The overall objective of this study is to investigate the role of intracellular free calcium (Ca)i as a messenger of neurohumoral action in the regulation of intestinal fluid and electrolyte transport. Several key questions regarding the stimulation of cytosolic calcium and the cellular mechanisms that are involved in calcium-dependent stimulus-secretion coupling will be addressed. These investigations will have relevance not only to physiological systems in other epithelia but also to the understanding of pathophysiological mechanisms of diarrheal disease. Furthermore, specific inhibitory mechanisms of Ca-mediated intestinal secretion will be studied to determine potential sites for future pharmacological intervention in the treatment of diarrheal disorders. Previous investigations in this area have been limited by the lack of suitable methodology to measure intracellular free calcium. With the development of the Ca-fluorescence indicator quin-2, it is now possible to reliably determine resting and agonist-stimulated (Ca)i in isolated chicken enterocytes. This technique and methods to determine phosphatidylinositol metabolism will be used to investigate different types of agonist-activated calcium entry, membrane transduction mechanisms involved in the stimulation of (Ca)i, tachyphylaxis, and the potential role for calcium in the actions of cyclic nucleotides. Finally, improvements and alternatives to the presently used methods have been proposed. A major objective will be the development of a system to simultaneously measure quin-2 fluorescence and ion transport changes in intact tissues. This system will have wide application not only to the study of physiological mechanisms but also to disorders of intestinal ion transport and their treatment.
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