INTRACELLULAR CALCIUM CONTROL OF CAMP SYNTHESIS

CAMP 合成的细胞内钙控制

• 批准号: 2669002
• 负责人: DERMOT M COOPER
• 金额: $ 25.26万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2669002
• 关键词: adenylate cyclase; calcium; calcium channel; calcium flux; calcium indicator; cyclic AMP; divalent cations; electrophysiology; isozymes; phosphodiesterases; second messengers; single cell analysis; tissue /cell culture

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.

描述(改编自申请人的摘要)： 细胞内Ca~(2+)、([Ca~(2+)]i)和cAMP提供了 协调两个主要第二使者的活动。虽然 这些途径在多个水平上相互作用的可能性是 原则上被广泛接受，没有具体的意识到 对这种相互作用的细胞的价值。钙离子对腺苷的刺激作用 环化酶似乎对海马区的可塑性模型有贡献- 长时程增强，但对这种相互作用是如何产生的知之甚少 调节细胞活动。克隆了一种腺酰环化酶，该酶 可以被细胞内钙离子浓度范围所抑制；这 类型V1(或其近亲类型V)的物种最突出 在纹状体、心脏和垂体前叶组织中形成。该实用程序 在这些来源的腺酰环化酶的钙抑制能力中 很清楚，尽管有人推测它提供了一种装置来产生 (或增强)心肌细胞的起搏器活动。然而，我们 才刚刚开始探索这一现象。研究项目： 不可兴奋的细胞已经显示出生理性的[钙]i升高 会通过这些酶抑制cAMP的合成。同样明显的是， [Ca~(2+)]i升高的性质是决定这种循环如何进行的关键 是受调控的；尤其是对钙离子敏感的循环酶是高度 对钙离子进入敏感，而不是从商店释放。这个 对钙离子进入的要求表明 进入部位和腺苷环化酶，这可能表明一些亚细胞 这些要素的组织。目前尚不清楚这种周期是否 受发生在可兴奋状态的快速[Ca~(2+)]i瞬变调节 细胞。如果这些周期酶可以如此调节，那么它们的效用是 大大增加，因为这可能会导致cAMP的振荡。这个 本申请中提出的步骤是询问(主要在GH3中 可兴奋细胞模型)i)是否存在潜在的负反馈 存在于[Ca~(2+)]i-动态平衡之间，受钙离子抑制物控制 2)磷酸二酯酶的影响。 加强对cAMP合成的抑制作用，以及iii) 腺苷环化酶是否出现高[Ca~(2+)]i。 此外，[Ca~(2+)]_i和[Ca~(2+)]_i负反馈的重要性 CAMP将通过分子生物学操作进行探测，再加上 [Ca~(2+)]的单细胞分析。最后，给出了一个程序 确定单细胞内cAMP水平是否迅速变化 腺酰环化酶对钙的抑制作用。那么这个提议呢， 在细胞层面迈出第一步，迈向有形的 了解[Ca~(2+)]i-和cAMP信号如何相互交织在一起 控制可兴奋的细胞活动。