Levin A, Blanck T J
Department of Anesthesiology, Cornell University Medical College, New York, New York, USA.
Anesthesiology. 1995 Jul;83(1):120-6. doi: 10.1097/00000542-199507000-00015.
Ca2+ plays an important role in signal transduction and anesthetic mechanisms. To date, no one has observed a direct effect of volatile anesthetics on a Ca(2+)-binding protein. We therefore examined the effects of halothane and isoflurane on the Ca(2+)-binding properties of bovine brain calmodulin.
The fluorescence emission of calmodulin was obtained over a range of Ca2+ concentrations (10(-7)-10(-4)M) in the presence and absence of halothane and isoflurane. The intrinsic tyrosine fluorescence of calmodulin was measured at an excitation wavelength of 280 nm and an emission wavelength of 320 nm. Fluorescence measurements were carried out in 50 mM hydroxyethylpiperazineethane sulfonic acid, 100 mM KC1, and 2 mM ethyleneglycol-bis-(beta-aminoethyl ether) tetraacetic acid at pH 7.0 and 37 degrees C. Experiments were performed in polytetrafluorethylene-sealed cuvettes so that the volatile anesthetic concentrations remained constant. The titration data were analyzed in two ways. The data were fit to the Hill equation by using nonlinear regression analysis to derive the Hill coefficient and the dissociation constant. The data were also analyzed by two-way analysis of variance with multiple comparisons to determine statistically significant effects. Volatile anesthetic concentrations were measured by gas chromatography.
The presence of volatile anesthetics altered the Ca(2+)-binding affinity of calmodulin in a dose-dependent fashion. At 0.57% (0.25 mM) halothane and 1.7% (0.66 mM) isoflurane, the affinity of calmodulin for Ca2+ relative to control was decreased. However, at higher concentrations of both anesthetics, the affinity for Ca2+ was increased. When the volatile anesthetics were allowed to evaporate from the experimental solutions, the observed rightward shift of the calmodulin-Ca2+ binding curve for Ca2+ at low concentrations of the anesthetics returned to the control position. The leftward shift seen at high concentrations of the anesthetics was irreversible after evaporation of 8.7% (3.3 mM) isoflurane and 5.7% (2.5 mM) halothane.
These data demonstrate a complex interaction of two hydrophobic volatile anesthetics with calmodulin. A biphasic effect was observed both for halothane and for isoflurane. Calmodulin, an EF-hand Ca(2+)-binding protein, undergoes a conformational shift when binding Ca2+, exposing several hydrophobic residues. These residues may be sites at which the anesthetics act.
钙离子在信号转导和麻醉机制中发挥着重要作用。迄今为止，尚未有人观察到挥发性麻醉剂对钙结合蛋白的直接作用。因此，我们研究了氟烷和异氟烷对牛脑钙调蛋白钙结合特性的影响。
在存在和不存在氟烷及异氟烷的情况下，于一系列钙离子浓度（10⁻⁷ - 10⁻⁴M）范围内获取钙调蛋白的荧光发射。在激发波长280nm和发射波长320nm下测量钙调蛋白的固有酪氨酸荧光。荧光测量在pH 7.0和37℃的50mM羟乙基哌嗪乙烷磺酸、100mM氯化钾和2mM乙二醇双（β - 氨基乙醚）四乙酸中进行。实验在聚四氟乙烯密封的比色皿中进行，以使挥发性麻醉剂浓度保持恒定。滴定数据采用两种方法分析。通过非线性回归分析将数据拟合到希尔方程，以得出希尔系数和解离常数。数据还通过具有多重比较的双向方差分析进行分析，以确定统计学上的显著效应。挥发性麻醉剂浓度通过气相色谱法测量。
挥发性麻醉剂的存在以剂量依赖方式改变了钙调蛋白的钙结合亲和力。在0.57%（0.25mM）氟烷和1.7%（0.66mM）异氟烷时，钙调蛋白对钙离子的亲和力相对于对照降低。然而，在两种麻醉剂的较高浓度下，对钙离子的亲和力增加。当挥发性麻醉剂从实验溶液中蒸发时，在低浓度麻醉剂下观察到的钙调蛋白 - 钙离子结合曲线向右的位移在钙离子低浓度时恢复到对照位置。在8.7%（3.3mM）异氟烷和5.7%（2.5mM）氟烷蒸发后，在高浓度麻醉剂下观察到的向左位移是不可逆的。
这些数据表明两种疏水性挥发性麻醉剂与钙调蛋白之间存在复杂的相互作用。氟烷和异氟烷均观察到双相效应。钙调蛋白是一种EF手型钙结合蛋白，在结合钙离子时会发生构象变化，暴露出几个疏水性残基。这些残基可能是麻醉剂作用的位点。
