Intrinsic cytosolic calcium buffering properties of single rat cardiac myocytes.
Intracellular passive Ca2+, buffering was measured in voltage-clamped rat ventricular myocytes. Cells were loaded with indo-1 (K+ salt) to an estimated cytosolic concentration of 44 +/- 5 microM (Mean +/- SEM, n = 5), and accessible cell volume was estimated to be 24.5 +/- 3.6 pl. Ca2+ transport by the sarcoplasmic reticulum (SR) Ca-ATPase and sarcolemmal Na-Ca exchange was inhibited by treatment with thapsigargin and Na-free solutions, respectively. Extracellular [Ca2+] was maintained at 10 mM and, in some experiments, the mitochondrial uncoupler "1799" was used to assess the degree of mitochondrial Ca2+ uptake. To perform single cell titrations, intracellular Ca2+ ([Ca2+]i) was increased progressively by a train of depolarizing voltage clamp pulses from -40 to +10 mV. The total Ca2+ gain with each pulse was calculated by integration of the Ca current and then analyzed as a function of the rapid change in [Ca2+]i during the pulse. In the range of [Ca2+]i from 0.1 to 2 microM, overall cell buffering was well described as a single lumped Michaelis-Menten type species with an apparent dissociation constant, KD, of of 0.63 +/- 0.07 microM (n = 5) and a binding capacity, Bmax, of 162 +/- 15 mumol/l cell H2O. Correction for buffering attributable to cytosolic indo-1 gives intrinsic cytosolic Ca2+ buffering parameters of KD = 0.96 +/- 0.18 microM and Bmax = 123 +/- 18 mumol/l cell H2O. The fast Ca2+ buffering measured in this manner agrees reasonably with the characteristics of known rapid Ca buffers (e.g., troponin C, calmodulin, and SR Ca-ATPase), but is only about half of the total Ca2+ buffering measured at equilibrium. Inclusion of slow Ca buffers such as the Ca/Mg sites on troponin C and myosin can account for the differences between fast Ca2+ buffering in phase with the Ca current measured in the present experiments and equilibrium Ca2+ buffering. The present data indicate that a rapid rise of [Ca2+]i from 0.1 to 1 microM during a contraction requires approximately 50 microM Ca2+ to be added to the cytosol.
Berlin
J R
JR
Bockus Research Institute, Graduate Hospital, Philadelphia, Pennsylvania 19146.
Bassani
J W
JW
Bers
D M
DM
D000818:Animals; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002352:Carrier Proteins / Q000037:antagonists & inhibitors; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D006321:Heart / Q000502:physiology*; D006352:Heart Ventricles; D066298:In Vitro Techniques; D007211:Indoles; D007700:Kinetics; D008297:Male; D018408:Patch-Clamp Techniques; D051381:Rats; D012508:Sarcolemma / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger; D013050:Spectrometry, Fluorescence / Q000379:methods; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D002021:Buffers; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D007211:Indoles; D019831:Sodium-Calcium Exchanger; D013729:Terpenes; D019284:Thapsigargin; D012964:Sodium; D000252:Calcium-Transporting ATPases; C048960:indo-1; D002118:Calcium