Calcium ion cycling in rat liver mitochondria

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Abstract
Addition of N-ethylmaleimide (NEM) to rat liver mitochondria respiring with succinate as substrate decreases both the initial rate of Ca2+ transport and the ability of mitochondria to retain Ca2+. As a result, Ca2+ begins to leave the mitochondria soon after it has entered. Half-maximal effects occur at an NEM concentration of about 100 nmol/mg of protein. The efflux of Ca2+ induced by NEM is not prevented by Mg2+ or by Ruthenium Red at concentrations known to prevent Ca2+ efflux when exogenous phosphate also is present. Swelling of mitochondria does not accompany NEM-induced Ca2+ efflux. Addition of Ca2+ to rat liver mitochondria in the presence of NEM produces an immediate decrease in .DELTA.E (membrane potential), which decreases further to only a slight extent over the next 8 min. Concomitant with this is an immediate increase and then levelling off of the -59 .DELTA.pH (transmembrane pH gradient). Preincubation of rat liver mitochondria with p-chloromercuribenzenesulfonate, which by contrast with NEM is unable to penetrate the inner mitochondrial membrane, also prevents Ca2+ retention. The .DELTA.E and -59 .DELTA.pH respond to Ca2+ addition in a manner similar to that which occurs when NEM is present. Subsequent addition of mercaptoethanol produces an immediate increase in both .DELTA.E and -59 .DELTA.pH. At the same time Ca2+ is rapidly accumulated by the organelles. Under the conditions of Ca2+ efflux seen here, the mitochondria apparently retain their functional integrity. This contrasts with the uncoupling effect of Ca2+ seen in the presence of Pi, which generally leads to a loss of mitochondrial integrity. A unique mechanism of Ca2+ cycling may take place when mitochondria have been treated with NEM.