Local Ca2+ Signaling in Smooth Muscle

描述（由申请人提供）：钙离子是普遍的信使，调节细胞内的许多过程，包括脑血管平滑肌（VSM）细胞，其中单层排列在小动脉血管壁上。这些脑小动脉VSM细胞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中至关重要。在VSM高度定位，短暂的细胞质Ca2+瞬态（Ca2+火花），通过ryanodine受体（RyRs）从肌浆网（SR）发出，控制附近的Ca2+激活离子通道，包括大电导K+(BK)通道和CI- (CI(Ca))通道。在火花“微域”中激活一簇BK通道会产生自发瞬态外向电流（STOC）；激活附近的CI（Ca）通道导致自发瞬态内向电流（STIC）。几乎所有的平滑肌细胞都显示STOCs，很多平滑肌细胞都显示stic，包括本文研究的小鼠动脉VSM细胞。该提案有两个基本目标：第一，促进对火花，STOCs， STICS及其产生的火花微域性质的基本生物物理理解；第二，更清楚地了解这些重要的脑小动脉VSM细胞的生理学。火花微畴将通过一种独特的高速宽视场成像系统进行研究，该系统将与同步膜片钳记录相结合。火花的性质和潜在的Ca2+电流将使用一种新的“信号质量”方法进行分析。SR Ca2+储存对微域内事件的影响将通过直接测量游离SR [Ca2+]和使用磷蛋白KO小鼠进行检查。在由不同基因编码的三种类型的RyRs中，3型（RyR3）仅在某些平滑肌细胞中发现，其中包括本文使用的动脉状VSM细胞。RYR3对微域内事件（即火花，STOCs和STICs）的影响将通过使用RYR3 - ko小鼠进行检查。STOCs被认为使VSM细胞超极化导致弛豫，而stic应该具有相反的效果。由于同样的火花可以引起这两种情况，这是一个明显的矛盾，我们假设火花对膜电位有稳定作用，从而对VSM细胞的收缩状态有稳定作用。我们进一步假设，那些影响火花的放松或收缩剂就像开关一样，以相反的方式影响tic和stocks。这一假设将通过检查内皮素和一氧化氮的作用机制来评估，它们分别是关键的收缩剂和松弛剂。功能性Ca2+微域的存在强烈表明Ca2+火花通过微域内的事件进行局部控制，这在心脏细胞的调节中是至关重要的。我们假设在VSM细胞中也有类似的局部调控。我们将研究Ca2+火花是如何被附近电压激活的ca2v通道调节的，以及Ca2+火花的反馈如何反过来影响Ca2+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.