Calcium ion cycling in rat liver mitochondria

1. Addition of N-ethylmaleimide to rat liver mitochondria respiring with succinate as substrate decreases both the initial rate of Ca(2+) transport and the ability of mitochondria to retain Ca(2+). As a result, Ca(2+) begins to leave the mitochondria soon after it has entered. Half-maximal effects occur at an N-ethylmaleimide concentration of about 100nmol/mg of protein. 2. The efflux of Ca(2+) induced by N-ethylmaleimide is not prevented by Mg(2+) or by Ruthenium Red at concentrations known to prevent Ca(2+) efflux when exogenous phosphate also is present. Swelling of mitochondria does not accompany N-ethylmaleimide-induced Ca(2+) efflux. 3. Addition of Ca(2+) to rat liver mitochondria in the presence of N-ethylmaleimide produces an immediate decrease in DeltaE (membrane potential), which decreases further to only a slight extent over the next 8min. Concomitant with this is an immediate increase and then levelling off of the -59DeltapH (transmembrane pH gradient). 4. Preincubation of rat liver mitochondria with p-chloromercuribenzenesulphonate, which by contrast with N-ethylmaleimide is unable to penetrate the inner mitochondrial membrane, also prevents Ca(2+) retention. The DeltaE and -59DeltapH respond to Ca(2+) addition in a manner similar to that which occurs when N-ethylmaleimide is present. Subsequent addition of mercaptoethanol produces an immediate increase in both DeltaE and -59DeltapH. At the same time Ca(2+) is rapidly accumulated by the organelles. 5. The above data are interpreted as indicating that under the conditions of Ca(2+) efflux seen here, the mitochondria retain their functional integrity. This contrasts with the uncoupling effect of Ca(2+) seen in the presence of P(i), which generally leads to a loss of mitochondrial integrity. We suggest that a unique mechanism of Ca(2+) cycling is able to take place when mitochondria have been treated with N-ethylmaleimide.