Passive Ca buffering and SR Ca uptake in permeabilized rabbit ventricular myocytes

L. Hove-Madsen, Donald M Bers

Research output: Contribution to journalArticle

80 Scopus citations

Abstract

Passive Ca binding was measured with a Ca-selective minielectrode in suspensions of permeabilized rabbit ventricular myocytes equilibrated with 5 μM thapsigargin and 30 μM 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 μM, n1 = 1.27 nmol/mg cell protein and K2 = 79 μM, 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 μM 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 μM. In the presence of 5 μM thapsigargin and no ruthenium red, a significant Ca uptake attributed to mitochondria was measured between 10 and 100 μM 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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume264
Issue number3 33-3
StatePublished - 1993
Externally publishedYes

Keywords

  • calcium electrodes
  • cardiac muscle
  • indo-1
  • mitochondria
  • thapsigargin

ASJC Scopus subject areas

  • Cell Biology
  • Clinical Biochemistry
  • Physiology

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