Activation of muscarinic acetylcholine receptors induces Ca(2+) mobilization in FRT cells.

The effect of the muscarinic receptors agonist carbachol (Cch) on intracellular calcium concentration ([Ca2+](i)) and cAMP level was studied in polarized Fischer rat thyroid (FRT) epithelial cells. Cch provoked a transient increase in [Ca2+](i), followed by a lower sustained phase. Thapsigargin, a specific microsomal Ca2+-ATPase inhibitor, caused a rapid rise in [Ca2+](i) and subsequent addition of Cch was without effect. Removal of extracellular Ca2+ reduced the initial transient response and completely abolished the plateau phase. Ryanodine. an agent that depletes intracellular Ca2+ stores through stimulation of ryanodine receptors (RyRs), had no effect on [Ca2+](i). However, the transitory activation of [Ca2+](i) was dose-dependently attenuated in cells pretreated with U73122, a specific inhibitor of phospholipase C (PLC). These data suggest that the Cch-stimulated increment of [Ca2+](i) required IP3 formation and binding to its specific receptors in Ca2+ stores. Further studies were performed to investigate whether the effect of Cch on Ca2+ entry into FRT cells was via L-type voltage-dependent Ca2+ channels (L-VDCCs). Nicardipine, a nonspecific L-type Ca2+ channel blocker, decreased Cch-induced increase on [Ca2+](i), while Bay K-8644, an L-type Ca2+ channel agonist, slightly increased [Ca2+](i) in FRT cells. These data indicate that Ca2+ entry into these nondifferentiated thyroid cells occurs through an L-VDCC, and probably through another mechanism such as a capacitative pathway. Cch did not affect the intracellular cAMP levels, but its effects on [Ca2+](i) were significantly reduced when cells were pretreated with forskolin, suggesting the existence of an intracellular cross-talk between PLC and cAMP mechanisms in the regulation of intracellular Ca2+ mobilization in neoplastic FRT cells.