LOCAL CA++ SIGNALING IN SMOOTH MUSCLE

长期以来，细胞内钙离子浓度的全球性变化调节着大量的细胞过程。最近，细胞内钙浓度的高度局部变化已经变得很明显，这些变化往往是构成全球钙变化的基本事件，也起到调节其邻近过程的作用。在平滑肌中，肌浆网(SR)短暂的、局部的钙释放被称为钙火花，通过激活细胞内的钙敏感钾通道来触发自发的瞬时外向电流(STOCs)。正因为如此，通过研究，平滑肌中的钙火花可能具有额外的临床意义和优点。此外，有迹象表明，钙火花可能还有额外的功能。尽管它们很重要，但对平滑肌中钙火花的性质和调节还不是很清楚。该方案的目的是利用独特的超高速数字成像系统，在使用膜片钳技术记录表面膜事件的同时，研究单个新鲜分离的平滑肌细胞中的钙电火花。我们的目标是了解各种因素是如何调节火花的大小和频率的，这些因素包括细胞内存储的钙水平；细胞内钙的浓度；第二信使系统，如由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；调节和免疫亲和素，FK506结合蛋白(FKBP)，它与从SR释放钙的通道结合。此外，还将研究钙火花与其STOC之间的精确关系，以及火花焦点在细胞内的空间分布以及高频或“热点”焦点的产生机制。将使用各种平滑肌细胞制剂，但特别强调一种新开发的小鼠主动脉细胞制剂。这将允许我们在研究火花调节时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一个基线。对血管平滑肌中的钙火花和局部钙信号的了解应该有助于洞察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.