Measurement of mitochondrial free Ca2+ concentration in living single rat cardiac myocytes.
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.
Laboratory of Cardiovascular Science, National Institute on Aging, Baltimore 21224.
H
H
Miyata
H S
HS
Silverman
S J
SJ
Sollott
E G
EG
Lakatta
M D
MD
Stern
R G
RG
Hansford
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002469:Cell Separation; D002614:Chelating Agents / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D004558:Electric Stimulation; D007211:Indoles / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D009994:Osmolar Concentration; D051381:Rats
D002614:Chelating Agents; D007211:Indoles; C059587:Indo-1 pentaacetoxymethyl ester; D008274:Magnesium; D004072:Digitonin; D002118:Calcium