LOCAL CA++ SIGNALING IN SMOOTH MUSCLE

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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.