The longterm goal of the investigator is the understanding of cellular mechanisms underlying colonic motility disorders. This understanding may allow the design of specific therapies for a wide variety of gastrointestinal disorders, such as the Irritable Bowel Syndrome, Intestinal Pseudo-obstruction and Inflammatory Bowel Syndrome, Intestinal Pseudo-obstruction and Inflammatory Bowel Disease. The basis for the development of future therapies is the characterization of physiological mechanisms of myocyte activation. The proposed studies are designed to test the correctness of a model for activation which includes opening of different types of Ca channels (receptor-operated and voltage-sensitive), cycylical increases in free intracellular Ca2+ ([Ca2+]), and cyclical activation of Ca-activated K channels. Freshly isolated myocytes from the rabbit longitudinal colon will be prepared by short collagenase digestion. These cells will be studied a, using the fluorescent dye Fura- 2 to determine changes in [Ca2+] ic and b, using the patchclamp technique to determine activation of plasma membrane channels for Ca and K. [Ca2+]ic will be determined in the resting state, in response to agonist- induced activation, and during pharmacologic modulation in cell suspensions and single myocytes. These studies will allow to characterize source and kinetics of activator Ca. Whole cell and single channel currents through Ca and K channels will be characterized in response to substance P activation. The regulation of these membrane conductances by agonist-induced second messengers (Ca2+, phophoinositol metabolites) and their pharmacological modulation will be characterized. If the results of the proposed studies indicate significant differences in the activation mechanisms of the longitudinal colon compared to other gastrointestinal muscle tissues, they may provide the basis for target-specific therapies of colonic motility disorders.
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