A novel methylxanthine-sensmve ca²⁺ influx mechanism is dependent on ca²⁺ pool emptying

Ca²⁺ influx through store-operated channels (SOCs) activated by Ca21 pool depletion represents an important component of Ca²⁺ signals generated in cells. We described a new and distinct Ca²⁺ influx component induced in cells after Ca²⁺ pools are emptied with thapsigargin (TG) (Ufret-Vincenty, C.A., et al., JBC 270, 26790-26793, 1995). This novel influx pathway can be activated by caffeine and other methylxanthines. We have further characterized this novel influx mechanism in DDT,MF-2 smooth muscle cells. The SOC-mediated Ca²⁺ influx component after emptying Ca²⁺ pools in DDT,MF-2 cells was short-lived and appeared to be rapidly deactivated. After treatment of DDT,MF-2 cells with 10 nM of the Ca²⁺locker, 2,5-di-fert-butylhydroquinone (DBHQ), 10 mM caffeine induced a rapid, large, but transient influx of Ca²⁺. This influx was distinct from SOC-mediated Ca²⁺ entry. Caffeine added to untreated DDT,MF-2 cells had no effect on cytosolic Ca²⁺ concentration, thus the caffeine-induced Ca²⁺ influx is active solely in the pool-depleted state. In DBHQ-treated cells, bradykinin-sensitive Ca ²⁺ pools quickly refilled and cells became insensitive to caffeine immediately after DBHQ removal. With TG-treated cells, reversal of TG-induced pool depletion required a high (20%) serum treatment, in addition to removal of TG, to allow refilling of agonist-sensitive Ca²⁺ pools concomitantly with the disappearance of caffeine-induced Ca²⁺ influx. Caffeine-activated Ca²⁺ influx was also induced by emptying intracellular pools using the Ca²⁺ ionophpre, ionomycin (10 nM), suggesting that emptying intracellular Ca²⁺ stores induces caffeine sensitivity, regardless of the means used for depletion. The results show that the appearance of caffeine-sensitive Ca²⁺ influx is directly correlated with depletion of intracellular Ca²⁺ pools.