Mouse -TC6 Insulinoma Cells: High Expression of Functional 3 4 Nicotinic Receptors Mediating Membrane Potential, Intracellular Calcium, and Insulin Release

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 4 2 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 [H]epibatidine binding was 7-fold higher in membranes from -TC6 cells than in rat brain membranes. No binding of I-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. A wide array of nicotinic acetylcholine receptors occurs in the mammalian nervous system and other organs (Dani, 2001), and there have been extensive efforts to define potent and selective agonists for such subtypes (Bunnelle et al., 2004; Toma et al., 2004; Daly, 2005). Often, multiple subtypes of nicotinic receptors exist in the same cell. Thus, cells that only express one subtype or have been selectively transfected with one subtype represent valuable model systems. The rat pineal gland is one such system, because it expresses the 3 4 subtype virtually exclusively (Hernandez et al., 2004). The neuromuscular subtype ( 1 1 ) is expressed in human TE-671 rhabdomyosarcoma cells (Lukas, 1989). The ganglionic subtypes ( 3 2* and 3 4*) are expressed in rat PC-12 pheochromocytoma cells (Avila et al., 2003). Human IMR-32 (Nelson et al., 2001) and human SH-SY5Y neuroblastoma cells (Dajas-Bailador et al., 2002), express a ganglionic subtype, but the subunit compositions are not known with certainty, because several subunits are expressed in these cells. Mammalian cells, stably transfected with different combinations of and nicotinic subunits, have proven useful for many studies (Whiting et al., 1991; Gopalakrishnan et al., 1996; Stauderman et al., 1998; Wang et al., 1998; Meyer et al., 2001; Eaton et al., 2003; Xiao and Kellar, 2004). However, the properties of receptors in transfected cells may not be fully equivalent to the properties in native systems because of ancillary proteins or other membrane components. In an effort to define insulinoma cell lines as models for pancreatic islet cells, the effects of carbamylcholine and other agonists on insulin release, membrane potential, and intracellular calcium were investigated with mouse -TC6, hamThis work was supported in part by MEXT-HAITEKU and by a grant from the Japan Society for the Promotion of Science (to M.O.). The research at the National Institutes of Health was supported by the intramural program of the National Institute on Diabetes and Digestive and Kidney Diseases. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.014902. ABBREVIATIONS: DMPP, dimethylphenylpiperazinium; A-85380, 3-(azetidinylmethoxy)pyridine; FCCP, carbonylcyanide 4-(trifluoromethoxy)phenylhydrazone; MLA, methyllycaconitine; MRS 1845, N-propargylnitrendipine; PCR, polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; SKF 96365, 1-[ -(3-(4-methoxy-phenyl)propoxy)-4-methoxyphenethyl]1H-imidazole. 0026-895X/06/6903-899–907 MOLECULAR PHARMACOLOGY Vol. 69, No. 3 U.S. Government work not protected by U.S. copyright 14902/3087829 Mol Pharmacol 69:899–907, 2006 Printed in U.S.A. 899 at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from ster HIT-T15, and rat RINm5F cells. Muscarinic agonists are well known to elicit an elevation in calcium and insulin release in pancreatic islets and insulinoma cell lines (Iismaa et al., 2000; Gilon and Henquin, 2001), as was confirmed in preliminary studies with mouse -TC6, hamster HIT-T15, and rat RINm5F cells (data not shown). In contrast, nicotinic agonists have not been reported to have such effects, and none were seen in the hamster and rat cell lines (data not shown). However, in vivo, both nicotine and dimethylphenylpiperazinium (DMPP), apparently through ganglionic activation, can elicit insulin secretion (Karlsson and Ahren, 1988). Nicotine did elicit marked increases in calcium in the mouse -TC6 cell line. Here, we report a detailed study of the effects of cholinergic agonists and antagonists on the mouse -TC6 insulinoma cells. We found that muscarinic (oxotremorine M), nicotinic (nicotine, epibatidine, A-85380, DMPP, and cytisine), and mixed cholinergic (carbamylcholine) agonists elevated intracellular calcium and caused insulin release in these cells. High levels of functional nicotinic receptors with characteristics of the 3 4 subtype were present. Thus, the -TC6 cells represent a new model system for the study of nicotinic receptors and their involvement in the calciumdependent release of insulin. Materials and Methods Materials and Cells. Nicotine, cytisine, dimethylphenylpiperazinium (DMPP) iodide, carbamylcholine, oxotremorine M, mecamylamine, dihydro-erythroidine, methyllycaconitine, atropine, scopolamine, nifedipine, A-85380, SKF 96365, and MRS 1845 were obtained from the Sigma Chemical Co. (St. Louis, MO). ( )-Epibatidine was from Tocris Cookson Inc. (Ellisville, MO). Dulbecco’s modified Eagle’s medium and RPMI 1640 culture medium, fetal bovine serum, penicillin/streptomycin, trypsin/EDTA, and TRIzol were from Invitrogen (Carlsbad, CA). DNase I was from Ambion (Austin, TX), and bovine serum albumin was from ICN Biochemicals (Irvine, CA). ( )-[H]Epibatidine, I-epibatidine, I-A-85380, I-bungarotoxin, and [Rb]rubidium chloride (Rb ) were supplied by PerkinElmer Life Sciences (Boston, MA). Mouse -TC6 cells and other cell lines were purchased from American Type Culture Collection (Manassas, VA). Cell Culture. Mouse -TC6 cells were cultured in Dulbecco’s modified Eagle’s medium containing 20 mM glucose at 37°C under 5% CO2 condition. The cells were subcultured every week. Cells from passages 30 to 80 were used for all experiments. When the cells had grown to 90 to 95% confluence in a cell culture flask (162 cm), the cells were stripped from the bottom of the flask by adding trypsin/ EDTA solution, and an aliquot of cell suspension was transferred into a new flask filled with new medium. Membrane Potential. The -TC6 cells were seeded in 96-well plates and cultured for 3 to 4 days. After reaching 90 to 95% confluence (1–2 10 cells/well), the cells were washed with Hanks’ balanced salt solution/HEPES buffer twice and loaded with the membrane potential kit dye (Molecular Devices Corporation, Sunnyvale, CA) for 60 min at a room temperature in the darkness. The components of Hanks’/HEPES buffer were as follows: 137 mM NaCl, 5.4 mM KCl, 0.34 mM KH2PO4, 1.26 mM CaCl2, 0.5 mM MgCl2, 0.41 mM MgSO4, 0.34 mM Na2HPO4, 5.5 mM D-glucose, and 20 mM HEPES, pH 7.4. Temporal changes in the membrane potential were monitored using a FLEX Station fluorescence microplate reader (Molecular Devices) with excitation at 535 mm and emission at 560 nm and then were calculated as a relative fluorescence intensity based on analyses by SoftMax Pro software (Molecular Devices). Maximum depolarization was elicited with 40 mM KCl as a calibrant at the end of each assay. Data obtained from each well were normalized by use of these maximum values as described previously (Fitch et al., 2003). Intracellular Calcium. Intracellular calcium measurements with -TC6 cells were carried out essentially as described above for the membrane potential assay, except for the fluorescence dye. The cells were loaded with the calcium ion kit dye (Molecular Devices) in the Hanks’/HEPES buffer for 60 min at room temperature in darkness. Temporal changes in the intracellular calcium concentration were monitored using excitation at 485 nm and emission at 525 nm and were calculated as a relative fluorescence intensity based on analyses by SoftMax Pro software. Maximum calcium ion levels were elicited with 5 M ionomycin/20 M FCCP/100 M carbamylcholine as a calibrant at the end of each assay. Data obtained from each well were normalized by the use of these maximum values as described previously (Fitch et al., 2003). Insulin Release. After reaching 80 to 90% confluence, mouse -TC6 cells were washed with glucose-free Krebs/HEPES Ringer solution (115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, and 25 mM HEPES, pH 7.4) twice and preincubated at 37°C for 30 min with the glucose-free Krebs/HEPES Ringer solution. After the preincubation, the cells were incubated in the Krebs/ HEPES Ringer solution containing 1 mg/ml bovine serum albumin and the indicated concentration of glucose in the presence or absence of test agents. The antagonists and channel blockers were applied 3 min before the addition of nicotine. An aliquot of supernatant was collected for radioimmunoassay. The amount of insulin released was measured with a radioimmunoassay kit (Linco Research, St. Charles, MO). Rb Efflux Assays. Functional properties of the nicotinic receptors expressed in the -TC6 cells were assessed by measurements Fig. 1. Concentration-dependent responses of nicotine on membrane potential in -TC6 cells: A, time course for the effect of nicotine at 1, 10, and 100 M in a representative assay. B, calculated values (mean S.E.M., n 7) for membrane depolarization changes as a percentage of the maximal response induced by the calibrant KCl. Responses at 10 and 100 M nicotine compared with the absence of nicotine, P 0.005. 900 Ohtani et al. at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from of nicotinic agonist-stimulated Rb efflux, as described previously (Xiao et al., 1998). In brief, aliquots of cells in the selection growth medium were plated into 24-well plates coated with poly(D-lysine). The plated cells were grown in medium at 37°C for 18 to 24 h to reach 70 to 95% of confluence. The cells were then loaded with RbCl by incubating them in growth medium (0.5 ml/well) containing RbCl (2 Ci/ml) for 4 h at 37°C. The loading mixture was then aspirated, and the cells were washed four times with 1 ml/well HEPES buffer (140 mM NaCl, 2 mM KCl, 1 mM MgSO4, 1.8 mM CaCl2, 11 mM glucose, and 15 mM HEPES, pH 7.4). One milliliter of buffer, with or without agonists, was then added to each well. After incubation for 2 min, the assay buffer was collected, and the amount of Rb efflux into the buffer was determined. Cells were then lysed by adding 1 ml of 100 mM NaOH to each well, and the lysate was collected for determination of the amount of Rb in the cells at the end of the efflux assay. Radioactivity of the assay buffer samples and lysates was measured by liquid scintillation counting. The total amount of Rb loaded was calculated as the sum of the Rb in the assay buffer sample and in the lysate of each well. The amount of Rb efflux was expressed as a percentage of total Rb loaded. Agoniststimulated Rb efflux was defined as the difference between efflux in the presence of nicotinic agonists and basal efflux measured in the absence of agonists. Nonlinear regression analyses and statistical analyses were performed using Prism 3 software (GraphPad Software, San Diego, CA). [H]Epibatidine Binding Assay. Binding of [H]epibatidine to nicotinic receptors was measured as described previously (Xiao et al., 1998) with minor modifications. In brief, cultured cells at 80% confluence were removed from their flasks (80 cm) with a disposable cell scraper and placed in 10 ml of 50 mM Tris-HCl buffer, pH 7.4, at 4°C. The cell suspension was centrifuged at 1000g for 5 min, and the pellet was collected. The cell pellet was then homogenized in 10 ml of buffer with a Brinkmann polytron homogenizer (model PT2100, 12 mm generator, 26,000 rpm, 20 s; Brinkmann Instruments, Westbury, NY) and centrifuged at 36,000g for 10 min at 4°C. The membrane pellet was resuspended in fresh Tris buffer, and aliquots of the membrane preparation equivalent to 30 to 200 g of protein were used for binding assays. Membrane preparations were incubated with [H]epibatidine for 4 h at 24°C. The volumes for saturation and competition binding assays were 1 and 0.5 ml/tube, respectively. Nonspecific binding was assessed in parallel incubations in the presence of 300 M nicotine. Bound and free ligands were separated by vacuum filtration through Whatman GF/C filters treated with 0.5% polyethylenimine (Whatman, Clifton, NJ). The filter-retained radioactivity was measured by liquid scintillation counting. Specific binding was defined as the difference between total binding and nonspecific binding. Data from saturation and competition binding assays were analyzed by nonlinear least-squares regressions using Prism 3 software. RT-PCR. The total RNA of -TC6 cells (1 10 cells) was extracted with TRIzol, precipitated with isopropyl alcohol, and then treated with DNase I. One microgram of RNA was reverse-transcribed to cDNA using a GeneAMP RNA PCR kit (Applied Biosystems, Foster City, CA) and then amplified by PCR with 30 cycles. The oligonucleotide primers for nicotinic subunits 3, 4, 2, 3, and 4 and for -actin (internal control) were synthesized commercially Fig. 2. Effect of nicotine and antagonists on intracellular calcium in -TC6 cells. A, time course for the effect of nicotine at 10 and 100 M in a representative assay. B, calculated values (mean S.E.M., n 4) for intracellular calcium changes as a percentage of the maximal response induced by ionomycin/FCCP/carbamylcholine. Responses at 10 and 100 M nicotine relative to no nicotine, P 0.005. C, time course for the atropine or mecamylamine in a representative assay. Both antagonists at 10 M were added together with nicotine (100 M) as indicated. D, calculated values (mean S.E.M., n 4) for intracellular calcium as the percentage of the maximal response induced by ionomycin/FCCP/carbamylcholine. Inhibition by mecamylamine, , P 0.001. Functional Nicotinic Receptors in an Insulinoma Cell Line 901 at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from (Bioserve Biotechnology, Laurel, MD), according to sequences used previously for nicotinic subunits (Kuo et al., 2002) and for -actin (Knaack et al., 1994). These primer sequences were as follows: nicotinic subunit 3: sense, 5 -TGGGGATTTCCAAGTGGA-3 ; antisense, 5 -CATGACCCTGGGGAGAAGGTT-3 ; nicotinic subunit 4: sense, 5 -GAATGTCACCTCCATCCGCATC-3 ; antisense, 5 -CCGGCA(A/G)TTGTC(C/T)TTGACCAC-3 ; nicotinic subunit 2: sense, 5 CTCCAACTCTATGGCGCTGCT-3 ; antisense, 5 -GAGCGGAACTTCATGGTGCAG-3 ; nicotinic subunit 3: sense, 5 -CTCCTCAGACATTTGTTCCAAGG-3 ; antisense, 5 -AATGAGGTCAACCATGGT-3; nicotinic subunit 4: sense, 5 -TCTGGTTGCCTGACATCGTG-3 ; antisense, 5 -GGGTTCACAAAGTACATGGA-3 ; and -actin: sense, 5 GATGACGATATCGCTGCGCTGGTCGTC-3 ; antisense, 5 -GACCCTCAGGGCATCGGAACCGCTCG-3 . Annealing temperatures for nicotinic subunits 3, 4, 2, 3, and 4, and for -actin in PCR were 52, 53, 62, 49, 52, and 65°C, respectively. A portion of the PCR products were separated on a 1.5% agarose gel containing ethidium bromide (67 ng/ml) by electrophoresis. Possible contamination of genomic DNA was assessed by performing the RT-PCR in the absence of a reverse transcriptase. Data Analysis. Data were expressed as the mean S.E.M. Statistical significance was assessed by the Student’s t test. A P value 0.05 was considered to be statistically significant.