Local Ca2+ Signaling in Smooth Muscle

平滑肌中的局部 Ca2 信号传导

• 批准号: 6731841
• 负责人: JOHN V WALSH
• 金额: $ 46.55万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6731841
• 关键词: arterioles; calcium channel; calcium flux; calcium indicator; calcium ion; cerebrovascular system; genetically modified animals; laboratory mouse; membrane potentials; phospholamban; potassium channel; protein structure function; sarcoplasmic reticulum; vascular smooth muscle; voltage /patch clamp; voltage gated channel

DESCRIPTION (provided by applicant): Calcium ions are universal messengers, regulating many processes within cells, including cerebral vascular smooth muscle (VSM) cells, a single layer of which lines the walls of arteriolar blood vessels. These cerebral arteriolar VSM cells, the subject of this study, are key determinants of blood pressure and local blood flow and thus are crucial in the pathophysiology of hypertension, subarachnoid hemorrhage, vasospasm and stroke. In VSM highly localized, brief cytosolic Ca2+ transients (Ca2+ sparks), emanating from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs), govern nearby Ca2+-activated ion channels, both large-conductance K+(BK) channels and CI- (CI(Ca)) channels. Activation of a cluster of BK channels in the spark "microdomain" causes a spontaneous transient outward current (STOC); activation of nearby CI(Ca) channels results in a spontaneous transient inward current (STIC). Virtually all smooth muscle cells display STOCs, and very many display STICs, including the mouse pial arteriolar VSM cells to be studied here. This proposal has two fundamental objectives: first, to advance basic, biophysical understanding of sparks, STOCs, STICS and the nature of the spark microdomain from which they arise; and second, to more clearly understand the physiology of these important cerebral arteriolar VSM cells. The spark micoromain will be studied with a unique, high-speed widefield imaging system in conjunction with simultaneous patch clamp recordings. The nature of the spark and the underlying Ca2+ current will be analyzed using a novel "signal mass" methodology. The influence of SR Ca2+ stores on events within the microdomain will be examined using direct measurements of free SR [Ca2+] and the use of a phospholamban KO mouse. Of the three types of RyRs, encoded by different genes, type 3 (RyR3) is found only in certain smooth muscle cells, among them arteriolar VSM cells employed here. The effect of RYR3 on events within the microdomain (i.e., sparks, STOCs and STICs) will be examined by use of a RyR3-KO mouse. STOCs are thought to hyperpolarize VSM cells leading to relaxation, whereas STICs should have the opposite effect. Since the same sparks can elicit both, an apparent paradox, we have postulated that sparks have a stabilizing effect on membrane potential and hence on the contractile state of VSM cells. We hypothesize further that those relaxing or contractile agents which affect sparks act like a switch by affecting STICs and STOCs in opposite ways. This hypothesis will be evaluated by examining the mechanisms of action of endothelin and nitric oxide, key contractile and relaxing agents, respectively. The existence of functional Ca2+ microdomains strongly suggests local control of Ca2+ sparks by events within the microdomain, which is critical in regulation of cardiac cells. We postulate an analogous local regulation in VSM cells. We will examine how Ca2 +,sparks are regulated by nearby voltage-activated Ca2vchannels and the how feedback from Ca2+ sparks in turn affects the Ca2+ channels.

描述（由申请人提供）：钙离子是通用的使者，调节细胞内的许多过程，包括脑血管平滑肌（VSM）细胞，其中一层是小动脉血管的壁。这些研究的主题是血压和局部血流的关键决定因素，因此对于高血压，蛛网膜下腔出血，血管痉挛和中风至关重要。在VSM中，通过ryanodine受体（RYRS）从肌浆网（SR）散发出高度局部的，简短的胞质Ca2+瞬变（Ca2+火花），治理了附近的Ca2+激活的离子通道，这两个通道都是大型的k+（bk）通道和CI（CI（CI）（CI（CA））。在火花“微区域”中激活BK通道簇会导致自发的瞬态向外电流（STOC）；附近CI（CA）通道的激活导致自发的瞬态内向电流（STIC）。实际上，所有平滑肌细胞都会显示stocs，并且显示了许多表现的Stics，包括在此处研究的小鼠小动脉VSM细胞。该提议具有两个基本目标：首先，要促进对火花，Stoc，Stics和Spark Microdomain的性质的基本生物物理理解；其次，更清楚地了解这些重要的脑小动脉VSM细胞的生理。 Spark Micoromain将与同时的贴片夹一起录制，并使用独特的高速广场成像系统进行研究。将使用新颖的“信号质量”方法分析火花和基础Ca2+电流的性质。 SR Ca2+存储对微域中事件的影响将使用游离SR [Ca2+]的直接测量和使用磷团小鼠的使用。在由不同基因编码的三种类型的RYR中，仅在某些平滑肌细胞中发现3型（RYR3），其中包括此处使用的小动脉VSM细胞。 RYR3对微区内事件（即火花，StoC和Stics）的影响将通过使用RYR3-KO小鼠进行检查。 stocs被认为会导致过极化的VSM细胞导致放松，而Stics应该具有相反的效果。由于同样的火花可以引起两者，这是一个明显的悖论，因此我们假设火花对膜电位具有稳定作用，因此对VSM细胞的收缩状态具有稳定的作用。我们进一步假设，那些放松或收缩的药物会通过相反的方式影响STIC和Stoc，就像开关一样。该假设将通过研究内皮素和一氧化氮，关键收缩和放松剂的作用机制来评估。功能性Ca2+微区的存在强烈表明微区内事件对Ca2+火花的局部控制，这对于调节心脏细胞至关重要。我们假设VSM细胞中的类似局部调节。我们将研究CA2+如何受到附近电压激活的CA2VCHANNEL的调节，以及CA2+火花的反馈反过来影响CA2+通道。