The Na+-and K+-induced Ca++ release was measured isotopically by millipore filter technique in pig heart mitochondria. With EGTA-quenching technique, the characteristics of mitochondrial Ca++-pool and the sources of Ca++ released from mitochondria by Na+ or K+ were analyzed. The mitochondrial Ca++-pool could be distinctly divided into two components: internal and external ones which were represented either by uptake through inner membrane, or by energy independent passive binding to external surface of mitochondria, respectively. In energized mitochondria, a large portion of Ca++was transported into internal pool with little external binding, while in de-enerigzed state, a large portion of transported Ca++ existed in the external pool with limited amount of Ca++ in the internal pool which was possibly transported through the Ca++-carrier present in the inner membrane. Na+ induced the Ca++ release from both internal pool and external pool and external binding pool of mitochondria. In contrast, K+ did not affect Ca++ of the internal pool, but, displaced Ca++ bound to external surface of the mitochondria. When the Ca++-reuptake was blocked by EGTA, the Ca++ release from the internal pool by Na+ was rapid; the rate of Ca++-efflux appeared to be a function of [Na+]2 and about 8mM Na+ was required to elicit half-maximal velocity of Ca++-efflux. So it was revealed that Ca++-efflux velocity was particulary sensitive to small changes of the Na+ concentration in physiological range. Energy independent Ca++-binding sites of mitochondrial external surface showed unique characteristics. The total number of external Ca++-binding sites of pig heart mitochondria was 29 nmoles per mg protein and the dissociation constant(Kd) was 34μM. The Ca++-binding to the external sites seemed to be competitively inhibited by Na+ and K+; the inhibition constant(Ki) were 9.7 mM and 7.1 mM respectively. Considering the intracellular ion concentrations and large proportion of Ca++ uptake in energized mitochondria, the external Ca++-binding pool of the mitochondria did not seem to play a significant role on the regulation of intracellular free Ca++ concentration. From this experiment, it was suggested that a small change of intracellular free Na+ concentration might play a role on regulation of free Ca++ concentration in cardiac cell by influencing Ca++-efflux from the internal pool of mitochondria.
