MCH-induced pigment aggregation in teleost melanophores is associated with a cAMP reduction.

It has previously been shown that alpha 2-adrenoceptors are involved in noradrenaline-induced pigment aggregation within fish melanophores. In the present investigation, melanin concentrating hormone (MCH) elicited pigment aggregation (EC50 approximately 1 x 10(-7) M) that was associated with a significant reduction in the cAMP content; 1 x 10(-7) M MCH reduced the cAMP content from a basal level of 50.4 +/- 2.8 pmol/mg protein to 36.9 +/- 3.8 pmol/mg protein. Like the alpha 2-adrenoceptor-induced pigment aggregation, the MCH response was effectively blocked by the adenylate cyclase stimulator forskolin. These findings suggest that attenuation of cAMP may serve as an intracellular signal transduction mechanism for both MCH and noradrenaline.     

Melanin concentrating hormone analogues: contraction of the cyclic structure. II. Antagonist activity

Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, melanin concentrating hormone (MCH), is a cyclic hormone possessing both MCH-like (melanin granule aggregating effect) and melanocyte stimulating hormone (MSH)-like (melanin granule dispersing effect) activities. Nine ring-contracted analogues were synthesized and characterized for their melanotropic activity on the fish (Synbranchus marmoratus) and frog (Rana pipiens) bioassays. In most cases, these analogues were totally devoid of MCH-like agonist activity, demonstrating the essential role of the disulfide bridge between residues 5 and 14 of the hormone. [Ala5, Cys10]MCH, for example, was totally devoid of MCH-like activity. This analogue, like alpha-MSH, however, antagonized the melanosome aggregating actions of MCH on fish melanocytes. The antagonistic activity of the analogue, like that of alpha-MSH, was Ca2+-dependent. Evidence suggested that this antagonism of MCH activity was related to the intrinsic MSH-like activity of the analogue. These results suggest that MCH and alpha-MSH may be structurally and, therefore, evolutionarily related.     

Effect of salmon melanin-concentrating hormone and mammalian gonadotrophin-releasing hormone on somatolactin release in pituitary culture of Cichlasoma dimerus

We detected a close morphological association between melanin-concentrating hormone (MCH)-immunoreactive (ir) fibers and somatolactin (SL)-ir cells in the pars intermedia of the cichlid fish Cichlasoma dimerus by double-label immunofluorescence. Male pituitaries obtained from adult C. dimerus were incubated with 0.1-10 muM salmon MCH, and the amount of SL released into the culture medium was semi-quantified by Western blot. This assay showed an increase of SL release in a dose-dependent manner (linear regression: P < 0.05). A close association of GnRH-ir fibers with SL-ir cells was also detected at the pars intermedia level. Male pituitaries were also incubated with 0.1-10 muM of mammalian GnRH, and SL release was semi-quantified by Western blot, showing an increase of released SL levels in a dose-dependent manner (linear regression: P < 0.05). In contrast, SL release was unaffected from female pituitaries incubated with salmon MCH; however, an increasing tendency was observed when mammalian GnRH was used. Hypothalamic close association of MCH-ir perikarya and GnRH-ir fibers was found by double-label immunofluorescence indicating a possible relationship between them. These results suggest that SL, like other pituitary hormones, is under hypothalamic control and is involved in diverse physiological processes including background adaptation and reproduction. This study has also shown that the in vitro culture of a single C. dimerus pituitary is a feasible method for studying the control of SL release and other pituitary hormones.     

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 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 (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.     

Protein-kinase C mediates MCH signal transduction in teleost, synbranchus marmoratus, melanocytes

MCH (melanin concentrating hormone) is a heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, which stimulates melanosome (melanin granule) aggregation to a perinuclear position within teleost fish integumental melanocytes, resulting in lightening of the skin. The mechanisms of action of MCH are unknown. Drugs that affect the diacylglycerol/inositol triphosphate pathway were used to investigate the possible roles of this pathway in the mechanisms of action of MCH on Synbranchus marmoratus (teleost) melanocytes. The shift of the dose-response curve to MCH in the presence of various concentrations of 4-bromophenacyl bromide and neomycin sulphate, phospholipase C inhibitors, suggests that phospholipase C is stimulated after MCH receptor activation. Low concentrations (10(-9) to 10(-8) M) of the phorbol ester TPA exhibited MCH-like activity, eliciting a dose-dependent melanosome aggregation. Higher doses, however, displaced to the right the dose-response curve to MCH, as did the protein kinase C inhibitors, dibucaine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). These results support the assumption that protein kinase C mediates the pigment aggregating activity of MCH. Both MCH and norepinephrine lightening actions were abolished by beta-glycerophosphate, a phosphatase inhibitor, suggesting that a protein dephosphorylation occurs during melanosome aggregation, and is, therefore, a common event triggered by MCH and norepinephrine, although both agonists act through separate receptors and exhibit different transduction mechanisms.     

Synthesis and bioactivity studies of two isosteric acyclic analogues of melanin concentrating hormone.

Salmon melanin concentrating hormone (MCH) is a cyclic heptadecapeptide. MCH stimulates perinuclear aggregation of melanosomes within integumental melanocytes of teleost fishes resulting in skin blanching. MCH contains a disulfide bridge forming a 10-residue ring [sequence: see text]. It has been proposed that the ring is necessary for maintenance of potency. In order to test this proposal, we have synthesized two pseudo-isosteric analogues of MCH that cannot cyclize. They differed only in the polarity of the side chain group of positions 5 and 14. Serine was substituted for Cys5 and Cys14 in one peptide and L alpha-aminobutyrate (Abu) was the substitution at the two positions in the other peptide. Using a fish skin bioassay we determined that these analogues exhibit less than 1/10,000th the potency of the native hormone. These results suggest that the disulfide bridge is necessary to maintain the correct conformational and topographical features of the hormone for receptor binding and transmembrane signal transduction.     

Melanin concentrating hormone (MCH) effects on teleost (Chrysiptera cyanea) melanophores

The in vitro biological actions of synthetic chum salmon melanin concentrating hormone (MCH) on melanophores of the blue damselfish (a teleost), Chrysiptera cyanea, were studied. This cyclic heptadecapeptide stimulated melanosome (melanin granule) aggregation (centripetal migration) within melanophores at a threshold concentration of about 10(-10) M. The action of this putative hormone was not blocked by alpha- or beta-adrenoceptor antagonists. It was concluded that the effects of MCH were direct and were not mediated indirectly through the actions of adrenergic neurotransmitters released from nerve terminals. Further evidence for this view comes from the observation that, unlike the case of neurotransmitter release, melanosome aggregation in response to MCH proceeded in the absence of calcium. The possible role of MCH in the control of color change of teleost fishes is discussed.     

Synthesis and biological actions of melanin concentrating hormone

A melanin (melanosome) concentrating hormone, MCH, was synthesized and the methodology for its synthesis is detailed. This heptadecapeptide, H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH , stimulated melanosome concentration (centripetal aggregation) within melanophores of all species of teleost fishes studied. Melanosome aggregation in response to MCH was not blocked by Dibenamine as was the response to norepinephrine (NE), demonstrating that melanosome aggregating responses to MCH and NE are mediated through separate receptors. Melanosome aggregation in response to MCH was reversed by an equimolar concentration of alpha-melanocyte stimulating hormone (alpha-MSH). In contrast, MCH stimulated melanosome dispersion (centrifugal movement) within melanophores of a frog (Rana pipiens) and a lizard (Anolis carolinensis). Therefore, MCH exhibits both melanosome concentrating and dispersing actions depending upon the species studied.     

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.     

Design and synthesis of substituted quinolines as novel and selective melanin concentrating hormone antagonists as anti-obesity agents

A novel series of substituted quinoline analogs were designed and synthesized as potent and selective melanin concentrating hormone (MCH) antagonists. These analogs show potent (nM) activity (12a-k) with a moderate selectivity. Conversely, the conformationally constrained thienopyrimidinone analogs (18a-g) showed improved activity in MCH-1R and selectivity over 5HT2C.     

Specific hybridization of a synthetic oligonucleotide in rat hypothalamic neurons immunoreactive to immune serum against salmon melanin-concentrating hormone

An oligonucleotide probe corresponding to the 9 C-terminal residues encoded by a complementary DNA of a rat peptide related to salmon melanin concentrating hormone (MCH) was synthetized. It specifically hybridized to the neurons stained by antisera to MCH in the rat posterior hypothalamus, as seen by coupling in situ hybridization and immunocytochemical methods. This result validates our sequence determination. This oligonucleotide will be useful to establish the complete sequence of the rat MCH precursor molecule. It will also constitute a valuable tool to study physiological or experimentally-induced changes in the expression of the rat MCH gene.     

Ontogenetic data on the peptidergic interneuronal population in immunoreactivity of the GRF 37 type serum of the human posterolateral hypothalamus. Immunocytochemical studies using anti-GRF 37 and anti-MCH (melanin-concentrating hormone) immune sera

Human posterolateral hypothalamic neurons are revealed with an anti GRF 37 serum as soon as the 7th week of fetal life. The same neuronal population can be observed in the adult brain even in hypothalami from old subjects, with the same distribution, and similar immunoreactivity than in fetal stages. These neurons are revealed using a melanin concentrating hormone (MCH) antiserum; the MCH immunoreactivity appears at the same stage of fetal development than GRF 37 immunoreactivity. The two antisera recognize two epitopes on one or two molecules. Those new facts agree with an hypothesis about the very important and permanent functional role of that new human hypothalamic interneuronal system.     

Effect of melanin concentrating hormone on pigment and adrenal cells in vitro

Highly purified synthetic salmonid melanin concentrating hormone (MCH) and some analogs were investigated for their ability to concentrate the pigment in scale melanophores of the Chinese grass carp, Ctenopharyngodon idellus, to produce melanin dispersion in frog or lizard melanophores and to inhibit alpha-MSH in its action on mouse melanoma and rat adrenal glomerulosa cells in vitro. In the grass carp, MCH produced half-maximal pigment aggregation at 6 X 10(-11) M and its oxidized form at 7 X 10(-11) M. Replacement of the two methionines at position 3 and 6 with norvaline lowered the potency by a factor of 2.7 and with propargylglycine by a factor of about 7. Linear, Cys5,14-Acm-protected MCH was a full agonist of MCH but with a 345-fold lower potency. Iodinated MCH showed similar, low activity. In tetrapods, salmonid MCH and its analogs displayed only marginal pigment dispersion at concentrations greater than 10(-5) M. Alkali-treatment of MCH increased the pigment-dispersing potency by a factor of about 30 whereas the activity for pigment aggregation in the grass carp was destroyed. At high concentrations (10(-6), 10(-5) M) MCH also stimulated tyrosinase activity in B-16 mouse melanoma cells but did not modify the effects of alpha-MSH in this system. By contrast, when tested on rat adrenal glomerulosa cells, salmonid MCH had no effect alone but at a concentration of greater than 10(-10) M it slightly reduced corticosterone production by an alpha-MSH concentration of 10(-7) M. Aldosterone production was not affected and MCH did not influence the response to ACTH.     

Functions of melanin-concentrating hormone in fish

Melanin-concentrating hormone (MCH) was originally discovered in fish, in which it causes aggregation or concentration of melanin granules in melanophores, thus regulating body color. MCH is a cyclic neuropeptide synthesized as a preprohormone in the hypothalamus of all vertebrates. Mammalian MCH plays an important role as a neurotransmitter or neuromodulator in regulating food intake and energy homeostasis. MCH signaling system may involve in regulating food intake also in fish. This neuropeptide binds to G-protein-coupled seven transmembrane receptor[s] to mediate its functions. This article reviews MCH and MCH receptor signaling systems in body color change and food intake in fish.     

Intracerebroventricular injection of neuropeptide EI increases serum LH in male and female rats

Melanin concentrating hormone (MCH) precursor-derived neuropeptide EI (NEI) has not yet been extensively studied. The aim of this study was to determine the effect of neuropeptide EI on serum levels of LH in normal male rats and chronically ovariectomized (CHR-OVX) female rats treated with estrogen benzoate (EB) and with a low dose of progesterone. The peptide, administered intracerebroventricularly in male and chronically ovariectomized female rats, increased LH serum levels compared to the controls injected with artificial cerebrospinal fluid. It is important to note that there is some relation between neuropeptide EI-melanin concentrating hormone and alpha-melanocyte stimulating hormone (alpha-MSH) indicating that all three peptides are associated in a complex inter-relationship. Therefore, the question that arises is if neuropeptide EI could also be related with the receptors for melanin concentrating hormone or alpha-melanocyte stimulating hormone.     

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.     

Tris buffer effects on melanophore aggregating responses

The adverse effects of tris (hydroxymethyl)-aminomethane (Tris) are for the first time reported on melanophore responses to agonists and potassium chloride. Melanophore responses in Tris- or bicarbonate-buffered solutions were compared. In the presence of Tris, the cumulative dose-response curve to norepinephrine was significantly shifted to the left, whereas methoxamine dose-response curves were similar in both buffers. The percentage aggregation in response to synthetic MCH (melanin concentrating hormone) was not affected by Tris in the bathing medium. The cumulative dose-response curve to potassium chloride was leftward shifted (one log case) in Tris-buffered solution. These results suggest that in fish melanophore preparations Tris might exert its actions on the presynaptic membrane and/or on the synaptic cleft enzyme COMT, drawing on a greater availability of neurotransmitters at the melanophore membrane receptors.