Melanotropin receptors demonstrated in situ in human melanoma.

Although some cultured human melanoma cell lines are responsive to melanotropins (melanocyte-stimulating hormones [MSH]), the prevalence and tissue distribution of MSH receptors in melanoma are unknown. We report here the use of an in situ binding technique to demonstrate specific MSH receptors in surgical specimens of human melanoma. The distribution and binding properties of specific MSH binding sites were determined by autoradiography and image analysis after incubation of frozen tumor tissue sections with a biologically active, radiolabeled analogue of alpha-MSH, [125I]iodo-Nle4, D-Phe7-alpha-MSH ([125I]NDP-MSH). In melanoma specimens from 11 patients, 3 showed high levels of specific binding, 5 showed low levels, and in 3 patients specific binding of [125I]NDP-MSH was not detectable. Specific MSH binding sites were present in melanoma cells, but not in adjacent connective or inflammatory tissues. Melanotropins, including alpha-MSH, NDP-MSH, and ACTH, inhibited [125I]NDP-MSH binding in a concentration-dependent manner, whereas unrelated peptides (somatostatin and substance P) did not. The apparent affinity of alpha-MSH for this binding site was in the nanomolar range (EC50 = 2 X 10(-9) M for inhibition of [125I]NDP-MSH binding in situ), similar to that recently described for the murine melanoma receptor. In one patient, analysis of multiple intratumor samples and tumors excised on three separate occasions revealed high levels of specific MSH binding in all samples. These results suggest that endogenous melanotropins may modulate the activities of human melanoma cells in vivo.

Melanotropin receptors of murine melanoma characterized in cultured cells and demonstrated in experimental tumors in situ.

Cultured melanoma cells are known targets for the pigment-inducing actions of melanotropins such as alpha-melanocyte-stimulating hormone (alpha-MSH). The objectives of the present studies were to determine the binding properties and functional relevance of MSH binding sites in a mouse melanoma cell line and to determine whether MSH receptors are expressed in situ. The binding properties of MSH receptors in intact cells of a highly metastatic, highly MSH-responsive mouse melanoma cell subline (B16-F10C23) were determined using a radiolabeled, biologically active preparation of the superpotent alpha-MSH analogue, [Nle4,D-Phe7]-alpha-MSH (125I-NDP-MSH). A single high-affinity class of binding site was detected (Kd for NDP-MSH, 5.6 x 10(-11) M; Kd for alpha-MSH, 2.6 x 10(-9) M as determined by Scatchard analysis and heterologous inhibition assays, respectively). alpha-MSH showed nearly identical concentration-response relationships in the radioreceptor assay (inhibition of 125I-NDP-MSH binding) and a bioassay (stimulation of intracellular cyclic AMP accumulation). Furthermore, the respective potencies of three melanotropins, NDP-MSH, alpha-MSH, and adrenocorticotropic hormone, in binding and biological assays were highly correlated. These results indicate that the 125I-NDP-MSH binding site represents the functional MSH receptor. Tumors were induced by inoculation of C57BL/6 mice with B16-F10C23 cells, and the presence of 125I-NDP-MSH binding sites was determined by in situ radiolabeling of frozen tissue sections followed by autoradiography. Specific MSH binding sites were distributed throughout the tumor tissue, but not in associated fibrovascular elements or in neighboring nonmelanoma tissues. As in cultured B16-F10C23 cells, melanotropins inhibited 125I-NDP-MSH binding to tissue sections in a concentration-dependent manner. These results support the hypothesis that functional MSH receptors are expressed in melanoma in situ, suggesting that the activities of melanoma cells in vivo may be subject to modulation by endogenous melanotropins. The methods described will be applicable for studies of the expression and regulation of MSH receptors in human melanoma and other target tissues.

Interaction of an alpha-melanocyte-stimulating hormone-diphtheria toxin fusion protein with melanotropin receptors in human melanoma metastases.

A hybrid toxin targeted to melanotropin receptors and selectively cytotoxic to melanoma cell lines in vitro has recently been developed. The toxin, a recombinant fusion protein (designated DAB389-MSH), contains the peptide sequences of alpha-melanocyte-stimulating hormone (alpha-MSH) and the catalytic (cytotoxic; Fragment A) and lipophilic (part of Fragment B) domains of diphtheria toxin. In the present study, binding of DAB389-MSH to melanotropin receptors in biopsy specimens of human and mouse melanoma metastases was assessed by measuring its ability to inhibit binding of a radiolabeled, superpotent analogue of alpha-MSH (125I-[Nle4,D-Phe7]-alpha-MSH; 125I-NDP-MSH) and comparing its potency in this system with those of the established ligands NDP-MSH and alpha-MSH. Radioligand binding to tissue sections in vitro was localized and quantified by autoradiography and image analysis. DAB389-MSH inhibited binding of 125I-NDP-MSH to experimental murine B16-F1C23 melanoma metastasis tissue and to melanoma metastases of three patients. In both mouse and human melanoma tissues, concentration-response relationships for DAB389-MSH-mediated inhibition of 125I-NDP-MSH binding were parallel, and its maximal effects were comparable in magnitude, to those of NDP-MSH and alpha-MSH. Half-maximal peptide concentrations for inhibition of 125I-NDP-MSH binding to mouse melanoma tissue sections were: NDP-MSH, 0.63 nM; alpha-MSH, 3.14 nM; and DAB389-MSH, 10.1 nM. In human melanoma tissues, the respective half-maximal peptide concentrations for inhibition of 125I-NDP-MSH binding to mouse melanoma tissue sections were: NDP-MSH, 1.80 nM; alpha-MSH, 2.43 nM; and DAB389-MSH, 11.9 nM. Taken together, these results suggest that NDP-MSH, alpha-MSH, and DAB389-MSH bind to a common melanotropin receptor in human metastatic melanoma cells. Since previous work has shown that melanotropin receptors are detectable in melanoma metastases of about 80% of human patients, malignant melanoma cells of many patients may be susceptible to killing by the melanotropin receptor-targeted cytotoxin DAB389-MSH.

Expression of melanocortin-5 receptor in secretory epithelia supports a functional role in exocrine and endocrine glands.

Melanocortins (alphaMSH and ACTH-related peptides) influence the physiological functions of certain peripheral organs, including exocrine and endocrine glands. This study was designed to determine the identity and anatomical localization of the melanocortin receptors (MC-R) expressed in these organs in the rat. MC5-R messenger RNA was found in exocrine glands, including lacrimal, Harderian, preputial, and prostate glands and pancreas, as well as in adrenal gland, esophagus, and thymus, as demonstrated by ribonuclease protection assays. In exocrine glands, MC5-R messenger RNA expression was restricted to secretory epithelia. MC-R protein was likewise present in secretory epithelia of exocrine glands, as determined by 125I-labeled [Nle4,D-Phe7]alphaMSH ([125I]NDP-MSH) binding and autoradiography in tissue sections. Specific [125I]NDP-MSH binding was also observed in adrenal cortex, thymus, spleen, and esophageal and trachealis muscle. MC receptors in these sites are accessible to circulating MC-R agonists in vivo, as specific binding of [125I]NDP-MSH was observed in exocrine and adrenal glands after systemic injection in vivo. Taken together, these findings show that the MC5 receptor is commonly and selectively expressed in exocrine glands and other peripheral organs. Based on these findings and compelling evidence from other studies, a functional coherence is suggested between central and peripheral actions of melanocortins and melanocortin receptors in physiological functions, including thermoregulation, immunomodulation, and sexual behavior.

Expression of melanocortin receptors and pro-opiomelanocortin in the rat spinal cord in relation to neurotrophic effects of melanocortins.

Although neurotrophic effects of alpha-melanocyte-stimulating hormone (alpha-MSH) are well established, the mechanism underlying these effects is unknown. To identify candidate components of the signaling system that may mediate these effects, in the present study rat spinal cord, dorsal root ganglia, sciatic nerve and soleus muscle were analysed for the expression of the neural MC3, MC4 and MC5 receptors and for the expression of the melanocortin precursor pro-opiomelanocortin (POMC). In rat lumbar spinal cord, the MC4 receptor was the only MC receptor subtype for which mRNA was detectable using RNAse protection assays. In situ binding studies using 125I-NDP-MSH, a synthetic alpha-MSH analogue, demonstrated MC receptor protein in the rat spinal cord, predominantly localised in substantia gelatinosa and area X, surrounding the central canal. Furthermore, POMC mRNA was demonstrated in rat spinal cord and dorsal root ganglia. These findings suggest a functional melanocortin system in the rat spinal cord, that might be involved in peripheral nerve repair. Regulation of POMC or MC receptor transcripts does not appear to be involved in the response to peripheral nerve crush in rats, since no change in mRNA expression patterns was detected after sciatic nerve crush, using quantitative RNAse protection assays. Nevertheless, subtle changes in melanocortin receptor binding did occur postsurgically in several regions of the spinal cord in both sham-operated and sciatic nerve-lesioned rats. The robust expression of MC receptor protein in spinal cord regions that are generally associated with nociception suggests a potentially broader involvement of endogenous melanocortins in spinal pathways which mediate the responses to peripheral injury, in addition to any direct melanocortin effects on sprouting and neurite outgrowth.

Characterization of functional melanotropin receptors in lacrimal glands of the rat.

The specific melanotropin (MSH) binding sites of rat lacrimal glands were characterized with respect to anatomic distribution, peptide specificity and selectivity, and coupling to a biological response. Tissue distribution of MSH binding sites was determined by autoradiography following in situ binding of a radiolabeled, biologically active preparation of a superpotent alpha-MSH analog, [125I]-[Nle4,D-Phe7]-alpha-MSH ([125I]-NDP-MSH). Intense, specific (i.e., alpha-MSH-displaceable) [125I]-NDP-MSH binding was observed throughout lacrimal acinar tissue, but not in ducts or stroma. In freshly isolated lacrimal acinar cells, specific binding of [125I]-NDP-MSH was maximal within 30 min and rapidly reversible, with a dissociation half-time of about 15 min. A number of melanotropins [alpha-MSH, [N,O-diacetyl-Ser1]-alpha-MSH, [des-acetyl-Ser1]-alpha-MSH, beta-MSH, ACTH(1-24) and ACTH(1-39)] were recognized by these binding sites, as assessed by their inhibition of [125I]-NDP-MSH binding; NDP-MSH was the most potent (IC50 = 1.3 x 10(-9) M). In contrast, other peptides, including ACTH(4-10) and the nonmelanotropic peptides VIP, substance P, somatostatin, and ACTH(18-39) (CLIP), had no effects on tracer binding. In isolated lacrimal acinar cells, alpha-MSH and NDP-MSH stimulated intracellular cyclic AMP accumulation. We conclude that lacrimal acinar cells express functional receptors recognizing melanotropins, suggesting that the lacrimal gland may be a target for physiological regulation by endogenous melanotropins.

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]Nle4,D-Phe7-alpha-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 alpha-MSH, gamma-MSH and ACTH1-39 compete with [125I]NDP-MSH for binding to brain tissue sites. In the present studies, each of the melanocortin peptides alpha-MSH, des-acetyl-alpha-MSH, beta-MSH and ACTH1-24 when present at 1 microM 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(O2),D-Lys,Phe9-alpha-MSH4-9 (Org2766), ACTH4-9, and the antipyretic peptide alpha-MSH11-13 did not affect [125I]NDP-MSH binding at concentrations up to 100 microM, 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 ACTH1-4, ACTH34-39 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 gamma-MSH-preferring melanocortin receptor type known as MC3, the relative order of binding affinities of melanocortins, determined in concentration-response studies, was NDP-MSH > or = ACTH1-24 > or = alpha-MSH > gamma-MSH > ACTH4-10 in all brain structures. This suggests that other melanocortin receptor type(s) in addition to MC3 probably account for most of the [125I]NDP-MSH binding detectable in the brain.(ABSTRACT TRUNCATED AT 400 WORDS)

Antipyretic role of endogenous melanocortins mediated by central melanocortin receptors during endotoxin-induced fever.

Bacterial infection causes fever, an adaptive but potentially self-destructive response, in the host. Also activated are counterregulatory systems such as the pituitary-adrenal axis. Antipyretic roles have also been postulated for certain endogenous central neuropeptides, including the melanocortins (alpha-MSH-related peptides). To test the hypothesis that endogenous central melanocortins have antipyretic effects mediated by central melanocortin receptors (MCRs), we determined the effect of intracerebroventricular injection of a synthetic MCR antagonist, Ac-Nle4,c-[Asp5,DNal(2')7,Lys10]alpha-MSH(4-10)-NH2 (SHU-9119) in endotoxin-challenged rats. The efficacy and specificity of SHU-9119 as an MCR antagonist in the rat was first validated in vitro and in vivo. In vitro, in heterologous cells expressing either rat MC3-R or MC4-R, the major MCR subtypes expressed in brain, SHU-9119 showed no intrinsic agonism, but it inhibited alpha-MSH-induced cAMP accumulation (IC50 = 0.48 +/- 0.19 and 0.41 +/- 0.28 nM, respectively) and [125I]-[Nle4,DPhe7]-alpha-MSH binding (IC50 = 1.0 +/- 0.1 and 0.9 +/- 0.3 nM, respectively). In vivo, exogenous alpha-MSH (180 pmol) inhibited fever in rats when administered intracerebroventricularly 30 min after Escherichia coli lipopolysaccharide (LPS) (25 microg/kg, i.p.). When co-injected with alpha-MSH, SHU-9119 (168 pmol, i.c.v.) prevented the antipyretic action of exogenous alpha-MSH. In contrast, neither alpha-MSH nor SHU-9119, alone or in combination, affected body temperatures in afebrile rats. In LPS-treated rats, intracerebroventricular injection of SHU-9119 significantly increased fever, whereas intravenous injection of the same dose of SHU-9119 had no effect. Neither intracerebroventricular nor intravenous SHU-9119 significantly affected LPS-stimulated plasma ACTH or corticosterone levels. The results indicate that endogenous central melanocortins exert an antipyretic influence during fever by acting on MCRs located within the brain, independent of any modulation of the activity of the pituitary-adrenal axis.

Identification of a receptor for gamma melanotropin and other proopiomelanocortin peptides in the hypothalamus and limbic system.

Corticotropin (ACTH) and melanotropin (MSH) peptides (melanocortins) are produced not only in the pituitary but also in the brain, with highest concentrations in the arcuate nucleus of the hypothalamus and the commisural nucleus of the solitary tract. We have identified a receptor for MSH and ACTH peptides that is specifically expressed in regions of the hypothalamus and limbic system. This melanocortin receptor (MC3-R) is found in neurons of the arcuate nucleus known to express proopiomelanocortin (POMC) and in a subset of the nuclei to which these neurons send projections. The MC3-R is 43% identical to the MSH receptor present in melanocytes and is strongly coupled to adenylyl cyclase. Unlike the MSH or ACTH receptors, MC3-R is potently activated by gamma-MSH peptides, POMC products that were named for their amino acid homology with alpha- and beta-MSH, but lack melanotropic activity. The primary biological role of the gamma-MSH peptides is not yet understood. The location and properties of this receptor provide a pharmacological basis for the action of POMC peptides produced in the brain and possibly a specific physiological role for gamma-MSH.