INTRACELLULAR CALCIUM CONTROL OF CAMP SYNTHESIS

描述（改编自申请人摘要）： 细胞内Ca 2+，[Ca 2 +]i和cAMP提供了 协调两个主要第二信使的活动。虽然 这些途径在多个水平上相互作用的可能性是 在原则上被广泛接受，没有具体的认识， 这种相互作用的细胞的价值。腺苷酸的Ca ~（2+）刺激 环化酶，似乎有助于海马可塑性模型- 长时程增强，但很少有人知道这种相互作用是如何发生的。 调节细胞活动。已克隆了腺苷酸环化酶， 可被细胞内Ca 2+浓度范围所抑制;这 V1型（或其近缘种V型）是最突出的 在纹状体、心脏和垂体前叶组织中形成。本实用 腺苷酸环化酶在这些来源中的Ca 2+吸收能力不是 很清楚，尽管已经推测它提供了一种用于产生 (or增强）心肌细胞中的起搏器活动。但我们 刚刚开始探索这一现象。研究 非兴奋性细胞已经表明，[Ca 2 +]i的生理升高， 将抑制这些酶的cAMP合成。同样清楚的是， [Ca 2 +] i上升的性质在决定这种环化酶如何 是受调节的;特别是，Ca 2+敏感的环化酶是高度受调节的。 对Ca ~（2+）的进入敏感，而对Ca ~（2+）的释放不敏感。的 对Ca 2+进入的要求表明， 进入位点和腺苷酸环化酶，这可能表明一些亚细胞 组织这些要素。不知道这种环化酶是否 由发生在可兴奋的细胞中的快速[Ca 2 +] i瞬变调节。 细胞如果这些环化酶可以如此调节，那么它们的效用是 这可能导致cAMP的振荡。的 在本申请中提出的步骤是询问（主要在GH 3中 可兴奋细胞模型）i）是否潜在的负反馈， [Ca 2 +] i-稳态之间存在的Ca 2 +-代谢控制 ii）磷酸二酯酶对腺苷酸环化酶的影响， 增强cAMP合成的抑制作用，和iii） 腺苷酸环化酶是否（通过水母发光蛋白标记）看到高[Ca 2 +]i。在 此外，[Ca ~（2+）]i和[Ca ~（2+）]i之间的负反馈的重要性 cAMP将通过分子生物学操作探测， [Ca2+]i.最后，一个程序概述， 确定cAMP水平是否在单个细胞中迅速变化， 腺苷酸环化酶的Ca 2+抑制作用。那么，这个提议， 在细胞水平上迈出了第一步， 了解[Ca 2 +]i-和cAMP-信号传导如何交织在一起， 控制可兴奋的细胞活动。

DESCRIPTION (Adapted from applicant's abstract): Interactions between intracellular Ca2+, ([Ca2+]i), and cAMP provide the possibility of coordinating the activities of the two major second messengers. Although the likelihood that these pathways interact at a number of levels is widely accepted in principle, there is no concrete awareness of the value to a cell of such interactions. Stimulation by Ca2+ of adenylyl cyclase, seems to contribute to the hippocampal model of plasticity - long term potentiation, but little is known about how this interaction modulates cellular activity. An adenylyl cyclase has been cloned which can be inhibited by the intracellular range of Ca2+ concentrations; this species, type V1 (or its close relative, type V) are the most prominent forms in striatum, cardiac and anterior pituitary tissue. The utility of the Ca2+ - inhibitability of adenylyl cyclase in these sources is not clear, although it has been speculated to provide a device for generating (or reinforcing) pacemaker activity in myocardial cells. However, we are just on the threshold of exploring this phenomenon. Studies in nonexcitable cells have shown that physiological elevation of [Ca2+]i will inhibit cAMP synthesis by these enzymes. It is also clear that the nature of the [Ca2+]i-rise is critical in determining how such cyclases are regulated; in particular, Ca2+-sensitive cyclases are highly sensitive to Ca2+ -entry, rather than release from stores. The requirement for Ca2+ -entry suggests a functional colocalization between entry sites and adenylyl cyclases, which may suggest some subcellular organization of these elements. It is not known whether such cyclases are regulated by the rapid [Ca2+]i-transients that occur in excitable cells. If these cyclases could be so regulated, then their utility is greatly increased, since this could result in oscillations in cAMP. The steps proposed in the present application are to ask (mainly in the GH3 excitable cell model) i) whether the potential negative feedback that exists between [Ca2+]i-homeostasis controlled by a Ca2+-inhibitable adenylyl cyclas, is utilized ii) the impact of phosphodiesterases at reinforcing the effects of inhibition of cAMP synthesis, and iii) whether (by aequorin-tagging) adenylyl cyclases see high [Ca2+]i. In addition, the importance of the negative feedback between [Ca2+]i and cAMP will be probed by molecular biological manipulations, coupled with single cell analysis of [Ca2+]i. Finally, a program is outlined to determine whether cAMP levels change rapidly in single cells, by virtue of the Ca2+ -inhibitability of adenylyl cyclase. This proposal, then, takes the first steps at a cellular level towards a tangible understanding of how [Ca2+]i- and cAMP-signalling are intertwined to control excitable cell activity.