The mitochondrial permeability transition and its inhibition by spermine: An investigation of mechanism

Abstract

Increased matrix-Ca{dollar}\sp{lcub}2+{rcub}{dollar}, in combination with a triggering agent/condition which may be oxidative stress, increased temperature or one of a large number of chemicals, induces the inner mitochondrial membrane to undergo a permeability transition allowing entry/efflux of small {dollar}(<{dollar}1500 dalton) solutes. The transition is associated with a cluster of Ca{dollar}\sp{lcub}2+{rcub}{dollar}-dependent events including mitochondrial swelling, uncoupling, collapse of the membrane potential, oxidation of pyridine nucleotides, and a transition from the aggregated to orthodox state. Although the transition has been implicated in hypoxia/reperfusion injury, heavy metal toxicity, Reyes syndrome, and the toxicity of redox active drugs and xenobiotics and is proposed to mediate non-shivering thermogenesis, its mechanistic details have yet to be clearly elucidated. The objective of this thesis was to clarify the biochemical process(es) underlying the mitochondrial permeability transition. We have identified spermine as a novel inhibitor of the transition in isolated mitochondria from rat heart and liver. The transition was induced by a series of triggering agents, t-butyl hydroperoxide (t-BH), Ca{dollar}\sp{lcub}2+{rcub},{dollar} carboxyatractylate, phenylarsine oxide and elevated inorganic phosphate (P{dollar}\sb{lcub}\rm i{rcub}){dollar} concentrations. Regardless of triggering agent, spermine inhibited three phenomena diagnostic for the transition: Ca{dollar}\sp{lcub}2+{rcub}{dollar}-release, mitochondrial swelling and pyridine nucleotide oxidation. Concentrations of spermine producing inhibition fell well within the physiological range. Spermine was then used together with the series of triggering agents under well defined conditions, to delineate more precisely the mechanism(s) by which spermine and selected other triggering agents and inhibitors modulate transition occurrence. Our results indicate that (1) Spermine inhibition is highly sensitive to the ionic composition of the medium; K{dollar}\sp+{dollar} antagonizes spermine action. (2) K{dollar}\sp+{dollar} and spermine act on sites external to the mitochondria. (3) The triggering agent P{dollar}\sb{lcub}\rm i{rcub}{dollar} induces the transition by decreasing the matrix concentration of ADP, an internal inhibitor of the transition. (4) Spermine inhibits the transition by enhancing the efficacy of ADP, most probably by increasing the affinity of the ADP binding site. (5) Ca{dollar}\sp{lcub}2+{rcub}{dollar} and t-BH induce the transition via mechanisms which are clearly distinct from the triggering action of P{dollar}\sb{lcub}\rm i{rcub}.{dollar}