Cytosolic free calcium concentration in individual cardiac myocytes in primary culture.
Cytosolic free Ca2+ concentration, [Ca2+]i, of single isolated Ca2+-tolerant rat ventricular myocytes in primary culture was determined by digital video imaging of intracellular fura-2 fluorescence. In deenergized myocytes in which contractile elements were uncoupled by 2,3-butanedione monoxime, the maximum and minimum fluorescence intensity ratio values of fura-2 in the cell were similar when compared with those of fura-2 solutions observed in the microscope. Through the use of in vitro calibration, [Ca2+]i in quiescent, rod-shaped myocytes was 90 +/- 6 nM. There was no detectable spatial heterogeneity in [Ca2+]i in resting myocytes. Localized regions of [Ca2+]i elevation were observed in cells undergoing spontaneous rhythmic contractions or when subjected to mild depolarization by KCl. Additions of gramicidin or veratridine resulted in massive increases in [Ca2+]i (greater than 1 microM) and immediate cell hypercontracture. Ruthenium red elicited a modest increase in [Ca2+]i but extracellular ATP or epinephrine had no effect. We conclude the following: 1) digital video imaging of resting cardiac cells did not reveal any subcellular Ca2+ gradients; 2) the fluorescence properties of intracellular fura-2 were similar to that in free solution; and 3) subcellular heterogeneity of [Ca2+]i in isolated myocytes was observed in cells undergoing spontaneous rhythmic contraction.
Cheung
J Y
JY
Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Tillotson
D L
DL
Yelamarty
R V
RV
Scaduto
R C
RC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004837:Epinephrine / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D006096:Gramicidin / Q000494:pharmacology; D006352:Heart Ventricles / Q000187:drug effects / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000378:metabolism*; D051381:Rats; D012430:Ruthenium Red / Q000494:pharmacology; D014701:Veratridine / Q000494:pharmacology
D001572:Benzofurans; D005456:Fluorescent Dyes; D012430:Ruthenium Red; D006096:Gramicidin; D014701:Veratridine; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2; D004837:Epinephrine