Passive Ca buffering in permeabilized rabbit ventricular myocytes was described by a two-site Michaelis curve (K1=0.42 microM, K2=79 microM), approximately twice the previously expected value.
New estimates of intracellular Ca buffering in cardiac myocytes reveal it is approximately twice the previously expected value, providing quantitative insights into cardiac Ca regulation.
Passive Ca binding was measured with a Ca-selective minielectrode in suspensions of permeabilized rabbit ventricular myocytes equilibrated with 5 microM thapsigargin and 30 microM ruthenium red to prevent sarcoplasmic reticulum (SR) or mitochondrial Ca uptake. Passive Ca binding was obtained by titration of the myocytes with Ca and subtraction of Ca binding in a blank titration without myocytes. Passive Ca binding could be described by a Michaelis binding curve with two sites: K1 = 0.42 microM n1 = 1.27 nmol/mg cell protein and K2 = 79 microM, n2 = 4.13 nmol/mg cell protein. The passive Ca buffering over the physiological Ca concentration was approximately twice the value expected from the values compiled by Fabiato A. Fabiato. Am. J. Physiol. 245 (Cell Physiol. 14): C1-C14, 1983. The maximal SR Ca uptake in the presence of 30 microM ruthenium red was fit by an uptake curve with a maximal uptake of 5.16 nmol/mg cell protein and a K 1/2 of 1.0 microM. In the presence of 5 microM thapsigargin and no ruthenium red, a significant Ca uptake attributed to mitochondria was measured between 10 and 100 microM free Ca. Rapid changes in free Ca concentration (Ca) measured with a Ca electrode were slower than simultaneous measurements of free Ca with indo-1 in permeabilized myocytes. However, oxalate, which buffers Ca and maximizes SR Ca uptake, increased the uptake rate and eliminated the difference in free Ca measured with Ca electrode and indo-1. This suggests that spatial gradients of Ca exist in permeabilized myocytes without Ca buffering. The new estimates of the buffering of intracellular Ca in cardiac myocytes should be valuable in developing quantitative insights into cardiac Ca regulation.
Hove‐Madsen et al. (Mon,) reported a other. Thapsigargin and ruthenium red was evaluated on Passive Ca binding and SR Ca uptake. Passive Ca buffering in permeabilized rabbit ventricular myocytes was described by a two-site Michaelis curve (K1=0.42 microM, K2=79 microM), approximately twice the previously expected value.