250 Biochimica et Biophysica Acta, 1056 (1991) 250-258 © 1991 Elsevier Science Publishers B.V. 0005-2728/91/$03.50 ADONIS 0005272891000672 BBABIO 43326 Alterations in mitochondrial Ca 2+ flux by the antibiotic X-537A (lasalocid-A) Regina V. Antonio, Lucia Pereira da Silva and Anibal E. Vercesi Departamento de Bioqu~mica, tnstituto de Biotogia, UN1CAMP, Campinas, SP (Brasil) (Received 25 April 1990) Key words: Mitochondrion; Calcium ion release; Lasalocid-A; Nigericin; (Liver); (Heart) A previous communication (Pereira da Silva, L., Bernardes, C.F. and Vercesi, A.E. (1984) Biochem. Biophys. Res. Commun. 124, 80-86) presented evidence that lasalocid-A, at concentrations far below those required to act as a Ca 2+ ionophore, significantly inhibits Ca ~+ efflux from liver mitochondria. In the present work we have studied the mechanism of this inhibition in liver and heart mitochondria. It was observed that lasalocid-A (25-250 nM), like nigericin, promotes the electroneutral exchange of K + for H + across the inner mitochondrial membrane and as a consequence can cause significant alterations in ApH and A~ k. An indirect effect of these changes that might lead to inhibition of mitochondrial Ca z+ release was ruled out by experiments showing that the three observed patterns of lasalocid-A effect depend on the size of the mitochondrial Ca ~+ load. At low Ca ~+ loads (5-70 nmol Ca2+/mg protein), under experimental conditions in which Ca 2+ release is supposed to be mediated by a Ca z +/2H + antiporter, the kinetic data indicate that lasalocid-A inhibits the efflux of the cation by a competitive mechanism. The Ca2+/2Na + antiporter, the dominant pathway for Ca z+ efflux from heart mitochondria, is not affected by lasalocid-A. At intermediate Ca 2+ loads (70-110 nmol Ca2+/mg protein), lasalocid-A slightly stimulates Ca z+ release. This effect appears to be due to an increase in membrane permeability caused by the displacement of a pool of membrane bound Mg z+ possibly involved in the maintenance of membrane structure. Finally, at high Ca 2+ loads (110-140 nmol Ca2+/mg protein) lasalocid-A enhances Ca 2+ retention by liver mitochondria even in the presence of Ca 2+-releasing agents such as phosphate and oxidants of the mitochondrial pyridine nucleotides. The maintenance of a high membrane potential under these conditions may indicate that lasalocid-A is a potent inhibitor of the Ca2+-induced membrane permeabilization. Nigericin, whose chemical structure resembles that of lasalocid-A, caused similar results. Introduction Calcium distribution across the inner mitochondrial membrane under steady-state conditions is kinetically determined by a continuous cyclic movement of the cat ion through independent pathways for influx and efflux (cf. Refs. 1-4). The influx pathway is well characterized as an electrophoretic uniporter [5,6], while the efflux of Abbreviations: At2H+, electrochemical proton gradient; A+, trans- membrane electrical potential; BSA, bovine serum albumin; NEM, N-ethylmaleimide; TPP +, tetraphenylphosphonium; RR, Ruthenium red; RLM, rat liver mitochondria; RHM, rat heart mitochondria: LA, lasalocid-A; Nig, nigericin; Mal, malonate; t-butOOH, tert-butyl- hydroperoxide. Correspondence: A.E. Vercesi, Departamento de Bioquimica, In- stituto de Biologia, UNICAMP, Caixa Postal 6109, 13081-Campinas, SP, Brazil. the cation is mediated by an Na+-dependent or an Na+-independent pathway [7]. The dominant pathway for Ca 2+ efflux in mitochondria from non-excitable tissues is independent of Na + [8] while the Na+-depen - dent pathway predominates in mitochondria from exci- table tissues [7]. The Na+-dependent release pathway appears to be an electroneutral 2Na+/Ca z+ exchanger [9]. Although an electroneutral CaZ+/2H+ antiporter has been pro- posed for the Na+-independent pathway, a wide varia- tion in Ca2+/H+ stoichiometry is observed if measure- ments are carried out over a range of medium pH [10]. Moreover, other observations indicate that the efflux of Ca 2+ does not increase with increasing pH gradient across the membrane, and no CaZ+-Ca 2+ exchange has been demonstrated to occur through this pathway [10]. These characteristics of the Na+-independent pathway argue against the mechanism being a passive Ca 2 +/2H + exchange [4].