INTERACTION OF MELANIN-CONCENTRATING HORMONE (MCH), NEUROPEPTIDE E-I (NEI), NEUROPEPTIDE G-E (NGE), AND α-MSH WITH MELANOCORTIN AND MCH RECEPTORS ON MOUSE B16 MELANOMA CELLS

Melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH) are known to exhibit mostly functionally antagonistic, but in some cases agonistic activities, e.g., in pigment cells and in the brain. Neuropeptide E-I (NEI) displays functional MCH-antagonist and MSH-agonist activity in different behavioral paradigms; the role of neuropeptide G-E (NGE) is not known. This study addressed the question of possible molecular interactions between α-MSH, MCH and the MCH-precursor-derived peptides NEI and NGE at the level of the pigment cell MCH receptor subtype (MCH-R_pc) and the different melanocortin (MC) receptors. Radioreceptor assays using [¹²⁵I]MCH, [¹²⁵I]α-MSH and [¹²⁵I]NEI as radioligands and bioassays were performed with MC1-R-positive and MC1-R-negative mouse B16 melanoma cells and with COS cells expressing the different MC receptors. The IC₅₀s of α-MSH and NEI or NGE for [¹²⁵I]MCH displacement from mouse MCH-R_pc were 80-fold and, respectively, > 300-fold higher than that of MCH, and the IC₅₀s for MCH and NEI or NGE for [¹²⁵I]α-MSH displacement from mouse MC1-R were 50,000-fold and > 200,000-fold higher than that of α-MSH. No high-affinity binding sites for NEI were detected on B16 melanoma cells and there was no significant displacement of [¹²⁵I]α-MSH by MCH, NEI or NGE with MC3-R, MC4-R and MC5-R expressed in COS cells. At concentrations of 100 nM to 10 μM, however, MCH, NEI and NGE induced cAMP formation and melanin synthesis which could be blocked by agouti protein or inhibitors of adenylate cyclase or protein kinase A. This shows that mammalian MCH-precursor-derived peptides may mimic MSH signalling via MC1-R activation at relatively high, but physiologically still relevant concentrations, as e.g. found in autocrine/paracrine signalling mechanisms.     

Antagonist and agonist activities of the mouse agouti protein fragment (91-131) at the melanocortin-1 receptor.

Antagonist and agonist activities of chemically synthetized mouse agouti protein fragment (91-131) (AP91-131) at the melanocortin type-1 receptor (MC1-R) were assessed using B 16-F1 mouse melanoma cells in vitro and the following assay systems: (i) receptor binding, (ii) adenylate cyclase, (iii) tyrosinase, (iv) melanin production, and (v) cell proliferation. In competition binding studies AP91-131 was about 3-fold less potent than the natural agonist alpha-melanocyte-stimulating hormone (alpha-MSH) in displacing the radioligand [125I]-[Nle4, D-Phe7]-alpha-MSH (Ki 6.5 +/- 0.8 nmol/l). Alpha-MSH-induced tyrosinase activation and melanin production were completely inhibited by a 100-fold higher concentration of AP9 l -131; the IC50 values for AP91-131 in thetwo assay systems were 91 +/- 22 nM and 95 +/- 15 nM respectively. Basal melanin production and adenylate cyclase activity in the absence of agonist were decreased by AP91-131 with IC50 values of 9.6+/-1.8 nM and 5.0+/-2.4 nM, respectively. This indicates inverse agonist activity of AP91-131 similar to that of native AP. The presence of 10 nM melanin-concentrating hormone (MCH) slightly potentiated the inhibitory activity of AP91-131 in the adenylate cyclase and melanin assays. On the other hand, AP91-131 inhibited cell growth similar to alpha-MSH (IC50 11.0 +/- 2.1 nM; maximal inhibition 1.8-fold higher than that of alpha-MSH). Furthermore, MC1-R was down-regulated by AP91-131 with about the same potency and time-course as with alpha-MSH. These results demonstrate that AP91-131 displays both agonist and antagonist activities at the MC1-R and hence that it is the cysteine-rich region of agouti protein which inhibits and mimics the different alpha-MSH functions, most likely by simultaneous modulation of different intracellular signalling pathways.     

Synthesis and characterization of new radioligands for the mammalian melanin-concentrating hormone (MCH) receptor

Melanin-concentrating hormone (MCH) is a neuropeptide present in the brain of all vertebrates. For the characterization of MCH receptors, a monoiodinated [Phe13, Tyr19]-MCH radioligand analogue was developed. The high susceptibility of [125I]-[Phe13, Tyr19]-MCH to oxidative damage and its very lipophilic nature made it necessary to develop new MCH radioligands. To increase the stability, native methionines were replaced by non-sulphur containing amino acid residues. In one analogue, the L-enantiomer of the phenylalanine residue at position 13 was substituted by the D-enantiomer, which increased the relative affinity of the ensuing [125I]-[D-Phe13, Tyr19]-MCH about 7-fold. The different analogues were iodinated by an enzymatic reaction and used for binding studies with mouse melanoma cells. [125I]-[Met(O)4,8, Phe13, Tyr19]-MCH and [125I]-[Hse4,8, Phe13, Tyr19]-MCH showed only about 19% of total binding and [125I]-[Ser4,8, Phe13, Tyr19]-MCH displayed about 44% of total binding when compared with [125I]-[Phe13, Tyr19]-MCH. Non-specific binding for all tracers was below 11% of total binding of [125I]-[Phe13, Tyr19]-MCH binding. [125I]-[D-Phe13, Tyr19]-MCH was used for saturation binding studies and revealed a KD of 122.7 +/- 15.3 pmol/l. This radioligand was further characterized by association and dissociation binding studies.     

SVK14 cells express an MCH binding site different from the MCH1 or MCH2 receptor

Melanin-concentrating hormone (MCH) is a cyclic peptide, mainly involved in the regulation of skin pigmentation in teleosts and feeding behavior in mammals. The human keratinocyte SVK14 cell line has been previously shown to express binding sites for the MCH analog [125I]-[Phe13,3-iodo-Tyr19]MCH. We report here that: (1) this binding site similarly recognized [125I]-[3-iodo-Tyr13]MCH; (2) its pharmacological profile clearly differed from those observed at the two human MCH receptor subtypes, MCH1-R and MCH2-R; (3) MCH did not induce any effect on second messenger systems (including cAMP, calcium, and MAP kinase signaling pathways), and (4) no mRNAs corresponding to the MCH receptors were found. In conclusion, the binding site characterized in the SVK14 cell line is distinct from the MCH1 and MCH2 receptors and deserves therefore further investigation.     

Effects and interactions between alpha-MSH and MCH/NEI upon striatal cAMP levels.

It is known that alpha-MSH augments cAMP levels in rat brain slices containing accumbens and caudate-putamen nuclei. In this study we examined: a) the effect of other neuropeptides: MCH and NEI, on this cyclic nucleotide; b) if the effects of alpha-MSH on cAMP production can be modulated by addition of MCH or NEI to the incubation medium. Both MCH and NEI (3.6 microM) increased the production of cAMP, whereas at doses of 0.6 microM exerted no effects. When alpha-MSH 0.6 microM was added with NEI or MCH (0.6 microM), only MCH blocked the increase in the cAMP induced by alpha-MSH. Neither MCH nor NEI at the highest dose used (3.6 microM) had any additive effect on AMPc when added together with alpha-MSH. We conclude that, at a high concentration, (MCH/NEI)-like peptides can use the intracellular signal transduction linked to cyclic nucleotides in the CNS.     

Different structural requirements for melanin-concentrating hormone (MCH) interacting with rat MCH-R1 (SLC-1) and mouse B16 cell MCH-R

Melanin-concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food-intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH-R1 and MCH-R2, are thought to mediate mainly the central effects of MCH, the MCH-R on pigment cells has not yet been identified, although in some studies MCH-R1 was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure-activity study in which 12 MCH peptides were tested on rat MCH-R1 and mouse B16 melanoma cell MCH-R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK-293 cells expressing rat MCH-R1 (SLC-1), the radioligand was [125I]-[Tyr13]-MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH-R, the analog [125I]-[D-Phe13, Tyr19]-MCH served as radioligand. The bioassay used for MCH-R1 was intracellular Ca2+ mobilization quantified with the FLIPR instrument, whereas for B16 MCH-R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrease of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side-chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N-terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5- to 10-fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH-R1 and B16 MCH-R was however observed with modifications at position 13 of MCH: whereas L-Phe13 in [Phe13, Tyr19]-MCH was well tolerated by both MCH-R1 and B16 MCH-R, change of configuration to D-Phe13 in [D-Phe13, Tyr19]-MCH or [D-Phe13]-MCH led to a complete loss of biological activity and to a 5- to 10-fold lower binding activity with MCH-R1. By contrast, the D-Phe13 residue increased the affinity of [D-Phe13, Tyr19]-MCH to B16 MCH-R about 10-fold and elicited MAP kinase activation as observed with [Phe13, Tyr19]-MCH or MCH. These data demonstrate that ligand recognition by B16 MCH-R differs from that of MCH-R1 in several respects, indicating that the B16 MCH-R represents an MCH-R subtype different from MCH-R1.     

[D‐(p‐Benzoylphenylalanine)13, tyrosine19]‐melanin‐concentrating hormone,a potent analogue for MCH receptor crosslinking

A photoreactive analogue of human melanin‐concentrating hormone was designed, [d‐ Bpa¹³,Tyr¹⁹]‐MCH, containing the d‐ enantiomer of photolabile p‐benzoylphenylalanine (Bpa) in position 13 and tyrosine for radioiodination in position 19. The linear peptide was synthesized by the continuous‐flow solid‐ phase methodology using Fmoc‐strategy and PEG‐PS resins, purified to homogeneity and cyclized by iodine oxidation. Radioiodination of [d ‐Bpa¹³,Tyr¹⁹]‐MCH at its Tyr¹⁹ residue was carried out enzymatically using solid‐ phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed‐ phase mini‐column and HPLC. Saturation binding analysis of [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH with G4F‐7 mouse melanoma cells gave a K_D of 2.2±0.2×10⁻¹⁰ mol/l and a B_max of 1047±50 receptors/cell. Competition binding analysis showed that MCH and rANF(1–28) displace [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH from the MCH binding sites on G4F‐7 cells whereas α‐MSH has no effect. Receptor crosslinking by UV‐irradiation of G4F‐7 cells in the presence of [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH followed by SDS‐polyacrylamide gel electrophoresis and autoradiography yielded a band of 45–50 kDa. Identical crosslinked bands were also detected in B16‐F1 and G4F mouse melanoma cells, in RE and D10 human melanoma cells as well as in COS‐7 cells. Weak staining was found in rat PC12 phaeochromocytoma and Chinese hamster ovary cells. No crosslinking was detected in human MP fibroblasts. These data demonstrate that [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH is a versatile photocrosslinking analogue of MCH suitable to identify MCH receptors in different cells and tissues; the MCH receptor in these cells appears to have the size of a G protein‐coupled receptor, most likely with a varying degree of glycosylation. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Effects of MCH/NEI in the striatum and interactions between them and with alpha-MSH on IP3 levels

In this study, we investigated: (a) the effect of melanocyte concentrating hormone (MCH) and neuropeptide glutamine (E)-isoleusine-(I) (NEI) on IP(3) production on an "in vitro" model using slices containing caudate putamen (CP) and accumbens nuclei (ACC); and (b) the interaction between these peptides and with alpha-melanocyte-stimulating-hormone (alpha-MSH) on the production of this second messenger. Only MCH at the highest dose studied (3.6 microM) increased the production of IP(3), whereas at the low concentration (0.6 microM) it did not affect IP(3) levels. NEI and alpha-MSH at both concentrations tested (0.6 and 3.6 microM), did not affect IP(3) production either. However, when NEI or alpha-MSH (at 3.6 microM) were added together with 3.6 microM MCH, the increase in the IP(3) content induced by this last peptide was blocked.     

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.     

Dimeric DOTA-alpha-melanocyte-stimulating hormone analogs: synthesis and in vivo characteristics of radiopeptides with high in vitro activity

Dimeric analogs of alpha-melanocyte-stimulating hormone (alpha-MSH) labeled with radiometals are potential candidates for diagnosis and therapy of melanoma by receptor-mediated tumor targeting. Both melanotic and amelanotic melanomas (over-)express the melanocortin-1 receptor (MC1-R), the target for alpha-MSH. In the past, dimerized MSH analogs have been shown to display increased receptor affinity compared to monomeric MSH, offering the possibility of improving the ratio between specific uptake of radiolabeled alpha-MSH by melanoma and nonspecific uptake by the kidneys. We have designed three linear dimeric analogs containing a slightly modified MSH hexapeptide core sequence (Nle-Asp-His-d-Phe-Arg-Trp) in parallel or antiparallel orientation, a short spacer, and the DOTA chelator for incorporation of the radiometal. In vitro, all three peptides were more potent ligands of the mouse B16-F1 melanoma cell melanocortin-1 receptor (MC1-R) than DOTA-NAPamide, which served as standard. The binding activity of DOTA-diHexa(NC-NC)-amide was 1.75-fold higher, that of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was 3.37-fold higher, and that of DOTA-diHexa(CN-NC)-amide was 2.34-fold higher. Using human HBL melanoma cells, the binding activity of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was sixfold higher than that of DOTA-NAPamide. Uptake by cultured B16-F1 cells was rapid and almost quantitative. In vivo, however, the data were less promising: tumor-to-kidney ratios 4 hr postinjection were 0.11 for [(111)In]DOTA-diHexa(NC-NC)-amide, 0.26 for diHexa(NC-NC)-Gly-Lys([(111)In]DOTA)-amide, and 0.36 for [(111)In]DOTA-diHexa(CN-NC)-amide, compared to 1.67 for [(111)In]DOTA-NAPamide. It appears that despite the higher affinity to the MC1-R of the peptide dimers and their excellent internalization in vitro, the uptake by melanoma tumors in vivo was lower, possibly because of reduced tissue penetration. More striking, however, was the marked increase of kidney uptake of the dimers, explaining the unfavorable ratios. In conclusion, although radiolabeled alpha-MSH dimer peptides display excellent receptor affinity and internalization, they are no alternative to the monomeric DOTA-NAPamide for in vivo application.     

Synthesis of 153N-6 analogues and structure-function analysis at murine melanocortin-1 (MC1) receptors.

153N-6 (H-[Met5,Pro6,D-Phe7,D-Trp9,Phe10]-MSH(5-13)) has emerged as the most potent antagonist of alpha-MSH activity on Xenopus laevis melanophores, from a library of 32 360 peptides based on alpha-MSH(5-13) [22]. A recent report has confirmed our observation that 153N-6 also binds to mammalian melanocortin receptors. Here we report the receptor-binding affinities and biologic activities of 153N-6 and 17 selected alpha-MSH analogues at the native MCI receptor expressed by murine B16 melanoma cells. Our intention is to determine the structural requirements for agonism and competitive antagonism of melanocortin activity at the MC1-R and to discover more potent antagonists. 153N-6 was able to inhibit the action of native alpha-MSH and the potent synthetic agonist, [Nle4,D-Phe7]alpha-MSH, at the murine MC1-R. However, the Ki of 153N-6 was 439 times higher than that of alpha-MSH and 4475 times higher than that of [Nle4,D-Phe7]alpha-MSH; too high to allow 153N-6 to be considered as a practical antagonist for use in vivo (Ki of 153N-6 = 9.0 X 10(-6) M). Because Met4 is an important component of alpha-MSH binding at the MC1-R, we investigated alpha-MSH(1-13) and alpha-MSH(4-13) analogues to produce compounds with higher MC1-R-binding affinity than 153N-6. The binding affinity of 153N-6 was not significantly different from alpha-MSH(5-13), but it was 232 times lower than alpha-MSH(4-13). Coupling of H-Nle (as an isosteric replacement for Met) or acetyl-Nle to the N-terminus of 153N-6 raised the binding affinity by a factor of 46, but this and all full-length alpha-MSH analogues with Met or Nle in position 4 were full agonists of the MC1-R. A full-length alpha-MSH(1-13) derivative of 153N-6 with Ala4 did not exhibit significantly greater binding affinity than 153N-6 and appeared to be a partial agonist at the MC1-R in the cAMP assay. These data suggest that Met4 is an important determinant of the intrinsic efficacy of melanocortins as well as their binding affinity at the MCI-R. Pro6 and Phe10 (with respect to alpha-MSH) were found to be the most influential substitutions that determined the antagonist activity of 153N-6.     

Anatomy, function and regulation of neuropeptide EI (NEI)

This review is focused on the anatomy, role and behavior of neuropeptide-glutamic acid-isoleucine (NEI), providing a general report on the neuropeptide. In addition to hormone release, this peptide also takes part in the regulation of grooming behavior and locomotor activity. NEI is produced by cleavage of prepro-MCH that probably takes place at the Lys(129)-Arg(130) and Arg(145)-Arg(146) sites (the glycine residue on the C-terminus of NEI strongly suggests that this peptide is amidated). This same prohormone is also the precursor of MCH, widely studied in relation to food and water intake, and NGE, of which little is known. NEI and MCH are extensively colocalized throughout the central nervous system (CNS), and NEI is also present in peripheral tissues. The latter is also effective in stimulating luteinizing hormone (LH) release and, to a lesser extent, FSH from primary pituitary cell cultures. In addition to releasing LH from the medial eminence, NEI also acts directly on gonadotropes. Lastly, this neuropeptide also acts at the CNS level on gonadotropin-releasing hormone (GnRH) neurons.     

Melanocyte stimulating hormone and melanin concentration hormone may be structurally and evolutionarily related

Two melanotropic peptides, melanin concentration hormone (MCH) and alpha-melanocyte stimulating hormone (alpha-MSH), exert opposing actions on melanosome (melanin granule) movements within teleost pigment cells, melanocytes (melanophores). MCH stimulates melanosome aggregation to the cell center whereas alpha-MSH stimulates pigment organelle dispersion out into the dendritic processes of the melanocytes. The actions of alpha-MSH are dependent upon extracellular calcium (Ca2+), whereas those of MCH are actually enhanced in the absence of the cation. At high concentrations (10(-5)-10(-8) M) MCH also exhibits MSH-like activity (autoantagonism), an effect which is abolished in the absence of Ca2+. Therefore, MCH exhibits MCH-like as well as MSH-like activity depending on the presence or absence of extracellular Ca2+. An analogue of MCH, [Ala5, Cys10]MCH, has been synthesized which is totally devoid of MCH activity but still exhibits MSH-like activity. These results suggest that the two melanotropic peptides share some component of structural similarity and may be evolutionarily related.     

Synthesis and lodination of human (phenylalanine13, tyrosine19) 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 Fmocstrategy 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 × 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 α-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.     

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.     

Identification,purification and characterization of a receptor for dengue virus-induced macrophage cytotoxin (CF2) from murine T cells

Dengue type-2 virus infection in mice induces a subpopulation of T lymphocytes to produce a cytokine cytotoxic factor, which induces macrophages (Mphi) to produce a biologically active cytotoxic cytokine, the Mphi cytotoxin (CF2). Previously we have identified the presence of intermediate-affinity receptors for CF2 on mouse peritoneal Mphi. The present study was undertaken to identify the CF2-receptors (CF2-R) on murine T cells followed by their purification and characterization. Receptor binding assay and Scatchard analysis revealed single, high-affinity (1.0309 nM) receptors for CF2 on T cells (22000 receptors per cell). The binding of [125I]CF2 on murine T cells was saturable and specific. Furthermore, CF2-R was purified from normal mouse T cell plasma membrane by affinity chromatography followed by reversed-phase high-pressure liquid chromatography. The presence of CF2-R was confirmed by indirect dot-blot assay and its binding with [125I]CF2. The purified CF2-R is a 90-95-kDa protein as characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. The chemical crosslinking of [125I]CF2 and its receptor complex showed a product of 100-110 kDa on different subpopulations of murine T cells. The pretreatment of target cells with anti-CF2-R antisera inhibited the cytotoxic activity of CF2 in a dose-dependent manner and thus confirmed the biological significance of CF2-R. Moreover, the presence of CF2-R was also identified on normal human peripheral blood mononuclear cells and T and B cells by crosslinking with [125I]CF2, thus revealing the possible role of CF2 and CF2-R in the immunopathogenesis of dengue virus disease.     

Use of an alpha-melanocyte-stimulating hormone analogue to improve alpha-melanocyte-stimulating hormone receptor binding assay in human melanoma

In order to optimize the detection and measurement of alpha-melanocyte-stimulating hormone (alpha-MSH) receptivity in human melanoma cells, and the authors replaced the natural hormone by [Nle4,D-Phe7]-alpha-MSH, a more stable and potent analogue in the receptor binding assay commonly performed with alpha-MSH. The following parameters were investigated: temperature, incubation time, number of cells, and ratio of labelled to unlabelled hormone. Optimal conditions for each assay were determined. The results demonstrate that the analogue has identical binding sites to alpha-MSH, as similar reciprocal displacements of each labelled (125I) hormone by serial dilutions of unlabelled alpha-MSH or [Nle4,D-Phe7]-alpha-MSH (10(-12) M to 10(-6) M) were obtained. To further compare the two hormones, we performed a screening of various human cell lines: ten melanomas and five nonmelanomas. The assay with [Nle4,D-Phe7]-alpha-MSH yielded more receptor expression on six of ten melanoma lines against only four of ten with the natural hormone. In conclusion, the use of radiolabelled [Nle4,D-Phe7]-alpha-MSH analogue instead of labelled alpha-MSH improved both sensitivity and reproducibility in this receptor binding assay on human melanoma lines.     

Changes in hypothalamic agouti-related protein (AGRP), but not alpha-MSH or pro-opiomelanocortin concentrations in dietary-obese and food-restricted rats.

Melanocortin-4 receptor (MC4-R) density is thought to be regulated by synaptic availability of endogenous agonist, alpha-melanocyte-stimulating hormone (alpha-MSH), and also by agouti-related protein (AGRP), which acts as a competitive antagonist. As hypothalamic MC4-R have been implicated in the regulation of energy balance, we examined concentrations of alpha-MSH and AGRP in hypothalami of dietary-obese and food-restricted rats. In dietary-obese rats, AGRP concentrations were significantly increased by 43% (p < 0.01) above lean controls, whereas a 91% (p < 0.01) reduction was observed in food-restricted rats. Surprisingly, hypothalamic concentrations of alpha-MSH and its precursor peptide, pro-opiomelanocortin (POMC), did not differ significantly from controls in either model. In conclusion, we suggest that MC4-R activity may not be regulated by changes in agonist (alpha-MSH) but by changes in the antagonist (AGRP) availability, which may modulate background activation of the receptor by tonic alpha-MSH release. AGRP may be an important modulator of feeding behaviour.     

beta-MSH: a functional ligand that regulated energy homeostasis via hypothalamic MC4-R?

alpha-Melanocyte stimulating hormone (MSH) has generally been assumed to be the endogenous ligand acting at the melanocortin-4 receptor (MC4-R), activation of which in the hypothalamus leads to reduced feeding. However, beta-MSH is also capable of activating MC4-R and inhibiting feeding. Here, we investigated the possibility that beta-MSH acts as an endogenous MC4-R agonist and that this melanocortin peptide plays a role in the regulation of feeding and energy balance. We found that beta-MSH had significantly higher affinities than alpha-MSH at both human MC4-R transfected into CHO cells (K(i): beta-MSH, 11.4+/-0.4 nmol/l versus alpha-MSH, 324+/-16 nmol/l, P<0.001) and MC4-R in rat hypothalamic homogenates (K(i): beta-MSH, 5.0+/-0.4 nmol/l versus alpha-MSH, 22.5+/-2.3 nmol/l, P<0.001). Incubation of brain slices with 5 microM beta-MSH significantly increased [35S]GTPgammaS binding by 140-160% (P<0.001), indicating activation of G-protein-coupled receptors (GPCRs), in the hypothalamic ventromedial (VMH), dorsomedial (DMH), arcuate (ARC) and paraventricular (PVN) nuclei. These sites match the distribution of beta-MSH immunoreactive fibres and also the distribution of MC4-R binding sites which we and others previously reported. Food-restriction significantly increased beta-MSH levels in the VMH, DMH and ARC (all P<0.05) above freely-fed controls, whilst alpha-MSH concentrations were unchanged. We propose that increased beta-MSH concentrations reflect blockade of the peptide's release in these sites, consistent with the increased hunger and the known up-regulation of MC4-R in the same nuclei. Thus, we conclude that (1). beta-MSH has higher affinity at MC4-R than alpha-MSH; (2). beta-MSH activates GPCR in these sites, which are rich in MC4-R; and (3). beta-MSH is present in hypothalamic nuclei that regulate feeding and its concentrations alter with nutritional state. We suggest that beta-MSH rather than alpha-MSH is the key ligand at the MC4-R populations that regulate feeding, and that inhibition of tonic release of beta-MSH is one mechanism contributing to hunger in under-feeding.