Nicotine and Ethanol Activate Protein Kinase A Synergistically via Gi Subunits in Nucleus Accumbens/Ventral Tegmental Cocultures: The Role of Dopamine D1/D2 and Adenosine A2A Receptors

Tobacco and alcohol are the most commonly used drugs of abuse and show the most serious comorbidity. The mesolimbic dopamine system contributes significantly to nicotine and ethanol reinforcement, but the underlying cellular signaling mechanisms are poorly understood. Nicotinic acetylcholine (nACh) receptors are highly expressed on ventral tegmental area (VTA) dopamine neurons, with relatively low expression in nucleus accumbens (NAcb) neurons. Because dopamine receptors D1 and D2 are highly expressed on NAcb neurons, nicotine could influence NAcb neurons indirectly by activating VTA neurons to release dopamine in the NAcb. To investigate this possibility in vitro, we established primary cultures containing neurons from VTA or NAcb separately or in cocultures. Nicotine increased cAMP response element-mediated gene expression only in cocultures; this increase was blocked by nACh or dopamine D1 or D2 receptor antagonists. Furthermore, subthreshold concentrations of nicotine with ethanol increased gene expression in cocultures, and this increase was blocked by nACh, D2 or adenosine A2A receptor antagonists, G or protein kinase A (PKA) inhibitors, and adenosine deaminase. These results suggest that nicotine activated VTA neurons, causing the release of dopamine, which in turn stimulated both D1 and D2 receptors on NAcb neurons. In addition, subthreshold concentrations of nicotine and ethanol in combination also activated NAcb neurons through synergy between D2 and A2A receptors. These data provide a novel cellular mechanism, involving G subunits, A2A receptors, and PKA, whereby combined use of tobacco and alcohol could enhance the reinforcing effect in humans as well as facilitate long-term neuroadaptations, increasing the risk for developing coaddiction. Alcoholism and nicotine addiction are significant public health problems (see Dani and Harris, 2005). The vast majority of alcoholics also smoke tobacco, with a significantly greater incidence of ethanol dependence in smokers versus nonsmokers (Miller and Gold, 1998). Early onset of smoking carries an increased risk for alcoholism later in life, in part because ethanol consumption is higher in smokers than in nonsmokers (Rimm et al., 1995). Smoking is also associated with increased risk for alcoholism relapse and other substance abuse (Sobell et al., 2002). Moreover, recent studies in PC12 cells show that ethanol up-regulates the expression of nicotinic acetylcholine receptors (nAChR) (Dohrman and Reiter, 2003), considered to reflect physical dependence on nicotine (Dani and Heinemann, 1996). However, despite frequent mutual reinforcement and comorbidity of alcoholism This work was supported by funds provided by the State of California for Medical Research on Alcohol and Substance Abuse through the University of California, San Francisco (to A.B.) and by the Department of the Army (Grant DAMD17-03-1-0061) (to I.D.). The United States Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD 21702-5014 is the awarding and administering acquisition office. The content of the information represented does not necessarily reflect the position or the policy of the United States Government, and no official endorsement should be inferred. 1 Current affiliation: Department of Psychiatry, Nara Medical University, Kashihara, Nara, Japan. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.120675. ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; A2AR, adenosine 2A receptor; MSN, medium spiny neuron; D1R, dopamine D1 receptor; D2R, dopamine D2 receptor; GAD, glutamic acid decarboxylase; NAcb, nucleus accumbens; CRE, cAMP-response element; PBS, phosphate-buffered saline; PKA, protein kinase A; SCH23390, R-( )-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride; eticlopride, S-( )-3-chloro-5-ethyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-hydroxy-2-methoxybenzamide hydrochloride; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide dihydrochloride; MSX-3, 3,7-dihydro-8-[(1E)-2-(3-methoxyphenyl)ethenyl]7-methyl-3-[3-(phosphonooxy)]-5-[propyl-1-(2-propynyl)]-1H-purine-2,6-dione disodium salt hydrate; FITC, fluorescein isothiocyanate; TH, tyrosine hydroxylase; VTA, ventral tegmental area; Luc, luciferase; ARK, -adrenergic receptor kinase. 0022-3565/07/3221-23–29$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 322, No. 1 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 120675/3225371 JPET 322:23–29, 2007 Printed in U.S.A. 23 at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from and smoking (Miller and Gold, 1998), the cellular molecular mechanisms that underlie simultaneous addiction to ethanol and nicotine remain unclear. Here we focus on the mesolimbic system because of its central role in the regulation of reward, motivation, and addiction (see Wise, 2004). Cell bodies of dopaminergic neurons originate in the VTA and substantia nigra and project to forebrain structures such as the striatum, including the nucleus accumbens (NAcb). Acute exposure to addictive substances such as nicotine and ethanol increases extracellular dopamine in the NAcb, and dopamine seems to mediate some of the reinforcing actions of these drugs (see Wise, 2004), including nicotine (Balfour et al., 2000) and ethanol (Hodge et al., 1997; Weiss and Porrino, 2002). The reinforcing actions of nicotine are probably mediated in part by the VTA, because activation of nAChRs on dopaminergic neurons in the VTA enhances their firing rate and causes dopamine release from nerve terminals in the NAcb/striatum (see Wonnacott et al., 2005). Furthermore, behavioral studies suggest that nAChRs on VTA neurons are necessary for the reinforcing effects of nicotine (Corrigall et al., 1994) and ethanol (Ericson et al., 1998). In addition, nAChRs are strongly expressed in VTA neurons and their axon terminals, but NAcb/striatal medium spiny GABAergic neurons (MSNs) express relatively few postsynaptic nAChRs (Pakkanen et al., 2005). Here, we used primary neuronal cultures prepared from the VTA/ventral midbrain (hereafter called VTA) and NAcb/ striatum (hereafter called NAcb) to identify signaling events underlying synergistic interactions between nicotine and ethanol. Our studies suggest that nicotine binding to VTA neurons enhances the release of dopamine, which in turn activates dopamine receptors on NAcb neurons. Activation of NAcb dopamine receptors induces CRE-mediated gene expression hours later. It is noteworthy that subthreshold concentrations of nicotine and ethanol were ineffective when applied separately in NAcb/VTA cocultures, but coapplication of nicotine and ethanol to cocultures enhanced gene expression synergistically via dopamine and adenosine A2A receptors (A2AR) on NAcb neurons. Materials and Methods Materials. Reagents were purchased from Sigma-Aldrich (St. Louis, MO) except where indicated, including R-( )-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390), S-( )-3-chloro-5-ethyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-hydroxy-2-methoxybenzamide hydrochloride (eticlopride), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89), and 3,7-dihydro-8-[(1E)-2-(3methoxyphenyl)ethenyl]-7-methyl-3-[3-(phosphonooxy)]-5-[propyl-1(2-propynyl)]-1H-purine-2,6-dione disodium salt hydrate (MSX-3). Neurobasal medium, B-27, GlutaMAX-I supplement, and Hanks’ balanced salt solution were from Invitrogen (Carlsbad, CA); papain was from Worthington Biochemicals (Freehold, NJ); Hibernate E was from Brain Bits LCC (Springfield, IL); polyclonal rabbit antiglutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) antibodies were from Chemicon International (Temecula, CA); and FITCor Texas Red-conjugated anti-rabbit or anti-mouse secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA); Luciferase assay system was from Promega (Madison, WI); -galactosidase assay system was from Stratagene (La