The general aim of this project is to investigate the role of intracellular free calcium levels ((Ca2+)i) in the regulation of vascular smooth muscle (VSM) contraction and relaxation. It is now well established that (Ca2+)i is involved in the mechanisms by which VSM contracts; however the precise role of this ion has not yet been defined. We will use aequorin to monitor (Ca2+) isimultaneously with isometric force form strips of ferret portal vein and aorta. Length-tension relationships will be determined while simultaneously monitoring (Ca2+)i in order to determine if length-dependent changes in active force in VSM are accompanied by and possibly caused by changes in (Ca2+)i. The mechanism of receptor-response coupling will be investigated by determining if different subtypes of alpha and beta adrenergic receptors produce different (Ca2+)i profiles or produce differential effects on calcium sensitivity. We will utilize phorbol esters as tools to activate protein kinase C (PKC) to investigate the possible role of PKC activation in the calcium dependent regulation of force maintenance. We will use two dimensional gel electrophoresis to measure the phosphorylation levels of the 20,000 dalton myosin light chains to see whether temporal changes in (Ca2+)i are accompanied by similar changes in the myosin light chain (MLC) phosphorylation levels, We will determine the effect of relatively specific inhibitors of protein kinase C and calmodulin antagonists on force generation, force maintenance (Ca2+)i and MLC phosphorylation. We will use calcium channel blockers as experimental tools to determine the source of the various components of the (Ca2+)i signals and to test the putative relationship between the calcium signals and the force profiles. We will also perform studies on enzymatically isolated smooth muscle cells using fura-2 to determine the degree to which the nonlinear properties and Mg2+ dependence of aequorin might have biased our perception of (Ca2+)i profiles. Finally, we plan to evaluate the use of fura-2 to study the mobilization of calcium from intracellular storage sites. The information obtained in this study should allow us to take a considerable step forward in understanding the role of (Ca2+)i in the regulation of normal (and eventually, abnormal) smooth muscle contraction.
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