LOCAL CA2+ IN ADRENERGIC CONTROL OF ARTERIAL RESISTANCE

描述（来自应用程序的逐字）：研究将集中在 局部Ca ~（2+）在肾上腺素能调节血管直径中的作用及机制 阻力动脉术语“局部Ca 2+”是指[Ca 2 +]振荡，波， 火花和可能的细胞间Ca 2+运动。这项研究的动机是 两个最近的实验结果：1）单个平滑肌内的[Ca 2 +] 完整的加压动脉的平滑肌细胞（SMC）在肾上腺素能 刺激，在一个模式完全不同的平均（壁）[Ca 2 +]，和2）在 相邻SMC，[Ca 2 +]振荡可以同步在同一时间， 频率，但通常是异步的，在不同的频率。的 这些事件与膜电位（Vm）振荡之间的关系， 肾上腺素能刺激引起的血管收缩和血管舒缩 知道的实验将利用最近的成像和控制能力 单个SMC和内皮细胞内的Ca 2+和其他信号分子 (EC)在完整的、加压的大鼠肠系膜小动脉的壁内。 具体目标是：1）确定细胞的机制， [Ca2+]-SMC振荡，并确定内皮是否调节这些 加压动脉中的机制，2）原位确定 平滑肌细胞缝隙连接和肌内皮细胞缝隙连接在Ca ~（2+）和 肌醇三磷酸（InsP 3）在细胞之间，3）确定如何激活 SMC上的α 1受体影响Ca 2+火花的频率，4）测试 同步[Ca 2 +]振荡机制的假说 振荡和血管舒缩。这些假设是由假定的 Vm、EC、缝隙连接、L型Ca ~（2+）通道、Ca ~（2+）激活的K ~+和Cl ~-的作用 通道，ryanodine受体和InsP 3受体。研究将利用 荧光Ca 2+指示剂和组合的共聚焦和多光子显微镜 用于局部[Ca 2 +]测量和笼状结构的衍射限制光解 动脉壁细胞内的化合物。缝隙连接在 细胞间信号传导将使用i）双光子光解 单个SMC中的笼状Ca 2+和笼状InsP 3，ii）特异性（肽）阻断剂 间隙连接通道，和iii）电测量。多焦点 多光子显微镜将用于快速光学切片的 动脉壁将测量动脉直径。动脉将 去内皮化和化学阻断运动。当地 单个SMC上的α 1受体的激活将通过双光子 笼状去甲肾上腺素（NE）的光解。全动脉激活将 通过暴露于α 1受体激动剂和神经元刺激来实现。通过 提供高分辨率图像的分子信使（Ca 2+）内， 完整的加压动脉壁，这项研究将提供一个新的， 细胞和细胞间机制的综合观点， 血管阻力

DESCRIPTION (Verbatim from the application): The research will focus on the role and mechanisms of local Ca2+ in adrenergic control of the diameter of resistance arteries. The term 'local Ca2+' means [Ca2+] oscillations, waves, sparks and possible inter-cellular Ca2+ movements. The research is motivated by two recent experimental results: 1) [Ca2+] within individual smooth muscle cells (SMC) of intact pressurized arteries oscillates during adrenergic stimulation, in a pattern quite different from average (wall) [Ca2+], and 2) in adjacent SMC, the [Ca2+] oscillations may be synchronized at the same frequency, but are often asynchronous, at different frequencies. The relationship between these events and oscillations of membrane potential (Vm), vasoconstriction, and vasomotion induced by adrenergic stimulation are not known. The experiments will exploit the recent ability to image and control Ca2+ and other signaling molecules within individual SMC and endothelial cells (EC) within the wall of an intact, pressurized rat mesenteric small artery. Specific goals to be achieved are: 1) Determine the cellular mechanisms of the [Ca2+]-oscillations in SMC and determine whether endothelium modulates these mechanisms in pressurized arteries, 2) Determine the effectiveness, in situ, of SMC gap junctions and myo-endothelial gap junctions in transmitting Ca2+ and inositol tris-phosphate (InsP3) between cells, 3) Determine how activation of alpha1-receptors on SMC affects the frequency of Ca2+ sparks, 4) Test hypotheses on the mechanisms of synchronized [Ca2+] oscillations, Vm oscillations, and vasomotion. The hypotheses are distinguished by the putative roles of Vm, EC, gap-junctions, L-type Ca2+-channels, Ca2+-activated K+ and Cl- channels, ryanodine receptors and InsP3 receptors. The research will utilize fluorescent Ca2+ indicators and a combined confocal and multi-photon microscope for local [Ca2+] measurements and diffraction-limited photolysis of caged compounds within cells of the arterial wall. The role of gap junctions in intercellular signaling will be investigated using i) two-photon photolysis of caged Ca2+ and caged InsP3 in individual SMC, ii) specific (peptide) blockers of gap-junction channels, and iii) electrical measurements. A multi-focal multi-photon microscope will be used for fast optical sectioning of the arterial wall. Arterial diameter will be measured. Arteries will be de-endothelialized and motion blocked chemically, as appropriate. Local activation of alpha1-receptors on individual SMC will be achieved by two-photon photolysis of caged norepinephrine (NE). Whole-artery activation will be achieved by exposure to alpha1-receptor agonists and neuronal stimulation. By providing high-resolution images of molecular messengers (Ca2+) within the walls of intact pressurized arteries, the research will provide a new, more integrated view of the cellular and inter-cellular mechanisms that control vascular resistance.