VOLATILE ANESTHETICS AND NMDA RECEPTORS

这项拟议研究的目标是了解挥发性物质的影响。 麻醉药对N-甲基-D-天冬氨酸(NMDA)受体-通道复合体和 了解这些操作在开发和维护过程中的作用 处于麻醉状态。 我们已经证明了2种挥发性麻醉药(安氟醚和氟烷) 选择性抑制谷氨酸刺激的[~3H]MK-801与NMDA的结合 受体离子通道及阻断NMDA受体介导的~(45)Ca流入大鼠 脑部微泡。此外，这种抑制作用通过 变构NMDA调节剂甘氨酸。这些发现提出了耐人寻味的 挥发性麻醉药抑制NMDA区突触传递的可能性 通过破坏谷氨酸激活(开放)固有离子的受体 频道。 需要检验的假设是，挥发性麻醉剂会抑制 通过干扰激动剂实现NMDA受体的兴奋性神经传递 与谷氨酸识别位点的结合，2)与甘氨酸的配体结合 和/或3)谷氨酸、甘氨酸、阳离子和多胺 激活NMDA受体离子通道。这些假说将得到检验。 使用放射性标记探针进行激动剂、调节性和离子通道结合 NMDA受体复合体上的位点，以及直接测量 受体产生的细胞内钙信号。 因此，具体目标是确定以下几个方面的影响 挥发性麻醉剂(氟烷、安氟烷、异氟烷、乙醚、 环丙烷、一氧化二氮和三氯甲烷) 大鼠脑(海马体)： 1)与NMDA受体复合体上谷氨酸结合部位的配基结合， 包括使用[~3H]CGS的受体密度、亲和力和动力学常数 19755作为特异性探针。 2)与甘氨酸调节位点结合的配体，使用[~3H]CGS 19755作为 特定的探头。 3)谷氨酸、甘氨酸、二价阳离子和亚精胺对NMDA的激活 受体离子通道，以[~3H]MK-801为探针。[~3H]MK-801结合 受谷氨酸、甘氨酸和多胺(如 亚精胺)，并被二价阳离子(如锌离子)抑制。 4)NMDA受体介导的大鼠脑组织钙含量变化 由于通过跨膜通道的通量和从 细胞内储存，使用荧光染料和45Ca。 不同麻醉剂对NMDA受体结合的影响 功能将与其1)麻醉效力进行比较，2) 物理化学性质，以及3)膜扰动作用，以便 评估这些行动与制定 麻醉状态。 这项研究将增加我们对挥发性麻醉作用的理解 大脑中的信号转导过程，并探索一种机制 可能有助于麻醉状态的发展。

The goal of the proposed research is to understand the effects of volatile anesthetics on the N-methyl-D-aspartate (NMDA) receptor-channel complex and to appreciate the role of these actions in the development and maintenance of the anesthetic state. We have shown that 2 volatile anesthetics (enflurane and halothane) selectively inhibit glutamate-stimulated [3H]MK-801 binding to NMDA receptor ion channels and block NMDA receptor-mediated 45Ca flux into rat brain microvesicles. Moreover, this inhibition is attenuated by the allosteric NMDA modulator, glycine. These findings raise the intriguing possibility that volatile anesthetics depress synaptic transmission at NMDA receptors by disrupting glutamate activation (opening) of the intrinsic ion channel. The hypotheses to be tested are that volatile anesthetics depress excitatory neurotransmission at NMDA receptors by disrupting 1) agonist binding to the glutamate recognition site, 2) ligand binding to the glycine modulatory site, and/or 3) glutamate, glycine, cation and polyamine activation of NMDA receptor ion channels. These hypotheses will be tested using radiolabelled probes for agonist, regulatory and ion channel binding sites on the NMDA receptor complex, as well as direct measurement of receptor-generated intra-cellular calcium signals. Accordingly, the specific aims are to determine the effects of several volatile anesthetics (halothane, enflurane, isoflurane, diethyl ether, cyclopropane, nitrous oxide and chloroform) on the following processes in rat brain (hippocampus): 1) Ligand binding to glutamate binding sites on the NMDA receptor complex, including receptor density, affinity and kinetic constants using [3H]CGS 19755 as a specific probe. 2) Ligand binding to the glycine modulatory site, using [3H]CGS 19755 as a specific probe. 3) Glutamate, glycine, divalent cation and spermidine activation of NMDA receptor ion channels, using [3H]MK-801 as the probe. [3H]MK-801 binding is allosterically stimulated by glutamate, glycine and polyamines (such as spermidine) and inhibited by divalent cations (such as Zn2+). 4) NMDA-receptor mediated changes in calcium content of rat brain microvesicles due to flux through transmembrane channels and release from intracellular stores, using a fluorescent dye and 45Ca. The potency of the different anesthetics in affecting NMDA receptor binding functions will be compared to their 1) anaesthetic potency, 2) physiochemical properties, and 3) membrane-perturbing actions, in order to assess the relationship of these actions to the elaboration of the anesthetic state. This research will increase our understanding of volatile anesthetic action on signal transduction processes in the brain, and explore a mechanism that may contribute to the development of the anesthetic state.