Cytoplasmic Ca²⁺ concentration changes evoked by muscarinic cholinergic stimulation in primary and metastatic melanoma cell lines

Experiments were performed to explore differences between cultured primary and metastatic melanoma cell lines in their muscarinic acetylcholine receptor-mediated intracellular Ca²⁺ signalization. The expression of type 1 and type 3 muscarinic receptors was detected and compared at the protein level using both immunocytochemistry and semiquantitative western blotting. The functionality of muscarinic receptors was tested by applying carbamylcholine (CCh; 1 mmol/l) and by recording the associated increases in cytoplasmic Ca²⁺ using Ca²⁺ imaging with the application of the Ca²⁺ indicator dye, fluo-4. These data indicate that the expression levels of the receptor proteins were not significantly different in the metastatic (HT199, HT168-M1) and the primary (WM35) cell lines. Although Ca²⁺ transients were evoked in all the three cell lines by CCh, the proportion of the CCh-positive cells was smaller amongst the WM35 cells. The Ca²⁺ transients could be effectively blocked by atropine (0.1 mmol/l). The time courses of the Ca²⁺ transients were highly variable, and in some instances they showed a late (plateau-like) component whose presence crucially depended on the influx of extracellular Ca²⁺. When the extracellular Ca²⁺ concentration was reduced, the duration of the CCh-evoked transients was considerably decreased; a phenomenon that was more pronounced in the metastatic cell lines. Although there are no fundamental differences in the muscarinic receptor-mediated Ca²⁺ signalization of the primary and metastatic cell lines, the quantitative differences showed in this study may partially explain the increased malignancy and migratory potential of the metastatic cells.