ANESTHETIC ALTERATION OF RYANODINE BINDING BY CARDIAC CALCIUM-RELEASE CHANNELS

Differential cardiac contractile depression by volatile anesthetics is well documented, and evidence points to differing actions on the myocardial sarcoplasmic reticulum (SR). Since the Ca2+-release channel (CaRC) of the SR binds ryanodine with high-affinity when opened by micromolar Ca2+ 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 similar to 450 kDa protein consistent with CaRC; similar to 3-fold enhancement of vesicular Ca-45(2+) uptake by ruthenium red; Ca2+-activated [H-3]ryanodine binding. Specific binding of 10 nM ryanodine was activated by > 0.5 mu M Ca2+ and was maximal at similar to 6 pmol/mg protein in greater than or equal to 20 mu M Ca2+. Halothane (1.5%), but not isoflurane, shifted the Ca2+-dependence of ryanodine binding to lower [Ca2+]. With submaximal activation by 5 mu M ca(2+), 1.5% and 0.75% halothane enhanced binding of 10-80 mu M 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 Ca2+ retention in the SR so that less Ca2+ will be available to activate contractions, consistent with halothane's depressant action.