Store-Operated Ca2+ Entry and Hypertension

商店操作的 Ca2 输入和高血压

• 批准号: 7858347
• 负责人: VERA GOLOVINA
• 金额: $ 37.13万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7858347
• 关键词: Address; Affect; Affinity; Animal Model; Blood Vessels; Caffeine; Carrier Proteins; Cell membrane; Cells; Chelating Agents; Chronic; Data; Development; Diamines; Dyes; Essential Hypertension; Ethylenes; Fluorescence Microscopy; Functional disorder; Genes; Homeostasis; Human; Hypertension; Image; Immunoblotting; Immunoprecipitation; In Vitro; Inositol; Lead; Link; Location; Measures; Membrane; Membrane Microdomains; Mesenteric Arteries; Mus; Muscle Contraction; Na(+)-K(+)-Exchanging ATPase; Ouabain; Peripheral; Play; Proteins; Pump; RNA Interference; Rattus; Regulation; Research Project Grants; Resolution; Rest; Role; Ryanodine; Sarcoplasmic Reticulum; Signal Transduction; Site; Smooth Muscle; Smooth Muscle Myocytes; Staining method; Stains; TRPC1 protein; Testing; Vascular resistance; Work; base; cyclopiazonic acid; digital imaging; immunocytochemistry; in vivo; innovation; insight; link protein; normotensive; novel; novel therapeutics; protein expression; receptor; research study; vasoconstriction; voltage

Chronic hypertension is typically associated with increased peripheral vascular resistance. This is due, in part, to enhanced arterial smooth muscle contractility, which is regulated by cellular Ca 2¿. Recent findings suggest that Ca 2¿ influx through TRPC-encoded store-operated channels (SOCs) in the plasma membrane (PM) may regulate myogenic tone. This project tests the hypothesis that TRPC-encoded SOCs are localized to PM microdomains adjacent to underlying junctional sarcoplasmic reticulum (jSR) and are functionally linked to distinct SR Ca 2+ stores [(IP3)/cyclopiazonic acid (CPA)- and caffeine/ryanodine (CAF/RY)-sensitive)]. I propose that the altered Ca 2¿ homeostasis in arterial smooth muscle cells (ASMCs) from ouabain hypertensive (OH)rats is duel in part, to upregulated expression of TRPC-encoded SOCs. Changes in TRPC expression under conditions of o_2Na + pump inhibition may result from elevated SR Ca 2¿ ([Ca 2 ]sR). The Specific Aims address the following questions: 1. Which TRPC proteins (TRPC1-7) are involved in Ca 2+ entry following unloading of distinct SR stores in freshly isolated ASMCs? Specific TRPC proteins will be downregulated by antisense oligos for TRPC genes to identify those associated with CPA- and/or CAF/RY-induced store-operated Ca 2¿ entry (SOCE). The latter will be probed by measuring SOCE- evoked changes in cytosolic Ca 2¿concentration ([Ca2+]c_) using fluorescence microscopy. TRPC expression in ASMCs will be identified by PCR and immunoblotting. 2. Is Ca 2+ entry through TRPC-encoded SOCs localized to PM microdomains adjacent to underlying jSR in ASMCs? a) High resolution imaging with near- membrane Ca 2+indicators will be used to localize the sites of SOCE; b) Immunocytochemistry on the same cell will determine the location of TRPC proteins within PM microdomains. 3. Which Ca 2¿ transport and storage mechanisms are altered in ASMCs from OH rats? Ca 2¿ transport proteins will be quantified and compared with resting [Ca2+]_, [Ca2*]sa and SOCE in ASMCs from control and OH rats. 4. Does nanomolar ouabain affect expression of TRPC-encoded SOCs in ASMCs in vitro, and what is the role of [Ca2+]sR? Mice with genetically altered Na + pumps and Na/Ca exchanger will be used. The results of this project will likely lead to new insights into the role of localized Ca 2+ transport mechanisms in the regulation of vascular tone and may suggest innovative approaches for the development of novel therapeutics for vascular dysfunction. i

慢性高血压通常与周围血管阻力增加有关。这是应得的 部分，增强了动脉平滑肌收缩力，受细胞Ca 2的调节。最新发现 建议通过TRPC编码的商店操作通道（SOC）在质膜中影响Ca 2。 （PM）可以调节肌源音。该项目检验了TRPC编码的SOC的假设是 位于基本连接肌质网（JSR）附近的PM微区域（JSR） 在功能上与不同的Sr Ca 2+商店相关[（IP3）/环皮二唑酸（CPA）和咖啡因/ryanodine （CAF/RY） - 敏感）]。我提出，动脉平滑肌细胞中的Ca 2稳态改变（ASMC） 从乌巴因高血压（OH）大鼠的部分原因是由于更新了TRPC编码的SOC的表达。 在O_2NA +泵抑制条件下，TRPC表达的变化可能导致SR Ca 2升高导致 （[CA 2] SR）。具体目的解决以下问题：1。哪种TRPC蛋白（TRPC1-7）为 在新鲜隔离的ASMC中卸载不同的SR商店后，参与了Ca 2+进入？特定的TRPC TRPC基因的反义寡素将下调蛋白质，以鉴定与CPA相关的基因 和/或CAF/RY诱导的商店经营的Ca 2？进入（SOCE）。后者将通过测量Soce-探测 使用荧光显微镜诱发了胞质Ca 2的浓度（[Ca2+] C_）的变化。 TRPC表达 在ASMC中，将通过PCR和免疫印迹来识别。 2。是通过TRPC编码的SOC进入Ca 2+进入 位于ASMC中基础JSR附近的PM微区域？ a）高分辨率成像，接近 膜Ca 2+指标将用于定位SOCE的位置； b）相同的免疫细胞化学 细胞将确定TRPC蛋白在PM微区中的位置。 3。哪个Ca 2？ OH大鼠的ASMC中的存储机制会改变吗？ Ca 2？将量化运输蛋白，并 与静止的[Ca2+] _，[Ca2*] SA和SOCE相比，来自对照和OH大鼠的soce。 4。纳摩尔 ouabain会影响TRPC在体外ASMC中编码的SOC的表达，[Ca2+] SR的作用是什么？老鼠 将使用一般更改的Na +泵和Na/Ca交换器。这个项目的结果可能会 导致对局部Ca 2+运输机制在调节血管音调中的作用的新见解 并可能提出用于开发新型血管功能障碍疗法的创新方法。 我