Melanin-concentrating Hormone Peptide Research Articles

17β-Estradiol regulation of melanin-concentrating hormone and neuropeptide-E-I contents in cynomolgus monkeys: a preliminary study

Melanin-concentrating hormone (MCH) and neuropeptide-E-I (NEI) regulate several behaviors and neuroendocrine functions in rats. Possible influence of these peptides on sexual behavior and reproduction in mammals other than rodents prompted us to investigate: 1) The sites of synthesis of MCH and NEI in the brain of a non-human primate ( M. fascicularis); 2) The effect of 17β-estradiol (E 2) benzoate (E 2B) on pro-MCH-derived peptide concentrations in the hypothalamus of the ovariectomized (OVX) cynomolgus monkeys ( M. fascicularis). Expression of MCH mRNA and peptides was examined by Northern blotting, RT-PCR and RP-HPLC/RIA. Our results demonstrate that the MCH gene is predominantly expressed in hypothalamus of macaque. E 2B exposure of OVX monkeys provoked parallel phasic variations in the MCH-immunoreactivity (IR) and NEI-IR. NEI-IR and to a lesser extent MCH-IR, showed a transient increase (associated with the estradiol peak) at 30 h with a final rise of both MCH-IR and NEI-IR observed at the time (72 h post E 2B) of the luteinizing hormone (LH) surge. RP-HPLC analysis of peptide extracts revealed the presence, in addition to mature MCH and NEI, of different MCH-IR and NEI-IR forms in the hypothalami of control and E 2B-treated monkeys. Taken together, our results indicated that hypothalamic MCH and NEI contents are regulated after E 2B treatment and they suggest the possible involvement of these peptides in the regulation of the pre-ovulatory midcycle LH surge in primates.

Melanin-Concentrating Hormone Binding Sites in Human SVK14 Keratinocytes

Melanin concentrating hormone (MCH) is a cyclic peptide which regulates a broad array of functions in the mammalian brain and it may act as a paracrine factor in peripheral organs. In these studies a radiolabeled MCH derivative, the [125I]-[Phe13, Tyr19]-MCH, was synthesized and used as a tracer to perform binding experiments. A number of human or rodent cell lines displayed specific binding with [125I]-[Phe13, Tyr19]-MCH, the highest binding capacity being observed with human SVK14 keratinocytes. Saturation binding analysis with SVK14 cells indicated about 10,000 MCH binding sites per cell and a Kd of 0.7 nM for [125I]-[Phe13, Tyr19]-MCH. Surprisingly, the iodinated [Phe13, Tyr19]-MCH displayed about 10-fold higher affinity (Ki ∼ 3.0 nM) for the putative MCH receptor than the noniodinated form (Ki ∼ 25-30 nM). Competition binding analyses comparing various MCH-related peptides revealed a similar low binding potency for all these peptides (Ki ∼ 65-160 nM). Strikingly, rat ANP and rat/human CNP but not rat BNP displaced [125I]-[Phe13, Tyr15]-MCH with Ki ∼ 210-365 nM and may be due to topological similarities instead of partial sequence identities between MCH and some of the natriuretic peptides. However, other peptides such as CRF, αMSH, Arg-vasopressin, and MGOP-peptide I did not compete with the radioligand. Finally, the molecular mass of the MCH binding sites on SVK14 cells was estimated to be 47 kDa by crosslinking and SDS-PAGE experiments. Taken together, our data revealed the widespread expression of MCH binding sites on mammalian cells, particularly on skin carcinoma cells. However, the low affinity of these sites for the native MCH and MCH-related peptides as well as competitivity with ANP and CNP indicates that further biochemical and functional characterizations are needed to validate them as genuine physiological MCH receptors.

Developmental and stage-dependent expression of melanin-concentrating hormone in mammalian germ cells.

Melanin-concentrating hormone (MCH) is a cyclic peptide predominantly expressed in the hypothalamus of mammals. This peptide modulates the stress response and regulates many goal-oriented behaviors in the rat brain. MCH mRNA and peptides generated from the precursor, namely MCH and neuropeptide (N) glutamic acid (E) isoleucine (I) amide (NEI), were also found in rodent peripheral tissues including those in adult testis. In the present study, we first examined the cellular distribution and content of MCH gene products and peptide in the testes of adult rats. Using reverse-transcription polymerase chain reaction with MCH gene primers and in situ hybridization with specific 33P-labeled oligoprobes, we characterized the MCH RNA species. Pro-MCH products were revealed through use of immunoperoxidase detection and an RIA with specific MCH and NEI antisera. Both MCH gene transcripts and MCH peptide were found within germ cells at the periphery of some of the seminiferous tubules of adult rats. We further investigated temporal expression of MCH in 7-microns sections of testes from adult rats and mice. MCH was predominantly found in nuclei of spermatogonia at stages II-IV of spermatogenesis and in nuclei of leptotene and zygotene spermatocytes (at stages IX- XIII). MCH was markedly absent in preleptotene spermatocytes at the stage VII-VIII, and MCH immunoreactivity was no longer detectable as spermatocytes underwent the pachytene step. MCH immunoreactivity was also found in some peritubular cells, mainly at stages II-IV. Finally, we studied MCH expression during puberty in the rat, in sterile mutant mice, and in one adult man. We found predominant staining with MCH antiserum over nuclei of immature germ cells as early as 10 days postpartum in the rat and further confirmed the absence of staining except for that in spermatogonia and early spermatocytes during rat postnatal development. MCH distribution was found to be similar in normal and sterile mutant mice, suggesting that MCH expression is not dependent upon the early steps of spermiogenesis. MCH immunoreactivity was found to be confined to the nuclei of spermatogonia and early spermatocytes in the adult man. Our results indicate that MCH is present predominantly within nuclei of spermatogonia and primary spermatocytes in three mammalian species and that its expression is under strong stage-specific and developmental regulation. This peptide may play a role during stem cell renewal and/or differentiation of early spermatocytes.

Synthesis and iodination of human (phenylalanine 13, tyrosine 19) melanin-concentrating hormone for radioreceptor assay.

An analogue of human melanin-concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr13 and Val19 of the natural peptide were replaced by phenylalanyl and tyrosyl residues: [Phe13, Tyr19]-MCH. The peptide was synthesized by the continuous-flow solid-phase methodology using Fmoc-strategy and polyhipe PA 500 and PEG-PS resins. The linear MCH peptides with either acetamidomethyl-protected or free cysteinyl residues were purified to homogeneity and cyclized by iodine oxidation, yielding the final product with the correct molecular weight of 2434.61. Radioiodination of the C-terminal tyrosine was carried out enzymatically using solid-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and by high-pressure liquid chromatography. The resulting [125I]-[Phe13, Tyr19]-MCH tracer was the first radiolabelled MCH peptide suitable for radioreceptor assay: saturation binding analysis using mouse G4F-7 melanoma cells demonstrated the presence of 1090 MCH receptors per cell. The dissociation constant (KD) was 1.18 x 10(-10) M, indicating high-affinity MCH receptors on these cells. MCH receptors were also found in other cell lines such as mouse B16-F1 and G4F and human RE melanoma cells as well as in PC12 and COS-7 cells. Competition binding analyses with a number of other peptides such as alpha-MSH, neuropeptide Y, substance P and pituitary adenylate cyclase activating peptide, demonstrated that the binding to the MCH receptor is specific. Atrial natriuretic factor was found to be a weak competitor of MCH, indicating topological similarities between MCH and ANF when interacting with MCH receptors.

Melanin-concentration hormone updated functional considerations

The melanin-concentrating hormone (MCH) is a vertebrate neuropeptide produced in hypothalamic perikarya whose fibers project to most regions of the brain and into the spinal cord. Its role as a neurohypophyseal color-change hormone is peculiar to teleost fish, but recent studies in mammals suggests that MCH itself, and other peptides derived from the same precursor, may participate in multiple functions in the central nervous system, modulating behavior and the perception of sensory information. Recent hybridization studies in mammals have greatly increased our understanding of the response of the MCH system to environmental factors, such as osomotic challenge, lactation, stress, and changes in corticosteroid levels. Further studies in lower vertebrates are needed to highlight the physiologically important functions that have led to the structural conservation of the MCH peptide during vertebrate evolution

Cloning and sequence analysis of hypothalamus cDNA encoding tilapia melanin-concentrating hormone

Melanin-concentrating hormone (MCH) is a neuroendocrine peptide involved in the regulation of skin pigmentation in teleosts. We isolated and sequenced a 543 bp hypothalamic cDNA encoding the MCH-preprohormone of tilapia (Oreochromis mossambicus). Initially, polymerase chain reaction (PCR) experiments were performed on hypothalamic RNA with a synthetic oligonucleotide primer corresponding to a conserved region of salmon and mammalian MCH peptide and an oligo dT primer. A 0.2 kb PCR fragment was obtained and found to have low but significant nucleotide sequence similarity with the 3' ends of known MCH-mRNAs. Subsequently, the PCR fragment was used to screen λZAP cDNA libraries constructed from tilapia hypothalamic poly(A(+)) RNA. The cloned tilapia MCH preprohormone cDNA encodes a 133-amino acid protein of which 17 amino acids belong to the signal peptide. The MCH peptide sequence is located at the carboxy terminus of the preprohormone structure and is preceded by a pair of arginine residues which can serve as a proteolytic cleavage site. 23 to 25 amino acids further upstream in the prohormone structure three consecutive basic residues are present. Cleavage at this site would yield a 22-amino acid MCH gene-related peptide (Mgrp), which is much larger than (12- to 13-amino acid) salmon and mammalian Mgrp. A comparative structural analysis between tilapia preproMCH and its salmon and mammalian counterparts revealed that the MCH peptide sequence is very well conserved (100% identity with salmon and 75% identity with both rat and human MCH). In contrast, the remaining parts of the preproMCH structures have diverged considerably. Northern blot analysis revealed the presence of tilapia preproMCH mRNA in the hypothalamus and not in other brain regions nor in several peripheral tissues.

Novel Hypothalamic and Preoptic Sites of Prepro‐Melanin‐Concentrating Hormone Messenger Ribonucleic Acid and Peptide Expression in Lactating Rats

The deduced structure of the rat melanin-concentrating hormone (MCH) precursor predicted the existence of at least two peptides that may be processed from it, one similar to teleost MCH and a second novel neuropeptide, NEI. Cellular localization studies confirmed that prepro-MCH (ppMCH) mRNA and the MCH and NEI peptides are expressed predominantly in cells in the zona incerta and caudal lateral hypothalamic area with minor contingents seen in the olfactory tubercle and pons. A moderate MCH-and NEI-immunoreactive axonal projection to the median eminence and, particularly, to oxytocin-rich regions of the posterior pituitary suggested some anatomical heterogeneity of ppMCH-expressing neurons in the hypothalamus, and an involvement in neuroendocrine function. In the present study, immunohistochemical and hybridization histochemical methods were used to follow MCH gene and peptide expression as a function of reproductive status in female rats. Nursing dams sacrificed after 8 to 21 days of lactation consistently displayed ppMCH mRNA and MCH and NEI immunoreactivity in a discrete and contiguous band, encompassing the ventral aspect of the medial part of the medial preoptic nucleus, the periventricular preoptic nucleus, and the most rostral aspects of the paraventricular nucleus of the hypothalamus (PVH). Combined immunohistochemical (for oxytocin) and hybridization histochemical (for ppMCH mRNA) staining failed to reveal a significant degree of congruence in the two chemically-specified cell populations in the PVH of lactating dams. The apparent induction of ppMCH-derived peptides and mRNA in the preoptic area and PVH was not apparent in animals sacrificed 4 to 8 days after weaning, during late pregnancy, or at any point in the estrous cycle. Moreover, no frank alterations in ppMCH mRNA were evident in the principal sites at which ppMCH is expressed at constitutively high levels, i.e. in the lateral hypothalamic area and zona incerta, as a function of reproductive status. The loci and apparent state-dependency of the induction of ppMCH mRNA and peptide expression suggests a role for these gene products in the control of lactation.

The melanin-concentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterization.

In addition to a nonadecapeptide homologous to the teleost melanin-concentrating hormone (MCH), the amino acid sequence predicted from a rat prepro-MCH (ppMCH) cDNA suggested that at least one (neuropeptide EI, or NEI), and possibly a second (NGE), additional neuropeptide may be encoded by this precursor. Cross-reactivity with epitopes of NEI or NGE can account for reported localization of alpha-MSH, rat CRF, and human GRF in rat dorsolateral hypothalamic neurons. We have used antisera raised against rat MCH and NEI in immunohistochemical studies at the light and electron microscopic levels, along with hybridization histochemical localization of ppMCH mRNA, to define the organization of this system. As expected, ppMCH mRNA is prominently expressed in cells in the lateral hypothalamic area and zona incerta. The MCH and NEI peptides were extensively colocalized in neurons in both of these areas. In addition, smaller cell groups in the olfactory tubercle and pontine tegmentum were also positively hybridized for ppMCH mRNA and immunostained for MCH and NEI. Fibers stained for MCH and NEI were similarly, and very broadly, distributed throughout the central nervous system in patterns that generally conformed with known projection fields of the lateral hypothalamic area and zona incerta. A differential distribution was seen in at least one region, the interanterodorsal nucleus of the thalamus, which contained a prominent terminal field stained for MCH but not NEI. At the electron microscopic level, MCH-stained perikarya displayed a prominent staining associated with the Golgi apparatus; this was not encountered in NEI-stained cells. Both peptides were distributed similarly in terminals in the lateral hypothalamic area and median eminence, with staining associated principally with dense-cored vesicles. The results suggest that ppMCH-derived peptides may serve as neurotransmitters or modulators of prominence in a surprisingly expansive projection field of incerto-hypothalamic neurons. The terminal distributions of this system seem most compatible with functional roles in generalized arousal and sensorimotor integration, processes previously implicated as being subject to modulation by the lateral hypothalamic area.

Identification of a Single Melanin‐Concentrating Hormone Messenger Ribonucleic Acid in Coho Salmon: Structural Relatedness with 7SL Ribonucleic Acid

Abstract Melanin-concentrating hormone (MCH) is a cyclic neuropeptide possessing antagonistic function to alpha-melanocyte-stimulating hormone and corticotropin-releasing factor in the control of melanosome dispersion within melanophores and adrenocorticotropin release in fish. We have isolated and characterized MCH cDNAs from coho salmon (Oncorhyncus kisutch). The precursor protein predicted by the longest cDNA consists of 132 amino-acids with a characteristic signal peptide at the N-terminus and the biologically active salmon MCH (sMCH) peptide at the C-terminus. The coho sMCH mRNA and protein sequences are very similar but not identical to the previously reported chum or chinook salmon counterparts, suggesting the existence of species polymorphism. Sequence similarities were revealed between alpha-melanocyte-stimulating hormone and part of the C-terminal domain of sMCH precursor. Two sMCH genes were found in coho salmon. By contrast to other salmon species, only one major sMCH mRNA was detected in coho species suggesting that differential MCH gene expression might occur in salmon. In addition, under low stringency oligoprobes complementary to the sMCH RNA recognize a 0.3 kb RNA which was identified as the 7SL RNA. The regions conserved between those RNAs fold in a similar secondary structure. These similarities might reflect common ancestry which may have functional significance.

Structure of the Human Melanin Concentrating Hormone mRNA

The melanin-concentrating hormone (MCH) is a cyclic neuropeptide which induces skin paling and may be involved in the control of the pituitary adrenal axis in teleost fishes. We have recently cloned and characterized the salmon and rat MCH mRNAs and we report in the present paper the cloning and sequencing of the human counterpart. The deduced human MCH (hMCH) precursor is 165 amino acids long and as for rat and salmon, encodes the MCH peptide at the C-terminus. The human and rat MCH precursors are very similar to one another but differ extensively from the salmon counterpart. Strong sequence conservation was found in the regions of mammalian prohormones encoding the novel putative neuropeptides named NGE and NEI which we had originally identified in the rat MCH precursor. Furthermore, sequence identities, with perhaps functional implications, were found among the MCH, human ANF, and aplysia peptide A hormone precursors.