Author:
Comelli Marina,Metelli Giuliana,Mavelli Irene
Abstract
Similar to ischemic preconditioning, diazoxide was documented to elicit beneficial bioenergetic consequences linked to cardioprotection. Inhibition of ATPase activity of mitochondrial F0F1ATP synthase may have a role in such effect and may involve the natural inhibitor protein IF1. We recently documented, using purified enzyme and isolated mitochondrial membranes from beef heart, that diazoxide interacts with the F1sector of F0F1ATP synthase by promoting IF1binding and reversibly inhibiting ATP hydrolysis. Here we investigated the effects of diazoxide on the enzyme in cultured myoblasts. Specifically, embryonic heart-derived H9c2 cells were exposed to diazoxide and mitochondrial ATPase was assayed in conditions maintaining steady-state IF1binding (basal ATPase activity) or detaching bound IF1at alkaline pH. Mitochondrial transmembrane potential and uncoupling were also investigated, as well as ATP synthesis flux and ATP content. Diazoxide at a cardioprotective concentration (40 μM cell-associated concentration) transiently downmodulated basal ATPase activity, concomitant with mild mitochondria uncoupling and depolarization, without affecting ATP synthesis and ATP content. Alkaline stripping of IF1from F0F1ATP synthase was less in diazoxide-treated than in untreated cells. Pretreatment with glibenclamide prevented, together with mitochondria depolarization, inhibition of ATPase activity under basal but not under IF1-stripping conditions, indicating that diazoxide alters alkaline IF1release. Diazoxide inhibition of ATPase activity in IF1-stripping conditions was observed even when mitochondrial transmembrane potential was reduced by FCCP. The results suggest that diazoxide in a model of normoxic intact cells directly promotes binding of inhibitor protein IF1to F0F1ATP synthase and enhances IF1binding indirectly by mildly uncoupling and depolarizing mitochondria.
Publisher
American Physiological Society
Subject
Physiology (medical),Cardiology and Cardiovascular Medicine,Physiology
Cited by
32 articles.
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