Hollmann Markus W, Herroeder Susanne, Kurz Katrin S, Hoenemann Christian W, Struemper Danja, Hahnenkamp Klaus, Durieux Marcel E
Department of Anesthesiology and Intensive Care Medicine, University Hospital Heidelberg, Germany.
Anesthesiology. 2004 Apr;100(4):852-60. doi: 10.1097/00000542-200404000-00015.
Several beneficial effects of local anesthetics (LAs) were shown to be due to inhibition of G protein-coupled receptor signaling. Differences in exposure time might explain discrepancies in concentrations of LAs required to achieve these protective effects in vivo and in vitro (approximately 100-fold higher). Using Xenopus oocytes and human neutrophils, the authors studied time-dependent effects of LAs on G protein-coupled receptor signaling and characterized possible mechanisms and sites of action.
Measurement of agonist-induced Ca2+-activated Cl currents, using a two-electrode voltage clamp technique, and determination of superoxide anion production by cytochrome c assay were used to assess the effects of LAs on G protein-coupled receptor signaling in oocytes and primed and activated human neutrophils, respectively. Antisense knockdown of G alpha q protein and inhibition of various proteins within the signaling pathway served for defining mechanisms and sites of action more specifically.
LAs attenuated G protein-coupled receptor signaling in both models in a time-dependent and reversible manner (lidocaine reduced lysophosphatidic acid signaling to 19 +/- 3% after 48 h and 25 +/- 2% after 6 h of control response in oocytes and human neutrophils, respectively). Whereas no effect was observed after extracellularly applied or intracellularly injected QX314, a lidocaine analog, using G alpha q-depleted oocytes, time-dependent inhibition also occurred after intracellular injection of QX314 into undepleted oocytes. Inhibition of phosphatases or protein kinases and agonist-independent G-protein stimulation, using guanosine 5'-O-3-thiotriphosphate or aluminum fluoride, did not affect time-dependent inhibition by LAs.
Inhibition of G protein-coupled receptor signaling by LAs was found to be time dependent and reversible. Critically requiring G alpha q-protein function, this effect is located downstream of guanosine diphosphate-guanosine triphosphate exchange and is not dependent on increased guanosine triphosphatase activity, phosphatases, or protein kinases.
局部麻醉药（LAs）的几种有益作用被证明是由于抑制了G蛋白偶联受体信号传导。暴露时间的差异可能解释了在体内和体外实现这些保护作用所需的局部麻醉药浓度的差异（体外浓度大约高100倍）。作者使用非洲爪蟾卵母细胞和人类中性粒细胞，研究了局部麻醉药对G蛋白偶联受体信号传导的时间依赖性影响，并对可能的作用机制和作用位点进行了表征。
分别使用双电极电压钳技术测量激动剂诱导的Ca2+激活的Cl电流，以及通过细胞色素c测定法测定超氧阴离子的产生，以评估局部麻醉药对卵母细胞和预激活及激活的人类中性粒细胞中G蛋白偶联受体信号传导的影响。对Gαq蛋白进行反义敲低以及抑制信号通路中的各种蛋白质，以更具体地确定作用机制和作用位点。
局部麻醉药在两种模型中均以时间依赖性和可逆的方式减弱G蛋白偶联受体信号传导（利多卡因在48小时后将溶血磷脂酸信号传导降低至对照反应的19±3%，在6小时后分别降低至25±2%，在卵母细胞和人类中性粒细胞中）。使用缺乏Gαq的卵母细胞时，细胞外应用或细胞内注射利多卡因类似物QX-314后未观察到效果，而将QX-314细胞内注射到未耗尽的卵母细胞中后也出现了时间依赖性抑制。使用鸟苷5'-O-3-硫代三磷酸或氟化铝抑制磷酸酶或蛋白激酶以及激动剂非依赖性G蛋白刺激，并不影响局部麻醉药的时间依赖性抑制作用。
发现局部麻醉药对G蛋白偶联受体信号传导的抑制是时间依赖性和可逆的。这种效应严重依赖Gαq蛋白功能，位于二磷酸鸟苷-三磷酸鸟苷交换的下游，并且不依赖于三磷酸鸟苷酶活性、磷酸酶或蛋白激酶的增加。
