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 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
acetylcholine was shifted to the right. The inhibition constant of
pirenzepine was calculated as 3.9 x 10(-7) M, of
methoctramine as 6.8 x 10(-7) M and of 4-DAMP-mustard as 1.9 X 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.