Snyder Gretchen L, Galdi Stacey, Hendrick Joseph P, Hemmings Hugh C
Department of Neuropharmacology, Intra-Cellular Therapies, Inc. (ITI), Audubon Business and Technology Center, 3960 Broadway, New York, NY 10032, USA.
Neuropharmacology. 2007 Oct;53(5):619-30. doi: 10.1016/j.neuropharm.2007.07.008. Epub 2007 Jul 24.
Isoflurane, propofol and ketamine are representative general anesthetics with distinct molecular mechanisms of action that have neuroprotective properties in models of excitotoxic ischemic damage. We characterized the effects of these agents on neuronal glutamate and dopamine signaling by profiling drug-induced changes in brain intracellular protein phosphorylation in vivo to test the hypothesis that they affect common downstream effectors. Anesthetic-treated and control mice were killed instantly by focused microwave irradiation, frontal cortex and striatum were removed, and the phosphorylation profile of specific neuronal signaling proteins was analyzed by immunoblotting with a panel of phospho-specific antibodies. At anesthetic doses that produced loss of righting reflex, isoflurane, propofol, and ketamine all reduced phosphorylation of the activating residue T183 of ERK2 (but not of ERK1); S897 of the NR1 NMDA receptor subunit; and S831 (but not S845) of the GluR1 AMPA receptor subunit in cerebral cortex. At sub-anesthetic doses, these drugs only reduced phosphorylation of ERK2. Isoflurane and ketamine also reduced phosphorylation of spinophilin at S94, but oppositely regulated phosphorylation of presynaptic (tyrosine hydroxylase) and postsynaptic (DARPP-32) markers of dopaminergic neurotransmission in striatum. These data reveal both shared and agent-specific actions of CNS depressant drugs on critical intracellular protein phosphorylation signaling pathways that integrate multiple second messenger systems. Reduced phosphorylation of ionotropic glutamate receptors by all three anesthetics indicates depression of normal glutamatergic synaptic transmission and reduced potential excitotoxicity. This novel approach indicates a role for phosphorylation-mediated down-regulation of glutamatergic synaptic transmission by general anesthetics and identifies specific in vivo targets for focused evaluation of anesthetic mechanisms.
异氟烷、丙泊酚和氯胺酮是具有独特分子作用机制的代表性全身麻醉剂，在兴奋性毒性缺血损伤模型中具有神经保护特性。我们通过分析药物诱导的体内脑内细胞内蛋白质磷酸化变化，来表征这些药物对神经元谷氨酸和多巴胺信号传导的影响，以检验它们影响共同下游效应器的假说。经麻醉处理的小鼠和对照小鼠通过聚焦微波辐射立即处死，取出额叶皮质和纹状体，并用一组磷酸特异性抗体通过免疫印迹法分析特定神经元信号蛋白的磷酸化谱。在产生翻正反射消失的麻醉剂量下，异氟烷、丙泊酚和氯胺酮均降低了ERK2（而非ERK1）激活残基T183的磷酸化；NR1 NMDA受体亚基的S897；以及大脑皮质中GluR1 AMPA受体亚基的S831（而非S845）的磷酸化。在亚麻醉剂量下，这些药物仅降低了ERK2的磷酸化。异氟烷和氯胺酮还降低了亲环蛋白在S94处的磷酸化，但对纹状体中多巴胺能神经传递的突触前（酪氨酸羟化酶）和突触后（DARPP - 32）标志物的磷酸化有相反的调节作用。这些数据揭示了中枢神经系统抑制药物对整合多个第二信使系统的关键细胞内蛋白质磷酸化信号通路的共同作用和药物特异性作用。所有三种麻醉剂均降低离子型谷氨酸受体的磷酸化，表明正常谷氨酸能突触传递受到抑制，潜在兴奋性毒性降低。这种新方法表明全身麻醉剂通过磷酸化介导的谷氨酸能突触传递下调发挥作用，并确定了用于麻醉机制重点评估的特定体内靶点。
