Non-neuronal cells express """"""""neuronal"""""""" nicotinic receptors (NAChR), suggesting that nicotine (Nic) toxicity may also result from a direct effect on these cells. NAChRs are present in keratinocytes (which line the mouth and nasal cavities, the upper respiratory tract and the oesophagus), bronchial epithelial cells, endothelial cells, and some smooth muscle cells. Block of NAChR in epithelial and endothelial cells causes cell-cell detachment. Long-term exposure to Nic may cause NAChR desensitization and cell-cell detachment of epithelial and endothelial cells, leading to bronchitis and esophagitis, atherosclerotic lesions, and facilitated entrance of carcinogenic compounds. A NAChR in the muscle cells of arteries may be related to modulation of their motility. The OVERALL GOAL of the proposed studies is to identify new molecular mechanisms mediating Nic toxicity through its binding to the NAChR of non- neuronal cells involved in pathology related tobacco abuse.
The SPECIFIC AIMS will be: 1) to demonstrate and/or quantify the presence of functional NAChR in non-neuronal tissues known to be affected by Nic abuse; in cell cultures by patch clamp and binding of snake peptide antagonists (alpha- and kappa-neurotoxins) specific for different NAChR subtypes; in tissue sections, by quantitative confocal microscopy using antibodies (Abs) specific for different NAChR subunits and marker Abs identifying the cell types; by immunoelectromicroscopy; 2) to investigate if NAChRs are expressed in other non-canonical locations; in organ sections by immunohistochemical approaches with subunit-specific Abs and in situ hybridization wit subunit specific probes; in transgenic mice, expressing a reporter gene driven by the promoter of the alpha subunit under study; 3) to identify the subunit composition of the NAChRs in non- neuronal locations; using NAChR subunit-specific Abs; by PCR using primers specific for the different subunits, followed by cloning and sequencing of the products; by Northern blots of the cell mRNA, probed with the cloned subunit sequences derived from the PCR experiments; 4) to demonstrate in these cells the presence of the enzymes that metabolize ACh; by immunohistochemical and by biochemical assays of the enzymes choline acetyltransferase and acetylcholinesterase; 5) to investigate the cellular functions modulated by Nic and ACh binding to these non-neuronal cells; in cultured cells found to express NAChRs, using functional assays of cell adhesion, motility, and proliferation; 6) to investigate the effects of acute and chronic Nic exposure in vitro; using the cell cultures and functional assays as in aim 5, after short- and long-term exposure to Nic; the amount and affinity of the NAChR expressed after exposure to Nic will be determined by 3H-Nic and 125/I-neurotoxin binding; by quantitative confocal microscopy using subunit-specific Abs; by RNAse protection assay, using subunit specific probes; 7) to investigate the effects of chronic Nic exposure in vivo on expression and function of NAChRs expressed by the cells/tissues described above; in mice after long-and short-term treatment with Nic intravenously; the NAChRs expressed in the different tissues will be quantified as in aim 6.
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