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 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.

Mitochondrial calcium transport

“…dient. When Ca*+ is accumulated by liver mitochondria in the presence of N-ethylmaleimide, to prevent Pi transport and concomitant ApH dissipation (section 4), accumulation is folfowed by a partial loss of Ca" [37], This has been interpreted as evidence for Ca*' cycling 1371; however, the loss of Ca*+ is paralleled by a time-dependent decrease in A$ which is sufficient to account for the observed loss of Ca2+ by re-equilibration via the uniporter (371.…”

Alloxan effects on mitochondria: study of oxygen consumption, fluxes of Mg2+, Ca2+, K+ and adenine nucleotides, membrane potential and volume change in vitro

“…Other thiol reagents, namely N-ethylmaleimide and diamide, have been reported to induce loss of Ca 2+, collapse of membrane potential and large amplitude swelling of isolated liver mitochondria [4,[30][31][32], whereas Ramachandran and Bygrave [33] found release of Ca 2+ but no swelling in rat liver mitochondria incubated with N-ethylmaleimide, possibly due to their use of potassium chloride as osmotic support instead of mannitol/ sucrose [34]. A requirement of external Ca 2+ for the release of Ca 2+ and the other events induced by Nethylmaleimide has also been suggested [31].…”

Inability of tributyltin-induced chloride/hydroxyl exchange to stimulate calcium transport in mitochondria isolated from flight muscle of the sheep blowfly Lucilia cuprina

“…Ca2+ transport was measured by the RuthenVol. 174 ium Red-EGTA quench technique (Reed & Bygrave, 1974) or by using combination Ca and pH electrodes (Ramachandran & Bygrave, 1978). Each of the experiments described was carried out at least twice and usually three times, with agreement between experiments within approx.…”