Anesthetic alteration of ryanodine binding by cardiac calcium release channels

Abstract

Differential cardiac contractile depression by volatile anesthetics is well documented, and evidence points to differing actions on the myocardial sarcoplasmic reticulum (SR). Since the Ca 2+-release channel (CaRC) of the SR binds ryanodine with high-affinity when opened by micromolar Ca 2+ concentrations, ryanodine binding to cardiac SR membrane vesicles was employed as an assay of anesthetic modulation of CaRC activity. Canine ventricle was homogenized, centrifuged preparatively and then differentially on a sucrose gradient. A fraction enriched with CaRCs was defined by: the presence of a ∼450 kDa protein consistent with CaRC; ∼3-fold enhancement of vesicular 45Ca 2+ uptake by ruthenium red; Ca 2+-activated [ 3H]ryanodine binding. Specific binding of 10 nM ryanodine was activated by > 0.5 μ M Ca 2+ and was maximal at ∼6 pmol/mg protein in ≥ 20 μM Ca 2+. Halothane (1.5%), but not isoflurane, shifted the Ca 2+-dependence of ryanodine binding to lower [Ca 2+]. With submaximal activation by 5 μM Ca 2+, 1.5% and 0.75% halothane enhanced binding of 10–80 μM ryanodine, while 2.5% isoflurane and 3.5% enflurane did not. A plot of bound/free vs. bound ryanodine suggests that halothane causes a dose-dependent increase in ryanodine binding to a high-affinity site, while isoflurane has no such action. In intact myocardium, this effect will decrease Ca 2+ retention in the SR so that less Ca 2+ will be available to activate contractions, consistent with halothane's depressant action.