doi:10.1111/j.1369-1600.2007.00072.x © 2007 The Authors. Journal compilation © 2007 Society for the Study of Addiction Addiction Biology, 13, 118–123 12•••• Original Article Endogenous morphine signaling regulates CYP2D6 and COMT Kirk J. Mantione et al. Correspondence to: George B. Stefano, Neuroscience Research Institute, SUNY College at Old Westbury, PO Box 210, Old Westbury, NY 11568, USA. E- mail: gstefano@sunynri.org CLINICAL STUDY Endogenous morphine signaling via nitric oxide regulates the expression of CYP2D6 and COMT: autocrine/paracrine feedback inhibition Kirk J. Mantione 1 , Patrick Cadet 1 , Wei Zhu 1 , Richard M. Kream 2 , Melinda Sheehan 1 , Gregory L. Fricchione 3 , Yannick Goumon 4 , Tobias Esch 5 & George B. Stefano 1 Neuroscience Research Institute, State University of New York—College at Old Westbury, USA 1 , Department of Biochemistry, Downstate Medical Center, State University of New York, USA 2 , Department of Psychiatry, Massachusetts General Hospital, USA 3 , HDR, CR1 Inserm U 575, Physiopathologie du Système Nerveux, France 4 and Division of Integrative Health Promotion, Coburg University of Applied Sciences, Germany 5 ABSTRACT We determined changes in mRNA expression in specific enzymes involved in the biosynthesis of morphine in human white blood cells via microarray. Leukocyte exposure to morphine down-regulated catechol-O-methyl transferase (COMT) and CYP2D6 by approximately 50% compared with control values. The treatment did not alter DOPA decar- boxylase and dopamine beta-hydroxylase expression, demonstrating the specificity of morphine actions. The verifica- tion of the microarray data was accomplished via real-time Taqman reverse transcriptase polymerase chain reaction (RT-PCR) focused on CYP2D6 and COMT expression in different blood samples treated with morphine. The analysis showed similar changes in the expression of CYP2D6 and COMT mRNA. The expression was reduced by 47 ± 7% for CYP2D6, substantiating the microarray finding of a 54% reduction. Furthermore, exposure of white blood cells to 10 -6 M S-nitroso-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, reduced the expression of CYP2D6 and COMT. Prior naloxone (10 -6 M) or N-nitro-L-arginine methyl ester (L-NAME) (10 -4 M) addition abrogated morphine’s down-regulating activity, demonstrating morphine was initiating its actions via stimulating constitutive NO synthase derived NO release via the μ 3 opiate receptor splice variant. In the past we demonstrated that UDP-glucurosyltrans- ferase is involved in metabolizing morphine to morphine 6-glucuronide in adrenal chromaffin cells. In the present study its expression was not found in controls and morphine-treated cells, suggesting that morphine 6-glucuronide may not be synthesized in white blood cells. Taken together, it appears that morphine has the ability to modulate its own synthesis via autocrine and paracrine signaling. Keywords COMT, CYP2D6, microarray, morphine, nitric oxide, tyrosine hydroxylase. INTRODUCTION Several research groups have actively investigated the integrative/regulatory role of opioid signaling in immune cell function over the last two decades (Sibinga & Gold- stein 1988; Stefano 1989; Stefano et al. 1996). Our recent contributions include the identification and clon- ing of a novel opioid receptor subtype, the μ 3 opiate recep- tor on human white blood cells (Stefano et al. 1993; Cadet, Mantione & Stefano 2003) that is selectively acti- vated by morphine and related morphinan alkaloids, but not by endogenous opioid peptides. Notably, μ 3 activation promotes synthesis and release of the free radical gas nitric oxide (NO) via Ca++ mobilization and stimulation of constitutive nitric oxide synthase (cNOS) in immune, as well as vascular and neural cells in invertebrate, mam- malian and human species (Stefano et al. 1995, 1996; Bilfinger et al. 2000; Magazine et al. 1996). The physiological relevance and unique regulatory role of the μ 3 receptor is further supported by the demon- stration of chemically authentic morphine by tandem mass spec analyses in human and invertebrate immune cells, vascular and nervous tissues, and cultured cells (Stefano et al. 1995, 1996; Bilfinger et al. 2000;