A novel Ca²⁺ entry mechanism is turned on during growth arrest induced by Ca²⁺ pool depletion

Ca²⁺ pool depletion with Ca²⁺ pump blockers induces growth arrest of rapidly dividing DDT₁MF-2 smooth muscle cells and causes cells to enter a stable, quiescent G₀-like growth state (Short, A. D., Bian, J., Ghosh, T. K., Waldron, R. T., Rybak, S. L., and Gill, D. L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4986-4990). Here we reveal that induction of this quiescent growth state with the Ca²⁺ pump blocker, thapsigargin, is correlated with the appearance of a novel caffeine-activated Ca²⁺ influx mechanism. Ca²⁺ influx through this mechanism is clearly distinct from and additive with Ca²⁺ entry through store-operated channels (SOCs). Whereas SOC-mediated entry is activated seconds after Ca²⁺ pool release, caffeine-sensitive influx requires at least 30 min of pool emptying. Although activated in the 1-10 mM caffeine range, this mechanism has clearly distinct methylxanthine specificity from ryanodine receptors and is not modified by ryanodine. It is also unaffected by the Ca²⁺ channel blockers SKF96365 or verapamil and is independent of modifiers of cyclic nucleotide levels. Growth arrest by thapsigargin-induced Ca²⁺ pool depletion can be reversed by treatment with 20% serum (Waldron, R. T., Short, A. D., Meadows, J. J., Ghosh, T. K., and Gill, D. L. (1994) J. Biol. Chem. 269, 11927-11933). The serum-induced return of functional Ca²⁺ pools and reentry of cells into the cell cycle correlates exactly with the disappearance of the caffeine-sensitive Ca²⁺ influx mechanism. Therefore, appearance and function of this novel Ca²⁺ entry mechanism are closely tied to Ca²⁺ pool function and cell growth state and may provide an important means for modifying exit from or entry into the cell cycle.