Bronco epithelial cells, endothelial cells and esophagus keratinocytes express nicotinic acetylcholine receptors of neuronal type (nAChR) sensitive to nicotine (Nic): the overall goal of the proposed studies is to identify new mechanisms of Nic toxicity resulting from a direct effect on those nAChRs. Block of the nAChR in bronchial epithelial and endothelial cells causes cell paralysis and cell-cell detachment. Long term exposure to Nic causes nAChR desensitization, and will likely result in cell-cell detachment, leading to bronchitis and esophagitis, atherosclerotic lesions, and facilitated entrance of carcinogenic compounds. Acute exposure of bronchial epithelial cells to Nic causes apoptosis, that might be an additional mechanism of Nic toxicity. The proposed studies will have three major (1 to 3), and three minor (4-6) specific aims: 1) to investigate the structural and functional properties of nAChRs expressed by bronchial epithelial cells, endothelial cells and esophagus keratinocytes; to extend these studies to lung alveolar epithelial cells; by patch clamp and binding studies using ligands specific for different nAChR subtypes; by immunohistochemistry, using antibodies and protein probes specific for different nAChR subtypes; by PCR using subunit specific primers, followed by cloning and sequencing of the products; by in situ hybridization using subunit specific probes. 2) to demonstrate in these cells the presence of the enzymes that metabolize ACh; by immunohistochemical and to demonstrate in these cells the presence of the enzymes that metabolize ACh; by immunohistochemical and biochemical assays of the enzymes choline acetyltransferase and acetylcholinesterase. 3) to investigate the cellular functions modulated by Nic and ACh binding , and the effects of acute and chronic Nic exposure in vitro, using functional assays of cell adhesion, motility, and proliferation, and by measuring the rate of cell death and apoptosis after exposure to Nic. 4) to investigate the possibility that the nAChRs expressed by bronchial epithelial cells are a port of entry for rabies virus, explained rabies cases that result from airborne exposure. 5) to investigate the characteristics of the endothelia nAChRs expressed in patients with Buerger's disease, a vasculitis of the limb blood vessels caused or triggered by tobacco usage. 6) to do pilot investigations to test whether nAChRs are expressed at other non-neuronal locations involved in tobacco toxicity.
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