Calcium Compartmentation and Regulation in Myocytes
The cytosolic free Ca2+ concentration of isolated rat myocytes that were resistant to addition of external Ca2+ (Ca2+-tolerant) has been measured by two independent methods, the null point titration technique and by use of Quin 2 as an intracellular Ca2+ probe. Values obtained for quiescent cells were in the range of 170 to 270 nM. Using Quin 2-Ca2+ fluorescence to monitor changes of cytosolic free Ca2+ ([Ca2+]i) in the presence of 0.65 mM external Ca2+, separate additions of the Ca2+ ionophore ionomycin, the mitochondrial uncoupling agent 1799 or the respiratory inhibitor KCN each caused an increase of [Ca2+]i of about 3-fold. The Quin 2 loaded myocytes responded to electrical stimulation by a transient increase of [Ca2+]i, which peaked about 75% above the resting level. The rise of [Ca2+]i was complete within 50 ms and declined gradually to the resting level. The β-agonist isoproterenol caused up to a 100% increase in the amplitude of the Quin 2-Ca2+ fluorescence change, with a half maximal effect at 130 µM. The stimulation-induced [Ca2+]i transient was abolished by addition of 100 µM propanolol after 10 µM isoproterenol.
The distribution of calcium within the myocyte was measured by addition of the mitochondrial uncoupling agent FCCP to release mitochondrial calcium followed by the Ca2+ ionophore A23187 to release calcium from other vesicular pools, using arsenazo III as an extracellular Ca2+ indicator. Over the range of total releasable cell calcium from 0.5 to 4.5 nmol/mg cell dry weight, the ratio of the distribution of mitochondrial to sarcoplasmic reticulum was approximately constant at 1:2. The role of mitochondria in regulating and buffering cytosolic free Ca2+ and possible regulation of mitochondrial Ca2+-dependent dehydrogenases by the intramitochondrial free Ca2+ during the cardiac contraction cycle is discussed.
KeywordsPyruvate Dehydrogenase Heart Mitochondrion Positive Inotropic Agent Ionophore Ionomycin Pyruvate Dehydrogenase Phosphatase
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