Wu Q Y, Feher J J
Virginia Commonwealth University, Medical College of Virginia, Department of Physiology, Richmond 23298, USA.
J Mol Cell Cardiol. 1995 Sep;27(9):1965-75. doi: 10.1016/0022-2828(95)90018-7.
The effect of 15 min of global, normothermic ischemia on 3H-ryanodine binding and the oxalate-supported Ca2+ uptake of cardiac sarcoplasmic reticulum (SR) was investigated in parallel using ventricular homogenates of isolated perfused rat hearts. Ischemia increased the Ca2+ efflux under the uptake assay conditions, as demonstrated by the greater stimulation of Ca2+ uptake by high concentrations of ryanodine (+RY) to close the SR Ca2+ channel. This effect was partially reversed by reperfusion. Ischemia depressed Ca2+ uptake rate -RY at free [Ca2+] of 0.4 microM and above, while the depression + RY was significant only above 10 microM Ca2+. 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 Ca2+ uptake rate. Thapsigargin or cyclopiazonic acid proportionally depressed Ca2+ uptake rate +RY and -RY and produced distinctly different effects of ischemia. Ischemia did not change the Bmax or Kd for equilibrium 3H-ryanodine binding, or the Hill coefficient or KCa for the [Ca2+]-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 Ca2+ uptake rates measured in the homogenate.
利用离体灌注大鼠心脏的心室匀浆，并行研究了15分钟全心常温缺血对3H-ryanodine结合以及心脏肌浆网（SR）草酸盐支持的Ca2+摄取的影响。缺血在摄取试验条件下增加了Ca2+外流，高浓度ryanodine（+RY）对Ca2+摄取的更大刺激证明了这一点，以关闭SR Ca2+通道。再灌注可部分逆转这种效应。在游离[Ca2+]为0.4 microM及以上时，缺血降低了Ca2+摄取率 -RY，而+RY时的降低仅在Ca2+高于10 microM时显著。我们检验了以下假设：通过添加毒胡萝卜素或环匹阿尼酸单独抑制Ca-ATPase，可以重现缺血对匀浆Ca2+摄取率的影响。毒胡萝卜素或环匹阿尼酸成比例地降低了Ca2+摄取率 +RY和 -RY，并产生了与缺血明显不同的效应。缺血并未改变平衡3H-ryanodine结合的Bmax或Kd，也未改变平衡3H-ryanodine结合的[Ca2+]依赖性的Hill系数或KCa。在摄取条件下测量的ryanodine结合速率因缺血而增加，并因再灌注而进一步增加。缺血对与高亲和力ryanodine结合位点平衡结合的速率和程度的影响与在匀浆中测量的对SR Ca2+摄取率的高度可重复效应无关。
