(S)-(2)-Cotinine, the Major Brain Metabolite of Nicotine, Stimulates Nicotinic Receptors to Evoke [H]Dopamine Release from Rat Striatal Slices in a Calcium-Dependent Manner

Cotinine, a major peripheral metabolite of nicotine, has recently been shown to be the most abundant metabolite in rat brain after peripheral nicotine administration. However, little attention has been focused on the contribution of cotinine to the pharmacological effects of nicotine exposure in either animals or humans. The present study determined the concentration-response relationship for (S)-(2)-cotinine-evoked H overflow from superfused rat striatal slices preloaded with [H]dopamine ([H]DA) and whether this response was mediated by nicotinic receptor stimulation. (S)-(2)-Cotinine (1 mM to 3 mM) evoked H overflow from [H]DA-preloaded rat striatal slices in a concentration-dependent manner with an EC50 value of 30 mM, indicating a lower potency than either (S)-(2)-nicotine or the active nicotine metabolite, (S)-(2)-nornicotine. As reported for (S)-(2)-nicotine and (S)-(2)-nornicotine, desensitization to the effect of (S)-(2)-cotinine was observed. The classic nicotinic receptor antagonists mecamylamine and dihydro-b-erythroidine inhibited the response to (S)-(2)-cotinine (1–100 mM). Additionally, H overflow evoked by (S)-(2)-cotinine (10–1000 mM) was inhibited by superfusion with a low calcium buffer. Interestingly, over the same concentration range, (S)-(2)-cotinine did not inhibit [H]DA uptake into striatal synaptosomes. These results demonstrate that (S)-(2)-cotinine, a constituent of tobacco products and the major metabolite of nicotine, stimulates nicotinic receptors to evoke the release of DA in a calcium-dependent manner from superfused rat striatal slices. Thus, (S)-(2)-cotinine likely contributes to the neuropharmacological effects of nicotine and tobacco use. The alkaloidal tobacco constituent (S)-(2)-cotinine is the major peripheral oxidative metabolite of (S)-(2)-nicotine in several animal species, including humans, and is able to pass the blood-brain barrier from the periphery (Gorrod and Wahren, 1993; Benowitz et al., 1994; Crooks et al., 1997). (S)-(2)-Cotinine has been detected in mouse, rat, and cat brain after peripheral nicotine administration (Applegren et al., 1962; Schmiterlow et al., 1967; Stalhandske, 1970; Petersen et al., 1984; Deutsch et al., 1992; Crooks et al., 1995, 1997; Crooks and Dwoskin, 1997) and has been shown to be the most abundant (S)-(2)-nicotine metabolite in the central nervous system after acute s.c. administration of nicotine to rats (Crooks et al., 1997). Interestingly, (S)-(2)-cotinine does not undergo significant biotransformation in brain tissue in vivo and has a much longer half-life in the central nervous system than does (S)-(2)-nicotine (Crooks et al., 1997). The origin of (S)-(2)-cotinine in brain has not been elucidated and could arise via two different mechanisms: formed oxidatively from nicotine locally in the brain or formed in the periphery and then redistributed to the brain. Although hepatic metabolism of (S)-(2)-nicotine to (S)-(2)-cotinine has been suggested to involve cytochromes P-4502D6, P-4502B6, P-4502E1, P-4502C9, and P-4502A6 (Cashman et al., 1992; McCracken et al., 1992; Flammang et al., 1992; Cholerton et al., 1994), recent evidence demonstrates that P-4502A6 is the major isozyme involved in hepatic C-oxidation of (S)-(2)nicotine to (S)-(2)-cotinine in humans (Nakajima et al., 1996; Messina et al., 1997). It is important to note that the regional localization of P-4502A6 and its role in local (S)-(2)-nicotine metabolism in brain have not been established to date. Interestingly, it has recently been reported that an individual’s inherent ability to metabolize nicotine to cotinine via CYP2A6 in part determines their tobacco dependence liability (Pianezza et al., 1998). In contrast to the plethora of studies investigating the neuropharmacological effects of (S)-(2)-nicotine, few studies have investigated the effects of (S)-(2)-cotinine. (S)-(2)-Nicotine has been reported to have intrinsic reinforcing properties suggested to be the result of activation of dopamine (DA) pathways in brain (Fibiger and Phillips, 1987; Corrigall et al., 1992, 1994; Balfour and Benwell, 1993). Nicotine facilitates DA release from striatal nerve terminals in in vivo Received for publication May 26, 1998. 1 This research was supported by grants from the National Institute on Drug Abuse (DA08656) and the Tobacco and Health Research Institute (Lexington, KY). ABBREVIATIONS: DHbE, dihydro-b-erythroidine; DA, dopamine; MEC, mecamylamine; ANOVA, analysis of variance. 0022-3565/99/2882-0905$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 288, No. 2 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 288:905–911, 1999 905 at A PE T Jornals on July 8, 2017 jpet.asjournals.org D ow nladed from studies using microdialysis in striatum (Imperato et al., 1986; Toth et al., 1992) and in in vitro superfusion studies using striatal slices (Westfall, 1974; Arqueros et al., 1978; Giorguieff-Chesselet et al., 1979; Westfall et al., 1987; Izenwasser et al., 1991; Harsing et al., 1992; Schulz et al., 1993, Sacaan et al., 1995) and synaptosomes (Takano et al., 1983; Chesselet, 1984; Rowell et al., 1987; Rapier et al., 1988, 1990; Grady et al., 1992; Rowell and Hillebrand, 1994; ElBizri and Clarke, 1994; Rowell, 1995). Concentrations (0.1–1 mM) of nicotine that correspond to plasma levels in moderate smokers (Russell et al., 1980; Kogen et al., 1981; Benowitz, 1990; Henningfield et al., 1993) evoked DA release in the latter in vitro studies. Moreover, nicotine-evoked striatal DA release was calcium dependent and was inhibited by mecamylamine or dihydro-b-erythroidine (DHbE) (Westfall et al., 1987; Rapier et al., 1988, 1990; Grady et al., 1992; El-Bizri and Clarke, 1994; Sacaan et al., 1995; Teng et al., 1997). Mecamylamine is a centrally active, noncompetitive nicotinic receptor antagonist that blocks the open ion channel of the nicotinic receptor more effectively than the closed channel (Varanda et al., 1985; Loiacono et al., 1993; Peng et al., 1994). DHbE is a selective, competitive nicotinic receptor antagonist that displaces nicotine from its binding site (Reavill et al., 1988; Grady et al., 1992) and inhibits its electrophysiological effects (Vidal and Changeux, 1989; Alkondon and Albuquerque, 1991; Mulle et al., 1991). Relatively little is known about the effects of (S)-(2)-cotinine on either DA-mediated behaviors or DA neurochemistry. (S)-(2)-Cotinine has a reported Ki of 1 mM for the [H]nicotine binding site in rat brain, which is approximately 1000-fold weaker affinity than that reported for (S)-(2)-nicotine (Abood et al., 1981). The purposes of the present study were to determine whether (S)-(2)-cotinine evokes H overflow from rat striatal slices preloaded with [H]DA in a concentrationand calcium-dependent manner and whether (S)-(2)-cotinine-evoked H overflow was inhibited by mecamylamine and DHbE, providing evidence for a nicotinic receptor-mediated mechanism. Experimental Procedures Materials. (S)-(2)-Cotinine and pargyline hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO). Nomifensine maleate, mecamylamine HCl, and DHbE were purchased from Research Biochemicals, Inc. (Natick, MA). [H]DA (3,4-ethyl-2[N-H]dihydroxyphenylethylamine; specific activity, 25.6 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Ascorbic acid and a-D-glucose were purchased from AnalaR (BHD Ltd., Poole, U.K.) and Aldrich Chemical Co. (Milwaukee, WI), respectively. TS-2 tissue solubilizer was purchased from Research Products International (Mount Prospect, IL). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA). Subjects. Male Sprague-Dawley rats (200–250 g) were obtained from Harlan Laboratories (Indianapolis, IN) and were housed two per cage with free access to food and water in the Division of Lab Animal Resources at the College of Pharmacy, University of Kentucky. Experimental protocols involving the animals were in strict accordance with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” and were approved by the Institutional Animal Care and Use Committee at the University of Kentucky. [H]DA Release Assays. Effects of drug on H overflow from rat striatal slices preloaded with [H]DA were determined using a previously published method (Dwoskin and Zahniser, 1986). Briefly, rat striatal slices (500 mm, 6–8 mg) were incubated for 30 min in Krebs’ buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 1.0 mM NaH2PO4, 1.3 mM CaCl2, 11.1 mM glucose, 25 mM NaHCO3, 0.11 mM L-ascorbic acid, and 0.004 mM ethylenediaminetetraacetic acid, pH 7.4, saturated with 95% O2/5% CO2 at 34°C). Slices were then incubated for an additional 30 min in buffer containing 0.1 mM [H]DA. Each slice was transferred to a superfusion chamber and superfused (1 ml/min) with Krebs’ buffer containing nomifensine (10 mM), a DA uptake inhibitor, and pargyline (10 mM), a monoamine oxidase inhibitor, to ensure that the H overflow primarily represented [H]DA rather than H metabolites (Cubeddu et al., 1979; Zumstein et al., 1981; Rapier et al., 1988). When basal outflow was stabilized after 60-min superfusion, two 5-min (5-ml) samples were collected to determine basal H outflow followed by superfusion with different concentrations of drugs. For all experiments, slices from a given rat were randomly assigned to all drug concentrations. For concentration-response studies, (S)-(2)-cotinine (1 mM to 3 mM) was added to the superfusion buffer after the collection of the second 5-min sample and remained in the buffer for 60 min. Each superfusion chamber containing one slice was exposed to only one concentration of (S)-(2)-cotinine. Thus, striatal tissue from each rat was exposed to all concentrations of (S)-(2)-cotinine, a repeated-measures design. In each experiment, in addition to slices exposed to (S)-(2)-cotinine, a control slice was superfused in the absence of (S)-(2)-cotinine (i.e., buffer control). The ability of mecamylamine (100 mM) and DHbE (10 mM) to inhibit (S)-(2)-cotinine (1–100 mM)-evoked H overflow was determined in two separate studies. These concentrations of mecamylamine and DHbE were chosen because they were found previously to maximally inhibit (S)-(2)-nicotine-evoked H overflow from [H]DA-preloaded striatal slices (Teng et al., 1997). In one series of experiments, six slices from one rat were superfused in the absence or presence of mecamylamine in each experiment, and in the second series of experiments, six slices from one rat were superfused in the absence or presence of DHbE. Mecamylamine or DHbE was superfused for 60 min before the addition of (S)-(2)-cotinine to the superfusion buffer. Superfusion continued for 60 min in the presence of (S)-(2)-cotinine plus mecamylamine or DHbE. Slices superfused in the absence of mecamylamine or DHbE constituted the (S)-(2)cotinine control condition. An additional striatal slice from each rat was superfused in the absence of exposure to any drug in each experiment and was referred to as buffer control. Because the purpose of these two studies was to determine the inhibitory effects of the antagonists against (S)-(2)-cotinine [i.e., (S)-(2)-cotinine exposure alone served as control], comparisons were made between the drug-exposure condition and the (S)-(2)-cotinine control rather than between the drug-exposure condition and the buffer control. To determine whether the effect of (S)-(2)-cotinine was dependent on extracellular calcium, in a separate series of experiments, (S)-(2)cotinine concentration-response curves were generated in Krebs’ buffer (control buffer) and concurrently in a low-calcium buffer. For the low-calcium buffer, 0.5 mM ethylene glycol bis(b-aminoethyl ether)-N,N,N9,N9-tetraacetic acid was added and CaCl2 was omitted from the Krebs’ buffer. At the end of the experiment, each slice was solubilized with TS-2. The radioactivity in the superfusate and tissue samples was determined by liquid scintillation counting (model B1600 TR Scintillation Counter; Packard, Meriden, CT) with an efficiency of 59%. To normalize potential differences in radioactivity between slices of varying weight, fractional release for each sample was calculated by dividing the tritium collected in superfusate by the total tissue tritium at the time of collection and was expressed as percentage of tissue; thus, the unit of fractional release is percentage. Basal outflow was calculated from the average of the fractional release of the two samples just before drug addition. (S)-(2)-Cotinine-evoked total H overflow was calculated by summing the increases in fractional release due to drug exposure after subtracting the basal outflow for an equivalent period of drug exposure. Calculation of total H overflow also takes 906 Dwoskin et al. Vol. 288 at A PE T Jornals on July 8, 2017 jpet.asjournals.org D ow nladed from into account differences among tissue weights, and the unit of total H overflow is percent. Illustrating fractional release as a function of time provides the duration and time course of the effect of drug, and each curve represents the effect of one concentration of the drug. Illustrating the results as total H overflow as a function of drug concentration provides the concentration-response curves allowing determination of pharmacological parameters, which describe the drug-receptor interaction. Statistical Analyses. Repeated-measures two-way analysis of variance (ANOVA) was used to analyze the concentration dependence of (S)-(2)-cotinine-evoked H overflow. The EC50 value for cotinine to evoke H overflow was determined using an iterative nonlinear least-squares curve-fitting program (Prism; GraphPAD, San Diego, CA). Repeated-measures two-way ANOVAs also were performed to analyze the time course of the (S)-(2)-cotinine-induced increase in fractional release. The tritium remaining in the striatal slice after (S)-(2)-cotinine exposure was analyzed by repeated-measures one-way ANOVA. Studies determining both the ability of mecamylamine or DHbE to antagonize the effect of (S)-(2)-cotinine and the dependence on external calcium were analyzed by repeatedmeasures, two-way ANOVA. A protected version of Fisher’s LSD test (i.e., only preplanned comparisons were considered to limit the overall type 1 error rate) was used for post hoc analysis. Results were considered statistically significant when P , .05. [H]DA Uptake Assay. [H]DA uptake was determined using minor modifications of a previously published method (Masserano et al., 1994). Striata were homogenized in 20 ml of ice-cold sucrose solution (0.32 M sucrose and 5 mM sodium bicarbonate, pH 7.4) with 12 passes of a Teflon-pestle homogenizer (clearance, approximately 0.003 in). The homogenate was centrifuged at 2000g at 4°C for 10 min. The supernatant was centrifuged at 12,000g at 4°C for 20 min. The resulting pellet was resuspended in 1.5 ml of ice-cold assay buffer (125 mM NaCl, 5 mM KCl, 1.5 mM KH2PO4, 1.5 mM MgSO4, 1.25 mM CaCl2, 10 mM glucose, 0.1 mM L-ascorbate, 25 mM HEPES, 0.1 mM ethylenediaminetetraacetic acid, and 0.1 mM pargyline, pH 7.4). The final protein concentration was 400 mg/ml. Assays were performed in duplicate in a total volume of 500 ml. Aliquots (50 ml of synaptosomal suspension containing 20 mg of protein) were added to assay tubes containing 350 ml of buffer and 50 ml of one of nine concentrations (final concentration, 1 nM to 1 mM) of (S)-(2)-cotinine or vehicle (1 mM HCl). Synaptosomes were preincubated at 34°C for 10 min before the addition of 50 ml of [H]DA (30.1 Ci/mmol, final concentration 10 nM) and accumulation proceeded for 10 min at 34°C. High-affinity uptake was defined as the difference between accumulation in the absence and the presence of 10 mM GBR 12909. Preliminary studies demonstrate that at 10 min, [H]DA uptake is within the linear range of the time-response curve when experiments are performed at 34°C. Accumulation was terminated by the addition of 3 ml of ice-cold assay buffer containing pyrocatechol (1 mM) and rapid filtration through a Whatman GF/B glass-fiber filter paper (presoaked with buffer containing 1 mM pyrocatecol) using a Brandel Cell Harvester (model MP-43RS; Biochemical Research and Development Laboratories, Inc., Gaithersburg, MD). The filters were washed three times with 3 ml of ice-cold buffer containing 1 mM pyrocatechol and then transferred to scintillation vials and radioactivity determined (model B1600TR scintillation counter, Packard). Protein concentration was determined using bovine serum albumin as the standard (Bradford, 1976).