Neural reflexes within the enteric nervous control secretion and motility patterns necessary for appropriate digestive function. Mechanical stimulation release 5- hydroxytryptamine (5-HT) from enterochromaffin cells and binds to 5-HT1P/4 receptors on submucosal afferent neurons in the reflex to cause epithelial chloride secretion. The overall goal is to understand the mechanisms by which a mechanical stimulus applied to the colon is transduced into chloride secretion. Three mechanical stimuli will be used: mucosal stroking, rotational shaking and cell deformation. The 1st aim uses an integrative approach to examine 5-HT release and chloride secretion in muscle-stripped colon from guinea pig, human or nucleotide- receptor deficient mice n response to mechanical stimulation or in a carcinoid tumor cell line, BON. Nucleotides released will be identified, the purinoreceptor subtype classified by agonist/antagonist potencies and the plasma membrane signaling stations known as caveolae identified by electron microscopy. The 2nd aim is to determine whether the purinoceptor in question is dependent on caveolae structure for appropriate signaling during mechanical stimulation. The 3rd aim is to determine if G proteins of the Gq family co-localize with purinoceptors in caveolae to mediate mechanically-evoked 5-HT release in BON cells. For these and subsequent studies immunoprecipitation and immunoblotting techniques will identify the presence of signaling molecules in fractions enriched in caveolae. The 4th aim examines the mechanism involved in mechanical deformation of single BON cells, and determines the role of phospholipase C and intracellular calcium stores in mechanically-evoked 5-HT release. The 5th aim identifies mechanisms by which protein kinase C in modulates mechanically-evoked 5-HT release by examining calcium stores and phosphorylation of phospholipase C. This study with its mechanistic approach from examining integrated systems to the single cell will provide new insights into the regulation of 5-HT release in normal human and animal tissue. It will provide new information about the basic physiology of carcinoid tumors and 5- HT release that may benefit the management of carcinoid syndrome. Understanding the regulation of 5-HT may be a key to management of functional bowel disease associated with an excess or reduced number of enterochromaffin cells.
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