REGULATION OF PHOSPHOINOSITIDE-LINKED CNS RECEPTORS

生物化学、药理学和分子生物学方法是 整合在磷脂酰肌醇（PPI）介导的信号的研究中 CNS中的转导，重点是PPI连接的毒蕈碱受体 （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。体内 研究提供了生理和解剖学上的优势， 实验准备中的关系基本上保持不变。他们 通过对培养的神经源性细胞系的研究， 这些制备方法简单得多，实验上也可以实现。 这两种方法结合在一起， 有说服力的实验问题。 根据已知的mRNA分布 对于脑中的AChR亚型，我们将使用 受体放射自显影、免疫沉淀和 mRNA杂交方法。氟磷酸二异丙酯-和 苯海索治疗的大鼠将用于研究慢性 胆碱能刺激培养细胞研究将探索分布 同源激动剂诱导的受体机制 螯合，包括G蛋白在其中的可能作用 过程表达多个PPI连接受体的细胞系（在 除了腺苷酸环化酶连接受体）将用于确定 不同受体之间的串扰程度和细胞内 信号系统。我们将探索IP 3受体功能的程度 可能受环腺苷酸依赖性蛋白激酶的调节。的影响 信号转导事件中关键酶和受体的过度表达 将被调查。培养细胞中的证据将被证明 支持或反对Li+产生治疗作用的假设 在躁狂抑郁性精神病中， 肌醇一磷酸磷酸酶（IP-Pl），被认为是 导致细胞内肌醇的消耗， 磷脂酰肌醇（PI）合酶。我们将测量肌醇和 肌醇磷酸、肌醇脂质及其前体： 磷脂酸、DAG和CDP-DAG，在静息和毒蕈碱条件下 在Li+存在和不存在下的刺激。分析和贴标 对PPI循环脂质的研究将利用其已知的 硬脂酰花生四烯酸DAG物质的富集。我们还将进一步 探索在体内和体外的调控和关键属性 脂质酶，PI合成酶。后者的基因将被克隆， 将制备用于免疫组织化学研究的抗体， 将探索多个基因编码PI合酶的可能性。的 这种酶的mRNA水平的增强或耗尽，以及 肌醇3 β-合酶和IP-β将通过北方印迹法测量，和 在适当的情况下，在全脑切片中，通过原位杂交。 各个子项目将共同为实现共同目标作出贡献 了解信号转导途径的调节机制 中枢神经系统的异常行为

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.