Local Ca2+ Signaling in Smooth Muscle

描述(申请人提供)：钙离子是通用的信使，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起着至关重要的作用。在VSM中，从肌浆网(SR)通过兰尼定受体(RyRs)发出的高度局部化的短暂胞浆钙瞬变(Ca2+)支配着附近的钙激活离子通道，包括大电导K(BK)通道和CI-(CI(Ca))通道。激活火花“微域”中的一簇BK通道会导致自发的瞬时外向电流(STEC)；激活附近的CI(Ca)通道会引起自发的瞬时内向电流(STIC)。实际上，所有的平滑肌细胞都显示STOCs，很多细胞显示STICs，包括这里要研究的小鼠软膜小动脉VSM细胞。这项提议有两个基本目标：第一，促进对火花、STOC、STIC及其产生的火花微域的性质的基本的生物物理理解；第二，更清楚地了解这些重要的脑小动脉VSM细胞的生理学。我们将利用一种独特的高速广域成像系统，结合同步的膜片钳记录来研究火花微区。火花的性质和潜在的钙电流将使用一种新的“信号质量”方法进行分析。通过直接测量游离SR[Ca~(2+)]和使用磷蛋白KO小鼠来检验SR[Ca~(2+)]存储对微域内事件的影响。在由不同基因编码的三种类型的RyRs中，3型(RyR3)仅存在于某些平滑肌细胞中，其中微动脉VSM细胞就是其中之一。RYR3对微域内事件(即火花、STOC和STIC)的影响将通过使用RyR3-KO小鼠来检测。STOCs被认为使VSM细胞超极化，导致松弛，而STICs应该有相反的作用。由于相同的火花可以引发两者，这是一个明显的悖论，我们假设火花对VSM细胞的膜电位具有稳定作用，从而对VSM细胞的收缩状态具有稳定作用。我们进一步假设，那些影响火花的松弛或收缩药剂通过以相反的方式影响STIC和STOC而起到开关的作用。这一假说将通过研究内皮素和一氧化氮的作用机制来评估，这两种物质分别是关键的收缩和松弛药物。功能钙微域的存在强烈暗示了微域内事件对钙火花的局部控制，这在心肌细胞的调节中是至关重要的。我们假设在VSM细胞中存在类似的局部调节。我们将研究钙火花是如何被附近的电压激活的钙离子通道调节的，以及来自钙离子火花的反馈如何反过来影响钙离子通道。

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.