Nicotine is a drug of abuse that presumably exerts its effects through its interactions with specific nicotine receptors in the brain. Chronic exposure to nicotine results in an increase in the abundance of nicotine binding sites in the brains of both rats and humans. We have discovered, cloned, and sequenced ten members of the gene family that encodes subunits of the neuronal nicotinic receptors. In this application we propose a means of using these clones to study the metabolism and regulation of the nicotine binding sites in the brain. This approach uses our cDNA clones as templates for the synthesis of the extracellular domains of each receptor subunit in bacteria and then uses these bacterially expressed proteins to generate antibodies in rabbits. Affinity chromatography on columns containing subunit specific peptides results in antibodies able to distinguish between the extracellular domains of each receptor subunit. These antibodies and radioactive peptides are used to create quantitative assays sufficiently sensitive to determine amounts of specific receptor subunits in milligram quantities of brain. We will use these assays to explore the metabolism of the nicotinic receptor and the mechanisms that produce the up-regulation of nicotine binding sites following chronic nicotine use. Our goal is to distinguish between the different mechanisms that might generate more nicotine binding sites. We will also use the antibodies in a more qualitative fashion to determine the location on neurons of nicotinic receptors of specific subunit composition. The long-term goal of this research is to understand how the location of the binding sites that are up-regulated during chronic drug treatment relates to the processes that lead to addiction. These experiments will address issues fundamental to nicotine addiction and to the cellular mechanisms that lead to dependence on the drug.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DA004077-09
Application #
2116956
Study Section
Drug Abuse Biomedical Research Review Committee (DABR)
Project Start
1987-07-01
Project End
1998-05-31
Budget Start
1995-07-01
Budget End
1996-05-31
Support Year
9
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Neurosciences
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
Wooltorton, Julian R A; Pidoplichko, Volodymyr I; Broide, Ron S et al. (2003) Differential desensitization and distribution of nicotinic acetylcholine receptor subtypes in midbrain dopamine areas. J Neurosci 23:3176-85
Franceschini, Davide; Paylor, Richard; Broide, Ron et al. (2002) Absence of alpha7-containing neuronal nicotinic acetylcholine receptors does not prevent nicotine-induced seizures. Brain Res Mol Brain Res 98:29-40
Broide, R S; Orr-Urtreger, A; Patrick, J W (2001) Normal apoptosis levels in mice expressing one alpha7 nicotinic receptor null and one L250T mutant allele. Neuroreport 12:1643-8
Orr-Urtreger, A; Broide, R S; Kasten, M R et al. (2000) Mice homozygous for the L250T mutation in the alpha7 nicotinic acetylcholine receptor show increased neuronal apoptosis and die within 1 day of birth. J Neurochem 74:2154-66
Xu, W; Gelber, S; Orr-Urtreger, A et al. (1999) Megacystis, mydriasis, and ion channel defect in mice lacking the alpha3 neuronal nicotinic acetylcholine receptor. Proc Natl Acad Sci U S A 96:5746-51
Chen, D; Dang, H; Patrick, J W (1998) Contributions of N-linked glycosylation to the expression of a functional alpha7-nicotinic receptor in Xenopus oocytes. J Neurochem 70:349-57
Chen, D; Patrick, J W (1997) The alpha-bungarotoxin-binding nicotinic acetylcholine receptor from rat brain contains only the alpha7 subunit. J Biol Chem 272:24024-9
Goldner, F M; Dineley, K T; Patrick, J W (1997) Immunohistochemical localization of the nicotinic acetylcholine receptor subunit alpha6 to dopaminergic neurons in the substantia nigra and ventral tegmental area. Neuroreport 8:2739-42
Orr-Urtreger, A; Goldner, F M; Saeki, M et al. (1997) Mice deficient in the alpha7 neuronal nicotinic acetylcholine receptor lack alpha-bungarotoxin binding sites and hippocampal fast nicotinic currents. J Neurosci 17:9165-71
Colquhoun, L M; Patrick, J W (1997) Pharmacology of neuronal nicotinic acetylcholine receptor subtypes. Adv Pharmacol 39:191-220

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