Action of ionophore A23187 at the cellular level. Separation of effects at the plasma and mitochondrial membranes.

Bovine epididymal spermatozoa incubated aerobically in vitro in the presence of 0.1 to 0.2 mM CaCl2 accumulate 25 to 50 nmol of calcium/10(8) cells. The addition of low concentrations of the ionophore A23187 (0.01 to 0.5 nmol/mg of sperm protein) induces efflux of this accumulated calcium. At high ionophore concentrations (0.5 to 5.0 nmol/mg of sperm protein), calcium release is followed by an influx of up to 25 nmol of calcium/10(8) cells that is not dependent on mitochondrial energization. A selective increase in the permeability of the sperm plasma membrane produced by treatment with the polyene antibiotic, filipin, results in the release of that calcium which is accumulated in the presence of high concentrations of A23187. Sperm first treated with filipin possess the ability to accumulate and retain calcium (in the presence of an oxidizable substrate) but release Ca2+ without subsequent reaccumulation after the addition of 3 nmol of A23187/mg of protein. These observations are explained by the existence of competing calcium pumps operating within the mitochondrial and plasma membranes of the spermatozoan. Treatment with high concentrations of A23187 allows calcium influx into a non-mitochondrial compartment of the sperm cell as a consequence of the equilibration of this cation across both mitochondrial and plasma membranes. The amount of calcium uptake and its sensitivity to filipin indicate that calcium binding to soluble, intracellular components is also involved. The ability of low concentrations of A23187 to induce calcium efflux is explained as a result of the continued operation of the plasma membrane pump coincident with ionophore-induced decay of the concentration gradient across the mitochondrial membrane. This hypothetical action of low levels of the ionophore on the mitochondria is supported by the observation of net movements of calcium with filipin-treated cells and the respiratory responses and movements of phosphate and membrane-associated calcium with intact sperm. It is suggested that the basis of this apparent selectivity of ionophore action lies in the relative activities and kinetic properties of the competing calcium pumps in the plasma and mitochondrial membranes of these cells. Ionophore-induced influx of calcium into the extramitochondrial space results in a stimulation of respiration and kinetic activity of the sperm. This activation of motility is observed also with cells made entirely dependent upon glycolysis (by treatment with respiratory inhibitors) and suggests a direct involvement of calcium in the regulation of flagellar function.