Morphine and Endomorphins Differentially Regulate -Opioid Receptor mRNA in SHSY-5Y Human Neuroblastoma Cells XIN YU, XIN MAO, ALLAN D. BLAKE, WEN XIN LI, and SULIE L. CHANG Department of Biology, Seton Hall University, South Orange, New Jersey (X.Y., A.D.B., S.L.C.); and Department of Virology, Wuhan University, Wuhan, China (X.M., W.X.L.) Received January 2, 2003; accepted April 21, 2003 ABSTRACT A sensitive quantitative-competitive reverse transcriptase-poly- merase chain reaction method was developed to measure -opioid receptor (MOR) mRNA expression in SHSY-5Y neu- roblastoma cells. Differentiation of SHSY-5Y cells with either retinoic acid (RA) or 12-o-tetradecanoyl-phorbol-13-acetate (TPA) significantly increased MOR mRNA levels. Morphine treatment (10 M) for 24 h decreased MOR mRNA levels in control, as well as RA- and TPA-differentiated cells. In contrast, chronic exposure to the opioid peptides endomorphin-1 or endomorphin-2 significantly increased MOR mRNA levels in undifferentiated and RA-differentiated cells. An opioid antago- nist, naloxone, reversed the morphine and endomorphin-1 and -2 effects on MOR mRNA levels in undifferentiated SHSY-5Y cells, but naloxone had differential reversing effects on the agonists’ regulation of MOR mRNA in RA- or TPA-differentiated cells. To investigate whether the changes in MOR mRNA ex- pression paralleled changes in MOR receptor function, intra- cellular cAMP accumulation in SHSY-5Y cells was measured. After chronic treatment with morphine, forskolin-induced cAMP levels in SHSY-5Y cells were significantly higher than those of untreated control cells. In contrast, forskolin-induced cAMP accumulation levels were lower in cells treated with endomor- phin-1 or -2 than in untreated control cells. Together, our stud- ies indicate that the opioid alkaloid morphine and the opioid peptides endomorphin-1 and -2 differentially regulate MOR mRNA expression and MOR function in SHSY-5Y cells. Opioid receptors belong to the G protein-coupled receptor superfamily. In the nervous system, opioid agonists stimu- late heterotrimeric proteins of the G i/o family and control neuronal function through several receptor-effector mecha- nisms, including activation of receptor-operated potassium channels, inhibition of voltage-gated calcium channels and the inhibition of adenylyl cyclase (Ueda et al., 1988). Phar- macological studies using highly selective ligands have clas- sified opioid receptors into three subtypes: , , and  based upon a characteristic pharmacology (Loh and Smith, 1990). Molecular cloning studies over the past 10 years have con- firmed the pharmacological classification of the opioid recep- tors (Evans et al., 1992; Kieffer et al., 1992; Chen et al., 1993a,b; Meng et al., 1993; Wang et al., 1994). The -opioid receptor (MOR) is the principal target of the clinically effica- cious opioids, such as morphine, as well as opioid peptides, such as the endomorphins, the only endogenous opiates known to bind selectively and with high affinity to the MOR. The clinical usefulness of opiates in controlling pain is com- promised by the development of opioid tolerance and depen- dence, which remain significant drawbacks to the use of opiate drugs, including morphine, as pain relievers (Harrison et al., 1998). Although intensively investigated, the molecu- lar mechanisms underlying opioid tolerance and dependence are poorly understood. SHSY-5Y cells are human neuroblastoma cells, a subclone of the SK-N-SH cell line (Kohl et al., 1980; Kuramoto et al., 1981). SHSY-5Y cells are of sympathetic adrenergic gangli- onic origin (Scott et al., 1986) and express both - and -opi- oid receptors (Yu and Sadee, 1988) in a ratio of 5:1 based on quantitative receptor binding studies. A series of biological and morphological changes have been shown to occur in SHSY-5Y cells during differentiation (Pahlman et al., 1990, 1995). Differentiation of SHSY-5Y cells with either retinoic acid (RA) or 12-o-tetradecanoyl-phorbol-13-acetate (TPA) is associated with considerable reduction in the proliferation rate and with induction of neuritic processes (Scott et al., 1986; Pahlman et al., 1990; Kohring and Zimmermann, 1998). Of particular interest is that opioid receptor-mediated signal transduction pathways are reportedly associated with RA- and TPA-induced differentiation (Kohring and Zimmer- This study was partially supported by Public Health Service National Institutes of Health R01 DA-07058 and K02 DA-016149 (to S.L.C.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.048694. ABBREVIATIONS: MOR, -opioid receptor; RA, retinoic acid; TPA, 12-o-tetradecanoyl-phorbol-13-acetate; QC-RT-PCR, quantitative-compet- itive reverse transcriptase-polymerase chain reaction; bp, base pair; rcRNA, reconstructed RNA from cDNA; IS, internal standard; ANVOA, analysis of variance; EtOH, ethanol. 0022-3565/03/3062-447–454$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 306, No. 2 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 48694/1077451 JPET 306:447–454, 2003 Printed in U.S.A. 447 at ASPET Journals on February 5, 2016 jpet.aspetjournals.org Downloaded from