Melanocortin Analog Research Articles

Melanocortin analogue Org2766 binds to rat Schwann cells, upregulates NGF low-affinity receptor p75, and releases neurotrophic activity

Binding of the stable melanocortin(4-9) analogue, Org2766 [Met(O2)-Glu-His-Phe-D-Lys-Phe] to cultured rat sciatic nerve Schwann cells was demonstrated using a biotinylated derivative in semiquantitative histochemical and CELISA assays. Org2766 bound to Schwann cells, but not to fibroblasts, and was displaced maximally by unlabeled Org2766, alpha-MSH and ACTH(1-24). Displacement of Org2766 from the binding sites was considerably reduced by N- and C-truncation of the peptide. Specific binding of Org2766 was also demonstrated in the immortal rat Schwann cell line SCL4.1/F7 and was more pronounced in cells displaying a differentiated morphology. Org2766 and alpha-MSH increased cyclic AMP content of Schwann cells but neither stimulated DNA synthesis when applied alone. However, in the presence of a priming (subthreshold) concentration of the mitogen, cholera toxin, Org2766 and alpha-MSH caused a delayed increase in DNA synthesis. Org2766 did not modulate the expression of several differentiation-related Schwann cell markers. However, Org2766 increased immunoreactivity for p75 low-affinity NGF receptor on Schwann cells and evoked the release of neurotrophic factor(s) that synergized with NGF in stimulating neurite outgrowth in rat DRG neurons. The results indicate that Schwann cells are a primary target for the action of Org2766 and provide evidence for an indirect mechanism by which melanocortins might stimulate neurite sprouting in regenerating peripheral nerve axons.

Identification of antagonists for melanocortin MC 3, MC 4 and MC 5 receptors

Antagonists for the melanocortin receptor family were identified by analysis of the effects of four melanocortin analogues on α-MSH(α-melanocyte-stimulating hormone)-induced cAMP accumulation in 293 human embryonal kidney (HEK) cells that expressed either the rat melanocortin MC 3 receptor, the human melanocortin MC 4 receptor or the ovine melanocortin MC 5 receptor. Two peptides, [ D-Arg 8]ACTH(adrenocorticotrope hormone)-(4–10) and [Pro 8,10, Gly 9]ACTH-(4–10), antagonized the action of α-MSH on the melanocortin MC 4 and MC 5 receptors, but not the melanocortin MC 3 receptor. [Ala 6]ACTH-(4–10) inhibited the α-MSH activation of the melanocortin MC 3 and MC 5, but only weakly antagonized the activation of the melanocortin MC 4 receptor. [Phe-I 7]ACTH-(4–10) antagonized the melanocortin MC 3, MC 4 and MC 5 receptors equally well. These antagonists were also tested to block a behavioral response induced by α-MSH. α-MSH-induced excessive grooming behavior in rats was inhibited by [Phe-I 7]ACTH-(4–10), [ D-Arg 8]ACTH-(4–10) and [Pro 8,10,Gly 9]ACTH-(4–10), but not by [Ala 6]ACTH-(4–10). This suggests that α-MSH-induced excessive grooming behavior is mediated by melanocortin MC 4 receptors.

Heterogeneity of brain melanocortin receptors suggested by differential ligand binding in situ

The existence of multiple brain melanocortin receptor types has been postulated, based on the complex pharmacology of intracerebrally administered melanocortin (melanocyte-stimulating hormone-related) peptides. In this study, this hypothesis was tested by determining whether different brain melanocortin receptor populations can be discriminated on a pharmacologic or neuroanatomic basis. The abilities of various pharmacologically active native melanocortins and structural analogs, as well as other test substances, to compete with biologically active [ 125I]Nle 4, d-Phe 7-α-MSH([ 125I]NDP-MSH) for binding to melanocortin receptors was determined, by in vitro binding and autoradiography in frozen rat brain tissue sections. We have previously shown that native melanocortins including α-MSH, γ-MSH and ACTH 1–39 compete with [ 125I]NDP-MSH for binding to brain tissue sites. In the present studies, each of the melanocortin peptides α-MSH, des-acetyl-α-MSH, β-MSH and ACTH 1–24 when present at 1 μM virtually eliminated [ 125I]NDP-MSH binding in each of a series of brain structures, including medial preoptic area, caudate putamen, olfactory tubercle, bed nucleus of the stria terminalis, ventral part of the lateral septal nucleus, hypothalamic periventricular and paraventricular nuclei, dorsal anterior amygdaloid area, substantia innominata and thalamic paraventricular nucleus; as well as in extraorbital lacrimal gland, a peripheral melanocortin target. In contrast, the behaviorally and neurotrophically active melanocortin analogs Met(O 2), d-Lys,Phe 9-α-MSH 4–9 (Org2766), ACTH 4–9 and the antipyretic peptide α-MSH 11–13 did not affect [ 125I]NDP-MSH binding at concentrations up to 100 μM, implying that the receptors or receptor binding sites which mediate the actions of these analogs must comprise additional types, distinct from those which bind [ 125I]NDP-MSH. Binding of [ 125I]NDP-MSH was also unaffected by the nonmelanotropic peptides ACTH 1–4, ACTH 34–39 and vasoactive intestinal polypeptide (VIP) and by the antipyretic drugs acetaminophen and lysine-salicylate. Although some of the brain structures are known to express mRNA encoding a γ-MSH-preferring melanocortin receptor type known as MC3, the relative order of binding affinities of melanocortins, determined in concentration-response studies, wasNdp-MSH≥ACTH 1–24⩾α-MSH>γ-MSH>ACTH 4–10 probably account for most of the [ 125I]NDP-MSH binding detectable in the brain. Furthermore, the potency relationships between these respective peptides and the binding activity of D-Trp 7, D-Phe 10-α-MSH 6–11 amide, a synthetic α-MSH antagonist, varied considerably among the brain sites and peripheral (lacrimal and melanoma) tissues studied, suggesting some degree of heterogeneity of ligand binding properties among [ 125I]NDP-MSH-binding melanocortin receptor populations in different regions of the brain. Considered together with the available data on the pharmacologic actions of melanocortins and the molecular biology of melanocortin receptors, the present results provide evidence for the existence of multiple, pharmacologically distinct classes of melanocortin receptors in the brain, potentially providing a basis for the pharmacological targeting of specific populations of central melanocortin receptors.

Early effect of an ACTH 4–9 analog (Org. 2766) on regenerative sprouting demonstrated by the use of neurofilament-binding antibodies isolated from a serum raised by α-MSH immunization

Antibodies binding to the 150 kDa neurofilament protein NF150 have been purified from a serum raised by immunizing a rabbit with α-MSH. The NF150-binding antibodies were purified by affinity chromatography on a column of cytoskeletal proteins coupled to CNBr-activated Sepharose-4B. Immunocytochemical application of these antibodies, followed by a FITC-coupled second antibody, labels axons in intact and regenerating nerves and provides a means of identifying and counting small regenerating sprouts from 48 h after nerve crush. We have been able to demonstrate that treatment with the neurotrophic melanocortin analog. Org. 2766, increases the number of regenerating axons present in the nerve as early as 72 h after nerve crush.