phase induced with the ram effect in anoestrous ewes. In the first experiment, CSF was collected from 8 adult ewes during spontaneous oestrous and during the mid-luteal phase (8-10 days after natural oestrus). In the second experiment, performed during the mid non-breeding season, a follicular phase was induced with the ram effect. After isolating a group of 16 ewes from rams, CSF was obtained from 5 of such ewes (control group). Three rams were joined with the ewes, and samples were obtained 12h (n=6) and 24h (n=5) later. In Experiment 1, there were no differences in MCH concentrations in CSF measured during the mid-luteal phase and spontaneous oestrus (0.14 ± 0.04 vs. 0.16 ± 0.05 ng/mL respectively). In Experiment 2, MCH concentrations tended to increase 12h after rams introduction (0.15 ± 0.08 vs. 0.35 ± 0.21 ng/mL, P=0.08), and increased significantly 24h after rams introduction (0.37 ± 0.15 ng/mL, P=0.02). We concluded that MCH concentration measured in the CSF from ewes increased markedly during the response to the ram effect but not during the natural oestrous cycle of ewes.     

Reciprocal changes in sensitivity to MCH and noradrenaline after denervation of teleost melanophores.

Melanophores of isolated fish scales survive for weeks in a culture medium. During this isolation period a progressive increase in sensitivity to noradrenaline (NA) takes place. In the present study, a 100-fold increase in sensitivity to NA was found after 9 days. However, at the same time, a 12-fold decrease in sensitivity to MCH was detected.     

Melanin concentrating hormone. II. Structure and biosynthesis of melanin-concentrating hormone

Melanin-concentrating hormone is a neuropeptide produced in teleost hypothalami and transferred to the neurohypophysis. Salmon MCH was a novel cyclic heptadecapeptide capable of inducing melanin aggregation of integumentary melanophores at picoto nano-molar concentrations in all teleosts tested. The MCH gene is intronless and the exon encodes a 132 amino acid precursor protein, in which the heptadecapeptide of MCH locates at the C-terminal end. Immunohistochemical surveys with anti-salmon MCH antiserum strongly suggest that an MCH-like peptide is present in the hypothalami of higher vertebrates. Biological effects of salmon MCH on other vertebrates are found to be versatile.     

Melanin concentrating hormone increase hippocampal synaptic transmission in the rat

A retrograde facilitation has been demonstrated in the one trial step-down inhibitory avoidance of melanin-concentrating hormone (MCH), when it was infused into rat hippocampal formation. Considering the high density of specific binding sites for the MCH peptide on the hippocampus and the participation of this structure on learning and memory processes we have studied the effects of MCH on the hippocampal synaptic transmission. For this purpose, slices of rat hippocampus were perfused with different concentration of MCH. The main result of the present study was a long-lasting potentiation on the hippocampal evoked response on dentate gyrus induced by MCH (4-11 microM) at 30, 60 and 120 min with a maximum effect at 120 min. Previous perfusion of DL - 2- amino - 5 phosphonovaleric acid (APV, 20 microM) was unable to impair the increased hippocampal evoked response induced by MCH 4 microM. On the other hand, the channel blocker Dizocilpine (MK-801, 10 microM) completely impaired the increased hippocampal synaptic plasticity induced by MCH perfusion. We postulate the increased hippocampal synaptic efficacy induced by MCH as one of the mechanisms underlying the retrograde facilitation on the inhibitory avoidance paradigm, observed after MCH hippocampal microinjection. We cannot rule out other MCH neurochemical mechanism and other areas of the brain involved in the MCH effects.     

Melanin concentrating hormone. IV. Development of a sensitive solid-phase radioimmunoassay for melanin-concentrating hormone

A two-step solid-phase radioimmunoassay for melanin-concentrating hormone (MCH) was developed for direct determination of the hormone in plasma samples. To this end, synthetic MCH was coupled to bovine thyreoglobulin and the complex was injected into rabbits. Specific antisera of high titer were obtained which did not crossreact with other hormones. The IgGs were chemically linked to immunobeads, an acrylamide/acrylic acid polymer matrix. In the first step, plasma MCH was immunoextracted by incubation of diluted plasma samples with anti-MCH immunobeads. In the second step, the washed polymer was incubated with radioiodinated MCH tracer for titration of non-occupied sites. This procedure made it possible to determine as little as 4 pg MCH per ml of plasma. Application of the radioimmunoassay to plasma levels of black or white background-adapted trout showed a marked difference in circulating MCH: while trout on a black background contained a mean value of 29 +/- 5.6 pg/ml, animals on a white background had 106 +/- 19 pg/ml. These findings strengthen the hypothesis that MCH is directly involved in the control of color change of teleost fishes. By contrast, there was no detectable salmonid MCH immunoreactivity in rat or human plasma.     

Differential structural requirements for the MSH and MCH activities of melanin concentrating hormone

H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH , melanin concentrating hormone (MCH), exhibits both melanin granule concentrating and dispersing (MSH-like) activities. Fragment analogues of MCH were synthesized as described herein and the melanotropic activities of the peptides were determined. In the frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays, the 5-17 and 5-14 fragments of MCH were inactive (at concentrations of 10(-5)M or less), whereas the 1-14 sequence exhibited minimal (about 10%) MSH-like activity compared to MCH, which, as reported previously, was about 600 times less active than alpha-MSH. In the teleost (fish) skin bioassay, the MCH5-17 analogue was equipotent to MCH, whereas the 1-14 analogue was 10-30 times and the cyclic N- and C- terminal truncated analogue, MCH5-14, was about 300 times less active than MCH. These results suggest that the N-terminal sequence is particularly critical to MSH-like activity in the tetrapod species studied, whereas other structural regions of MCH, particularly in the C-terminal, are more related to MCH activity in teleosts.     

Melanin-concentrating hormone (MCH) reverts the behavioral effects induced by inescapable stress

The aim of this work was to investigate if MCH modifies the feeding and freezing responses in rats exposed to stressful stimuli. We used a basic version of contextual fear, where one group of rats were placed in a novel environment and two different groups were exposed to footshock paradigms, one of them escapable and the other one inescapable. At the end of each treatment, freezing and feeding were measured. Only the animals exposed to inescapable footshock paradigm showed significant increase in the food intake and freezing behavior in comparison to the control animals. The MCH administration (intra-hippocampal or intra-amygdaline) reverted these effects elicited by inescapable footshock. Results presented in this paper lead us to the assumption that the anxiolytic effect of the peptide is responsible for the reversion of the IS effects.     

Melanin concentrating hormone induces hippocampal acetylcholine release via the medial septum in rats

Among various actions of melanin concentrating hormone (MCH), its memory function has been focused in animal studies. Although MCH neurons project to various areas in the brain, one main target site of MCH is hippocampal formation for memory consolidation. Recent immunohistochemical study shows that MCH neurons directly project to the hippocampal formation and may indirectly affect the hippocampus through the medial septum nucleus (MS). It has been reported that sleep is necessary for memory and that hippocampal acetylcholine (ACh) release is indispensable for memory consolidation. However, there is no report how MCH actually influences the hippocampal ACh effluxes in accordance with the sleep–wake cycle changes. Thus, we investigated the modulatory function of intracerebroventricular (icv) injection of MCH on the sleep–wake cycle and ACh release using microdialysis techniques. Icv injection of MCH significantly increased the rapid eye movement (REM) and non-REM episode time and the hippocampal, not cortical, ACh effluxes. There was a significant correlation between REM episode time and hippocampal ACh effluxes, but not between REM episode time and cortical ACh effluxes. Microinjection of MCH into the MS increased the hippocampal ACh effluxes with no influence on the REM episode time. It appears that the effect sites of icv MCH for prolongation of REM episode time may be other neuronal areas than the cholinergic neurons in the MS. We conclude that MCH actually increases the hippocampal ACh release at least in part through the MS in rats.     

Melanin concentrating hormone. V. Isolation and characterization of alpha-melanocyte-stimulating hormone from frog pituitary glands

The structure of alpha-melanocyte-stimulating hormone (alpha-MSH) has been determined in the pars intermedia of the frog Rana ridibunda. Pulse-chase labeling of frog neurointermediate lobes with selective amino acids revealed that the composition of frog alpha-MSH is similar to that of alpha-MSH from all mammalian species yet studied. Tryptic mapping of nexly synthetized alpha-MSH generated two fragments with the following amino acid composition: (T1) Trp, Pro, Lys, Gly, Val and (T2) Tyr, Arg, Phe, His, Ser, Glu. Concurrently, alpha-MSH was purified from 100 neurointermediate lobes to apparent homogeneity by reverse-phase HPLC. The sequence of the peptide determined by automated Edman degradation was Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val. The structure of frog alpha-MSH is thus identical to mammalian des-N alpha-acetyl alpha-MSH and differs from the sequence of toad (Xenopus laevis) alpha-MSH only by the first residue (Ser instead of Ala). These results confirm that the sequence of alpha-MSH has been highly preserved during evolution.     

Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1).

To identify possible ligands of the orphan somatostatin-like receptor 1 (SLC-1), rat brain extracts were analyzed by using the functional expression system of Xenopus oocytes injected with cRNAs encoding SLC-1 and G protein-gated inwardly rectifying potassium channels (GIRK). A strong inward current was observed with crude rat brain extracts which upon further purification by cation exchange chromatography and high performance liquid chromatography (HPLC) yielded two peptides with a high agonist activity. Mass spectrometry and partial peptide sequencing revealed that one peptide is identical with the neuropeptide melanin concentrating hormone (MCH), the other represents a truncated version of MCH lacking the three N-terminal amino acid residues. Xenopus oocytes expressing the MCH receptor responded to nM concentrations of synthetic MCH not only by the activation of GIRK-mediated currents but also by the induction of Ca(2+) dependent chloride currents mediated by phospholipase C. This indicates that the MCH receptor can couple either to the G(i)- or G(q)-mediated signal transduction pathway, suggesting that MCH may serve for a number of distinct brain functions including food uptake behavior.