Lim Kelvin H H, Javadov Sabzali A, Das Manika, Clarke Samantha J, Suleiman M-Saadeh, Halestrap Andrew P
The Bristol Heart Institute, Bristol Royal Infirmary, Malborough Street, Bristol BS2 8HW, UK.
J Physiol. 2002 Dec 15;545(3):961-74. doi: 10.1113/jphysiol.2002.031484.
Studies with different ATP-sensitive potassium (K(ATP)) channel openers and blockers have implicated opening of mitochondrial K(ATP) (mitoK(ATP)) channels in ischaemic preconditioning (IPC). It would be predicted that this should increase mitochondrial matrix volume and hence respiratory chain activity. Here we confirm this directly using mitochondria rapidly isolated from Langendorff-perfused hearts. Pre-ischaemic matrix volumes for control and IPC hearts (expressed in microl per mg protein +/- S.E.M., n = 6), determined with (3)H(2)O and [(14)C]sucrose, were 0.67 +/- 0.02 and 0.83 +/- 0.04 (P < 0.01), respectively, increasing to 1.01 +/- 0.05 and 1.18 +/- 0.02 following 30 min ischaemia (P < 0.01) and to 1.21 +/- 0.13 and 1.26 +/- 0.25 after 30 min reperfusion. Rates of ADP-stimulated (State 3) and uncoupled 2-oxoglutarate and succinate oxidation increased in parallel with matrix volume until maximum rates were reached at volumes of 1.1 microl ml(-1) or greater. The mitoK(ATP) channel opener, diazoxide (50 microM), caused a similar increase in matrix volume, but with inhibition rather than activation of succinate and 2-oxoglutarate oxidation. Direct addition of diazoxide (50 microM) to isolated mitochondria also inhibited State 3 succinate and 2-oxoglutarate oxidation by 30 %, but not that of palmitoyl carnitine. Unexpectedly, treatment of hearts with the mitoK(ATP) channel blocker 5-hydroxydecanoate (5HD) at 100 or 300 microM, also increased mitochondrial volume and inhibited respiration. In isolated mitochondria, 5HD was rapidly converted to 5HD-CoA by mitochondrial fatty acyl CoA synthetase and acted as a weak substrate or inhibitor of respiration depending on the conditions employed. These data highlight the dangers of using 5HD and diazoxide as specific modulators of mitoK(ATP) channels in the heart.
使用不同的ATP敏感性钾（K(ATP)）通道开放剂和阻滞剂进行的研究表明，线粒体K(ATP)（mitoK(ATP)）通道的开放与缺血预处理（IPC）有关。据预测，这应该会增加线粒体基质体积，从而提高呼吸链活性。在此，我们使用从Langendorff灌注心脏快速分离的线粒体直接证实了这一点。用（3）H(2)O和[(14)C]蔗糖测定的对照心脏和IPC心脏的缺血前基质体积（以微升每毫克蛋白质±标准误表示，n = 6）分别为0.67±0.02和0.83±0.04（P < 0.01），在30分钟缺血后分别增加到1.01±0.05和1.18±0.02（P < 0.01），在30分钟再灌注后增加到1.21±0.13和1.26±0.25。ADP刺激的（状态3）以及解偶联的2-氧代戊二酸和琥珀酸氧化速率与基质体积平行增加，直到在体积达到1.1微升毫升(-1)或更大时达到最大速率。mitoK(ATP)通道开放剂二氮嗪（50微摩尔）导致基质体积有类似增加，但对琥珀酸和2-氧代戊二酸氧化起抑制而非激活作用。将二氮嗪（50微摩尔）直接添加到分离的线粒体中也会使状态3的琥珀酸和2-氧代戊二酸氧化受到30%的抑制，但对棕榈酰肉碱的氧化无抑制作用。出乎意料的是，用100或300微摩尔的mitoK(ATP)通道阻滞剂5-羟基癸酸（5HD）处理心脏，也会增加线粒体体积并抑制呼吸。在分离的线粒体中，5HD被线粒体脂肪酰辅酶A合成酶迅速转化为5HD-CoA，并根据所采用的条件作为呼吸的弱底物或抑制剂起作用。这些数据突出了将5HD和二氮嗪用作心脏中mitoK(ATP)通道特异性调节剂的危险性。
