Intracellular Ca2+ sequestration in cultured mouse fibroblasts: ATP-dependent Ca2+ uptake by saponin-permeabilized Swiss 3T3 cells.

Abstract

Intracellular Ca2+ sequestration was studied by determining 45Ca2+ uptake by saponin-permeabilized Swiss 3T3 mouse fibroblasts. Uptake was enhanced by addition of ATP and was dependent on the concentrations of ATP and saponin. ADP and UTP also enhanced uptake, but less than did ATP. Mitochondrial Ca2+ uptake was distinguished from nonmitochondrial uptake by the use of antimycin A plus oligomycin at various Ca2+ concentrations. Major sites for sequestration at 10(-5) M Ca2+ are probably mitochondrial and those at 10(-7) M Ca2+ nonmitochondrial, because these drugs inhibited ATP-dependent Ca2+ uptake at 10(-5) M but not at 10(-7) M. A23187 completely inhibited uptake, regardless of the Ca2+ concentration. Effects of various agents on sequestration were tested; ruthenium red, 2,4-dinitrophenol, monovalent cation ionophores, and melittin markedly decreased ATP-dependent Ca2+ uptake at 10(-5) M Ca2+. The calmodulin antagonists trifluoperazine (TFP) and W-7 inhibited Ca2+ uptake at both 10(-5) and 10(-7) M Ca2+, TFP more so than W-7, especially at 10(-5) M. These results suggest that intracellular Ca2+ in Swiss 3T3 cells is regulated by mitochondrial and nonmitochondrial Ca2+ stores in an ATP-dependent way and that in a physiological resting state the nonmitochondrial Ca2+ store, probably the endoplasmic reticulum, is the major site for the sequestration.