GLUTAMATERGIC AND IP3 SIGNALING IN CNS AND PERIPHERY

磷酸肌醇信号系统是一种广泛存在的机制 用于从神经递质、激素和生长 影响细胞内过程的因素。此应用程序的重点是 两个与磷酸肌醇有关新发现的研究 信号传导：（1）在高等动物物种（豚鼠、兔、恒河猴）中 在人神经母细胞瘤细胞中，锂增加 乙酰胆碱刺激的第二信使Ins（1，4，5）P3。(2)在 在没有添加激动剂的情况下， 大脑皮层切片，通过激活NMDA受体， 提高Ins（1，4，5）P3。本提案的目的是了解Li + 提高激动剂刺激的ins（1，4，5）P3，Li+如何提高细胞外 谷氨酸，以及什么是生理相关性和后果， 细胞信号Li+对突触谷氨酸调节的影响将 通过研究切片、神经胶质细胞和 突触体，PKC调节的胞吐作用（DAG水平和 选择性PKC抑制剂）和谷氨酸释放的内源性抑制剂。 Li+对突触体谷氨酸释放的影响 前突触模型）将被确定。Li+之间的连接 谷氨酸和Ins（1，4，5）P3的变化。将使用抑制剂 谷氨酸释放和NMDA受体激动剂。一般假设 Li+可提高任何激活受体产生的Ins（1，4，5）P3 将测试通过G蛋白或通过增加的Ca2+流入与PLC偶联的 通过测定Li+对皮层脑片Ins（1，4，5）P3的影响， 在大量合适的神经递质的存在下。在 与灵长类动物中心的寺泽荣博士合作， 在未麻醉和未应激的恒河猴大脑皮层中的释放 将通过推拉灌注和 在存在和不存在锂的情况下进行微透析。 初步 该模型的研究非常有前途。 释放其他 还将研究神经递质。 锂的作用 皮质下结构中Ins（1，4，5）P3和Ins（1，3，4，5）P4的积聚 将被进一步研究，主要是在海马体中。的影响 急性和慢性锂对人神经母细胞瘤的各种参数的影响 细胞，即，Ins（1，4，5）P3和Ins（1，3，4，5）P4（慢性），数量和 Ins（1，4，5）P3受体、DAG和胞质Ca 2+的亲和力将 接受检查。这些效应也将在分化细胞中进行研究， 更接近于成年神经元。的分子机制 锂升高的Ins（1，4，5）P3最好在胰腺中研究， 进一步研究;即，是由于PLC增加引起的标高 活性或抑制Ins（1，4，5）P3 5-磷酸酶，或两者兼而有之？最后， NMDA和Ins（1，4，5）P3受体可能下调， 将研究用锂长期治疗的豚鼠。初步 数据表明小鼠中Ins（1，4，5）P3受体的下调。 这些 研究可能揭示治疗的分子机制， 锂的作用，从长远来看可能会带来医疗效益。

The phosphoinositide signaling system is a widespread mechanism for transducing signals from neurotransmitters, hormones, and growth factors to intracellular processes. The focus of this application is an investigation of two recent discoveries related to phosphoinositide signaling: (1) In higher animal species (guinea pig, rabbit, rhesus monkey) and in human neuroblastoma cells, lithium increases acetylcholine-stimulated second messenger Ins(1,4,5)P3. (2) in the absence of added agonist, lithium raises extracellular glutamate in cerebrocortical slices, which, via activation of the NMDA receptor, raises Ins(1,4,5)P3. The objectives of this proposal are to learn how Li+ elevates agonist-stimulated ins(1,4,5)P3, how Li+ raises extracellular glutamate, and what are the physiological relevance and consequences for cell signaling. The effect of Li+ on synaptiC glutamate regulation will be probed by studies of uptake of glutamate by slices, glia and synaptosomes, PKC-modulated exocytosis (levels of DAG and effect of selective PKC inhibitors) and endogenous inhibitors of glutamate release. The effect of Li+ on glutamate release from synaptosomes (an isolated presynaptic model) will be determined. The connection between Li+ linked changes in glutamate and Ins(1,4,5)P3. will be explored using inhibitors of glutamate release and NMDA receptor agonists. The general hypothesis that Li+ elevates Ins(1,4,5)P3 generated by any activated receptor coupled to PLC via G proteins or via increased Ca2+ influx will be tested by measuring the effect of Li+ on Ins(1,4,5)P3 in cerebrocortical slices in the presence of a wide selection of appropriate neurotransmitters. In collaboration with Dr. Ei Terasawa at the Primate Center, glutamate release in cerebral cortex of unanesthetized and unstressed rhesus monkeys will be measured in vivo by push-pull perfusion and by microdialysis in the presence and absence of lithium. preliminary studies in this model are very promising. The release of other neurotransmitters will also be studied. The effects of lithium on Ins(1,4,5)P3 and Ins(1,3,4,5)P4 accumulation in subcortical structures will be further explored, primarily in the hippocampus. The effects of acute and chronic lithium on various parameters in human neuroblastoma cells, i.e., Ins(1,4,5)P3 and Ins(1,3,4,5)P4 (chronic), number and affinities of the Ins(1,4,5)P3 receptor, DAG, and cytosolic Ca2+, will be examined. These effects will also be studied in differentiated cells, which more closely resemble adult neurons. The molecular mechanism of lithium-elevated Ins (1,4,5)P3 can best be studied in pancreas and will be further investigated; i.e., is the elevation due to increased PLC activity or inhibition of Ins(1,4,5)P3 5-phosphatase, or both? Lastly, possible down-regulation of the NMDA- and Ins(1,4,5)P3-receptors in guinea pigs chronically treated with lithium will be studied. Preliminary data suggest down-regulation of the Ins(l,4,5)P3 receptor in mice. These studies may throw light on the molecular mechanism of the therapeutic action of lithium, which in the long run may lead to medical benefit.