Protective effects of verapamil and diltiazem against inorganic phosphate induced impairment of oxidative phosphorylation of isolated heart mitochondria

The effects of verapamil and diltiazem on oxidative phosphorylation of isolated rabbit heart mitochondria were related to the experimental conditions employed. In an assay medium containing 250 mM sucrose, 1 mM pyruvate and 5 mM potassium phosphate buffer (pH 7) at 37° (sucrose medium), only a high concentration of verapamil (200–800 μM) or diltiazem (400–600 μM) affected mitochondria. State 4 respiration was stimulated, state 3 respiration was inhibited, and the ADP: O ratio was decreased by these drugs in sucrose medium. These effects resulted in a depression of the respiratory control index (RCI) and oxidative phosphorylation rate (OPR). On the other hand, in an assay medium containing 150 mM KCl, 1 mM pyruvate and 2 mM potassium phosphate buffer (pH 7) at 37° (KCl medium), the high rate of state 3 respiration and the normal value of the ADP: O ratio were not influenced significantly by diltiazem (400–800 μM) or verapamil (200–400 μM). These data indicate that neither verapamil nor diltiazem has an effect on the normal, functioning, isolated mitochondria in KCl medium. Elevation of inorganic phosphate (P₁) from 2 to 5 mM in the KCl medium induced a swelling of the mitochondria, inhibition of state 3 respiration, and a decrease in the ADP: O ratio, RCI and OPR. Under these conditions, a low concentration of verapamil (25–200 μM) or diltiazem (50–800 μM) inhibited the swelling effect of P_i and at the same time prevented the P_i-induced decrease in state 3 respiration, and the ADP: O ratio, RCI and OPR. In a medium containing 150 mM KCl, 1 mM pyruvate, 2 mM ADP and 10 μM palmitoyl-CoA, the addition of 5 mM P_i induced swelling of mitochondria and a decreased rate of state 3 respiration. Under these conditions, even a low concentration of verapamil (6–200 μM) or diltiazem (25–400 μM) inhibited swelling and prevented the inhibition of state 3 respiration. It is concluded that low concentrations of verapamil and diltiazem had no effect on unswollen heart mitochondria. An increase in the free P_i concentration induced swelling of mitochondria and resulted in an inhibition of oxidative phosphorylation, provided that the extramitochondrial potassium concentration was as high as that normally found in the cytosol. Under these conditions, a low concentration of verapamil and diltiazem was able to affect the mitochondrial membranes so as to prevent P_i-induced swelling and the related inhibition of oxidative phosphorylation.