The enteric nervous system (ENS) contains many interneurons arranged in microcircuits that mediate intrinsic reflexes. The physiology of enteric interneurons is poorly understood. 5-Hydroxytryptamine 95-HT), which is also present in mucosal epithelial (EC) cells, is the neurotransmitter of an enteric interneuron. We have characterized a novel enteric neuronal 5- HT receptor. This """"""""5-HT1P"""""""" receptor is coupled to a G protein and is responsible for mediating a slow EPSP. This proposal is designed to study the function of serotonergic neurons and 5-HT1P receptors. We will test the hypothesis that EC cells are pressure transducers that release 5-HT, which initiates the peristaltic reflex by activating 5-HT1P and/or 5-HT3 receptors on intrinsic sensory nerves in the lamina propria. We will determine whether pressure directly causes EC cells to release 5-HT, whether this release is Ca2+-dependent, and whether EC cell secrete 5-HT apically or basally. We will also determine whether 5-HT1P and/or 5-HT3 antagonists block the peristaltic reflex and whether the reflex depends on epithelial 5-HT. We will visualize the neurons that become active during the peristaltic reflex by histochemically measuring neuronal cytochrome oxidase activity or nuclear c-fos immunoreactivity. We will utilize available anti-idiotypic antibodies to localize 5-HT1P receptors in relation to identified serotonergic synapses on neurons shown to be 5-HT- responsive and on mucosal nerves in relation to EC cells. Electrophysiological studies will be done on myenteric neurons to test the hypothesis that 5-HT1P responses are mediated by cAMP. Effects of cholera and pertussis toxins will be compared and the ability of phosphodiesterase inhibition or intracellular injection of GTP-gamma-S to mimic or enhance responses to 5-HT will be assessed. We will evaluate the action of a permanent inhibitor of cAMP-dependent protein kinase, Rp-adenosine 3', 5'- phophosphorothioate, and antibodies directed against the C terminal (cytosol facing) domains of the alpha subunits of Gs and other G proteins. The effects of 5-HT1P receptor agonists and antagonists on the level of cAMP in isolated myenteric ganglia will be analyzed. Finally, we propose to clone a full length cDNA encoding the 5-HT1P receptor. We will first screen a library constructed from isolated myenteric ganglia at reduced stringency with probes that are likely to be homologous to the 5-HT1P receptor. Hybridizing cDNA clones will be sequenced and expressed in mammalian cells to determine if the clones have the typical ligand-binding characteristics of the 5-HT1P receptor. Effector coupling mechanisms of the cloned receptor will also be studied in transfected cells. If necessary, as alternative cloning strategies, we will utilize the polymerase chain reaction or the screening of a library expressed in mammalian cells with anti-idiotypic antibodies. Since functional disorders of Gl motility are common, often disabling, and the cause of suffering for which there is currently no adequate therapy, these studies have considerable clinical as well as basic significance.
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