REGULATION OF PHOSPHOINOSITIDE-LINKED CNS RECEPTORS

生化、药理学和分子生物学方法有 整合于磷脂酰肌醇(PPI)介导的信号研究 中枢神经系统中的转导，重点是PPI连接的M受体 (MAChR)S，朝着建立细胞基础的最终目标 它们在正常和功能失调的大脑状态下的调节。活体内 研究提供了生理学和解剖学的优势 实验准备过程中的关系基本保持不变。他们 与之相辅相成的是对培养的神经源性细胞系的研究 准备工作要简单得多，而且可以通过实验获得。 这两种方法结合在一起，可以让人摆出直截了当的姿势 令人信服的实验问题。根据已知的mRNAs分布 对于大脑中的AChR亚型，我们将使用 受体放射自显影、免疫沉淀和放射自显影的联合方法 信使核糖核酸杂交方法。二异丙基氟磷酸盐-和 用苯海索治疗的大鼠将被用来研究慢性 胆碱能刺激。培养细胞研究将探索分布 MAChRs与同源激动剂诱导受体的作用机制 隔离，包括G蛋白在这一过程中的可能作用 进程。表达多个PPI连接受体的细胞系(In 除了腺苷环化酶连接的受体)将被用来确定 不同受体之间和细胞内的串扰程度 信号系统。我们将探索IP3受体在多大程度上发挥作用 可能受环磷酸腺苷依赖的蛋白激酶调节。的效果 关键酶和受体在信号转导事件中的过度表达 将会被调查。在培养的细胞中的证据将被证明 支持或反对Li+产生治疗效果的假设 在躁郁症精神病患者中作为其抑制的结果 肌醇一磷酸酶(IP-Pase)，它被认为是 导致细胞内可用肌醇耗尽 磷脂酰肌醇(PI)合成酶。我们将测量肌醇和 肌醇磷酸盐、肌醇脂类及其前体： 静息和毒扁豆碱条件下的磷脂酸盐、DAG和CDP-DAG Li+的存在和不存在时的刺激。分析和标记 对PPI周期脂类的研究将利用其已知的 在硬脂酰基花生四烯酰基DAG物种中的富集物。我们还将进一步 探讨关键蛋白在体内和体外的调控及性质 脂肪酶、PI合成酶。后者的基因将被克隆， 将为免疫组织化学研究准备抗体，并 将探索编码PI合成酶的多个基因的可能性。这个 该酶的信使核糖核酸水平的增强或减少，以及 肌醇3P-合成酶和IP-Pase将通过Northern blotts进行测定，以及 在适当的情况下，通过原位杂交在整个大脑的部分组织中进行。 各个子项目共同为共同目标作出贡献。 对信号转导途径调控机制的认识 在构成异常行为状态的中枢神经系统中。

Biochemical, pharmacological and molecular biological approaches are integrated in an investigation of phosphoinositide (PPI)-mediated signal transduction in the CNS, with emphasis on PPI-linked muscarinic receptors (mAChR)s, toward the eventual goal of establishing the cellular basis of their regulation in normal and dysfunctional brain states. In vivo studies offer the advantage that physiological and anatomical relationships in the experimental preparation remain largely intact. They are complemented by studies in cultured cell lines of neural origin preparations that are vastly simpler and experimentally accessible. Together, the two approaches permit one to pose straightforward yet cogent experimental questions. Based on the known distribution of mRNAs for AChR isoforms in brain, we will explore their regulation using the combined approaches of receptor autoradiography, immunoprecipitation and mRNA hybridization methods. Diisopropylfluorophosphate- and trihexyphenidyl-treated rats will be used to study effects of chronic cholinergic stimulation. Cultured cell studies will explore distribution of mAChRs and the mechanism of homologous agonist-induced receptor sequestration, including the possible role for G-proteins in this process. A cell line expressing multiple PPI-linked receptors (in addition to adenyl cyclase-linked receptors) will be used to determine the extent of cross-talk among different receptors and intracellular signalling systems. We will explore to what degree IP3 receptor function may be regulated by cyclic AMP-dependent protein kinase. Effects of overexpression of key enzymes and receptors on signal transduction events will be investigated. Evidence in cultured cells will be brought to bear for or against the hypothesis that Li+ produces its therapeutic effect in manic depressive psychoses as a consequence of its inhibition of inositol monophosphate phosphatase (IP-Pase), which has been presumed to result in depletion of intracellular inositol available to phosphatidylinositol (PI) synthase. We will measure inositol and the inositol phosphates, the inositol lipids and their precursors: phosphatidate, DAG, and CDP-DAG, under conditions of rest and muscarinic stimulation in the presence and absence of Li+. Analytical and labeling studies on the PPI cycle lipids will take advantage of their known enrichment in the stearoyl arachidonoyl DAG species. We will also further explore in vivo and in vitro the regulation and properties of the key lipid enzyme, PI synthase. The gene for the latter will be cloned, antibodies will be prepared for immunohistochemistry studies, and the possibility of multiple genes encoding PI synthase will be explored. The enhancement or depletion of mRNA levels for this enzyme, as well as inositol 3P-synthase and IP-Pase, will be measured by northern blots, and where appropriate in sections of whole brain, by in situ hybridization. Together, the individual subprojects will contribute to the common goal of understanding regulatory mechanisms of signal transduction pathways in the CNS that underlie abnormal behavioral states.