LOCAL CA++ SIGNALING IN SMOOTH MUSCLE

长期以来，人们已经认识到，细胞溶质钙（Ca）浓度的整体变化调节了大量的细胞过程。 最近，它已经变得很明显，高度本地化的变化，细胞溶质钙浓度往往是基本的事件，使全球变化的钙，也有助于调节过程中，他们的近邻。 在平滑肌中，肌浆网（SR）中钙的短暂局部释放（称为钙火花）通过激活细胞中的钙敏感性钾通道触发自发瞬时外向电流（STOCs）。仅出于这个原因，平滑肌中的钙火花可能具有额外的临床意义和研究价值。此外，有迹象表明，钙火花可能有额外的功能。 尽管它们的重要性，平滑肌中钙火花的性质和调节还没有很好地理解。 本建议的目的是研究钙火花在单一的，新鲜解离的平滑肌细胞使用一个独特的超高速数字成像系统在同一时间的表面膜事件记录使用膜片钳技术。 我们的目标将是了解火花的大小和频率是如何受到各种因素的调节，包括细胞内钙储存的水平;钙的胞质浓度;第二信使系统，如一氧化氮和各种蛋白激酶介导的系统;钙通过细胞表面的电压门控钙通道进入;调节和亲免素，FK 506结合蛋白（FKBP），此外，将研究Ca火花与其STOC之间的精确关系以及火花焦点在细胞中的空间分布和负责的机制。高频病灶或“热点”。 将采用各种平滑肌细胞制剂，但特别强调将放在一个新开发的小鼠主动脉细胞制剂。 这将使我们能够在火花调节的研究中使用受磷蛋白缺陷的敲除细胞，并为转基因技术的未来研究提供基线。 了解血管平滑肌中的钙火花和局部钙信号传导，可以深入了解血管张力的调节和各种疾病，如高血压、血管痉挛和中风。

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.