Local Ca2+ Signaling in Smooth Muscle

描述（由申请人提供）：钙离子是通用信使，调节细胞内的许多过程，包括脑血管平滑肌（VSM）细胞，其单层排列在小动脉血管的壁上。这些脑小动脉VSM细胞，本研究的主题，是血压和局部血流量的关键决定因素，因此在高血压，蛛网膜下腔出血，血管痉挛和中风的病理生理学中至关重要。在VSM中，通过兰尼碱受体（RyR）从肌浆网（SR）发出的短暂的胞质Ca 2+瞬变（Ca 2+火花）支配附近的Ca 2+激活的离子通道，包括大电导K+（BK）通道和CI-（CI（Ca））通道。在火花“微区”中的BK通道簇的激活引起自发的瞬时外向电流（STOC）;附近的Cl（Ca）通道的激活引起自发的瞬时内向电流（STIC）。几乎所有的平滑肌细胞都显示STOC，很多显示STIC，包括这里要研究的小鼠软脑膜小动脉VSM细胞。该提案有两个基本目标：第一，推进火花，STOC，STICS的基本，生物物理学的理解和火花微域的性质，它们产生的;第二，更清楚地了解这些重要的大脑小动脉VSM细胞的生理。将使用独特的高速宽视野成像系统结合同步膜片钳记录研究火花微区。火花的性质和潜在的Ca 2+电流将使用一种新的“信号质量”的方法进行分析。将使用游离SR [Ca 2 +]的直接测量和使用受磷蛋白KO小鼠来检查SR Ca 2+储存对微域内事件的影响。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（RyR 3），其中在本文中使用的小动脉VSM细胞中。RYR 3对微域内事件的影响（即，火花、STOC和STIC）将通过使用RyR 3-KO小鼠进行检查。STOC被认为是导致松弛的VSM细胞过度增殖，而STIC应该具有相反的效果。由于相同的火花可以引起这两个，一个明显的矛盾，我们假设，火花有一个稳定的影响膜电位，从而对VSM细胞的收缩状态。我们进一步假设，那些影响火花的放松或收缩剂就像一个开关，以相反的方式影响STIC和STOC。这一假设将通过检查内皮素和一氧化氮的作用机制进行评估，分别是关键的收缩剂和舒张剂。功能性Ca 2+微区的存在强烈表明微区内事件对Ca 2+火花的局部控制，这在心肌细胞的调节中至关重要。我们假设一个类似的本地调节VSM细胞。我们将研究如何Ca 2+，火花是由附近的电压激活的Ca 2 v通道调节，以及如何反馈从Ca 2+火花反过来影响Ca 2+通道。

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.