METABOLIC AND HORMONAL CONTROL OF CARDIAC CONTRACTION

描述(改编自申请人的摘要)：研究内容为 建议解开短时间内涉及的细胞内机制 钙离子对C6-2B大鼠cAMP积聚的长期调节作用 胶质瘤细胞。在前一次授权期内，申请人 证明钙离子对cAMP有明显的抑制作用 腺苷环化酶在C6-213细胞中的积聚和作用。这些细胞是 一个有用的模型来研究钙离子对腺酰环化酶的抑制，因为它们 几乎只含有VI型腺酰环化酶，是一种最近克隆的 这种形式被亚微摩尔浓度的钙离子抑制。这个 申请人提出，钙离子在短时间内起着重要作用 长期调节C6-2B细胞内cAMP浓度。 第一个焦点将是钙离子调节cAMP的机制。 新陈代谢。在该项目的后期，申请者将延长这些研究 确定钙离子参与激素和/或cAMP诱导 耐火性。基于申请人以前工作的方法和 对于高分辨率的同步高分辨率，提出了一种新的高分辨率同步高分辨率算法 活细胞内Ca~(2+)和cAMP多平面速度比成像 使用卷积-去卷积技术来改善空间 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- 关于两者相互依赖的动力学的浓度信息 第二个信使将帮助申请人审查更本地化的 标准成像可能无法检测到的第二信使变化 技巧。CAMP和钙离子的浓度都将是 经过实验修改，以检查短期和长期 两者在稳定状态方面的相互依赖 浓度和振荡行为。研究将涉及 直接微量注射Ca~(2+)和cAMP的药理作用 第二信使或它们的修饰物或类似物，或修改 VI型腺苷反义RNA催化cAMP合成 环化酶是C6-2B细胞中腺苷酸环化酶的主要形式，含量丰富 在心脏组织中。进一步的研究旨在确定这一点 腺酰环化酶或G蛋白环化酶的作用机制 相互作用，通过研究钙离子抑制腺苷的机制 G蛋白重组制剂中的环化酶和环化酶 磷脂小泡。

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.