Biochimica etBiophysicaActa 850 (1986) 41-48 41 Elsevier BBA42013 t-Butylhydroperoxide-induced Ca 2÷ efflux from liver mitochondria in the presence of physiological concentrations of Mg 2+ and ATP Celene Fernandes Bernardes, Lucia Pereira da Silva and Anlbal EugSnio Vercesi * Departamento de BioquJmica, Instituto de Biologia, Universidade Estadual de Campinas, C.P. 6109, 13100 Campinas, SSo Paulo (Brazil) (Received November22nd, 1985) Key words: Ca 2+ efflux; NAD(P)H oxidation; Hydroperoxide; Ruthenium red; Acetate; (Rat liver mitochondria) Isolated rat liver mitochondria, energized either by succinate oxidation or by ATP hydrolysis, present a transient increase in the rate of Ca 2+ efflux concomitant to NAD(P)H oxidation by hydroperoxides when suspended in a medium containing 3 mM AT]P, 4 mM Mg 2÷ and acetate as permeant anion. This is paralleled by an increase in the steady-state concentration of extramitochondrial Ca 2÷, a small decrease in d~ and an increase in the rate of respiration and mitochondrial swelling. With the exception of mitochondrial swelling all other events were found to be reversible. If Ca 2÷ cycling was prevented by ruthenium red, the changes in A~, the rate of respiration and the extent of mitochondrial swelling were significantly diminished. In addition, there was no significant decrease in the content of mitochondrial pyridine nucleotides. Mitochondrial coupling was preserved after a cycle of Ca 2 ÷ release and re-uptake under these experimental conditions. It is concluded that hydroperoxide-induced Ca 2+ efflux from intact mitochondria is related to the redox state of pyridine nucleotides. Introduction Energized liver mitochondria, particularly in the presence of membrane stabilizers such as ATP and Mg 2÷, show the ability to buffer ex- tramitochondrial Ca 2+ at concentrations between 0.25 and 1.0 #M, depending on the medium com- position [1-5]. This is the result of Ca 2÷ flux through two operative pathways: an uniporter that promotes an electrophoretic Ca 2÷ influx in re- * To whom correspondence should be addressed. Abbreviations: t-BuOOH, t-butylhydroperoxide; Hepes, 4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid; TPP +, tetra- phenylphosphonium; FCCP, carbonyl cyanide p-trifluoro- methoxyphenylhydrozone; A~k, transmembrane potential; EGTA, ethylene glycolbis(fl-aminoethylether)-N,N, N', N'-te- traacetic acid. sponse to the electrical membrane protential [6-11] and an electroneutral Ca 2+ efflux through an anti- port that exchanges one internal Ca 2+ for two external H + [12-14], but see also Ref. 15. This model of Ca 2+ transport in mitochondria was first suggested by Drahota et al. [16]. The concept of Ca 2+ cycling through two independent pathways became established after experiments showing net Ca 2+ efflux in the presence of ruthenium red, a specific inhibitor of the Ca 2+ uniporter [17-19]. The steady-state extramitochondrial Ca 2+ con- centration can be altered in the presence of differ- ent agents which modify the rate of either Ca 2+ influx or efflux [11,14,20]. It was first shown by Lehninger et al. [21] that Ca 2+ efflux from isolated mitochondria could be stimulated by the oxidized state of mitochondrial pyridine nucleotides. This 0005-2728/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)