METABOLIC AND HORMONAL CONTROL OF CARDIAC CONTRACTION

描述（改编自申请人的摘要）：研究是 提出要解开细胞内的机制，涉及在短 Ca ~（2+）对C6-2B大鼠cAMP积累的长期调节作用 胶质瘤细胞在上一个补助期内，申请人 表明Ca ~（2+）对cAMP有显著的抑制作用 C6-213细胞中的积累和腺苷酸环化酶作用。这些细胞 一个有用的模型，研究钙抑制腺苷酸环化酶，因为他们 几乎只含有VI型腺苷酸环化酶，最近克隆的 这种形式被亚微摩尔浓度的Ca 2+抑制。的 申请人提出Ca 2+在短时间内起重要作用， 以及对C6-2B细胞中cAMP浓度的长期调节。 第一个焦点将是Ca 2+调节cAMP的机制 新陈代谢.在项目后期，申请人将扩展这些研究 为了确定Ca ~（2+）参与激素和/或cAMP诱导的细胞凋亡， 耐火度基于申请人以前工作的方法， 的其他人，提出了高分辨率同时高 活细胞内Ca ~（2+）和cAMP多平面速度比成像 使用卷积-去卷积技术来提高空间分辨率 基于微观点扩散知识的图像分辨率 功能因此，详细的三维细胞内时间- 浓度信息的相互依赖的动力学， 第二信使将帮助申请人审查更多的本地化 标准成像可能无法检测到的第二信使变化 技术. cAMP和Ca 2+的浓度将是 经过实验修改，以检查短期和长期 两者在稳定状态下的相互依赖性 浓度和振荡行为。研究将涉及 钙离子和cAMP的药理学操作，直接微量注射 第二信使或其修饰物或类似物，或其修饰物， 用与VI型腺苷酰互补的反义RNA合成cAMP 腺苷酸环化酶是C6-2B细胞中腺苷酸环化酶的主要形式， 在心脏组织中。进一步的研究旨在确定这一点 在腺苷酸环化酶水平或G蛋白环化酶水平， 相互作用，通过检查腺苷酸的Ca 2+抑制机制， 重组G蛋白制剂中的环化酶和重组G蛋白制剂中的环化酶 磷脂囊泡。

DESCRIPTION (adapted from the applicant's abstract): Studies are proposed to unravel the intracellular mechanisms involved in the short and long term regulation by Ca2+ of cAMP accumulation in C6-2B rat glioma cells. During the previous grant period the applicant demonstrated that Ca2+ exerts a dramatic inhibitory action on cAMP accumulation and upon adenylyl cyclase in C6-213 cells. These cells are a useful model to study Ca2+ inhibition of adenylyl cyclase, since they almost exclusively contain type VI adenylyl cyclase, a recently cloned form which is inhibited by submicromolar concentrations of Ca2+. The applicant has proposed that Ca2+ plays an important role in the short and long term regulation of the concentration of cAMP in C6-2B cells. The first focus will be upon the mechanism by which Ca2+ regulates cAMP metabolism. Later in the project the applicant will extend these studies to ascertain the involvement of Ca2+ in hormone and/or cAMP induced refractoriness. Methodology based upon the applicant's previous work and that of others, is proposed for the high resolution simultaneous high speed ratio imaging of Ca2+ and cAMP in living cells at multiple planes of focus using convolution-deconvolution techniques to improve spatial image resolution based upon knowledge of the microscopic point spread function. Thus, detailed three dimensional intracellular time- concentration information about the interdependent kinetics of both second messengers will help the applicant to examine more localized second messenger changes which might not be detected by standard imaging techniques. The concentrations of both cAMP and Ca2+ will be experimentally modified to examine the short and long term interdependency between the two in terms of their steady state concentration and oscillatory behavior. Studies will involve pharmacological manipulation of Ca2+ and cAMP, direct microinjection of the second messengers or their modifiers or analogs, or modification of cAMP synthesis with antisense RNA complementary to type VI adenylyl cyclase, the major form of adenylyl cyclase in C6-2B cells and abundant in cardiac tissue. Further studies are aimed at defining this mechanism, at the level of adenylyl cyclase or on G protein-cyclase interactions, by examining the mechanism of Ca2+ inhibition of adenylyl cyclase in reconstituted preparations of G proteins and cyclase in phospholipid vesicles.