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 cholinergic receptors are present. Muscarinic receptors were shown to be expressed in morphogenetically active embryonic cells. Therefore, the possibility exists that in melanomas an embryonic trait is re-expressed after transformation. In the present study, we demonstrated the presence of muscarinic receptors in the human melanoma cell line SK-Mel-28 by immunofluorescence with the monoclonal antibody M 35 and characterized the receptors further by measuring calcium mobilization after muscarinic stimulation. Cell suspensions were stained with fura-2 and fluorescence was followed at 380 nm excitation in a fluorimeter cuvette. After the addition of acetylcholine or carbachol a steep decrease in fluorescence intensity indicated calcium mobilization from intracellular stores (peak reaction), which was followed by a constantly lowered fluorescence level indicating a steady influx of extracellular calcium in the presence of agonist. By quantitative evaluation, dose–response curves were obtained from which an ED of 4.3×10−6 M was calculated for acetylcholine and an ED of 2.2×10−5 M was calculated for carbachol. After preincubation with antagonists the dose–response curve of acetylcholine was shifted to the right. The inhibition constant of pirenzepine was calculated as 3.9×10−7 M, of methoctramine as 6.8×10−7 M and of 4-DAMP-mustard as 1.9×10−8 M. Comparison with the data from the literature and those obtained in the chick embryo indicates that the muscarinic receptor in SK-Mel-28 melanoma cells pharmacologically behaves as the M3 type and corresponds to the embryonic muscarinic receptor characterized by us in earlier studies.