Alkali metal cation release and respiratory inhibition induced by nigericin in rat liver mitochondria.

Nigericin is a monobasic acid antibiotic originally isolated by Harned et al.1' 2 The antibiotic analyzed for C39H69011 and the molecular weight of the sodium salt was found to be 736. Lardy et al.3 found nigericin and dianemycin to have the unusual property of inhibiting mitochondrial oxidation of glutamate and most other DPN-linked substrates and not inhibiting oxidation or phosphorylation with ,3-hydroxybutyrate or succinate. Nigericin and dianemycin were also observed to inhibit the exchange of P32 between inorganic orthophosphate and ATP. These antibiotics did not uncouple oxidative phosphorylation. 3 In recent studies, nigericin4 and dianemycin were found to inhibit the uptake of K+ induced by monactin or valinomycin in mitochondria. Nigericin and dianemycin cause a rapid loss of alkali metal cations which have accumulated in mitochondria under the influence of a variety of agents. The following is a report of the effect of nigericin on alkali metal cation movements and substrate oxidation in rat liver mitochondria. Experiments with this antibiotic have disclosed an unexpected difference between two groups of substrates with respect to alkali metal cation requirement for oxidation. Experimental Materials and Methods.-Preparation of mitochondria: Mitochondria were obtained from the liver of male, white rats of the Sprague-Dawley strain by standard procedures,5 except that the homogenization medium contained 0.25 M mannitol, 0.08 M sucrose, and 0.01 M EDTA. The mitochondria were washed 3 times and resuspended to a concentration of 1 ml per gm of original liver in a medium of 0.25 M mannitol and 0.08 M sucrose. The experiments were begun as soon as the mitochrondria were prepared, and completed within 4 hr. The experiments were performed using an apparatus which simultaneously monitors and records concentrations of 02, K+, or Na+, and H+, light scatter, and fluorescence.6' 7It was designed, developed, and constructed by B. Chance, D. Mayer, and B. Pressman at the Johnson Foundation, University of Pennsylvania. Light (520 mu) scatter was monitored at 180°. The concentrations of K+ and Na+ were monitored with Beckman cation-sensitive electrodes #39046 and 39047. The concentration of H+ was monitored with a Beckman 39030 combination pH electrode. Oxygen was monitored with a collodion-covered, vibrating platinum electrode. After suitable amplification the data were recorded using a 6-channel Honeywell 1508 Visicorder. The cuvette was maintained at 280 with a constant temperature jacket. The cuvette volume was 5 ml. Reagents: The Tris salt of ATP was obtained from the Sigma Co. The antibiotics used were obtained from the following sources: monactin Prof. V. Prelog, Zurich; gramicidin B-Dr. Bernard Witkop, National Institutes of Health; antimycin A-Dr. T. Kagawa, Kyowa Fermentation Ind. Co.; nigericin-Dr. R. Harned, Commercial Solvents Corporation; and valinomycin Dr. J. C. McDonald, Saskatoon, Saskatchewan. Results.-The introduction of monactin or valinomycin into a medium containing mitochondria, K+, Mg++, P04, or acetate and an oxidizable substrate induces the