Andrienko T, Kuznetsov A V, Kaambre T, Usson Y, Orosco A, Appaix F, Tiivel T, Sikk P, Vendelin M, Margreiter R, Saks V A
Laboratory of Fundamental and Applied Bioenergetics, INSERM E0221, Joseph Fourier University, Grenoble, France.
J Exp Biol. 2003 Jun;206(Pt 12):2059-72. doi: 10.1242/jeb.00242.
Regulation of mitochondrial respiration both by endogenous and exogenous ADP in the cells in situ was studied in isolated and permeabilized cardiomyocytes, permeabilized cardiac fibers and 'ghost' fibers (all with a diameter of 10-20 micro m) at different (0-3 micro moll(-1)) free Ca(2+) concentrations in the medium. In all these preparations, the apparent K(m) of mitochondrial respiration for exogenous ADP at free Ca(2+) concentrations of 0-0.1 micro moll(-1) was very high, in the range of 250-350 micro moll(-1), in contrast to isolated mitochondria in vitro (apparent K(m) for ADP is approximately 20 micro moll(-1)). An increase in the free Ca(2+) concentration (up to 3 micro moll(-1), which is within physiological range), resulted in a very significant decrease of the apparent K(m) value to 20-30 micro moll(-1), a decrease of V(max) of respiration in permeabilized intact fibers and a strong contraction of sarcomeres. In ghost cardiac fibers, from which myosin was extracted but mitochondria were intact, neither the high apparent K(m) for ADP (300-350 micro moll(-1)) nor V(max) of respiration changed in the range of free Ca(2+) concentration studied, and no sarcomere contraction was observed. The exogenous-ADP-trapping system (pyruvate kinase + phosphoenolpyruvate) inhibited endogenous-ADP-supported respiration in permeabilized cells by no more than 40%, and this inhibition was reversed by creatine due to activation of mitochondrial creatine kinase. These results are taken to show strong structural associations (functional complexes) among mitochondria, sarcomeres and sarcoplasmic reticulum. Inside these complexes, mitochondrial functional state is controlled by channeling of ADP, mostly via energy- and phosphoryl-transfer networks, and apparently depends on the state of sarcomere structures.
在分离和透化的心肌细胞、透化的心脏纤维和“鬼”纤维（直径均为10 - 20微米）中，研究了原位细胞内内源性和外源性ADP对线粒体呼吸的调节作用，介质中游离Ca(2+)浓度不同（0 - 3微摩尔/升）。在所有这些制剂中，游离Ca(2+)浓度为0 - 0.1微摩尔/升时，线粒体呼吸对外源性ADP的表观K(m)非常高，在250 - 350微摩尔/升范围内，这与体外分离的线粒体不同（ADP的表观K(m)约为20微摩尔/升）。游离Ca(2+)浓度增加（高达3微摩尔/升，处于生理范围内），导致表观K(m)值显著降低至20 - 30微摩尔/升，透化完整纤维中呼吸的V(max)降低，肌节强烈收缩。在已提取肌球蛋白但线粒体完整的鬼心脏纤维中，在所研究的游离Ca(2+)浓度范围内，ADP的高表观K(m)（300 - 350微摩尔/升）和呼吸的V(max)均未改变，也未观察到肌节收缩。外源性ADP捕获系统（丙酮酸激酶 + 磷酸烯醇丙酮酸）对透化细胞中内源性ADP支持的呼吸抑制不超过40%，由于线粒体肌酸激酶的激活，肌酸可逆转这种抑制。这些结果表明线粒体、肌节和肌浆网之间存在强烈的结构关联（功能复合体）。在这些复合体内，线粒体功能状态主要通过ADP的通道化来控制，大多通过能量和磷酸转移网络，显然取决于肌节结构的状态。
