Morphine dependence in human neuroblastoma SH-SY5Y cells is associated with adaptive changes in both the quantity and functional interaction of PGE1 receptors and stimulatory G proteins.
Chronic exposure of all-trans-retinoic acid-differentiated SH-SY5Y cells to morphine (10 mu M; 2 days) results in sensitization of adenylate cyclase as characterized by a significant increase in both PGE1 receptor-mediated as well as receptor-independent (NaF, 10 mM; forskolin, 100 mu M) stimulation of effector activity. To investigate the underlying biochemical alterations, chronic opioid regulation of each of the components comprising the stimulatory PGE1 receptor system was examined. On receptor level, chronic morphine treatment was found to reduce PGE1 receptor number (Bmax) by approximately 40%, whereas their affinity slightly increased. Binding experiments performed in the presence of GTPgammaS (100 mu M) further indicate that the decrease in PGE1 receptor density is associated with a loss of functionally G protein-coupled receptors. On post-receptor level, chronic morphine treatment substantially increased the abundance and functional activity of stimulatory G proteins, as assessed by cholera toxin-catalyzed ADP-ribosylation of GSalpha and S49 cyc- reconstitution assays. No changes were found on the level of adenylate cyclase. Evaluation of the functional interaction between PGE1 receptors and GS in situ by application of a C-terminal anti-GSalpha antibody revealed a more intense coupling efficiency between these two entities, since a significant higher amount of antibody (2.3-fold) was required in morphine dependent cell membranes to half-maximally attenuate PGE1 receptor-stimulated adenylate cyclase activity. In addition, limitation of the amount of functionally available GSalpha within the PGE1 receptor/adenylate cyclase signal transduction cascade abolished the generation of a supersensitive adenylate cyclase response during the state of naloxone (100 mu M)-precipitated withdrawal. These data demonstrate that in human neuroblastoma SH-SY5Y cells chronic morphine-induced sensitization of adenylate cyclase is associated with distinct quantitative and qualitative adaptations within the stimulatory adenylate cyclase-coupled PGE1 receptor system. Thus, alterations in the functional activity of stimulatory receptor systems are suggested to contribute to the cellular mechanisms underlying opioid dependence.
Ammer
H
H
Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Germany.
Schulz
R
R
D000222:Adaptation, Physiological / Q000502:physiology*; D000262:Adenylyl Cyclases / Q000378:metabolism; D000527:Alprostadil / Q000378:metabolism*; D000595:Amino Acid Sequence; D001932:Brain Neoplasms / Q000378:metabolism; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008566:Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D009021:Morphine Dependence / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism; D018078:Receptors, Prostaglandin E / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013375:Substance Withdrawal Syndrome / Q000503:physiopathology; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D018078:Receptors, Prostaglandin E; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D000527:Alprostadil