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• W2024308977 abstract "Epibatidine and mecamylamine are ligands used widely in the study of nicotinic acetylcholine receptors (nAChRs) in the central and peripheral nervous systems. In the present study, we find that nicotine blocks only 75% of 125I-epibatidine binding to rat brain membranes, whereas ligands specific for serotonin type 3 receptors (5-HT3Rs) block the remaining 25%. 125I-Epibatidine binds with a high affinity to native 5-HT3Rs of N1E-115 cells and to receptors composed of only 5-HT3A subunits expressed in HEK cells. In these cells, serotonin, the 5-HT3R-specific antagonist MDL72222, and the 5-HT3R agonist chlorophenylbiguanide readily competed with 125I-epibatidine binding to 5-HT3Rs. Nicotine was a poor competitor for 125I-epibatidine binding to 5-HT3Rs. However, the noncompetitive nAChR antagonist mecamylamine acted as a potent competitive inhibitor of 125I-epibatidine binding to 5-HT3Rs. Epibatidine inhibited serotonin-induced currents mediated by endogenous 5-HT3Rs in neuroblastoma cell lines and 5-HT3ARs expressed in HEK cells in a competitive manner. Our results demonstrate that 5-HT3Rs are previously uncharacterized high affinity epibatidine binding sites in the brain and indicate that epibatidine and mecamylamine act as 5-HT3R antagonists. Previous studies that depended on epibatidine and mecamylamine as nAChR-specific ligands, in particular studies of analgesic properties of epibatidine, may need to be reinterpreted with respect to the potential role of 5-HT3Rs. Epibatidine and mecamylamine are ligands used widely in the study of nicotinic acetylcholine receptors (nAChRs) in the central and peripheral nervous systems. In the present study, we find that nicotine blocks only 75% of 125I-epibatidine binding to rat brain membranes, whereas ligands specific for serotonin type 3 receptors (5-HT3Rs) block the remaining 25%. 125I-Epibatidine binds with a high affinity to native 5-HT3Rs of N1E-115 cells and to receptors composed of only 5-HT3A subunits expressed in HEK cells. In these cells, serotonin, the 5-HT3R-specific antagonist MDL72222, and the 5-HT3R agonist chlorophenylbiguanide readily competed with 125I-epibatidine binding to 5-HT3Rs. Nicotine was a poor competitor for 125I-epibatidine binding to 5-HT3Rs. However, the noncompetitive nAChR antagonist mecamylamine acted as a potent competitive inhibitor of 125I-epibatidine binding to 5-HT3Rs. Epibatidine inhibited serotonin-induced currents mediated by endogenous 5-HT3Rs in neuroblastoma cell lines and 5-HT3ARs expressed in HEK cells in a competitive manner. Our results demonstrate that 5-HT3Rs are previously uncharacterized high affinity epibatidine binding sites in the brain and indicate that epibatidine and mecamylamine act as 5-HT3R antagonists. Previous studies that depended on epibatidine and mecamylamine as nAChR-specific ligands, in particular studies of analgesic properties of epibatidine, may need to be reinterpreted with respect to the potential role of 5-HT3Rs. Epibatidine is an alkaloid originally isolated from Ecuadorian poison frog skin (1Spande T.F. Garraffo H.M. Edwards M.W. Yeh H.J.C. Panne L. Daly J.W. J. Am. Chem. Soc. 1992; 114: 3475-3478Crossref Scopus (576) Google Scholar). Electrophysiological and ligand binding studies demonstrate that epibatidine is an agonist that binds with high affinity to a subset of nAChRs 2The abbreviations used are:nAChRnicotinic acetylcholine receptor5-HT3serotonin type 35-HT3Rserotonin type 3 receptorCPBGchlorophenylbiguanide.2The abbreviations used are:nAChRnicotinic acetylcholine receptor5-HT3serotonin type 35-HT3Rserotonin type 3 receptorCPBGchlorophenylbiguanide. throughout the central and peripheral nervous systems (2Badio B. Daly J.W. Mol. Pharmacol. 1994; 45: 563-569PubMed Google Scholar, 3Bonehaus D.W. Bley K.R. Broka C.A. Fontana D.J. Leung E. Lewis R. Wong S.A. J. Pharmacol. Exp. Ther. 1995; 272: 1199-1203PubMed Google Scholar, 4Houghtling R.A. Davila-Garcia M.I. Kellar K.J. Mol. Pharmacol. 1995; 48: 280-287PubMed Google Scholar, 5Ulrich Y.M. Hargreaves K.M. Flores C.M. Neuropharmacology. 1997; 36: 1119-1125Crossref PubMed Scopus (46) Google Scholar, 6Gerzanich V. Peng X. Wang F. Wells G. Anand R. Fletcher S. Lindstrom J. Mol. Pharmacol. 1995; 48: 774-782PubMed Google Scholar). Epibatidine administration has toxic effects, such as seizures and hypertension (7Dobelis P. Hutton S. Lu Y. Collins A.C. J. Pharmacol. Exp. Ther. 2003; 306: 1159-1166Crossref PubMed Scopus (47) Google Scholar, 8Boyce S. Webb J.K. Shepheard S.L. Russell M.G. N. Hill R.G. Rupniak N.M. J. Pain. 2000; 85: 443-450Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 9Fisher M. Huangfu D. Shen T.Y. Guyenet P.G. J. Pharmacol. Exp. Ther. 1994; 270: 702-707PubMed Google Scholar). However, at similar doses, epibatidine has antinociceptive effects that are blocked by nAChR antagonists, such as mecamylamine, consistent with the analgesic activity of epibatidine being mediated through nAChRs (2Badio B. Daly J.W. Mol. Pharmacol. 1994; 45: 563-569PubMed Google Scholar, 10Radek R.J. Curzon P. Decker M.W. Brain Res. Bul. 2004; 64: 323-330Crossref PubMed Scopus (5) Google Scholar, 11Damaj M.I. Creasy K.R. Grove A.D. Rosecrans J.A. Martin B.R. Brain Res. 1994; 664: 34-40Crossref PubMed Scopus (100) Google Scholar). Administration of mecamylamine alone also has pronounced physiological effects that alter pain perception and blood pressure (7Dobelis P. Hutton S. Lu Y. Collins A.C. J. Pharmacol. Exp. Ther. 2003; 306: 1159-1166Crossref PubMed Scopus (47) Google Scholar, 12Rashid M.H. Furue H. Yoshimura M. Ueda H. Pain. 2006; 125: 125-135Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 13Young J.M. Shytle R.D. Sandberg P.R. George T.P. Clin. Ther. 2001; 23: 532-565Abstract Full Text PDF PubMed Scopus (79) Google Scholar). nicotinic acetylcholine receptor serotonin type 3 serotonin type 3 receptor chlorophenylbiguanide. nicotinic acetylcholine receptor serotonin type 3 serotonin type 3 receptor chlorophenylbiguanide. 5-HT3Rs and nAChRs are members of the same ionotropic neurotransmitter receptor family that also includes GABAA and glycine receptors (14Connolly C.N. Wafford K.A. Biochem. Soc. Trans. 2004; 32: 529-534Crossref PubMed Scopus (156) Google Scholar, 15Ortells M.O. Lunt G.G. Trends Neurosci. 1995; 18: 121-127Abstract Full Text PDF PubMed Scopus (464) Google Scholar). Within this neurotransmitter receptor family, 5-HT3Rs are most similar to nAChRs, sharing up to 30% sequence homology (16Maricq A.V. Peterson A.S. Brake A.J. Myers R.M. Julius D. Science. 1991; 254: 432-437Crossref PubMed Scopus (880) Google Scholar, 17Werner P. Kawashima E. Reid J. Hussy N. Lundstrom K. Buell G. Humbert Y. Jones K.A. Mol. Brain Res. 1994; 26: 233-241Crossref PubMed Scopus (77) Google Scholar). Physiological studies indicate that 5-HT3Rs and nAChRs regulate many of the same pathways (18Bucaffusco J.J. Pharmacol. Rev. 1996; 48: 179-211PubMed Google Scholar, 19Costall B. Naylor R.J. Curr. Drug Targets CNS Neurol. Disord. 2004; 3: 27-37Crossref PubMed Scopus (118) Google Scholar, 20Iwamoto E.T. Marion L. J. Pharmacol. Exp. Ther. 1993; 265: 777-789PubMed Google Scholar, 21Saxena P.R. Villalon C.M. J. Cardiovasc. Pharmacol. 1990; 15: 17-34Crossref PubMed Scopus (241) Google Scholar). Co-expression of these two classes of receptors occurs in regions of the nervous system associated with pain processing, such as the dorsal horn of the spinal cord, nucleus of the solitary tract, raphe magnus nucleus, and nucleus dorsalis (22Ferezou I. Cauli B. Hill E.L. Rossier J. Hamel E. Lambolez B. J. Neurosci. 2002; 22: 7389-7397Crossref PubMed Google Scholar, 23Decker M.W. Curzon P. Holladay M.W. Nikkel A.L. Bitner R.S. Bannon A.W. Donnelly-Roberts D.L. Puttfarcken P.S. Kuntzweiler T.A. Briggs C.A. Williams M. Arneric S.P. J. Physiol. 1999; 92: 221-224Google Scholar, 24Cordero-Erausquin M. Marubio L.M. Klink R. Changeux J. Trends Pharmacol. Sci. 2000; 21: 211-217Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 25Farber L. Haus U. Spath M. Drechsler S. Scand. J. Rheumatol. 2004; 33: 2-8Crossref Scopus (82) Google Scholar). Local or systemic administration of nAChR agonists reduces the pain response in rodents (2Badio B. Daly J.W. Mol. Pharmacol. 1994; 45: 563-569PubMed Google Scholar, 20Iwamoto E.T. Marion L. J. Pharmacol. Exp. Ther. 1993; 265: 777-789PubMed Google Scholar, 26Bitner R.S. Nikkel A.L. Curzon P. Arneric S.P. Bannon A.W. Decker M.W. J. Neurosci. 1998; 18: 5426-5432Crossref PubMed Google Scholar, 27Cucchiaro G. Chaijale N. Commons G. J. Pharmacol. Exp. Ther. 2005; 313: 389-394Crossref PubMed Scopus (39) Google Scholar, 28Gilbert S.D. Clark T.M. Flores C.M. Pain. 2001; 89: 159-165Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 29Khan I.M. Buerkle H. Taylor P. Yaksh T.L. Neuropharmacology. 1998; 37: 1515-1525Crossref PubMed Scopus (93) Google Scholar, 30Lawand N.B. Lu Y. Westlund K.N. Pain. 1999; 80: 291-299Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). 5-HT3R ligands appear to be both pro- and antinociceptive, depending on the pain stimulus and route of administration (19Costall B. Naylor R.J. Curr. Drug Targets CNS Neurol. Disord. 2004; 3: 27-37Crossref PubMed Scopus (118) Google Scholar, 31Zhang L. Oz M. Weight F.F. Neuroreport. 1995; 8: 1464-1468Crossref Scopus (33) Google Scholar, 32Zeitz K.P. Guy N. Malmberg A.B. Dirajlal S. Martin W.J. Sun L. Bonehaus D.W. Stucky C.L. Julius D. Basbaum A.I. J. Neurosci. 2002; 22: 1010-1019Crossref PubMed Google Scholar, 33Moore K.A. Oh E.J. Weinreich D. J. Appl. Physiol. 2002; 92: 2529-2534Crossref PubMed Scopus (22) Google Scholar, 34Ali Z. Wu G. Kozlov A. Barasi S. Neurosci. Lett. 1996; 208: 203-207Crossref PubMed Scopus (97) Google Scholar, 35Alhaider A.A. Lei S.Z. Wilcox G.L. J. Neurosci. 1991; 11: 1881-1888Crossref PubMed Google Scholar). 5-HT3Rs and nAChRs also contribute to the modulation of blood pressure. Activation of nAChRs increases pressor and heart rate in normotensive and spontaneously hypertensive animals (18Bucaffusco J.J. Pharmacol. Rev. 1996; 48: 179-211PubMed Google Scholar, 36Holliday M.W. Wasicak J.T. Lin N. He Y. Ryther K.B. Bannon A.W. Buckley M.J. Kim D.J.B. Decker M.W. Anderson D.J. Cambell J.E. Kuntzweiler T.A. Donnelly-Roberts D.L. Piattoni-Kaplan M. Briggs C.A. Williams M. Arneric S.P. J. Med. Chem. 1998; 41: 407-412Crossref PubMed Scopus (133) Google Scholar, 37Hoffman W.E. Schmid P.G. Phillips M.I. J. Pharmacol. Exp. Ther. 1978; 206: 644-651PubMed Google Scholar, 38Tseng C-J. Appalsamy M. Robertson D. Mosqueda-Garcia R. J. Pharmacol. Exp. Ther. 1993; 265: 1511-1518PubMed Google Scholar). In spontaneously hypertensive rats, chronic inhibition of 5-HT3Rs lowers blood pressure (39Alkadhi, K. A., Otoom, S. A., Tanner, F. L., Sockwell, D., and Hogan, Y. H. (2001) 226, 1024–1030Google Scholar), whereas in normal rats, activation of 5-HT3Rs decreases arterial pressure (40Ferreira H.S. Silva E.C. Cointeiro C. Oliveira E. Faustino T.N. Fregoneze J.B. Brain Res. 2004; 1028: 48-58Crossref PubMed Scopus (16) Google Scholar). Inhibition of 5-HT3Rs likewise produced a significant decrease in blood pressure in obese rats prone to stress-induced hypertension (41Gerges N.Z. Aleisa A.M. Alhaider A.A. Alkadhi K.A. Neuropharmacology. 2002; 43: 1070-1076Crossref PubMed Scopus (25) Google Scholar). In addition to these effects, drug-induced and epilepsy-associated convulsive activity display an nAChR (42Stitzel J.A. Jimenez M. Marks M.J. Tritto T. Collins A.C. J. Pharmacol. Exp. Ther. 2000; 293: 67-74PubMed Google Scholar, 43Dani J.A. Bertrand D. Annu. Rev. Pharmacol. Toxicol. 2007; 47: 699-729Crossref PubMed Scopus (913) Google Scholar, 44Butt C.M. King N.M. Stitzel J.A. Collins A.C. J. Pharmacol. Exp. Ther. 2004; 308: 591-599Crossref PubMed Scopus (42) Google Scholar) and 5-HT3R (45Wada Y. Shiraishi J. Nakamura M. Koshino Y. Brain Res. 1997; 759: 313-316Crossref PubMed Scopus (26) Google Scholar, 46Grant K.A. Hellevuo K. Tabakoff B. Alcohol. Clin. Exp. Res. 1994; 18: 410-414Crossref PubMed Scopus (27) Google Scholar, 47Anuradha K. Hota D. Pandhi P. Indian J. Exp. Biol. 2004; 42: 368-372PubMed Google Scholar) sensitivity. These observations link 5-HT3R function with responses associated with administration of some nAChR-specific ligands. Experimental evidence indicates that select nAChR ligands cross-react with 5-HT3Rs as either agonists or antagonists (48Sepulveda M.I. Baker J. Lummis S.C. Neuropharmacology. 1994; 33: 493-499Crossref PubMed Scopus (21) Google Scholar, 49Lummis S.C. Kilpatrick G.J. Martin I.L. Eur. J. Pharmacol. 1990; 189: 22-27Google Scholar, 50Hope A.G. Belelli D. Mair I.D. Lambert J.J. Peters J.A. Mol. Pharmacol. 1999; 55: 1037-1043Crossref PubMed Scopus (40) Google Scholar, 51Gurley D.A. Lanthorn T.H. Neurosci. Lett. 1998; 247: 107-110Crossref PubMed Scopus (52) Google Scholar, 52Machu T.K. Hamilton M.E. Frye T.F. Shanklin C.L. Harris M.C. Sun H. Tenner T.E. Soti F.S. Kem W.R. J. Pharmacol. Exp. Ther. 2001; 299: 1112-1119PubMed Google Scholar). In this study, using a combination of neuroblastoma cell lines, heterologous expression of 5-HT3ARs, and rat brain preparations, we show that epibatidine binds with high affinity to 5-HT3Rs. In addition, both epibatidine and mecamylamine function as antagonists when bound to 5-HT3Rs. Thus, 5-HT3Rs may participate in physiological effects previously thought to be nAChR-specific. Cell Lines and Transfection—The human kidney epithelial cell line tsA201 (HEK cells) was maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum (Hyclone, Logan, UT). Hemagglutinin epitope-tagged mouse 5HT3A was subcloned into the pcDNA3.1 vector using standard methodology. HEK cells were transiently transfected with the subunit cDNA constructs using a calcium phosphate protocol (53Eertmoed A. Vajello Y.F. Green W.N. Methods Enzymol. 1999; 293: 564-585Crossref Scopus (22) Google Scholar). In some experiments, for comparison with mouse 5-HT3A receptors, HEK cells were transfected with cDNA encoding the human 5-HT3A receptor subunit. Mouse neuroblastoma N1E-115 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37 °C, 5% CO2. Mouse neuroblastoma NB41A3 cells (ATCC) were maintained in F-12 medium supplemented with 15% horse serum, 5% fetal calf serum. Cells were plated onto 35-mm culture dishes (Fisher) for use in electrophysiological experiments. All medium was supplemented with penicillin (100 IU/ml) and streptomycin (100 μg/ml). Rat Brain Membranes—Membranes were prepared as described previously (54Drisdel R.C. Green W.N. J. Neurosci. 2000; 20: 133-139Crossref PubMed Google Scholar). Briefly, adult rats were decapitated, and the entire brain was dissected and placed in ice-cold 50 mm NaPO4, pH 7.4, 50 mm NaCl, 2 mm EDTA, and 2 mm EGTA plus protease inhibitors (2 mm phenylmethylsulfonyl fluoride, 2 mm N-ethylmaleimide, chymostatin, pepstatin, 1-chloro-3-tosylamido-7-amino-2-heptanone, and leupeptin at 10 μg/ml). All chemicals were obtained from Sigma unless otherwise specified. The brain tissue was minced and homogenized in a Teflon-glass homogenizer. Homogenates were centrifuged at 100,000 × g for 1 h. Pellets were taken through one more cycle of homogenization and centrifugation. The resulting pellets were resuspended in 150 mm NaCl, 5 mm EDTA, 50 mm Tris, pH 7.4, 0.02% NaN3 plus protease inhibitors and frozen at –80 °C until needed. Ligand Binding Studies—Unlabeled epibatidine in phosphate-buffered saline was added to 125I-epibatidine (2200 Ci/mmol) or [3H]epibatidine (47.3 Ci/mmol) (PerkinElmer Life Sciences) to a total epibatidine concentration of 1.7 and 10 μm, respectively. Equilibrium binding to whole cell membrane fractions was measured by incubating at room temperature in phosphate-buffered saline with increasing amounts of radiolabeled epibatidine on a table top rotator for 20 min. For competitive binding experiments, cells were preincubated with different serotonergic or nicotinic ligands at the specified concentrations, followed by the addition of 50 nm 125I-epibatidine. Nonspecific binding was determined in the presence of 1 mm epibatidine, 100 μm MDL72222 or by performing binding on cells transfected with empty vector. Samples were washed three times rapidly with ice-cold phosphate-buffered saline in a Brandel apparatus. 125I- and [3H]epibatidine counts were measured in a Wallac 1470 automatic γ counter and a Beckman LS6500 scintillation counter, respectively. Equilibrium binding constants were calculated based on the least squares equation, amount of ligand bound = Bmax × [ligand]/KD + [ligand]. Competition binding curves were generated with the least squared fit to the Hill equation, fraction of maximum = 1/(1 + IC50/[ligand])n, where n represents the Hill coefficient. IC50 values were converted to KI values using the equation, KI = IC50/1 + ([epibatidine]/KD). The KD for epibatidine used to calculate KI values for nicotine and MDL72222 in Fig. 1 was 8 pm (55Gnadisch D. London E.D. Terry P. Hill G.R. Mukhin A.G. Neuroreport. 1999; 10: 1631-1636Crossref PubMed Scopus (35) Google Scholar) and 27 nm (see Fig. 2A), respectively.FIGURE 2Saturation and competition binding of serotonergic and nicotinic ligands to 5-HT3Rs. A, representative saturation binding curves showing specific 125I-epibatidine binding (KD = 27 ± 6 nm) and [3H]epibatidine binding (KD = 22 ± 5 nm) to N1E-115 cell membrane fractions. B, various nicotinic and serotonergic ligands compete for 125I-epibatidine binding sites in N1E-115 cell membranes. C, saturation binding of 125I-epibatidine (KD = 14 ± 4 nm) to 5-HT3ARs expressed heterologously in HEK cell membranes. D, competition binding experiments were performed as described in B on membrane fractions from HEK cells expressing the 5-HT3AR. The graphs represent the least squares fit to the Hill equation, fraction of maximum = 1/(1 + [ligand]/IC50)n, where n represents the Hill coefficient. KI values calculated from IC50 values and Hill coefficients are shown in Table 1. Each point represents the mean ± S.D. of three determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Electrophysiological Recording—The whole cell configuration of the patch clamp technique was used to record currents from cells expressing either recombinant 5-HT3A receptors (HEK cells) or native 5-HT3 receptors (N1E-115 and NB41A3 cells). The electrode solution contained 140 mm CsCl, 2 mm MgCl2, 0.1 mm CaCl2, 1.1 mm EGTA, and 10 mm HEPES (pH 7.4 with CsOH). The extracellular solution contained 140 mm NaCl, 2.8 mm KCl, 2 mm MgCl2, 1 mm CaCl2, 10 mm HEPES, and 10 mm glucose (pH 7.4 with NaOH). Cells were voltage-clamped at an electrode potential of –60 mV. 5-HT3 receptors were activated by locally applying agonists to the cell by pressure (10 p.s.i.) ejection (Picospritzer II; General Valve Corp.) from modified patch pipettes. The recording chamber was continuously perfused with extracellular solution (5 ml/min). Experiments took place at 20–22 °C. Currents were monitored using an Axopatch 200B, low pass-filtered at 2 kHz, digitized at 10 KHz using a Digidata 1320A interface, and acquired using pCLAMP8 software (all from Axon Instruments) onto the hard drive of a personal computer for off-line analysis. The peak amplitudes of agonist-activated currents were measured using pCLAMP8 software. Concentration-response relationships were fitted with modified logistic functions to determine EC50, IC50, and Hill slope values, as described previously (56Adodra S. Hales T.G. Br. J. Pharmacol. 1995; 115: 953-960Crossref PubMed Scopus (84) Google Scholar). High Affinity Epibatidine Binding in Rat Brain to Sites Other than nAChRs—When relatively high concentrations of 125I-epibatidine (50 nm) were used to measure binding to membranes prepared from rat brain homogenates, a component of the 125I-epibatidine binding was insensitive to nicotine competition (Fig. 1A). Although nicotine blocked 75% of the binding to rat brain membranes, 25% of the binding remained intact even at nicotine concentrations as high as 1 mm. Nicotine blocked 125I-epibatidine binding with a KI of 2 nm, which was derived from the fit of the Hill equation to the data in Fig. 1A. The KI value for nicotine is similar to other measurements using epibatidine binding to rat brain synaptosomal membranes (55Gnadisch D. London E.D. Terry P. Hill G.R. Mukhin A.G. Neuroreport. 1999; 10: 1631-1636Crossref PubMed Scopus (35) Google Scholar) and rat cerebral cortex (57Davila-Garcia M.I. Musachio J.L. Kellar K.J. J. Neurochem. 2003; 85: 1237-1246Crossref PubMed Scopus (37) Google Scholar). These data indicate that 25% of the 125I-epibatidine binding is to sites other than nAChRs. Since epibatidine has been shown to block serotonin-induced currents mediated by 5-HT3ARs in oocytes (51Gurley D.A. Lanthorn T.H. Neurosci. Lett. 1998; 247: 107-110Crossref PubMed Scopus (52) Google Scholar), and nAChRs and 5-HT3Rs share a high degree of sequence similarity (16Maricq A.V. Peterson A.S. Brake A.J. Myers R.M. Julius D. Science. 1991; 254: 432-437Crossref PubMed Scopus (880) Google Scholar), we tested the effects 5-HT3R-specific ligands on 125I-epibatidine binding to rat brain fractions. As shown in Fig. 1B, increasing concentrations of the 5-HT3R-specific antagonist, MDL72222, blocked 25% of the 125I-epibatidine binding in experiments parallel to those in Fig. 1A using the rat brain membranes. We obtained similar data using a 5-HT3R-specific agonist, chlorophenylbiguanide (CPBG) (data not shown). The effects of nicotine and the 5-HT3R-specific ligands were additive, as shown in Fig. 1C. Preincubation of the rat brain membranes with saturating concentrations of either nicotine and MDL72222 or nicotine and CPBG blocked all specific 125I-epibatidine binding to the membrane. These results indicate that 125I-epibatidine binds to two distinct subpopulations of receptors in rat brain: 1) a nicotine-sensitive subpopulation consisting of nAChRs and 2) a nicotine-insensitive subpopulation consisting of 5-HT3Rs. Epibatidine Binding to Native and Heterologous 5-HT3Rs—To further test whether there are 125I-epibatidine binding sites on 5-HT3Rs, we performed binding assays on preparations in which native and heterologously expressed 5-HT3Rs were present in the absence of any significant nAChR expression. To study native 5-HT3Rs, we used undifferentiated mouse neuroblastoma N1E-115 cells. These cells express endogenous 5-HT3A and 5-HT3B subunits (58Reeves D.C. Lummis C.R. BMC Neurosci. 2006; 710.1186/1471-2202-7-27Crossref PubMed Scopus (25) Google Scholar) but little to no nicotinic receptors (59Oortgiesen M. van Kleef R.G. Vijverberg H.P. Brain Res. 1997; 747: 1-8Crossref PubMed Scopus (7) Google Scholar). Consistent with an absence of nAChR high affinity sites, we detected no specific binding to membranes from undifferentiated N1E-115 cells at 125I-epibatidine concentrations of 0.5 nm. At higher concentrations, we observed an increasing level of specific, high affinity 125I-epibatidine binding to the neuroblastoma cell 5-HT3Rs with an estimated KD of 27 ± 6 nm (Fig. 2A). We obtained similar results (an estimated KD of 22 ± 5 nm) with [3H]epibatidine binding to neuroblastoma cell 5-HT3Rs (Fig. 2A). Serotonin, MDL72222, and CPBG were highly potent inhibitors of 125I-epibatidine binding to N1E-115 cell membranes as compared with nicotine in competition binding experiments (Fig. 2B). Surprisingly, mecamylamine, a noncompetitive antagonist of different nAChRs, blocked 125I-epibatidine binding to 5-HT3Rs. The relative potency in competing for 125I-epibatidine binding sites in N1E-115 cell membranes was MDL72222 > epibatidine > 5-HT = CPBG > mecamylamine ⋙ nicotine (Table 1).TABLE 1Inhibition of 125I-epibatidine binding to 5-HT3Rs by various serotonergic and nicotinic ligandsN1E-115 cellsMouse 5-HT3ARs in HEK cellsKIHill coefficientKIHill coefficientmmEpibatidine2.2 ± 0.5 × 10-60.66 ± 0.055.0 ± 1.0 × 10-70.38 ± 0.03Mecamylamine2.2 ± 0.6 × 10-50.53 ± 0.052.0 ± 0.2 × 10-60.93 ± 0.07Serotonin5.4 ± 1.2 × 10-60.38 ± 0.035.3 ± 1.4 × 10-60.53 ± 0.06MDL722223.0 ± 0.8 × 10-70.44 ± 0.051.2 ± 0.2 × 10-60.45 ± 0.06CPBG5.6 ± 1.0 × 10-60.70 ± 0.109.7 ± 1.4 × 10-60.64 ± 0.05Nicotine2.0 ± 0.3 × 10-40.47 ± 0.052.7 ± 1.4 × 10-40.34 ± 0.06 Open table in a new tab 5-HT3Rs native to neuroblastoma cells have functional properties similar to those of homomeric 5-HT3A receptors (60Stewart A. Davies P.A. Kirkness E.F. Safa P. Hales T.G. Neuropharmacology. 2003; 44: 214-223Crossref PubMed Scopus (37) Google Scholar). To compare 5-HT3Rs of neuroblastoma cells with those of known composition, we performed experiments on membranes from HEK cells expressing mouse 5-HT3A subunits. As shown in Fig. 2C, 5-HT3ARs bound 125I-epibatidine with a KD of 14 ± 4 nm, which is approximately half of the KD observed for native receptors in N1E-115 cells. Competition binding experiments were also performed on membranes from HEK cells expressing 5-HT3ARs. The relative potency in competing for 125I-epibatidine binding sites from the HEK cell membranes was epibatidine > MDL72222 > 5-HT = mecamylamine > CPBG ⋙ nicotine (Table 1). The Action of Epibatidine on 5-HT3R Function—Previous studies have shown that some nicotinic receptor agonists, including epibatidine, inhibit serotonin-induced currents in oocytes expressing 5-HT3ARs (51Gurley D.A. Lanthorn T.H. Neurosci. Lett. 1998; 247: 107-110Crossref PubMed Scopus (52) Google Scholar, 52Machu T.K. Hamilton M.E. Frye T.F. Shanklin C.L. Harris M.C. Sun H. Tenner T.E. Soti F.S. Kem W.R. J. Pharmacol. Exp. Ther. 2001; 299: 1112-1119PubMed Google Scholar). Therefore, we investigated the effect of epibatidine on 5-HT3R function in cells expressing native and recombinant 5-HT3 receptors. N1E-115 neuroblastoma cells express endogenous functional 5-HT3Rs (61Lambert J.J. Peters J.A. Hales T.G. Dempster J. Br. J. Pharmacol. 1989; 97: 27-40Crossref PubMed Scopus (111) Google Scholar). 5-HT (30 μm) activated inward currents recorded from N1E-115 cells voltage-clamped at –60 mV. Application of epibatidine to the recording chamber caused a concentration-dependent inhibition of 5-HT-evoked currents (Fig. 3A) with an IC50 of 3.3 ± 0.3 μm (Table 2). Epibatidine had a similar potency (IC50 = 4.7 ± 0.5 μm) as an inhibitor of 5-HT-evoked currents recorded from NB41A3 mouse neuroblastoma cells, which expresses native 5-HT3Rs with properties similar to those of homomeric 5-HT3ARs (60Stewart A. Davies P.A. Kirkness E.F. Safa P. Hales T.G. Neuropharmacology. 2003; 44: 214-223Crossref PubMed Scopus (37) Google Scholar). We tested the effect of epibatidine on recombinant mouse 5-HT3ARs expressed in HEK cells. Epibatidine inhibited 5-HT-evoked currents with an IC50 of 4.1 ± 0.3 μm (Table 2). 5-HT elicited a concentration-dependent activation of current mediated by recombinant 5-HT3ARs with an EC50 of 5.7 ± 0.7 μm (Fig. 3B). In the presence of epibatidine (30 μm), this response is shifted to the right (EC50 = 10.1 ± 0.3 μm) without altering the maximum current amplitude, indicating that epibatidine is a competitive antagonist at 5-HT3Rs (Fig. 3B).TABLE 2Inhibition by epibatidine of 5-HT-evoked currentsIC50Hill coefficientμm5-HT3Rs in neuroblastoma cells N1E-1153.3 ± 0.31.5 ± 0.2 NB41A34.7 ± 0.51.1 ± 0.1Ectopic 5-HT3ARs in HEK cells Mouse 5-HT3A4.1 ± 0.31.6 ± 0.1 Human 5-HT3A7.6 ± 0.71.1 ± 0.1 Open table in a new tab d-Tubocurarine, a plant alkaloid that inhibits nAChR function, also inhibits 5-HT3Rs (50Hope A.G. Belelli D. Mair I.D. Lambert J.J. Peters J.A. Mol. Pharmacol. 1999; 55: 1037-1043Crossref PubMed Scopus (40) Google Scholar). The potency of d-tubocurarine as an antagonist of 5-HT3R-mediated currents is highly species-dependent. Human 5-HT3ARs are >1000 times less sensitive to inhibition by d-tubocurarine than are rodent 5-HT3ARs. Therefore, we examined whether the antagonist potency of epibatidine also varies between mouse and human 5-HT3ARs. 5-HT (30 μm)-activated currents recorded from HEK cells expressing recombinant human 5-HT3ARs were inhibited by epibatidine with an IC50 of 7.6 ± 0.7 μm (n = 5), a value that is only slightly higher than the IC50 of epibatidine as an inhibitor of mouse 5-HT3ARs (Table 2). Since mecamylamine displaces binding of epibatidine to 5-HT3Rs (Fig. 2B and Table 1), we examined whether the nAChR antagonist affects 5-HT-activated currents mediated by recombinant 5-HT3ARs. Mecamylamine (10 μm) caused a 30 ± 4% (n = 4) inhibition of 5-HT-activated currents mediated by human 5-HT3ARs. In this study, we identify high affinity 125I-epibatidine binding sites in the brain with the pharmacological characteristics of 5-HT3Rs. These sites are distinctly serotonergic, since the 5-HT3R agonist (CPBG) and antagonist (MDL72222) effectively block 125I-epibatidine binding, whereas nicotine is ineffective at blocking binding. Nicotine is also a poor competitor against 125I-epibatidine binding to 5-HT3AR homomers expressed heterologously in HEK cells and endogenous 5-HT3Rs expressed by N1E-115 mouse neuroblastoma cells. In these cells, epibatidine blocks serotonin-induced currents in a competitive manner with IC50 values similar to that observed for the inhibition of recombinant 5-HT3R-mediated currents in oocytes (51Gurley D.A. Lanthorn T.H. Neurosci. Lett. 1998; 247: 107-110Crossref PubMed Scopus (52) Google Scholar). Thus, in addition to being an agonist at nAChRs, we conclude that epibatidine is a competitive antagonist of 5-HT3Rs. The affinity of epibatidine for 5-HT3R sites is approximately 3 orders of magnitude lower than for nAChR subtypes that bind epibatidine with high affinity. Epibatidine binds to these nAChRs with KD values in the 10–100 pm range (57Davila-Garcia M.I. Musachio J.L. Kellar K.J. J. Neurochem. 2003; 85: 1237-1246Crossref PubMed Scopus (37) Google Scholar, 62Parker S.L. Fu Y. McAllen K. Luo J. McIntosh J.M. Lindstrom J.M. Sharp B.M. Mol. Pharmacol. 2004; 65: 611-622Crossref PubMed Scopus (88) Google Scholar, 63Whiteaker P. Jimenez M. McIntosh J.M. Collins A.C. Marks M.J. Br. J. Pharmacol. 2000; 131: 729-739Crossref PubMed Scopus (101) Google Scholar). This variability might arise from ligand depletion during binding studies that can result in underestimation of the affinity of epibatidine for nAChRs. In studies that avoided ligand depletion, epibatidine binds to a single population of brain nAChRs with KD values in the 1–8 pm range (4Houghtling R.A. Davila-Garcia M.I. Kellar K.J. Mol. Pharmacol. 1995; 48: 280-287PubMed Google Scholar, 55Gnadisch D. London E.D. Terry P. Hill G.R. Mukhin A.G. Neuroreport. 1999; 10: 1631-1636Crossref PubMed Scopus (35) Google Scholar). Because of its extremely high affinity for nAChRs, virtually all studies measuring equilibrium binding have used epibatidine concentrations less than 1 nm, which would result in little, if any, binding to 5-HT3Rs. Thus, the failure to observe epibatidine binding to 5-HT3Rs in earlier studies is most likely due to the relatively low concentrations of radiolabeled epibatidine used to measure binding to nAChRs. When epibatidine concentrations much higher than 1 nm were used in radiolabeled epibatidine binding studies, a second population of binding sites with lower affinity for epibatidine was observed (4Houghtling R.A. Davila-Garcia M.I. Kellar K.J. Mol. Pharmacol. 1995; 48: 280-287PubMed Google Scholar, 64McCallum S.E. Collins A.C. Paylor R. Marks M.J. Psychopharmacology. 2006; 184: 314-327Crossref PubMed Scopus (59) Google Scholar, 65Marks M.J. Whiteaker P. Collins A.C. Mol. Pharmacol. 2006; 70: 947-959Crossref PubMed Scopus (49) Google Scholar). Based on the works of Marks et al. (65Marks M.J. Whiteaker P. Collins A.C. Mol. Pharmacol. 2006; 70: 947-959Crossref PubMed Scopus (49) Google Scholar), using nAChR subunit null mice, at least some of the lower affinity epibatidine sites consist of nAChRs. However, data presented in this study suggest that a portion of the lower affinity sites may also consist of 5-HT3Rs. Our results are consistent with earlier studies that have established significant pharmacological crossover between 5-HT3Rs and nAChRs. For example, the nAChR antagonist, d-tubocurarine, and 5-HT3R-specific antagonists, such as MDL72222 and GR65630, bind to 5-HT3Rs with comparable affinities. Furthermore, d-tubocurarine blocks 5-HT3R-mediated currents (49Lummis S.C. Kilpatrick G.J. Martin I.L. Eur. J. Pharmacol. 1990; 189: 22-27Google Scholar, 50Hope A.G. Belelli D. Mair I.D. Lambert J.J. Peters J.A. Mol. Pharmacol. 1999; 55: 1037-1043Crossref PubMed Scopus (40) Google Scholar). Similar actions are observed for the nAChR channel blockers, chlorpromazine and QX222 (48Sepulveda M.I. Baker J. Lummis S.C. Neuropharmacology. 1994; 33: 493-499Crossref PubMed Scopus (21) Google Scholar). Other studies also show that tropisetron, which is a selective 5-HT3R antagonist, binds with high affinity and is a potent partial agonist at the α7 nAChR (66Macor J.E. Gurley D.A. Lanthorn T.H. Loch J. Mack R.A. Mullen G. Tran O. Wright N. Gordon C. Bioorg. Med. Chem. Lett. 2001; 44: 319-321Crossref Scopus (143) Google Scholar), whereas the α7 agonist, PSAB-OFP, acts as a potent agonist at 5-HT3Rs (67Broad L.M. Felthouse C. Zwart R. McPhie G.I. Pearson K.H. Craig P.J. Wallace L. Broadmore R.J. Boot J.R. Keenan M. Baker S.R. Sher E. Eur. J. Pharmacol. 2002; 452: 137-144Crossref PubMed Scopus (34) Google Scholar). Relevant to our results, the nAChR agonists, anabaseine and epibatidine, are shown to block serotonin-activated currents in oocytes expressing 5-HT3ARs with IC50 values of 14 and 8 μm, respectively (51Gurley D.A. Lanthorn T.H. Neurosci. Lett. 1998; 247: 107-110Crossref PubMed Scopus (52) Google Scholar, 52Machu T.K. Hamilton M.E. Frye T.F. Shanklin C.L. Harris M.C. Sun H. Tenner T.E. Soti F.S. Kem W.R. J. Pharmacol. Exp. Ther. 2001; 299: 1112-1119PubMed Google Scholar). Similarly, we find that epibatidine effectively blocks rodent 5-HT3R-mediated currents in a competitive manner with IC50 values of 3.3 and 4.1 μm in neuroblastoma cells and HEK cells, respectively. The mechanisms underlying the physiological effects of epibatidine have not been fully characterized. The concentrations of epibatidine required for analgesia are in the micromolar range (27Cucchiaro G. Chaijale N. Commons G. J. Pharmacol. Exp. Ther. 2005; 313: 389-394Crossref PubMed Scopus (39) Google Scholar, 29Khan I.M. Buerkle H. Taylor P. Yaksh T.L. Neuropharmacology. 1998; 37: 1515-1525Crossref PubMed Scopus (93) Google Scholar, 30Lawand N.B. Lu Y. Westlund K.N. Pain. 1999; 80: 291-299Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 68Curzon P. Nikkel A.L. Bannon A.W. Arneric S.P. Decker M.W. J. Pharmacol. Exp. Ther. 1998; 287: 847-853PubMed Google Scholar, 69Rashid M.H. Ueda H. Brain Res. 2002; 953: 53-62Crossref PubMed Scopus (70) Google Scholar) and also produce adverse symptoms, including hypothermia, hypertension, ataxia, and seizures (8Boyce S. Webb J.K. Shepheard S.L. Russell M.G. N. Hill R.G. Rupniak N.M. J. Pain. 2000; 85: 443-450Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 70Sullivan J.P. Decker M.W. Brioni J.D. Donnelly-Roberts D.L. Anderson D.J. Bannon A.W. Kang C. Adams P. Piattoni-Kaplan M. Buckley M.J. Gopalakrishnan M. Williams M. Arneric S.P. J. Pharmacol. Exp. Ther. 1994; 271: 624-631PubMed Google Scholar). These concentrations are similar to the concentrations that we found antagonize 5-HT3Rs and are orders of magnitude higher than what is needed to saturate the high affinity binding site on nAChRs but in line with the epibatidine concentrations that activate nAChRs. As an agonist, epibatidine (10–100 nm) increases neurotransmitter release from hippocampal slice preparations (70Sullivan J.P. Decker M.W. Brioni J.D. Donnelly-Roberts D.L. Anderson D.J. Bannon A.W. Kang C. Adams P. Piattoni-Kaplan M. Buckley M.J. Gopalakrishnan M. Williams M. Arneric S.P. J. Pharmacol. Exp. Ther. 1994; 271: 624-631PubMed Google Scholar, 71Sacaan A.I. Menzaghi F. Dunlop J.L. Correa L.D. Whelan K.T. Lloyd G.K. J. Pharmacol. Exp. Ther. 1996; 276: 509-515PubMed Google Scholar) and current responses in hippocampal and retinal neurons (72Alkondon M. Albuquerque E.X. J. Pharmacol. Exp. Ther. 1996; 274: 771-782Google Scholar, 73Lecchi M. McIntosh J.M. Bertrand S. Safran A.B. Bertrand D. J. Neurosci. 2005; 21: 3182-3188Google Scholar). Using heterologous expression of α4β2 nAChRs, Buisson et al. found a similar dose dependence for epibatidine as an nAChR agonist (74Buisson B. Vallejo Y.F. Green W.N. Bertrand D. Neuropharmacology. 2000; 39: 2561-2569Crossref PubMed Scopus (47) Google Scholar). Further evidence that agonist activity of epibatidine underlies its physiological role is that the nicotinic antagonist mecamylamine blocks both the functional activity and antinociceptive effects of epibatidine (2Badio B. Daly J.W. Mol. Pharmacol. 1994; 45: 563-569PubMed Google Scholar, 10Radek R.J. Curzon P. Decker M.W. Brain Res. Bul. 2004; 64: 323-330Crossref PubMed Scopus (5) Google Scholar, 11Damaj M.I. Creasy K.R. Grove A.D. Rosecrans J.A. Martin B.R. Brain Res. 1994; 664: 34-40Crossref PubMed Scopus (100) Google Scholar). However, the fact that mecamylamine has pronociceptive properties (12Rashid M.H. Furue H. Yoshimura M. Ueda H. Pain. 2006; 125: 125-135Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) and our data showing that mecamylamine competes with the epibatidine binding to 5-HT3Rs (Table 1) indicate that the previous findings with mecamylamine are also consistent with these ligands interacting with 5-HT3R sites. Our findings that epibatidine binds with high affinity to and antagonizes 5-HT3Rs raise questions about where epibatidine elicits its physiological actions and indicate that 5-HT3Rs should be considered as a potential target. We are most grateful to Dr. Paul Vezna for supplying the rat brains used in this study." @default.
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