MITOCHONDRIAL CALCIUM-UPTAKE STIMULATED BY CIBACRON BLUE F3GA IN BOVINE SPERM

The triazine dye Cibacron Blue P3GA stimulated calcium uptake into an ionomycin-sensitive compartment of washed bovine sperm, Cibacron blue- stimulated uptake was not sensitive to nifedipine, diltiazem, or verap amil, but was blocked by transition metals, 1 mu M ruthenium red, rote none, and the protonophore CCCP. Uptake was dependent on the maintenan ce of energized mitochondria but not on oxidative or substrate level A TP production and was not supported by glycolysis. The cibacron blue-s timulated uptake was judged by these criteria to be mitochondrial. Upt ake in the absence of Cibacron blue was 0.046 +/- 0.008 and 0.268 +/- 0.068 nmol/min/10(8) cells with 0.1 mM Cibacron blue at 15 mu M Ca2+. Half-maximal stimulation occurred at 20-25 mu M Cibacron blue. Cibacro n blue also allowed efflux of mitochondrial calcium in the presence of external calcium. This efflux would be blocked by ruthenium red, Ni2, and La3+, but not by Co2+, which effectively blocked Cibacron blue-s timulated influx. Permeabilizing the plasma membrane with filipin indi cated that Cibacron blue stimulation of mitochondrial calcium uptake o perates directly on the mitochondria. Cibacron blue does not stimulate mitochondrial respiration and thus, calcium uptake was not the result of increased mitochondrial flux. (C) 1995 Academic Press, Inc.