Calcium accumulation and release by the sarcoplasmic reticulum of digitonin-lysed adult mammalian ventricular cardiomyocytes.
We have developed a model for characterizing calcium handling by the intact cardiac sarcoplasmic reticulum (SR) that yields data consistent with both mathematical simulations of in situ SR Ca2+ uptake and deduced behavior of the Ca2(+)-induced Ca2+ efflux channels in mechanically skinned single cardiac cells. In Na(+)-based media (37 degrees C, pH 7.2, 50 mM Pi, 10 mM MgATP, pMg 3.3, 10 mM phosphocreatine), SR 45Ca2+ uptake by digitonin-lysed rat myocytes as a function of free [Ca2+] peaked at pCa 6.2, declined until pCa 5.6 and increased again at lower pCa. When Ca2(+)-induced Ca2+ efflux was inhibited with 30 microM ruthenium red and 10 mM procaine, uptake was saturable with a Vmax of 160 +/- 5 nmol.min-1.mg-1, K0.5 of 500 nM free [Ca2+] and slope factor of 1.6. In K(+)-based media, maximum Pi- and oxalate-supported uptake increased to 220 and 260 nmol.min-1.mg-1, respectively. Without phosphocreatine, 45Ca2+ uptake declined under all conditions; this was correlated with a decrease in ATP/ADP. Vmax for 45Ca2+ uptake was increased 20% in hyperthyroid myocytes but depressed 30% in myocytes from heart failure-prone rats. In canine myocytes, Vmax was the same as in normal rat cells, but K0.5 was 830 nM. Without efflux inhibitors, ryanodine caused a concentration-dependent decline in net Pi-supported 45Ca2+ uptake at pCa 6.3 (K0.5 = 1 microM), while 10 microM ryanodine depressed uptake at all pCa between 7.2 and 5.6. Ruthenium red/procaine fully reversed this effect.
Department of Physiological Chemistry, Ohio State University Medical Center, Columbus 43210-1239.
D K
DK
Wimsatt
C M
CM
Hohl
G P
GP
Brierley
R A
RA
Altschuld
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods; D002478:Cells, Cultured; D004072:Digitonin / Q000494:pharmacology*; D006352:Heart Ventricles / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D011343:Procaine / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012016:Reference Values; D012430:Ruthenium Red / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D013974:Thyroxine / Q000494:pharmacology
D012430:Ruthenium Red; D011343:Procaine; D000255:Adenosine Triphosphate; D004072:Digitonin; D007545:Isoproterenol; D013974:Thyroxine; D002118:Calcium