MEASUREMENT OF MITOCHONDRIAL CALCIUM IN SINGLE LIVING CARDIOMYOCYTES BY SELECTIVE REMOVAL OF CYTOSOLIC INDO-1

The aim of the present study was to determine whether, in indo I aceto xymethyl ester (AM)-loaded rat cardiomyocytes, it was possible to remo ve cytosolic but not mitochondrial indo 1 by promoting loss of cytosol ic indo 1 through plasma membrane anion pumps (which are blocked by pr obenecid). Isolated rat ventricular myocytes were loaded with indo 1-A M under conditions (15 min at 30 degrees C) in which about half of the dye is located within mitochondria. Cells were then maintained at 25 degrees C for 2.5 h followed by incubation at 37 degrees C for 1.5 h. After this ''heat treatment,'' the myocyte fluorescence signal was 44% of the value of cells measured before heat treatment, and loss of flu orescence was prevented by 1 mM probenecid. The remaining fluorescence was shown to originate from mitochondria, since 1) Ca2+ uptake and ef flux could be inhibited by ruthenium red and clonazepam, respectively, and 2) low concentrations of digitonin, which release only cytosolic marker enzymes, decreased fluorescence of untreated myocytes but had l ittle effect on the fluorescence signal of heat-treated cells. We conc lude that heat treatment selectively removes cytosolic indo 1, leaving a signal due to mitochondrial indo 1 only.