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Mouse -TC6 Insulinoma Cells: High Expression of Functional 34 Nicotinic Receptors Mediating Membrane Potential, Intracellular Calcium, and Insulin Release

Chemical Biology (T.O.) and Laboratory of Bioorganic Chemistry (M.O., J.W.D.), National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, United States Department of Health and Human Services, Bethesda, Maryland; Faculty of Pharmacy, Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan (M.O., T.O.); and Department of Pharmacology, Georgetown University, School of Medicine, Washington, DC (M.B., Y.X., K.J.K.)

Nicotine elicited membrane depolarization, elevation of intracellular calcium, rubidium efflux, and release of insulin from mouse -TC6 insulinoma cells. Such responses were blocked by the nicotinic antagonist mecamylamine but not by the muscarinic antagonist atropine. Neither the selective ₄₂ antagonist dihydro--erythroidine nor the selective ₇ antagonist methyllycaconitine significantly blocked the nicotine-elicited depolarization or the calcium response. The elevation of intracellular calcium did not occur in calcium-free media, indicating that the increase in intracellular calcium was due to the influx of calcium. The rank order of potency for nicotinic agonists was as follows: epibatidine > nicotine = 3-(azetidinylmethoxy)pyridine (A-85380), cytisine, dimethylphenylpiperazinium (DMPP). Cytisine and DMPP seemed to be partial agonists. The density of nicotinic receptors measured by [³H]epibatidine binding was 7-fold higher in membranes from -TC6 cells than in rat brain membranes. No binding of ¹²⁵I-A-85380 was detected, indicating the absence of 2-containing receptors. Reverse transcription-polymerase chain reaction analyses indicated the presence of mRNA for 3 and 4 subunits and 2 and 4 subunits in -TC6 cells. The binding and functional data suggest that the major nicotinic receptor is composed of 3 and 4 subunits. The -TC6 cells thus provide a model system for pharmacological study of such nicotinic receptors.

Address correspondence to: Dr. John W. Daly, Building 8, Room 1A17, National Institutes of Health, Bethesda, MD 20892-0820. E-mail: jdaly{at}nih.gov