Protein Kinase A Activation Down-Regulates, Whereas Extracellular Signal-Regulated Kinase Activation Up-Regulates -1 Receptors in B-104 Cells: Implication for Neuroplasticity Gianfrancesco Cormaci, Tomohisa Mori, Teruo Hayashi, and Tsung-Ping Su Cellular Pathobiology Unit/Development and Plasticity Section/Cellular Neurobiology Research Branch, Intramural Research Program/National Institute on Drug Abuse/National Institutes of Health/Department of Health and Human Services, Baltimore, Maryland Received May 24, 2006; accepted October 17, 2006 ABSTRACT The -1 receptor (Sig-1R) can bind psychostimulants and was shown to be up-regulated in the brain of methamphetamine self-administering rats. Up-regulation of Sig-1Rs has been implicated in neuroplasticity. However, the mechanism(s) whereby Sig-1Rs are up-regulated by psychostimulants is un- known. Here, we employed a neuroblastoma cell line B-104, devoid of dopamine receptors and transporter, and examined the effects of psychostimulants as well as cAMP on the expres- sion of Sig-1Rs in this cell line, with a specific goal to identify signal transduction pathway(s) that may regulate Sig-1R ex- pression. Chronic treatments of B-104 cells with physiological concentrations of cocaine or methamphetamine failed to alter the expression of Sig-1Rs. N 6 ,2'-O-Dibutyryl-cAMP (dB- cAMP), when used at 0.5 mM, caused a down-regulation of Sig-1Rs that could be blocked by a protein kinase A (PKA) inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline- sulfonamide dihydrochloride (H-89). However, dB-cAMP, when used at 2 mM, caused an up-regulation of Sig-1Rs that was insensitive to the H-89 blockade but was partially blocked by an extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase inhibitor PD98059 (2'-amino-3'-methoxyfla- vone). Furthermore, 2 mM dB-cAMP induced an ERK phos- phorylation lasting at least 90 min, at which time the phosphor- ylation caused by 0.5 mM dB-cAMP had already diminished. PD98059, applied 90 min after addition of 2 mM dB-cAMP, attenuated the Sig-1R up-regulation. Our results indicate that cAMP is bimodal in regulating Sig-1R expression: a down- regulation via PKA and an up-regulation via ERK. Results also suggest that psychostimulants may manipulate the cAMP- PKA-Sig-1R and/or the cAMP-ERK-Sig-1R pathways to achieve a neuroplasticity that favors addictive behaviors. The -1 receptor (Sig-1R) is a unique intracellular nonopi- oid binding site with nano- to low-micromolar affinities for diverse classes of drugs including benzomorphans, antide- pressants, neuroleptics, steroid hormones including proges- terone, and psychostimulants such as cocaine and metham- phetamine (Sharkey et al., 1988; Su et al., 1988; Snyder and Largent, 1989; Quirion et al., 1992; Nguyen et al., 2005). Sig-1Rs exist in the central and peripheral nervous systems where they modulate neurotransmitter release and cell ex- citability via ion channels (Monnet et al., 1990; Hayashi and Su, 2001; Aydar et al., 2002; Nuwayhid and Werling, 2003). Behavioral studies implicated Sig-1Rs in higher ordered brain functions including mood and learning and memory (Maurice et al., 2001; Meunier et al., 2004). Recently, a number of studies suggested that psychostimu- lant-induced behaviors involve an up-regulation of Sig-1Rs. Ujike et al. (1996) demonstrated that cocaine-induced loco- motor sensitization was cross-sensitized to the Sig-1R ago- nist 3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and that the sensitization could be suppressed by a Sig-1R antagonist, This work was supported by the Intramural Research Program of National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services. G.C., T.M., and T.H. contributed equally to this work. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.108415. ABBREVIATIONS: Sig-1R, -1 receptor; BMY 14802, -(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazine; DA, dopamine; PKA, protein kinase A; ERK, extracellular signal-regulated kinase; dB-cAMP, N 6 ,2'-O-dibutyryl-cAMP; NE-100, N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)- phenyl]-ethylamine monohydrochloride; PD98059, 2'-amino-3'-methoxyflavone; FCS, fetal calf serum; DMEM, Dulbecco’s modified Eagle’s medium; MS-377, ((R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartate; H-89, N-[2-(p-bromocin- namylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; PAGE, polyacrylamide gel electrophoresis; TBS-T, Tris-buffered saline plus 0.05% Tween 20; ANOVA, analysis of variance; SCH23390, R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; Sp- cBIMP, Sp isomer of cyclic-5,6-dichloro-benzimidazol-riboside thiophosphate. 0022-3565/07/3201-202–210 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 320, No. 1 U.S. Government work not protected by U.S. copyright 108415/3164862 JPET 320:202–210, 2007 Printed in U.S.A. 202 by on February 14, 2009 jpet.aspetjournals.org Downloaded from