Downmodulation of mitochondrial F0F1 ATP synthase by diazoxide in cardiac myoblasts:: a dual effect of the drug
Downmodulation of mitochondrial F0F1 ATP synthase by diazoxide in cardiac myoblasts:: a dual effect of the drug
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DOI：
10.1152/ajpheart.00366.2006
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发表时间：
2007-02-01
影响因子：
4.8
通讯作者：
Mavelli, Irene
中科院分区：
文献类型：
--
作者：
Comelli, Marina;Metelli, Giuliana;Mavelli, Irene
与缺血预处理类似，二氮氧化物被证明可以引发与心脏保护相关的有益生物能量后果。抑制线粒体F0F1 ATP合酶的ATP酶活性可能在这种作用中起作用，并且可能涉及天然抑制剂蛋白IF1。我们最近用纯化的酶和从牛心脏分离的线粒体膜证明，二氮氧化物通过促进IF1结合和可逆地抑制ATP水解与F0F1 ATP合成酶的F-1区相互作用。本文研究了二氮氧化物对培养成肌细胞酶的影响。具体来说，将胚胎心脏来源的H9c2细胞暴露于二氮氧化物中，在维持稳态IF1结合（基础ATP酶活性）或在碱性ph下分离结合的IF1的条件下，检测线粒体ATP酶。还研究了线粒体跨膜电位和解偶联，以及ATP合成通量和ATP含量。心脏保护浓度（40 μ M细胞相关浓度）的二氮氧化物会短暂下调基础ATP酶活性，同时伴有轻度线粒体解偶联和去极化，但不影响ATP合成和ATP含量。从F0F1 ATP合酶中碱性剥离IF1在二氮唑处理的细胞中比在未处理的细胞中较少。格列苯脲预处理和线粒体去极化阻止了基础条件下而非IF1剥离条件下atp酶活性的抑制，表明二氮氧化物改变了碱性IF1的释放。在if1剥离条件下，即使FCCP降低了线粒体跨膜电位，也观察到二氮氧化物对atp酶活性的抑制作用。结果表明，在正常缺氧完整细胞模型中，二氮氧化物直接促进抑制蛋白IF1与F0F1 ATP合酶的结合，并通过线粒体轻度解偶联和去极化间接增强IF1的结合。
Similar to ischemic preconditioning, diazoxide was documented to elicit beneficial bioenergetic consequences linked to cardioprotection. Inhibition of ATPase activity of mitochondrial F0F1 ATP 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 F-1 sector of F0F1 ATP synthase by promoting IF1 binding 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 IF1 binding ( basal ATPase activity) or detaching bound IF1 at 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 mu M 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 IF1 from F0F1 ATP 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 IF1 release. 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 IF1 to F0F1 ATP synthase and enhances IF1 binding indirectly by mildly uncoupling and depolarizing mitochondria.