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.
- ca²⁺ withdrawal
- cell culture
- cytoplasmic ca²⁺
- muscarinic acetylcholine receptors