Isoflurane: Identification of Key New Targets

描述（由申请人提供）：尽管进行了大量的研究，但对全身麻醉剂的作用仍然没有完全的了解。这项研究的目的是促进我们对异氟烷的理解，希望对所有麻醉剂有更深入的了解。异氟醚是一种卤化挥发性麻醉剂，被认为是通过抑制中枢神经系统功能来产生麻醉的。许多麻醉剂，包括异氟醚，被认为可以调节和/或直接激活GABAA受体。异氟醚对其他通道和受体也有影响。在一系列初步研究中，我们观察到异氟醚在临床相关浓度下，显著抑制PC12细胞中的神经递质释放机制。依托咪酯和异丙酚也能抑制PC12细胞的释放机制，提示抑制神经递质释放可能是麻醉剂的一个重要的一般作用。由于神经递质释放机制在PC12和神经元之间高度保守，我们假设麻醉剂干扰了神经元的神经递质释放机制，这可能代表了麻醉在完整动物中的重要作用部位。本应用程序中概述的实验将在培养的海马神经元、PC12细胞和突变小鼠中进行。这项提议有三个目标。首先，我们将确定临床上相关浓度的异氟醚是否会干扰海马神经元的神经递质释放。接下来，将确定麻醉作用部位。我们首先检查syntaxin 1A和unc-13，但其他位点包括SNAP- 25， synaptobrevin和synaptotagmin 1，也将进行研究。关于为什么选择这些站点的详细信息在此应用程序中提供。一旦神经递质释放机制靶点被确定，它们将发生突变以抑制异氟烷与靶点之间的相互作用。这些信息将用于设计具有相同突变的敲入小鼠，这将使我们能够通过一系列生理和行为测试来确定这些动物是否仍然对异氟醚（或其他麻醉剂）有反应，或者它们对麻醉剂的反应是否发生了改变。项目简介：异氟烷是一种广泛使用的挥发性麻醉剂。我们的目标是找出异氟醚是如何产生麻醉的。特别是，我们将探索对神经递质释放机制的影响，因为我们的初步数据表明，这可能是异氟醚和其他麻醉剂的关键作用部位。这可能会在未来设计出新的更有益的药物。

DESCRIPTION (provided by applicant): Despite a great deal of research, a complete understanding of the actions of general anesthetics is still not available. The objective of this research is to advance our understanding of isoflurane, with the hope of gaining insights into all anesthetics. Isoflurane, a halogenated volatile anesthetic, is thought to produce anesthesia by depressing central nervous system function. Many anesthetics, including isoflurane, are thought to modulate and/ or directly activate GABAA receptors. Isoflurane is also known to have effects on other channels and receptors. In a set of preliminary studies we observed that isoflurane, at clinically relevant concentrations, dramatically inhibited the neurotransmitter release machinery in PC12 cells. Etomidate and propofol also inhibited the release machinery in PC12 cells, suggesting that inhibition of neurotransmitter release may be an important general action of anesthetics. Because neurotransmitter release mechanisms are strongly conserved between PC12 and neurons we hypothesize that anesthetics interfere with the neuronal neurotransmitter release machinery and this may represent an important site of action for anesthesia in intact animals. The experiments outlined in this application will be carried out in cultured hippocampal neurons, PC12 cells and mutant mice. This proposal has three goals. First, we will determine whether isoflurane interferes with neurotransmitter release in hippocampal neurons at clinically relevant concentrations. Next, the anesthetic site of action will be identified. We start by examining syntaxin 1A and unc-13, but other sites including SNAP- 25, synaptobrevin and synaptotagmin 1, will be investigated as well. Details about why these sites were selected are provided in this application. Once neurotransmitter release machinery targets are identified they will be mutated in order to suppress the interaction between isoflurane and the target. This information will be used to design knock-in mice with the same mutations, which will permit us to determine, using a battery of physiological and behavioral tests, whether these animals still respond to isoflurane (or other anesthetics) or whether their responses to the anesthetics are altered.Project Narrative: Isoflurane is a widely used volatile anesthetic. Our goal is to find out how isoflurane produces anesthesia. In particular, we will explore the effect on the neurotransmitter release machinery, as our preliminary data suggests this may be a critical site of action for isoflurane and other anesthetics. This may allow for the design of new more beneficial drugs in the future.