Roma Drozdz

INTERACTIONS OF ALPHA -MELANOTROPIN AND AGOUTI ON B16 MELANOMA CELLS : EVIDENCE FOR INVERSE AGONISM OF AGOUTI

alpha-Melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin) and agouti control the switch between eumelanin and pheomelanin synthesis in mammalian melanocytes. Here we investigated interactions between alpha-MSH, agouti protein, cAMP elevating agents and phorbol ester on mouse B16 melanoma cells. Agouti (Kd 3.7 nmol/l) and alpha-MSH (Kd 2.3 nmol/l) had similar affinities to the MC1 melanocortin receptor. Both alpha-MSH and agouti induced MC1 receptor down-regulation. Agouti antagonized melanogenesis induced by alpha-MSH, forskolin, cholera toxin (CT), and pertussis toxin (PT). It also reduced the constitutive melanin formation of long-term cultures. Cell proliferation was inhibited by agouti (43% at 100 nM). This effect was reversed by alpha-MSH, forskolin, or CT. B16-G4F cells, a cell variant that lacks the MC1 receptor, did not respond to agouti. From these results we conclude that agouti shows the characteristics of an inverse agonist acting through the MC1 receptor.

MELANIN-CONCENTRATING HORMONE BINDING TO MOUSE MELANOMA CELLS IN VITRO

An analogue of human melanin‐concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr13 was replaced by Phe and Val19 by Tyr. The resulting monoiodinated [125I][Phe13,Tyr19]‐MCH radioligand was biologically active and led to the discovery of high‐affinity binding sites on mouse B16‐F1, G4F and G4F‐7 melanoma cells. Saturation binding analysis with G4F‐7 cells revealed 1090 MCH receptors per cell and a K D of 1.18 × 10−10 mol/l. Receptors for MCH were also found on rat PC12 phaeochromocytoma cells, human RE melanoma cells and COS‐7 cells. Competition binding analyses with other peptides such as α‐MSH, NPY and PACAP demonstrated that MCH receptor binding is specific. rANF(1–28) was found to be a weak competitor of MCH, indicating topological similarities between MCH and rANF(1–28) when interacting with MCH receptors.

Binding Sites for Melanin-Concentrating Hormone (MCH) in Brain Synaptosomes and Membranes from Peripheral Tissues Identified with Highly Tritiated MCH

Melanin-concentrating hormone (MCH) is a neuropeptide occurring in the brain of all vertebrate species. In chromatophores of teleost fishes it induces pigment granule aggregation. In mammals, however, its physiological function is not yet clear. Attempts to identify the site(s) of its action by binding analysis failed because radioiodinated MCH with the natural sequence was devoid of biological activity. We have now synthesized an analogue of rat/human MCH, [Pra4,8,12,19]-MCH, containing four L-propargylglycine (Pra) residues in positions 4, 8, 12, and 19 for catalytic tritiation to norvaline ([3H4]Nva) residues, each of which containing four tritium atoms. The resulting [3H]-MCH ([(3H4)Nva4,8,12,19]-MCH) had a specific radioactivity of approx. 12,200 GBq/mmol (330 Ci/mmol) and retained a biological activity of 10% as compared to rat/human MCH when tested in the carp scale assay. A series of qualitative binding studies performed with rat crude membranes from brain and peripheal tissues as well as with rat brain synaptosomes using the [3H]-MCH radioligand provided the first evidence for the presence of MCH receptors in mammalian tissues. The data showed that specific binding is present in the hypothalamus, hippocampus and in the adrenal gland while none was detected in the brain cortex or spleen. Owing to the tendency of [3H]-MCH to non-specific binding to tissue, glass and plastic surfaces, a saturation binding analysis with this radioligand was not possible.

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.

[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 KD of 2.2±0.2×10−10 mol/l and a Bmax 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