CALCIUM SENSITIVE ADENYLYL CYCLASES

两个第二信使cAMP和Ca 2+的信号传导是细胞凋亡的核心。 通过激素控制许多生理过程， 神经传递素 Ca 2+敏感性（可刺激性和 心脏、离散脑区、垂体中的腺苷酸环化酶， 血小板等提供了一种可行的手段， 系统可以紧密协调。 这一潜在的重要作用， Ca 2+敏感的腺苷酸环化酶依赖于是否生理升高 细胞质中Ca 2+（[Ca 2 +]）的释放引起活性的变化， 在体外研究中预期。 当前的指导性前提 项目是需要清楚地了解[Ca 2 +]i是如何分布的 在细胞域和时间上。 这一基础极大地丰富了 提出的问题和获得的信息的质量。 电流 该实验室的研究表明，生理[Ca 2 +] i确实会升高， 抑制细胞系中的内源性Ca 2+可降解腺苷酸环化酶， C6-2B神经胶质瘤 然而，非常引人注目的是，当抑制功效 [Ca 2 +]i升高的进入和释放分量分别为 评估，只有Ca 2 +-进入抑制cAMP的合成，而释放， 没有效果。 此外，在HEK 293细胞中，用各种不同的细胞因子转染。 腺苷酸环化酶cDNA，Ca 2 +-进入足以调节Ca 2 +- 可复制和可刺激的腺苷酸环化酶。 所以现在 我想问，这种对进入的依赖是否是一种预先确定的结果 对于在C6-2B细胞中表达的环化酶，因为C6=2B中的Ca 2+进入 细胞在将[Ca 2 +]i递送至环化酶方面特别有效，或 这种敏感性是否是Ca 2+敏感腺苷酸的固有特性 环化酶，即使当它们在HEK 293细胞中转染时。 这些问题 将通过单个C6-B细胞的成像实验来解决，以确定 是否在血浆中获得不同浓度的Ca 2 + 细胞膜对Ca ~（2+）进入或释放的反应; 还将进行平行的免疫组织化学实验， 腺苷酸环化酶是否存在于高Ca 2+区域（所谓的 caveolae）。 是否同样依赖于进入与释放 在HEK 293细胞中表达的腺苷酸环化酶cDNA将被展示。 确定，作为探索体内敏感性是否 钙离子可通过分子生物学方法从体外敏感性中分离出来， 使用嵌合和缺失方法操纵环化酶cDNA。 这些研究将通过探索以下领域的实验来补充： 环化酶负责它们在体外对Ca 2+的敏感性。 这 然后，该项目结合了[Ca 2 +]i的细胞生物学和分子研究， cAMP，试图建立可能的关键作用，Ca 2 + - 敏感的腺苷酸环化酶。

Signalling by the two second messengers, cAMP and Ca2+, is central to the control of numerous physiological processes by hormones and neurotransmitters. The discovery of Ca2+-sensitive (stimulable and inhibitable) adenylyl cyclases in heart, discrete brain regions,pituitary, platelets, etc. provides a viable means whereby these two signalling systems could be tightly coordinated. This potentially important role for Ca2+- sensitive adenylyl cyclases rests on whether physiological elevation of cytosolic Ca2+ ([Ca2+]) causes the changes in activity that are anticipated from in vitro studies. A guiding premise in the present project is the need to understand clearly how [Ca2+]i is distributed both within cellular domains and over time. This foundation greatly enriches the questions asked and the quality of the information derived. Current research from this lab indicates that indeed physiological [Ca2+]i-rises inhibit the endogenous Ca2+-inhibitable adenylyl cyclase in cell lines such as C6-2B gliomas. However, very strikingly, when the inhibitory efficacy of the entry and release component of a rise in [Ca2+]i are separately evaluated, only Ca2+-entry inhibits cAMP synthesis, while release is without effect. In addition, in HEK 293 cells, transfected with various adenylyl cyclase cDNAs, Ca2+-entry is sufficient to regulate both Ca2+- inhibitable and -stimulable adenylyl cyclases. Therefore, it is now proposed to ask whether this dependence on entry is a predetermined outcome for a cyclase that is expressed in C6-2B cells, because Ca2+-entry in C6=2B cells is particularly efficacious in delivering [Ca2+]i to the cyclase, or whether this sensitivity is a property intrinsic to Ca2+-sensitive adenylyl cyclases, even when they are transfected in HEK 293 cells. These issues will be addressed by imaging experiments in single C6-B cells, to determine whether different concentrations of Ca2+ are achieved at the plasma membrane in response to Ca2+ -entry or -release, elicited by various means; parallel immunohistochemical experiments will also be performed to ask whether adenylyl cyclases are found in regions of high Ca2+ (so-called caveolae) in C6-2B cells. Whether the same dependence on entry vs. release is displayed by adenylyl cyclase cDNAs expressed in HEK 293 cells will be determined, as a prelude to exploring whether the in vivo sensitivity to Ca2 can be separated from the in vitro sensitivity by molecular biological manipulation of cyclase cDNAs, using chimeric and deletion approaches. These studies will be complemented by experiments that probe the domains of cyclases that are responsible for their in vitro sensitivity to Ca2+. This project then combines cell biological and molecular studies of [Ca2+]i and cAMP in an attempt to establish the likely critical roles played by Ca2+ - sensitive adenylyl cyclases in cellular signalling.