Rooney T A, Hager R, Stubbs C D, Thomas A P
Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
J Biol Chem. 1993 Jul 25;268(21):15550-6.
The ability of halothane to stimulate phospholipase C (PLC) was examined in turkey erythrocyte membranes prepared from [3H]inositol-labeled turkey erythrocytes by measuring [3H]inositol phosphate formation ([3H]InsP) in the presence and absence of G-protein activation. In the presence of guanosine 5'-3-O-(thio)triphosphate) (GTP gamma S), halothane (0.5-10 mM) caused a dose-dependent activation of PLC. The EC50 value for halothane-induced PLC activation was 2.8 +/- 0.3 mM. Halothane (0.1-30 mM) had no effect on PLC activity in the absence of G-protein activation and did not affect Ca(2+)-dependent PLC activity. The activation of PLC by GTP gamma S occurred after an initial lag period of 60 s which was followed by a linear increase in [3H]InsP. Halothane dose-dependently decreased the lag period for GTP gamma S-induced PLC activation (minimal value 15 s) and increased the rate of [3H]InsP formation at all time points following this lag. As a result, halothane shifted the EC50 value for GTP gamma S-induced PLC activation to the left (4-fold) and increased its maximal response. Halothane also caused a dose-dependent activation of PLC in the presence of AlF4-. Half-maximal stimulation of AlF4(-)-activated PLC occurred with an EC50 value of 2.9 +/- 0.4 mM halothane, which is similar to the halothane dose giving half-maximal stimulation of PLC in the presence of GTP gamma S. At low doses (0.1-0.3 mM) halothane inhibited both isoproterenol- and adenosine 5'-O-(2-thiodiphosphate) (ADP beta S)-induced [3H]InsP formation, whereas at higher concentrations it stimulated PLC independent of the presence of these agonists. At concentrations chosen to reflect their different membrane/buffer partition coefficients, both hexanol (5 mM) and benzyl alcohol (20 mM) fluidized turkey erythrocyte membranes to the same degree as halothane (5 mM). However, these agents had no effect on GTP gamma S- or AlF(4-)-induced PLC activity, indicating that halothane-induced PLC activation was not secondary to changes in bulk lipid fluidity properties. Halothane also stimulated [3H]inositol bisphosphate and [3H]inositol trisphosphate formation in intact erythrocytes. These data demonstrate that the anesthetic halothane can stimulate G-protein-dependent PLC activity and modify the responsiveness of this signaling system to activation by receptor-linked agonists.
通过在有无G蛋白激活的情况下测量[3H]肌醇磷酸形成（[3H]InsP），研究了氟烷刺激火鸡红细胞膜中磷脂酶C（PLC）的能力。这些火鸡红细胞膜是由[3H]肌醇标记的火鸡红细胞制备而来。在存在鸟苷5'-3-O-（硫代）三磷酸（GTPγS）的情况下，氟烷（0.5 - 10 mM）引起PLC的剂量依赖性激活。氟烷诱导PLC激活的EC50值为2.8±0.3 mM。在没有G蛋白激活的情况下，氟烷（0.1 - 30 mM）对PLC活性没有影响，并且不影响钙依赖性PLC活性。GTPγS对PLC的激活在最初60秒的延迟期后发生，随后[3H]InsP呈线性增加。氟烷剂量依赖性地缩短了GTPγS诱导PLC激活的延迟期（最小值15秒），并在该延迟后的所有时间点增加了[3H]InsP的形成速率。结果，氟烷将GTPγS诱导PLC激活的EC50值向左移动（4倍）并增加了其最大反应。在存在AlF4-的情况下，氟烷也引起PLC的剂量依赖性激活。AlF4-激活的PLC的半数最大刺激发生时，氟烷的EC50值为2.9±0.4 mM，这与在存在GTPγS时给予PLC半数最大刺激的氟烷剂量相似。在低剂量（0.1 - 0.3 mM）时，氟烷抑制异丙肾上腺素和腺苷5'-O-（2-硫代二磷酸）（ADPβS）诱导的[3H]InsP形成，而在较高浓度时，无论这些激动剂是否存在，它都能刺激PLC。在选择反映其不同膜/缓冲液分配系数的浓度下，己醇（5 mM）和苄醇（20 mM）使火鸡红细胞膜的流动性与氟烷（5 mM）相同程度地增加。然而，这些试剂对GTPγS或AlF4-诱导的PLC活性没有影响，表明氟烷诱导的PLC激活不是由于整体脂质流动性特性的变化所致。氟烷还刺激完整红细胞中[3H]肌醇二磷酸和[3H]肌醇三磷酸的形成。这些数据表明，麻醉剂氟烷可以刺激G蛋白依赖性PLC活性，并改变该信号系统对受体连接激动剂激活的反应性。
