Radioreceptor Assay for α;-Msh Using Mouse B16 Melanoma Cells

Abstract

A radioreceptor assay for alpha-MSH is described which is based on cultured mouse B16 melanoma cells and bioactive monoiodinated [Nle4]-alpha-MSH tracer. The assay was used (1) to study the binding characteristics of alpha-MSH to B16 cells, (2) to determine the relative binding activity of MSH peptides, and (3) to measure MSH in tissue extracts. The association of alpha-MSH to B16 cells reached a stable plateau after 3 h at 15 degrees C. At 25 degrees or 37 degrees C, the binding was transient and at 0-1 degree C, the association was very slow. The hormone-receptor complex was relatively stable between 0 degrees and 15 degrees C whereas a 50% dissociation was reached after 90 min at 25 degrees C and after 35 min at 37 degrees C. The mean KD for alpha-MSH of four saturation experiments was 1.3 nM and the number of receptors 9570 per cell. 1,10-Phenanthroline had a stabilizing effect in the binding assay when used at a 0.3 mM concentration. From the MSH peptides tested in the binding assay, some showed similar potencies in three bioassays (tyrosinase, melanin and Anolis skin), whereas others displayed considerably lower bioassay values than expected from the binding data. This shows that binding and bioactivity can be dissociated in some of the MSH peptides. The biological activity of MSH from the neurointermediate lobe of the rat pituitary as measured by its binding to B16 cells corresponds fairly well with RIA results; in the anterior lobe, alpha-MSH values are overestimated because of the large amount of ACTH present.

Radioiodination of Rhenium Cyclized α-Melanocyte-Stimulating Hormone Resulting in Enhanced Radioactivity Localization and Retention in Melanoma

“…The presence of ␣-MSH receptors on melanoma cells and the fact that the receptors undergo rapid internalization on ligand binding have led scientists to explore the possibility of using peptide ligands as carriers to deliver radionuclides for detection and treatment of melanoma (21). Because radioiodinated MAbs or peptides usually exhibit faster clearance rates from normal tissues compared with radiometallabeled ones, and a variety of radiohalogens are available for possible imaging and therapeutic purposes, halogenated proteins continue to play an important role in radiopharmaceutical development for cancer diagnosis and treatment (7,8).…”

“…␣-MSH receptors display nanomolar to subnanomolar affinities for ␣-MSH peptides and are rapidly internalized on ligand binding (21,22). High receptor affinity and specificity make ␣-MSH peptide analogs attractive vehicles for delivering radionuclides to melanoma cells (23)(24)(25).…”

Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors

“…A number of recent studies have therefore concentrated on determining the presence or absence of MSH receptors on various melanoma cells. Several groups of investigators have used radioreceptor binding assays and autoradiography techniques to identify these receptor in mouse (26)(27)(28)(29)(30)(31) and in several human melanoma cell lines (32)(33)(34)(35). These studies failed to detect the existence of MSH receptors on all human melanoma cell lines.…”

Design and characterization of α-melanotropin peptide analogs cyclized through rhenium and technetium metal coordination

“…Quantitative receptor-binding assays were carried out following a previously described method (28,29). Cells were seeded at a density of 5 ϫ 10 5 cells per well in 24-well tissue culture plates and exposed to various concentrations of peptides in 0.5 ml of binding media (25 mM Hepes͞NaOH, pH 7.4͞0.2% BSA͞0.3 mM 1, 10-phenanthroline͞Ϸ100,000 cpm of 125 ]-␣-MSH in 100 l of saline solution subcutaneously between the scapulae 30 min prior to the injection of 99m TcCCMSH.…”