Effect of ischemia and ischemia-reperfusion on ryanodine binding and Ca 2+ uptake of cardiac sarcoplasmic reticulum

Abstract

The effect of 15 min of global, normothermic ischemia on 3H-ryanodine binding and the oxalate-supported Ca 2+ uptake of cardiac sarcoplasmic reticulum (SR) was investigated in parallel using ventricular homogenates of isolated perfused rat hearts. Ischemia increased the Ca 2+ efflux under the uptake assay conditions, as demonstrated by the greater stimulation of Ca 2+ uptake by high concentrations of ryanodine (+RY) to close the SR Ca 2+ channel. This effect was partially reversed by reperfusion. Ischemia depressed Ca 2+ uptake rate -RY at free [Ca 2+] of 0.4 μ m and above, while the depression +RY was significant only above 10 μ m Ca 2+. We tested the hypothesis that inhibition of the Ca-ATPase alone. by adding thapsigargin or cyclopiazonic acid, could reproduce the effects of ischemia on the homogenate Ca 2+ uptake rate. Thapsigargin or cyclopiazonic acid proportionally depressed Ca 2+ uptake rate +RY and -RY and produced distinctly different effects of ischemia. Ischemia did not change the B max or K d for equilibrium 3H-ryanodine binding, or the Hill coefficient or K Ca for the [Ca 2+]-dependence of equilibrium 3H-ryanodine binding. The rate of ryanodine binding. measured under the uptake conditions, was increased by ischemia and further increased by reperfusion. The effect of ischemia on the rate and extent of equilibrium binding to the high-affinity ryanodine binding site were unrelated to the highly reproducible effects on SR Ca 2+ uptake rates measured in the homogenate.