Hemmings Hugh C, Yan Wayne, Westphalen Robert I, Ryan Timothy A
Department of Anesthesiology, Weill Medical College of Cornell University, New York, New York 10021, USA.
Mol Pharmacol. 2005 May;67(5):1591-9. doi: 10.1124/mol.104.003210. Epub 2005 Feb 22.
General anesthetics have marked effects on synaptic transmission, but the mechanisms of their presynaptic actions are unclear. We used quantitative laser-scanning fluorescence microscopy to analyze the effects of the volatile anesthetic isoflurane on synaptic vesicle cycling in cultured neonatal rat hippocampal neurons monitored using either transfection of a pH-sensitive form of green fluorescent protein fused to the luminal domain of VAMP (vesicle-associated membrane protein), (synapto-pHluorin) or vesicle loading with the fluorescent dye FM 1-43. Isoflurane reversibly inhibited action potential-evoked exocytosis over a range of concentrations, with little effect on vesicle pool size. In contrast, exocytosis evoked by depolarization in response to an elevated extracellular concentration of KCl, which is insensitive to the selective Na+ channel blocker tetrodotoxin, was relatively insensitive to isoflurane. Inhibition of exocytosis by isoflurane was resistant to bicuculline, indicating that this presynaptic effect is not caused by the well known GABA(A) receptor modulation by volatile anesthetics. Depression of exocytosis was mimicked by a reduction in stimulus frequency, suggesting a reduction in action potential initiation, conduction, or coupling to Ca2+ channel activation. There was no evidence for a direct effect on endocytosis. The effects of isoflurane on synaptic transmission are thus caused primarily by inhibition of action potential-evoked synaptic vesicle exocytosis at a site upstream of Ca2+ entry and exocytosis, possibly as a result of Na+ channel blockade and/or K+ channel activation, with the possibility of lesser contributions from Ca2+ channel blockade and/or soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated vesicle fusion.
全身麻醉药对突触传递有显著影响，但其突触前作用机制尚不清楚。我们使用定量激光扫描荧光显微镜，分析挥发性麻醉药异氟烷对培养的新生大鼠海马神经元中突触囊泡循环的影响，监测方法为转染与囊泡相关膜蛋白（VAMP）腔结构域融合的pH敏感型绿色荧光蛋白（突触pH荧光蛋白）或用荧光染料FM 1-43装载囊泡。异氟烷在一定浓度范围内可逆性抑制动作电位诱发的胞吐作用，对囊泡池大小影响较小。相比之下，细胞外高浓度KCl引起的去极化诱发的胞吐作用对异氟烷相对不敏感，这种去极化诱发的胞吐作用对选择性Na⁺通道阻滞剂河豚毒素不敏感。异氟烷对胞吐作用的抑制作用对荷包牡丹碱有抗性，表明这种突触前效应不是由挥发性麻醉药对众所周知的GABAA受体的调节引起的。刺激频率降低可模拟异氟烷对胞吐作用的抑制，提示动作电位起始传导或与Ca²⁺通道激活的偶联减少。没有证据表明异氟烷对内吞作用有直接影响。因此，异氟烷对突触传递的影响主要是由于在Ca²⁺内流和胞吐作用上游位点抑制动作电位诱发的突触囊泡胞吐作用，可能是由于Na⁺通道阻断和/或K⁺通道激活，也可能有较小程度的Ca²⁺通道阻断和/或可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体介导的囊泡融合的作用。
