Zhou, Z (reprint author), Chinese Acad Sci, Inst Neurosci, Shanghai Inst Biol Sci, Shanghai 200031, Peoples R China,
Mitochondrial Ca uptake is important for ATP production and intracellular Ca buffering. Several agents are often used to interfere with mitochondrial Ca transport, but the use of these agents in intact cells is complicated by lack of knowledge about how rapidly these agents gain access to intracellular mitochondria. We have developed a novel method to assess the time course with which extracellularly applied inhibitors gain access to mitochondria in intact ventricular myocytes preloaded with the fluorescent Ca indicator indo-1-AM. Cell contraction (assessed as the normalized change in cell length DeltaL(n)) was employed as an index of the cytosolic [Ca] ([Ca](c)), and was compared with the indo-1 ratio R-n. which reflects both mitochondrial [Ca] ([Ca](m)) and [Ca](c). Upon abrupt plasma membrane damage in control cells, the delay (t(k)) between the rise in L-n and R-n was < 10 s (reflecting the time lag between the change in [Ca]c and that in [Ca](m)). Exposure of cells to 50 muM ruthenium red (RR) increased t(k) as a monotonic function of preincubation time. In contrast, 10 muM Ru360, a selective and more potent Ca uniporter blocker (K-i similar to0.2 nM) reached a comparable maximal tk after only 10 min, making it practical to use in intact cells. Carbonylcyanide p(trifluoromethoxy)phenylhydrazone (FCCP) and carbonylcyanide m-chlorophenylhydrazone (CCCP) produced smaller maximal effects on tk, but did so almost immediately. These results are the first quantitative data on the time course of blockade of mitochondrial Ca uptake by the four most widely used mitochondrial Ca uptake antagonists in single ventricular myocytes.