INTERACTIONS BETWEEN CA & CAMP REGULATION IN MYOCYTES

这是一项研究钙在腺苷调节中的作用的建议。 鸡心肌细胞的环化酶(AC)活性。提出了证据 心肌细胞表达V和VI型AC亚型，这两种亚型都是 被钙离子抑制。PI已经发现，降低外部钙刺激 针对ISO或Forskolin的营地生产。申请人 建议使用异构体在蛋白质水平上鉴定AC异构体。 特异性抗体，并改变V和VI型的表达水平 腺病毒介导的鸡胚胎心肌细胞同工酶研究 表达技术(正义或反义)，用来检查 这些变化对钙-AC相互作用有影响。AC活动将是 在表达已知水平的AC V和VI的心肌细胞中测量，并且 钙的影响将在完整的细胞中测量(在 细胞外钙/硝苯地平)，在通透性细胞内和在分离的 膜。第二个具体目标将侧重于以下方面的长期影响 钙通道阻滞剂对心肌细胞AC水平的影响 荧光素酶报告基因的构建，以评估细胞内cAMP水平。第三 特定目标将研究TH AC亚型的潜在磷酸化 通过PKA和PKC，以及对活动水平的相关影响 在钙和坎普生产之间的谈话。

This is a proposal to examine the role of Ca in regulating adenylyl cyclase(AC) activity in chick cardiac myocytes. Evidence is presented that the myocytes express type V nd VI AC isoforms, both of which are inhibited by Ca. The PI has found that lowering external Ca stimulates cAMP production in response to either ISO or forskolin. The applicant proposes to identify the AC isoforms at the protein level using isoform- specific antibodies, and to alter expression levels of types V and VI isozymes in embryonic chick heart myocytes using adenovirus-mediated expression techniques (sense or antisense), an to examine the effects of these alterations on the Ca-AC interaction. AC activities will be measured in myocytes expressing known levels of AC V and VI, and the effects of Ca will be measured in intact cells (variations in extracellular Ca+/- nifedipine), in permeabilized cells and in isolated membranes. The second specific aim will focus on the long term effects of exposure to Ca channel blockers on myocyte AC levels, using a CRE- luciferase reporter construct, to assess cellular cAMP levels. The third specific aim will investigate potential phosphorylation of th AC isoforms by PKA and PKC, and associated effects on activity levels and the"cross- talk" between Ca and cAMP production.