Mitochondrial free Ca2+ concentration in unstimulated rat cardiac myocytes is low (<100 nM) but increases up to 600 nM during high-frequency pacing (4 Hz) with norepinephrine.
A novel technique using indo-1 allows continuous measurement of mitochondrial free Ca2+ in single living rat cardiac myocytes, revealing that [Ca2+]m increases with pacing frequency and norepinephrine, consistent with a signal to increase oxidative phosphorylation.
A technique that allows the continuous measurement of mitochondrial free Ca2+ (Ca2+m) in a single living cardiac myocyte is described. It involves the introduction of the fluorescent chelating agent indo-1 into the cell by exposure to the acetoxymethyl ester, followed by selective quenching of the fluorescence of indo-1 in the cytosol by Mn2+. The identity of the remaining fluorescence due to intramitochondrial indo-1 is established by its resistance to treatment of the cell with digitonin at concentrations that release cytosolic but not mitochondrial enzymes and by the finding that ruthenium red and carbonyl cyanide p-trifluoromethoxyphenylhydrazone prevent its response to elevated cytosolic free Ca2+ (Ca2+c). Ca2+m is found to be low (less than 100 nM) in unstimulated cells and to rise in procedures that chronically elevate Ca2+c, such as Na+ replacement. The gradient Ca2+m/Ca2+c is less than unity at values of Ca2+c of less than 500 nM but rapidly increases at higher values of Ca2+c. Although there is no detectable increase in Ca2+m during a single electrical stimulation, Ca2+m increases up to 600 nM as the pacing frequency is raised to 4 Hz in the presence of norepinephrine; this increase occurs over the course of many contractions. It is concluded that these findings are consistent with an increase in Ca2+m acting as a signal to increase dehydrogenase activity, and hence flux through oxidative phosphorylation, in response to increased work loads.
Miyata et al. (Tue,) reported a other. Measurement of mitochondrial free Ca2+ using indo-1 and Mn2+ quenching was evaluated on Mitochondrial free Ca2+ concentration ([Ca2+]m). Mitochondrial free Ca2+ concentration in unstimulated rat cardiac myocytes is low (<100 nM) but increases up to 600 nM during high-frequency pacing (4 Hz) with norepinephrine.