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 局部钙离子在肾上腺素能调控血管直径中的作用及机制 阻力动脉。术语“局部钙离子”指的是[钙离子]振荡、波动、 火花和可能的细胞间钙离子运动。这项研究的动机是 最近的两个实验结果：1)单个血管内的[Ca~(2+)] 完整加压动脉的细胞(SMC)在肾上腺素能过程中振荡 刺激，模式与平均(墙)[Ca~(2+)]非常不同，以及2) 相邻的SMC，可同时同步[Ca~(2+)]振荡 频率不同，但通常是异步的。这个 这些事件与膜电位(Vm)的振荡之间的关系， 肾上腺素能刺激引起的血管收缩和血管运动不是 为人所知。这些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Ca2+和其他信号分子 (EC)在完整、加压的大鼠肠系膜小动脉的管壁内。 要实现的具体目标是：1)确定细胞机制 SMC中的[Ca~(2+)]-振荡，并确定内皮是否对其进行调制 加压动脉中的机制，2)决定在原位的有效性 SMC缝隙连接和肌内皮细胞缝隙连接在钙离子和钙离子转运中的作用 细胞之间的肌醇三磷酸(InsP3)，3)决定如何激活 SMC上的α1受体影响钙离子放电频率，4)测试 关于[Ca~(2+)]同步振荡机制的假设 摆动和血管运动。这些假设是由推定的 Vm、EC、缝隙连接、L型钙通道、钙激活的K+和Cl-的作用 通道、兰尼定受体和InsP3受体。这项研究将利用 钙离子荧光指示剂与共焦多光子组合显微镜 笼状晶体的局域[Ca~(2+)]测量和衍射极限光解 动脉壁细胞内的化合物。缝隙连接在植物生长发育中的作用 细胞间信号的研究将使用i)双光子光解 笼状钙离子和笼状InsP3在单个SMC，II)特异性(肽)阻断剂中的作用 缝隙结通道的测量，以及iii)电测量。多焦点 多光子显微镜将用于快速光学切片 动脉壁。将测量动脉直径。动脉将会是 去内皮化和运动阻断，视情况而定。本地 单个SMC上α1受体的激活将通过双光子实现 笼状去甲肾上腺素(NE)的光解全动脉激活将是 通过暴露于α1受体激动剂和神经元刺激而实现。通过 提供分子信使(钙离子)的高分辨率图像 对于完整的增压动脉壁，这项研究将提供一种新的、更 控制细胞和细胞间机制的综合视图 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.