Dependence of cardiac mitochondrial pyruvate dehydrogenase activity on intramitochondrial free Ca2+ concentration

Abstract

(1) The free Ca2+ concentration of the matrix of rat heart mitochondria ([Ca2+](m)) was determined from the fluorescence of internalized indo-1. The value of the K(d) of indo-1-Ca2+ in the mitochondrial matrix was determined to be 95 nM, on the basis of equilibration of [Ca2+](m) with the extramitochondrial free Ca2+ ([Ca2+](o)) in the presence of rotenone, nigericin, valinomycin and Br-A23187. (2) [Ca2+](m) responded to energization/de-energization protocols, the inhibition of Ca2+-uptake by Ruthenium Red and the potentiation of Ca2+-efflux by Na+ in a manner which was consistent with the known kinetic properties of the mitochondrial Ca2+-transport processes. (3) The concentration gradient [Ca2+](m)/[Ca2+](o) was found to be near unity (0.82 ± 0.18) when mitochondria were incubated in media containing 10 mM-Na+; the additional presence of 1 mM-Mg2+ reduced the gradient to values below unity (0.26 ± 0.03). The polyamine spermine increased the Ca2+ concentration gradient in the presence of 1 mM-Mg2+. (4) The fraction of pyruvate dehydrogenase in the active form (PDH(A)) was found to increase with [Ca2+](m), with a K0.5 for activation of approximately 300 nM-Ca2+. This value of the activation constant was not affected by conditions, e.g. addition of Mg2+, which changed the [Ca2+](m)/[Ca2+](o) concentration gradient, and the presence of different oxidizable substrates, which changed the [NADH/NAD+](m) concentration ratio. Thus pyruvate dehydrogenase interconversion responds directly to changes in [Ca2+](m), as inferred in earlier work.