NADH-linked substrate dependence of peroxide-induced respiratory inhibition and calcium efflux in isolated renal mitochondria

Peroxide-induced state 3 respiratory inhibition and Ca²⁺ efflux in isolated renal mitochondria exhibited a NADH-linked substrate dependence. ADP-stimulated respiratory rates in the presence of various concentrations of tert-butyl hydroperoxide (tBOOH, 0-1000 nmol/mg protein) were determined using glutamate, β-hydroxybutyrate, or pyruvate as substrates. Pyruvatedriven respiration was most sensitive to inhibition (K_i-≈ 75 nmol of tBOOH/mg protein) followed by β-hydroxybutyrate and glutamate (K_i ≈ 150 nmol of tBOOH/mg protein for each). Calcium (5-10 nmol/mg protein) potentiated tBOOH-induced respiratory inhibition using all three substrates. Mitochondrial Ca²⁺ efflux, induced by tBOOH, was most pronounced with pyruvate as substrate. Glutamate prevented Ca²⁺ efflux while the efflux rate with β-hydroxybutyrate was intermediate between glutamate and pyruvate. The substrate-dependent pattern of tBOOH-induced NAD(P)H (NADH plus NADPH) and cytochrome 6 oxidation was similar to that seen for respiratory inhibition and Ca²⁺ efflux suggesting that NAD(P)H may be a common factor in both responses. Low tBOOH concentrations inhibited pyruvate dehydrogenase flux while higher concentrations enhanced pyruvate dehydrogenase flux and activation. The results are discussed in relation to currently proposed theories of reactive oxygen-induced respiratory inhibition, Ca²⁺ efflux, and reperfusion injury.