Involvement of 7 Nicotinic Acetylcholine Receptors in Gene Expression of Dopamine Biosynthetic Enzymes in Rat Brain

Brain dopaminergic systems are critical in mediating the physiological responses to nicotine. The effects of several concentrations of nicotine (0.08, 0.17, or 0.33 mg/kg body weight) and involvement of 7 nicotinic acetylcholine receptors (nAChRs) in gene expression of key enzymes in dopamine biosynthesis were evaluated in the ventral tegmental area (VTA) and substantia nigra (SN), cell bodies of the mesocorticolimbic and nigrostriatal pathways. Nicotine elicited a dose-dependent elevation of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis in VTA and SN. The VTA was more sensitive to lower concentrations of nicotine with maximal response observed with the lowest dose of nicotine. Nicotine also elevated mRNA levels of GTP cyclohydrolase I (GTPCH), rate limiting in biosynthesis of TH’s essential cofactor tetrahydrobiopterin in both dopaminergic locations. The changes in TH and GTPCH mRNAs were correlated. Pretreatment with the 7 nAChR antagonist methyllycaconitine prevented the nicotine-induced rise in TH or GTPCH mRNA in VTA and SN. Administration of 7 nAChR agonist 3-[2,4-dimethoxybenzilidene]anabaseine at 1 to 10 mg/kg or (E,E-3-(cinnamylidene)anabaseine at 0.3 to 1 mg/kg increased TH mRNA in VTA and SN, but not in peripheral catecholaminergic cells. Thus, agonists of 7 nAChRs have therapeutic potential for increasing TH gene expression in dopaminergic regions without some of nicotine’s disadvantages, such as its harmful effects on the cardiovascular system. The findings indicate that nicotine may regulate dopamine biosynthesis by alterations in gene expression of TH and its cofactor. The 7 nAChRs are involved in mediating these effects of nicotine. Catecholaminergic systems are critical in mediating the physiological effects of nicotine. The self-administering and reinforcing properties of nicotine seem to arise from direct action on the mesocorticolimbic dopamine system. This system, with cell bodies in VTA, plays a key role in mediating the reinforcing effects of natural rewards as well as of drugs of abuse, including nicotine, through increased dopaminergic transmission in the nucleus accumbens. The nigrostriatal dopaminergic system, with cell bodies in the SN, innervates the caudate nucleus and putamen of the corpus striatum. Activation of striatal neuronal acetylcholine receptors (nAChRs) triggers release of dopamine. These receptors are also involved in mediating nicotine’s effects on locomotion. Abnormalities of dopaminergic neurotransmission are associated with a number of neuropsychiatric disorders, including schizophrenia and Tourette’s syndrome. Nicotine binding in caudate nucleus and putamen is significantly reduced in Parkinson’s disease, Alzheimer’s disease, and dementia with Lewy bodies (Court et al., 2000). Nicotinic compounds have therapeutic potential for treatment of these disorders (Rusted et al., 2000). In addition to triggering release of catecholamines, nicotine also promotes their biosynthesis. Several studies reveal that injections of high concentrations (1–4 mg/kg, free base) of nicotine increase activity of adrenal TH, the rate-limiting enzyme in the catecholamine biosynthetic pathway (Slotkin et al., 1976; Fossom et al., 1991). This is reflected by a rise in TH protein resulting from increased mRNA levels (Fossom et al., 1991; Hofle et al., 1991; Hiremagalur and Sabban, 1995). In the brain, TH is elevated by lower concentrations of nicotine. Nicotine injections (0.8 mg/kg, free base) raised TH activity in SN and VTA as well as in noradrenergic cell bodies of the locus coeruleus (Smith et al., 1991; Mitchell et al., 1993). A single injection of nicotine led to a large increase in TH mRNA in locus coeruleus even 6 days later (Mitchell et al., 1993). We have shown previously that injections of nicotine (0.35 and 1.75 mg/kg) doubled TH mRNA levels in VTA and SN (Serova et al., 1999a). It is unclear what is the lowest effective dose of nicotine needed to stimulate TH gene expression in dopaminergic locations. This study was supported by National Institutes of Health Grant NS28869 (to E.S.), Office of Naval Research Grant N00014-021-1-0315 (to E.S.), a grant from Philip Morris, Inc. (to E.S.), and Scientist Development Grant 0130102N from the American Heart Association (to L.S.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.039198. ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; TH, tyrosine hydroxylase; SN, substantia nigra; VTA, ventral tegmental area; BH4, tetrahydrobiopterin; MLA, methyllycaconitine; DMXB, 3-[2,4-dimethoxybenzilidene]anabaseine; 3CA, E,E-3-(cinnamylidene)anabaseine; GTPCH, GTP cyclohydrolase I. 0022-3565/02/3033-896–903$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 303, No. 3 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 39198/1024589 JPET 303:896–903, 2002 Printed in U.S.A. 896 at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from Although TH is the rate-limiting step in biosynthesis of dopamine, as well as of other catecholamines, it is also dependent on levels of its essential cofactor, tetrahydrobiopterin (BH4) (Nagatsu and Ichinose, 1999). Intraventricular administration of BH4 increases the biosynthesis of dopamine in nigrostriatal neurons (Kettler et al., 1974). Moreover, patients with Parkinson’s disease and dementia not only have reduced levels of dopamine but also reduced levels of BH4. These observations suggest that nicotine’s effect on the dopaminergic system may be influenced by regulation of BH4 as well as of TH. The effect of nicotine on biosynthesis of BH4 has not been examined previously. Nicotine mediates its actions through nAChRs, which exist as a variety of subtypes. Diverse nAChRs are formed as pentamers from different combinations of -type and -type subunits (for review, see Leonard and Bertrand, 2001). Receptors containing 4 2 and 7 subunits are, respectively, the major highand low-affinity nAChRs in the brain. Both types of receptors are present in the SN and VTA (Tsuneki et al., 2000). Previously, we found that the 7 nAChR subtype is required for the nicotine-elicited elevation of TH mRNA in PC12 cells. Moreover, 7 receptor agonists were at least as effective as nicotine in triggering these effects (Gueorguiev et al., 2000). The 7 subunits can form a homopentameric receptor, which is reported to desensitize rapidly compared with other nAChR subtypes. Their high Ca conductance could enable 7 nAChRs to take part in synaptic mechanisms in which calcium acts as a second messenger (Pugh and Berg, 1994). Whether these receptors play a role in regulation of gene expression of dopamine biosynthetic enzymes in vivo is unclear. Information regarding their role in the rewarding properties of nicotine and the withdrawal syndrome are contradictory. Studies by Schilstrom et al. (1998), Nomikos et al. (1999), and Panagis et al. (2000) indicate involvement of 7 nAChRs in nicotine-induced dopamine release in the nucleus accumbens, and in locomotion, whereas studies by Grottick et al. (2000) suggest a negligible role for 7 nAChRs in nicotine-induced hyperlocomotion and reward. Herein, we examine nicotine’s effect on TH and GTPCH gene expression in the cell bodies of the nigrostriatal and mesocorticolimbic dopaminergic systems. The potential involvement of 7 nAChRs is evaluated, as well as the ability of specific nAChR agonists to elicit the effects of nicotine in central and peripheral catecholaminergic regions. Materials and Methods Animals, Drug Treatment, and Doses. All animal experiments were approved by the Animal Care and Use Committee. Adult, pathogen-free, male Sprague-Dawley rats (250 to 300 g) were purchased from Taconic Farms (Germantown, NY) and housed four per cage. They were maintained under controlled conditions on a 12-h light/dark cycle at 23 2°C. Animals were given food and water ad