Contractions in gastrointestinal (GI) smooth muscles provide the forces necessary to move food and waste through the GI tract. Contractions are initiated by electrical and biochemical events that increase the intracellular concentration of calcium, [Ca2+](i). Experiments proposed in this project will utilize recently developed fluorescence techniques to study changes in [Ca2+](i) and investigate how [Ca2+](i) regulates cellular events including: how [Ca2+(i) regulates its own entry into cells by controlling important ion channels in the plasma membrane, how [Ca2+](i) affects the rate of extrusion of Ca2+ from the cell, and how [Ca2+](i) regulates the sensitivity of the contractile apparatus to changes in [Ca2+](i). Parallel studies will be performed on spontaneously active, gastric smooth muscles and isolated gastric smooth muscle cells. In spontaneously active muscles, electrical activity (recorded with intracellular microelectrodes), Ca2+ transients (recorded by fluorescence photometry), and contractions will be simultaneous monitored. In isolated cells, membrane potential will be controlled using patch clamp techniques while optically monitoring [Ca2+](i). Using patch clamp methods, the actual influx of Ca2+ carried as Ca2+ current and the open probability of Ca2+-activated K+ channels can be precisely determined. These parallel studies in tissues and cells enhance the physiological relevance of both experimental approaches: Measurements made on cells and channels will increase our understanding of the basic mechanisms that produce and regulate changes in [Ca2+](i), and measurements made on intact muscles will help relate the more basic studies to physiological responses of the stomach. In the proposed studies, the regulation of voltage-dependent Ca2+ channels and Ca2+- activated K+ channels by [Ca2+](i) will be investigated. Ca2+ appears to affect these channels in a manner that decreases further entry of Ca2+ (i.e. [Ca2+](i) produces negative feedback). The extrusion of Ca2+ will be studied to determine factors that regulate restoration of basal [Ca2+](i) after a period of excitation. Finally, the question of how physiological agonists regulate Ca2+ influx and the sensitivity of the contractile elements to Ca2+ will be studied. These questions are central to an understanding of the physiology of gastric motility. An understanding of these mechanisms will aid in our understanding motility disorders and appropriate therapies for these disorders.
Showing the most recent 10 out of 23 publications
