MccJ25 induces swelling of rat mitochondria with cytochrome C leakage and NAD(P)H depletion

NIKLISON CHIROU MV; BELLOMIO A; MINAHK CJ; MORERO RD

Rosario, Santa Fe. Argentina.

Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica; 2006

Sociedad Argentina de Biofísica

Mitochondria are considered to play a central role in apoptotic cell death. The opening of the “mitochondrial permeability transitions pore” (MPT) is known to occur under conditions of oxidative stress and matrix calcium overload. Next, it swells and releases cytochrome c (cyt c) from the intermembrane space which is a critical early event in mitochondrially mediated apoptosis. Microcin J25 (MccJ25) is a 21 amino acid antimicrobial peptide with a distinctive lasso-structure. In Escherichia coli, the peptide inhibits both the RNA polymerase and the respiratory chain. In a previous study, we explored the effect of MccJ25 on “not activated” intact mitochondria derived from rat heart and we reported that the peptide inserts into the membrane modifying its permeability and provoking consequently an electrical potential dissipation. Moreover, it acts inhibiting the complex III. In the present work we studied the effect of MccJ25 on the liberation of cyt c from “not activated” mitochondria, the swelling and the oxidation of NAD(P)H induced by the peptide. These last two experiments were performed with succinate “activated” mitochondria and in the presence or absence of the electron transport chain inhibitors (rotenone, cyanide, antimycin A, myxothiazol and stigmatellin), the Ca2+ uniporter channel inhibitor (ruthenium red) and the MPT inhibitor (cyclosporine A). We observed that MccJ25, in a concentration dependent way, induce the liberation of the apoptotic inductor cyt c. Moreover, the mitochondrial swelling is inhibited by rotenone, cyanide, antimycin A, ruthenium red and cyclosporine A. On the other hand, the oxidation of NAD(P)H was only inhibited by antimycin A and ruthenium red but not by cyclosporine A, stigmatellin and myxothiazol. The fact that the mitochondrial swelling induced by MccJ25 was inhibited by cyanide and antimycin A, allow us to conclude that the peptide effect occurs only when the electron flow through the respiratory chain is operating. In addition, the swelling effect is highly increased in activated mitochondria and completely inhibited by rotenone (complex I inhibitor) in “not activated” mitochondria. The NAD(P)H oxidation occurs previously to the opening of the MPT pore and is coupled to the transport of calcium from the intermembrane space to the matrix, since is inhibited by antimycin A and ruthenium red but not by cyclosporine A.