Calcium ion cycling in rat liver mitochondria

Abstract

1. Addition of N-ethylmaleimide 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 N-ethylmaleimide concentration of about 100nmol/mg of protein. 2. The efflux of Ca²⁺ induced by N-ethylmaleimide 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 N-ethylmaleimide-induced Ca²⁺ efflux. 3. Addition of Ca²⁺ to rat liver mitochondria in the presence of N-ethylmaleimide produces an immediate decrease in ΔE (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 −59ΔpH (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 Ca²⁺ retention. The ΔE and −59ΔpH respond to Ca²⁺ addition in a manner similar to that which occurs when N-ethylmaleimide is present. Subsequent addition of mercaptoethanol produces an immediate increase in both ΔE and −59ΔpH. At the same time Ca²⁺ is rapidly accumulated by the organelles. 5. The above data are interpreted as indicating that under the conditions of Ca²⁺ efflux seen here, the mitochondria retain their functional integrity. This contrasts with the uncoupling effect of Ca²⁺ seen in the presence of P_i, which generally leads to a loss of mitochondrial integrity. We suggest that a unique mechanism of Ca²⁺ cycling is able to take place when mitochondria have been treated with N-ethylmaleimide.