LOCAL CA++ SIGNALING IN SMOOTH MUSCLE

人们早就认识到，胞质钙（Ca）浓度的全局变化调节了大量的细胞过程。最近发现，胞质钙浓度的高度局部变化往往是构成钙全局变化的基本事件，并有助于调节其附近的过程。在平滑肌中，钙从肌浆网（SR）短暂、局部释放，被称为钙火花，通过激活细胞中钙敏感的钾通道，触发自发的瞬时外向电流（STOCs）。因此，研究平滑肌中的钙火花可能具有额外的临床意义和价值。此外，有迹象表明Ca火花可能具有附加功能。尽管它们很重要，但平滑肌中钙火花的性质和调节尚不清楚。本提案的目的是使用独特的超高速数字成像系统研究单个新鲜解离的平滑肌细胞中的Ca火花，同时使用膜片钳技术记录表面膜事件。目标将是了解火花的大小和频率如何受到各种因素的调节，包括细胞内储存的钙水平；胞质钙浓度；第二信使系统，如由一氧化氮和多种蛋白激酶介导的信使系统；钙通过细胞表面的电压门控钙通道进入；此外，还将研究Ca火花与其STOC之间的确切关系，以及火花灶在细胞内的空间分布和高频或“热点”灶的形成机制。各种平滑肌细胞的制备将被采用，但特别强调将放在一个新开发的制备小鼠主动脉细胞。这将使我们能够在火花调控的研究中使用缺乏磷蛋白的敲除细胞，并为未来的转基因技术研究提供基础。对血管平滑肌中钙火花和局部钙信号的理解，将有助于深入了解血管张力和各种疾病（如高血压、血管痉挛和中风）的调节。

It has long been recognized that global changes in cytosolic calcium (Ca) concentration regulate a large number of cellular processes. More recently it has become apparent that highly local changes in cytosolic Ca concentration are often the elementary events that make up global changes in Ca and also serve to regulate processes in their immediate vicinity. In smooth muscle, brief, localized releases of Ca from the sarcoplasmic reticulum (SR), referred to as Ca sparks, trigger spontaneous transient outward currents (STOCs) by activating Ca-sensitive potassium channels in their cell. For this reason alone Ca sparks in smooth muscle may have additional clinical significance and merit through study. Moreover, there are indications that Ca sparks may have additional functions. Despite their importance, the nature and regulation of Ca sparks in smooth muscle is not well understood. The purpose of this proposal is to study Ca sparks in single, freshly dissociated smooth muscle cells using a unique ultra-high speed digital imaging system at the same time as surface membrane events are recorded using patch clamp technology. The goal will be to understand how the magnitude and frequency of the sparks are regulated by a variety of factors including the level of Ca in the intracellular stores; the cytosolic concentration of Ca; second messenger systems such as those mediated by nitric oxide and a variety of protein kinases; Ca entry through voltage-gated Ca channels at the cell surface; the regulatory and the immunophilin, FK506 binding protein (FKBP), that is bound to the channels releasing Ca from the SR. In addition the precise relationship between a Ca spark and its STOC will be studied as well as the spatial distribution of spark foci in the cell and the mechanisms responsible for foci of high frequency or "hot spots". A variety of smooth muscle cell preparations will be employed but special emphasis will be placed on a newly developed preparation of mouse aortic cells. These will permit us to use phospholamban-deficient knockout cells in the study of spark regulation and provide a baseline for future studies with transgenic technology. An understanding of Ca sparks and local Ca signaling in vascular smooth muscle should provide insight into the regulation of vascular tone and a wide variety of diseases such as hypertension, vasospasm, and stroke.