ADP-dependent palmitoylcarnitine sensitivity of mitochondria isolated from the perfused rabbit heart.

Abstract

The function of mitochondria as influenced by the depletion of oxidizable substances in the myocardium was examined during perfusion of the isolated rabbit heart. Mitochondria were isolated using proteinase and KCl–albumin medium, and their respiration was measured polarographically at 25 °C.(i) Mitochondria of nonperfused hearts (control) responded to repeated additions of ADP with repeated and reproducible "state 4 to state 3 transitions" in the presence of a variety of oxidizable substrates (glutamate, oxoglutarate, acetate, pyruvate, β-hydroxybutyrate, succinate, and palmitoyicamitine).(ii) The heart of a fed animal was perfused almost to exhaustion in a substrate-free medium and mitochondria were isolated and tested. The following findings were obtained (iii–viii).(iii) The respiratory control was gradually lost in a response to repeated additions of ADP, when palmitoylcamitine was used as the substrate. A larger quantity of mitochondria in a given assay partially counteracted this gradual loss of the respiratory control.(iv) The respiratory control and P/O ratios of mitochondria obtained from these hearts were slightly depressed as compared to those of the control, when measured using glutamate or oxoglutarate. Malate addition was required for pyruvate oxidation by mitochondria from perfused hearts.(v) The state 3 respiratory rate of the mitochondria obtained from an exhausted heart was about one-quarter that of the control mitochondria regardless of substrates used. The values were independent of the amount of mitochondria present in the assay system.(vi) With paimitoyicamitine as the substrate, the respiratory control ratio calculated from the first state 3 to state 4 transition was influenced by the amount of mitochondria present. The ratio was depressed at low concentrations of mitochondria.(vii) The rate of exogenous NADH oxidation in the absence of cytochrome c and the optical density of the mitochondrial suspension at 520 mμ, were similar with mitochondria prepared from nonperfused and perfused hearts. An addition of cytochrome c accelerated the rate of NADH oxidation by the latter more than the control.(viii) Unstimulated ATPase activity was high in mitochondria of the perfused heart; the enzyme was stimulated by DNP to a lesser degree and by a smaller quantity of paimitoyicarnitine than the control.(ix) The mitochondria prepared from the perfused heart of a fasted animal and from the fed rabbit's heart perfused in the presence of glucose, albumin, or 30 mM KCl (cardiac arrest) showed a normal respiratory function.(x) The mitochondria of the heart in anoxic arrest showed uncoupling of oxidative phosphorylation and a low respiratory rate regardless of the hydrogen donors used.(xi) It is concluded that a prolonged lack of hydrogen sources in perfused exhausted rabbit hearts results in the mitochondrial dysfunction.