Molecular Brain Research 81 (2000) 177–180 www.elsevier.com / locate / bres Shortcommunication Identification of morphine in the adrenal medullary chromaffin PC-12 cellline a a b a,c a Yannick Goumon , Wei Zhu ,Benjamin S. Weeks ,Federico Casares , Patrick Cadet , b c a , * Maria Bougaeva , Bruce Brownawell , George B. Stefano a Neuroscience Research Institute , State University of New York , College at Old Westbury , Old Westbury , NY 11568, USA b Adelphi University , Department of Biology , One South Avenue , Garden City , NY 11530, USA c Marine Sciences Research Center , State University of New York at Stony Brook , Stony Brook , NY 11794 - 5000, USA Accepted 13 June 2000 Abstract Morphine was identified in the adrenal medulla chromaffin PC-12 cell line by reversed-phase HPLC, following liquid and solid extraction. The morphine corresponding HPLC fractions (1.74660.615 ng of morphine / million cells) were further analyzed by gas chromatography–mass spectrometry and found to be identical to synthetic morphine. Furthermore, using primers derived fr neuronal m1 opiate receptor, we used RT-PCR to detect expression of m transcripts from this cell line.The transcript was absent. The study conclusively proves morphine, but not a m opiate receptor, is constitutively expressed in the adrenal medulla chromaff line.  2000 Elsevier Science B.V. All rights reserved. Theme : Neurotransmitters, modulators, transporters, and receptors Topic : Opioids: anatomy, physiology, and behavior Keywords : Rat;Endogenous morphine; Adrenal gland; Nitric oxide Morphinehas been demonstrated in the vertebrate (National Institute of Child Health and Human Develop- adrenalgland by way of immunocytochemistry and ment,National Institutes of Health,Bethesda, MD) and radioimmunoassay [5–8,11,12,14]. Recently,we also wereculturedin Dulbecco’sModificationof Eagle’s foundmorphinepresentin the rat adrenalglandand Medium (DMEM) containing 7.5% fetal bovine serum, 21 determined its identity by way of gaschromatography– 7.5% equine serum and 0.1 mg ml gentamycin. For the mass spectrometry (GC–MS) [9]. biochemical assay, PC-12 cells were collected and replated In the present study we sought to determine which cells at 50% confluence in 80 ml flasks in 12 ml of medium for may be responsible for morphine synthesis in the adrenal 48 h. The cells werecollected by agitation and were gland.We now demonstrate that the adrenal medullary pelleted at 1000 rpm for 10 min. The pellet was then stored chromaffin derived PC-12 cell line, which constitutes a cell at 2808C for later use. line derived from rat pheochromocytoma, has this ability, The extraction experiments using internal or external suggesting that it represents a site for the synthesis of the morphinestandards (Sigma,St. Louis,MO) wereper- opiate alkaloid in the adrenal gland. formed in different rooms to avoid morphine contamina- The PC-12 cells were kindly provided by Gordon Guroff tion ofPC-12 samples. Single-use siliconized tubes also were used to prevent the lossof morphine aswell as contamination between assays [16]. Cells were extensively Abbreviations: GC–MS, gas chromatography–mass spectrometry; washed with phosphate buffered saline (PBS) (3 times, 1 HPLC,high-performance liquid chromatography; MS,morphine sulfate; min)priorto extraction to avoid an additional potential TFA, trifluoroacetic acid source ofmorphine contamination. Cellswere counted, *Corresponding author.Tel.: 11-516-876-2732; fax: 11-516-876- homogenized in 2 ml of 1 N HCl, and then extracted with 2727. E-mailaddress : gbs11@banet.net (G.B.Stefano). 5 ml chloroform–isopropanol 9:1 [10].After 5 min at 0169-328X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 1 6 9 - 3 2 8 X ( 0 0 ) 0 0 1 4 1 - 8