Lynch C, Frazer M J
Department of Anesthesiology, University of Virginia Health Sciences Center, Charlottesville 22908.
Biochim Biophys Acta. 1994 Aug 24;1194(1):109-17. doi: 10.1016/0005-2736(94)90208-9.
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 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 approximately 450 kDa protein consistent with CaRC; approximately 3-fold enhancement of vesicular 45Ca2+ uptake by ruthenium red; Ca(2+)-activated [3H]ryanodine binding. Specific binding of 10 nM ryanodine was activated by > 0.5 microM Ca2+ and was maximal at approximately 6 pmol/mg protein in > or = 20 microM Ca2+. Halothane (1.5%), but not isoflurane, shifted the Ca(2+)-dependence of ryanodine binding to lower [Ca2+]. With submaximal activation by 5 microM Ca2+, 1.5% and 0.75% halothane enhanced binding of 10-80 microM 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.
挥发性麻醉剂对心脏收缩力的差异性抑制作用已有充分记录，且有证据表明其对心肌肌浆网（SR）有不同作用。由于当SR的Ca(2+)释放通道（CaRC）在微摩尔浓度的Ca2+作用下打开时，它会以高亲和力结合ryanodine，因此ryanodine与心脏SR膜囊泡的结合被用作检测麻醉剂对CaRC活性调节的方法。将犬心室匀浆，先进行制备性离心，然后在蔗糖梯度上进行差速离心。富含CaRC的组分通过以下方式定义：存在与CaRC一致的约450 kDa蛋白质；钌红使囊泡对45Ca2+的摄取增加约3倍；Ca(2+)激活的[3H]ryanodine结合。10 nM ryanodine的特异性结合在> 0.5 microM Ca2+时被激活，在>或= 20 microM Ca2+时，结合量在约6 pmol/mg蛋白质时达到最大值。1.5%的氟烷（而非异氟烷）使ryanodine结合的Ca(2+)依赖性向更低的[Ca2+]水平偏移。在5 microM Ca2+的亚最大激活作用下，1.5%和0.75%的氟烷增强了10 - 80 microM ryanodine的结合，而2.5%的异氟烷和3.5%的恩氟烷则没有。结合/游离ryanodine与结合ryanodine的关系图表明，氟烷导致ryanodine与高亲和力位点的结合呈剂量依赖性增加，而异氟烷则无此作用。在完整心肌中，这种效应将减少SR中Ca2+的潴留，从而使可用于激活收缩的Ca2+减少，这与氟烷的抑制作用一致。
