ANTAGONIST ACTIONS OF NICOTINE

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The objectives of the studies described in this proposal are to compare the characteristics of several subtypes of neuronal nicotinic receptors and to determine how nicotine affects the number and function of these receptors. Neuronal nicotinic receptors are assembled from alpha and beta subunits, and mammalian brain expresses mRNA encoding at least 6 different alpha and 3 different beta subunits. Despite this potential diversity, the properties of only two subtypes of neuronal nicotinic receptors in mammalian brain have been relatively well-characterized. Two important reasons for this lack of information about other neuronal nicotinic receptor subtypes are that l) until recently the radiolabeled nicotinic ligands that were available could reliably measure only receptors that contained either alpha4 and beta2 subunits or alpha7 subunits, and 2) there were no mammalian cell models that expressed a single defined subtype of neuronal nicotinic receptor. The studies described in this proposal are designed to overcome these limitations. We will use [3H)epibatidine ([3H]E3), a new ligand with very high affinity (approximately 5pM - 1 nM) for several different subtypes of neuronal nicotinic receptors, and measurements of nicotinic receptor-mediated 86Rb+ efflux to compare the characteristics and the regulation of nicotinic receptors in existing clonal cell lines and new, stably transfected mammalian cell lines that express defined subtypes of neuronal nicotinic receptors. The specific aims of this research are: 1) To compare the regulation of alpha4/beta2 nicotinic receptors in primary cultures of rat brain and in mammalian cell lines that stably express these receptors to the regulation of receptors in rat brain in vivo. 2) To characterize the pharmacological properties and the regulation of the two or more nicotinic receptor binding sites labeled by [3H]epibatidine in brain. 3) To compare the pharmacological properties and subunit composition of the non-alpha4 nicotinic receptors in rat PC12 cells, IMR-32 cells and SHSY-5Y cells. 4) To examine the regulation of the function and number of non-alpha4 nicotinic receptors. 5) To construct a library of mammalian cell lines stably transfected with combinations of at least two different neuronal nicotinic receptor subunit cDNA clones. 6) To use the stably transfected cells to study acute desensitization of each defined receptor subtype and to determine how each alpha and beta subunit influences acute desensitization. 7) To determine how chronic exposure to nicotine and other nicotinic drugs affects the regulation of the number and function of each defined receptor subtype.