Characterization of muscarinic receptors in the human melanoma cell line SK-Mel-28 via calcium mobilization

In melanoma cells of primary and metastatic human melanomas muscarinic chol inergic receptors are present. Muscarinic receptors were shown to be expres sed in morphogenetically active embryonic cells. Therefore, the possibility exists that in melanomas an embryonic trait is re-expressed after transfor mation. In the present study, we demonstrated the presence of muscarinic re ceptors in the human melanoma cell line SK-Mel-28 by immunofluorescence wit h the monoclonal antibody M 35 and characterized the receptors further by m easuring calcium mobilization after muscarinic stimulation. Cell suspension s were stained with fura-2 and fluorescence was followed at 380 nn excitati on in a fluorimeter cuvette. After the addition of acetylcholine or carbach ol a steep decrease in fluorescence intensity indicated calcium mobilizatio n from intracellular stores (peak reaction), which was followed by a consta ntly lowered fluorescence level indicating a steady influx of extracellular calcium in the presence of agonist. By quantitative evaluation, dose-respo nse curves were obtained from which an ED of 4.3 x 10(-6) M was calculated for acetylcholine and an ED of 2.2 x 10(-5) M was calculated for carbachol. After preincubation with antagonists the dose-response curve of acetylchol ine was shifted to the right. The inhibition constant of pirenzepine was ca lculated as 3.9 x 10(-7) M, of methoctramine as 6.8 x 10(-7) M and of 4-DAM P-mustard as 1.9 x 10(-8) M. Comparison with the data from the literature a nd those obtained in the chick embryo indicates that the muscarinic recepto r in SK-Mel-28 melanoma cells pharmacologically behaves as the M3 type and corresponds to the embryonic muscarinic receptor characterized by us in ear lier studies. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.