Abstract
The cytosolic free Ca2+ concentration of calcium-tolerant rat myocytes has been measured by the null point titration technique using arsenazo III as a Ca2+ indicator and digitonin to permeabilize the plasma membrane. The mean value obtained for 8 separate preparations was 270 +/- 35 nM. The distribution of releasable calcium between the mitochondrial and sarcoplasmic reticular compartments was measured by the successive additions of uncoupler and A23187 to cells pretreated with ruthenium red. The relative distribution of calcium in each pool was independent of the cell calcium content up to the maximum value of releasable calcium investigated (4.5 nmol/mg of cell dry weight) and was distributed in the approximate ratio of 2:1 in favor of the sarcoplasmic reticulum. The cells contained 1 nmol of calcium/mg of cell dry weight in a form nonreleasable by A23187, which was independent of the total cell calcium content as measured by atomic absorption spectroscopy. It is calculated that the calcium content of mitochondria in heart under physiological conditions is about 5 nmol/mg of mitochondrial protein. At this level, the mitochondria are likely to provide effective buffering of the cytosolic free Ca2+ concentration of quiescent heart cells. The corresponding intramitochondrial free Ca2+ is in a range above values needed to regulate the activity of Ca2+-dependent enzymes of the citric acid cycle in heart. The physiological calcium content of the sarcoplasmic reticulum in heart cells is estimated to be about 2.5 nmol/mg of cell dry weight, which is at least 5-fold greater than the amount of calcium release calculated to cause maximum tension development of cardiac muscle.
Highlights
Tribution of calcium in each pool was independent of Calcium-tolerant cardiac myocytes were prepared from Sprague-Dawley rats weighing 200-300 g releasable calcium investigated (4.5 nmol/mg of cell using a modification (6) of the procedure described by Kao et al (7)
Dry weight) and was distributed in the approximate The yield of cells was 10-20 mg dry weight/heart, and 70-80% were ratio of 2:l in favorof the sarcoplasmic reticulum.The cells contained1 nmol of calcium/mg of cell dry weight in a form nonreleasable by A23187, which was independent of the total cell calcium content as measured by atomic absorption spectroscopyI.t is calculated that the calciumcontent of mitochondria in heart under physiological conditions is about 5 nmol/mg of mitochondrial protein
Correspondingintramitochondrialfree Ca2+ is ina thermostated cuvette in an Aminco DW2a spectrophotometer and range above values needed to regulate the activity of changes in the absorbance of the Ca-arsenazo complex were measured phCleaaa2sr+mt .-idcTephreeenptidhceyunslituoemnlozgyiimncaelhs eccoaafelrctllitsuhmiesceicstortiinmcteantaet ocdfidtthoceybcselaercaiobn-outcduaodesindnnsicgtieitoynantwruaonatfivitosesmnle,fanutlglhllteahsmecpaxoaltueirra.nctFosefololr6uf7mlC5ae-ra6aC2s8+ua5or2ern+mEmceGonnTatctAeao'nfsttertohantcesioiotcvinyvetirwotytsahosoelfiacrd0aj.fn1urgesoeetepfCdtriocabma2yl+ 2.5 nmol/mg of cell dry weight, whichis at least 5-fold 0.1-1.5 pM free Ca2+,followed by addition of 10 pg of digitonin/mg of greater than the amounot f calcium release calculated cell dry weight (1).The amount of uncoupler-releasable calcium and tocausemaximumtensiondevelopment of cardiac A23187-releasable calcium was measured as described previously (4)
Summary
Tribution of calcium in each pool was independent of Calcium-tolerant cardiac myocytes (resistant to damage by 1 mM the cell calcium content up to the maximum value of Ca") were prepared from Sprague-Dawley rats weighing 200-300 g releasable calcium investigated (4.5 nmol/mg of cell using a modification (6) of the procedure described by Kao et al (7). Dry weight) and was distributed in the approximate The yield of cells was 10-20 mg dry weight/heart, and 70-80% were ratio of 2:l in favorof the sarcoplasmic reticulum.The cells contained nmol of calcium/mg of cell dry weight in a form nonreleasable by A23187, which was independent of the total cell calcium content as measured by atomic absorption spectroscopyI.t is calculated that the calciumcontent of mitochondria in heart under physiological conditions is about 5 nmol/mg of mitochondrial protein. At this level, the mitochondria are in the rod form showing clear striations underphase contrast microscopy.
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