ELSEVIER Brain Research 722 (1996) 125-131 BRAIN RESEARCH Research report Morphine stiimulates nitric oxide release from invertebrate microglia Yu Liu a,c, David Shenouda a Thomas V. Bilfinger a,b Michelle L. Stefano a,b Harold I. Magazine d George B. Stefano a,b, * a Neuroscience Research Institute, State University of New York at Old Westbury, PO Box 210, Old Westbury, NY 11568, USA b Cardiothoracic Division, Department of Surgery, State University of New York at Stony Brook, Stony Brook, NY 11794-8191, USA c Department of Biochemistry, Medical College, Jinan University, Guangzhou, 510632 China d Dept. Biology, Queens College and Graduate School of the City University of New York, 65-30 Kissena Blvd. Flushing, New York, NY 11367, USA Accepted 30 January 1996 Abstract Morphine stimulates nitric oxide (NO) release in human endothelial cells. To determine whether this mechanism also occurs in invertebrates, the mussel Mytilus edulis was studied. Exposure of excised ganglia to morphine for 24 h resulted in a significant dose-dependent decrease in rnicroglial egress that was naloxone sensitive. In coincubating the excised ganglia with morphine and the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), an increase in microglial egress was observed, suggesting that morphine may stimulate microglia to release NO. Morphine exposure to these cells in vitro resulted in NO release (39.4 ___ 4.9 nM), a phenomenon found to be naloxone sensitive (10 -6 M; NO level = 5.9 + 2.6 nM) and L-NAME sensitive (10 -4 M; NO level = 2.8 _ 1.8 nM). Opioid peptides did not stimulate NO release, indicating that the process was mediated by the opiate alkaloid selective /x3 receptor. Coincubation of microglia with L-arginine or the superoxide scavenger, superoxide dismutase, resulted in significantly higher NO levels observed following morphine stimulation. Taken together, the data demonstrate that morphine can stimulate NO release in cells obtained from an invertebrate that represents an animal 500 million years divergent in evolution from man, underscoring the significance of this process and further substantiating the critical importance of morphine as a naturally occurring signal molecule. Keywords: Mytilus edulis; Morphine; Nitric oxide; Microglia; Ganglion; Invertebrate 1. Introduction Morphine inhibits the movement of mammalian and invertebrate immunocytes 1128,37] and microglia from ex- cised vertebrate and invertebrate ganglia [36]. Recently, it has also been demonstrated that morphine can enhance constitutive nitric oxide (NO) levels in human and rat endothelial cells [38]. Whether morphine stimulates NO release from invertebrate r~dcroglia has not yet been estab- lished. Recent work demonstrating that both NO donors and morphine can cause invertebrate immunocytes to be- Abbreviations: DAMA, (D-AIa 2, MetS)-enkephalinamide; DADLE, (o-Ala 2, LeuS)-enkephalin; DPDPE, (D-Pen 2, o-PenS)-enkephalin; DAGO, (D-Ala 2, Me-Phe4, Gly(ol)5)-enkephalin); NO, nitric oxide; NOS, nitric oxide synthase; SNAP, S-nitroso-N-acetyl-DL-penicillamine; L-NAME, L-nitro-arginine methyl ester. * Corresponding author. Fax: (i[) (516) 876-2727. 0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0006-8993(96)00204-l come round as well as to inhibit phagocytosis [27] suggests that NO may be released from morphine-stimulated inver- tebrate cells. Studies are emerging that document the involvement of NO in various physiological functions of invertebrates, such as olfaction [10,14] and epithelial function [8]. Fur- thermore, nitric oxide synthase (NOS) immunoreactivity has been found in various invertebrate tissues [2,7,18,24,25,32]. The application of NO via NO-donors has also been demonstrated to have intracellular actions [1] as well as to influence immunocyte behavior [27]. The present study demonstrates for the first time, to our knowledge, that morphine can selectively induce constitu- tive NO release from invertebrate cells, specifically, the microglia of the mussel Mytilus edulis. In addition, we demonstrate that the inhibitory action of morphine on microglial egress from excised ganglia appears to be medi- ated by morphine-induced relase of NO.