VOLATILE ANESTHETICS AND NEURONAL CALCIUM

描述（由申请人提供）：挥发性麻醉剂（VA）介导的不动似乎主要是由于脊髓水平兴奋性神经传递的抑制。神经元通过调节细胞内Ca 2+浓度和区室化将电和化学刺激转化为有意义的生理信号。作用于特定Ca 2+调节位点的化学物质已被证明会改变VA的MAC。在细胞水平，VA已被证明可以抑制神经元细胞中的电压依赖性Ca 2+通道和Ca 2+瞬变。我们推测挥发性麻醉剂通过调节钙通道和信号传导抑制脊髓神经元产生不动性。我们将通过比较三种VA、异氟烷、氟烷和F3的作用与两种不能产生固定性的结构相关分子（非固定剂（NIM））F6和F8的作用来检验这一假设。将探索对电压依赖性（L型和N型）和配体（谷氨酸）激活的Ca 2+通道、谷氨酸介导的神经元兴奋性和突触体谷氨酸释放的影响。初步研究将涉及人类SH-SY 5 Y神经母细胞瘤细胞作为模型系统，以确定药物作用的预期靶点。我们还将研究背根神经节神经元（DRG），脊髓腹角（VH）运动神经元的原代培养以及突触体，所有分离自成年大鼠脊髓。具体目的将确定VA和NIM是否（1）在其对质膜L-和N-型电压依赖性Ca 2+通道的阻断方面不同;（2a）在其对容量性谷氨酸激活的阳离子Ca 2+通道的阻断方面不同;（2b）改变谷氨酸对引起细胞质Ca 2+瞬变的敏感性，以及对谷氨酸激活的离子型和/或代谢型受体的作用;和（2c）对脊髓突触体突触前谷氨酸释放的影响及其对Ca ~（2+）的依赖性不同。将采用全细胞和膜片钳和电流钳以及荧光方法，包括用于测量细胞内Ca 2+、质膜电位和谷氨酸释放的成像。这些研究的结果将产生重要的分子洞察到神经元中的Ca 2+信号传导，并澄清VA对Ca 2+信号传导的影响与VA介导的麻醉的不动性方面的相关性。

DESCRIPTION (provided by applicant): Immobility mediated by volatile anesthetics (VAs) appears to result largely from depression of excitatory neurotransmission at the spinal cord level. Neurons transform electrical and chemical stimuli into meaningful physiological signals by the regulation of intracellular Ca2+ concentration and compartmentalization. Chemicals that act at particular Ca2+ regulatory sites have been shown to alter the MAC of VAs. At the cellular level, VAs have been shown to inhibit voltage-dependent Ca2+ channels and Ca2+ transients in neuronal cells. We hypothesize that Volatile anesthetics produce immobility by inhibiting spinal cord neurons through the modulation of Ca2+ channels and signaling. We will test this hypothesis by comparing the effects of tree VAs, isoflurane, halothane, and F3, with that of two structurally related molecules that fail to produce immobility (non-immobilizers (NIMs)), F6 and F8. Effects on voltage-dependent (L- and N-type) and ligand (glutamate)-activated Ca2+ channels, glutamate-mediated neuronal excitability, and glutamate release from synaptosomes will be explored. Initial studies will involve human SH-SY5Y neuroblastoma cells as a model system to identify prospective targets of drug action. We will also study dorsal root ganglion neurons (DRG), and spinal ventral horn (VH)- motor neurons in primary culture as well as synaptosomes, all isolated from the adult rat spinal cord. The specific aims will determine whether VAs and NIMs (1) differ in their block of plasma membrane L- and N-type voltage dependent Ca2+ channels; (2a) differ in their block of capacitative- glutamate activated cationic Ca2+ channels; (2b) change the sensitivity of glutamate for evoking cytoplasmic Ca2+ transients, and action on glutamate-activated ionotropic and/or metabotropic receptors; and (2c) differ in their effect on the presynaptic release of glutamate from spinal cord synaptosomes and its dependence on Ca2+. Whole cell and patch voltage- and current-clamp, and fluorescence methodologies including imaging for measuring intracellular Ca2+, plasma membrane potential, and glutamate release will be employed. The results of these studies will yield important molecular insights into Ca2+ signaling in neurons and clarify the relevance of VA effects on Ca2+ signaling to the immobility aspect of VA-mediated anesthesia.