CALCIUM SENSITIVE ADENYLYL CYCLASES

由两个第二信使cAMP和Ca~(2+)传递的信号是 激素和激素对多种生理过程的控制 神经递质。发现钙敏感(可刺激和 可抑制的)心脏、脑不连续区域、脑下垂体、 血小板等提供了一种可行的手段，使这两种信号 系统可以紧密协调。这一潜在的重要角色 Ca~(2+)敏感的腺酰环化酶取决于生理性升高 细胞内钙离子([Ca~(2+)])引起的活性变化是 预期来自体外研究。当前的指导前提 项目是需要清楚地了解[Ca~(2+)]i是如何分布的 在蜂窝域内和随着时间的推移。这一基础大大丰富了 提出的问题和所获得的信息的质量。当前 这个实验室的研究表明，生理上的[钙]i-确实会升高 抑制内源性钙离子可抑制的腺苷酸环化酶 为C6-2B胶质瘤。然而，非常引人注目的是，当抑制效果 [Ca~(2+)]i升高的进入部分和释放部分是分开的 经评估，只有钙离子进入抑制cAMP的合成，而释放 没有效果。此外，在HEK 293细胞中，转染了不同种类的 腺苷环化酶cDNAs，钙离子内流足以调节两种钙离子- 可抑制和可刺激的腺酰环化酶。因此，现在是 提议询问这种对进入的依赖是否是预定的结果 对于在C6-2B细胞中表达的循环酶，因为C6中的钙离子进入=2B 细胞在将[Ca~(2+)]i输送到循环酶方面特别有效，或者 这种敏感性是否是钙敏感腺苷的固有特性 环化酶，即使它们在HEK 293细胞中被转染。这些问题 将通过在单个C6-B细胞中进行成像实验来解决，以确定 血浆中是否存在不同浓度的钙离子 膜对Ca~(2+)内流或释放的反应，通过各种方式引起； 并行的免疫组织化学实验也将被用来询问 是否在高钙区(即所谓的 C6-2B细胞中。对进入和释放的依赖程度是否相同 在HEK 293细胞中表达的腺苷环化酶cDNA 作为探索体内敏感性的前奏 用分子生物学方法可将CA2从体外敏感性中分离出来 使用嵌合体和缺失方法操纵环化酶cDNA。 这些研究将得到探索以下领域的实验的补充 在体外它们对钙离子敏感的环化酶。这 然后，项目结合了细胞生物学和分子生物学研究[Ca~(2+)]i和 CAMP，试图确定钙离子可能发挥的关键作用- 细胞信号转导中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.