INHIBITORY REGULATION OF ADENYLATE CYCLASE

这个项目的一个持续目标是勾勒出 腺苷环化酶在信号转导通路中的抑制作用。它有 近年来越来越明显的是，各种信号转导 细胞内的通路在多个水平上相互作用。因此，一个 直接修饰(例如通过GTP)的单个神经递质 调节蛋白)一种信号产生酶的活性，可以通过 一种信号的产生，调制了许多其他的活动 小路。这种相互作用使细胞活动得到很好的协调。 了解这些相互作用如何调节整个细胞 响应性和识别信号的分子方面 允许这种相互作用的转导系统是最多的 当前荷尔蒙和神经递质具有挑战性和洞察力的领域 研究。本提案旨在剖析一种新的互动模式 在两个主要的信令系统之间，即[Ca2+]i和cAMP。在一个 细胞类型的数量(包括脑垂体和心脏组织，但不包括 例如，肝脏)，以亚微摩尔浓度(即 生理上升高的细胞内浓度)产生了强大的 对质膜腺苷酸环化酶的抑制。虽然是初步的 有证据表明，这种抑制作用可能发生在催化单元。 在腺苷环化酶中，钙离子的作用机制尚不清楚。 因此，本提案采取三种办法：一) 用生物化学方法表征这种抑制作用，并确定 这种作用是直接在腺酰环化酶的催化单元上介导的，或者 通过一些其他改变催化活性的因素；ii)确定 在完整细胞中，升高[钙]i的激素是否能抑制cAMP 通过这一机制积累；以及iii)使用可用的探针 从目前克隆的腺酰环化酶中，克隆腺酰环化的种类 在NCB-20细胞中表达的循环酶，该酶可被 亚微摩尔[Ca~(2+)]，并确定c DNA的表达是否 表现出钙离子抑制作用。NCB-20细胞系是一种方便的模型系统 其中解决了许多与对抗性有关的问题 [Ca~(2+)]i与cAMP之间的相互作用，不受离子通道效应的影响。 这些不可兴奋的细胞表达阿片和α2受体，它们 抑制cAMP的产生，5-羟色胺和PGE1受体刺激 腺苷环化酶；以及缓激肽和M胆碱能受体 刺激磷脂酶C。这样的系统应该允许测定 亚微摩尔[Ca~(2+)]_i是否直接抑制腺苷环化酶 有助于肌醇磷酸之间的拮抗相互作用 途径和cAMP，以及涉及的机制。

A continuing goal of this project is to delineate the role of the inhibition of adenylyl cyclase in signal transduction pathways. It has become apparent in recent years that the various signal transduction pathways in cells interact at a number of levels. As a result, an individual neurotransmitter that directly modifies (e.g. by a GTP regulatory protein) the activity of one signal generating enzyme, may, via the generation of a signal, modulate the activities of a number of other pathways. Such interactions impart fine coordination of cellular activity. Understanding how these interactions modulate overall cellular responsiveness and identifying the molecular aspects of the signal transduction systems that permit such interactions is one of the most challenging and insightful areas of current hormone and neurotransmitter research. The present proposal aims to dissect a novel mode of interaction between the two major signalling systems, i.e. [Ca2+]i and cAMP. In a number of cell types (including pituitary and cardiac tissue, but not, for instance, liver) Ca2+, in submicromolar concentrations (i.e. physiologically elevated intracellular concentrations) exerts a powerful inhibition of plasma membrane adenylyl cyclase. Although preliminary evidence suggests that the inhibition may be exerted at the catalytic unit of adenylyl cyclase, the mechanism of the Ca2+ effect is unknown. Consequently, the present proposal takes three approaches: i) to characterize this inhibition biochemically and to determine whether the effect is mediated directly at the catalytic unit of adenylyl cyclase, or by some other factor that modifies catalytic activity; ii) to determine whether, in intact cells, hormones that elevate [Ca2+]i can inhibit cAMP accumulation by this mechanism; and iii) using probes that are available from currently-cloned adenylyl cyclases, to clone the species of adenylyl cyclase that is expressed in NCB-20 cells, which is potently inhibited by submicromolar [Ca2+], and determine whether expression of the cDNA will display Ca2+-inhibition. The NCB-20 cell line is a convenient model system in which to address many of the issues relating to antagonistic interactions between [Ca2+]i and cAMP, independent of ion channel effects. These non-excitable cells express opiate and alpha2-receptors, which inhibit cAMP production, serotonin and PGE1 receptors that stimulate adenylyl cyclase; and bradykinin and muscarinic cholinergic receptors that stimulate phospholipase C. Such a system should allow a determination of whether direct inhibition of adenylyl cyclase by submicromolar [Ca2+]i contributes to antagonistic interactions between the inositol phosphate pathway and cAMP, and the mechanisms involved.