This proposal describes a course of training and research designed to establish the candidate as an independent biomedical investigator. The candidate has chosen sponsors who are well suited as mentors and are experts in their field. Dr. Julio Vergara is an expert in calcium control mechanisms and imaging techniques and Dr. William J. Snape, Jr. is an expert in smooth muscle physiology and pathophysiology. The candidate seeks to answer the question: Does cAMP relax colonic smooth muscle through a primary effect on cytosolic calcium or directly on actin myosin cross- bridge formation? To accomplish this goal, two phases of research and training are proposed. During Phase I, the candidate will pursue formal course work in cell and muscle physiology as well as participating in the academically enriched environment of the UCLA Department of Physiology. The candidate will develop a kinetic model relating calcium and isometric force transients in rabbit colonic circular muscle strips. During Phase II, the proposed kinetic model will be used to quantitatively assess the role of cAMP on smooth muscle contraction and relaxation. The specific experimental aims are: 1) to measure intracellular calcium and isometric stress simultaneously, 2) to validate this method with two calcium sensitive dyes (fluo-3 and indo-1), 3) to measure the kinetic parameters of these calcium and force transients, 4) to assess the effect of photolytically released cAMP (caged cAMP) on the measured kinetic relationships, and 5) to correlate cAMP-mediated regulation of the contractile apparatus to biochemical studies of myosin light chain kinase. It is though that a better understanding of the mechanisms that control smooth muscle contraction and relaxation may lead to important, new treatments in patients with disorders of colonic transit and motility such as idiopathic constipation and the irritable bowel syndrome.
