title: Opioid peptide receptor studies. 17. Attenuation of chronic morphine effects after antisense oligodeoxynucleotide knock-down of RGS9 protein in cells expressing the cloned Mu opioid receptor.
abstract: RGS proteins are a recently described class of regulators that influence G-protein-mediated signaling pathways. We have shown previously that chronic morphine results in functional uncoupling of the mu opioid receptor from its G protein in CHO cells expressing cloned human mu opioid receptors. In the present study, we examined the effects of morphine treatment (1 microM, 20 h) on DAMGO-stimulated high-affinity [35S]GTP-gamma-S binding and DAMGO-mediated inhibition of forskolin-stimulated cAMP accumulation in HN9.10 cells stably expressing the cloned rat mu opioid receptor, in the absence and presence of the RGS9 protein knock-down condition (confirmed by Western blot analysis). RGS9 protein expression was reduced by blocking its mRNA with an antisense oligodeoxynucleotide (AS-114). Binding surface analysis resolved two [35S]GTP-gamma-S binding sites (high affinity and low affinity sites). In sense-treated control cells, DAMGO-stimulated [35S]GTP-gamma-S binding by increasing the B(max) of the high-affinity site. In sense-treated morphine-treated cells, DAMGO-stimulated [35S]GTP-gamma-S binding by decreasing the high-affinity Kd without changing the B(max). AS-114 significantly inhibited chronic morphine-induced upregulation of adenylate cyclase activity and partially reversed chronic morphine effects as measured by DAMGO-stimulated [35S]GTP-gamma-S binding. Morphine treatment increased the EC50 (6.2-fold) for DAMGO-mediated inhibition of forskolin-stimulated cAMP activity in control cells but not in cells treated with AS-114 to knock-down RGS9. These results provide additional evidence for involvement of RGS9 protein in modulating opioid signaling, which may contribute to the development of morphine tolerance and dependence.
authors.affiliation: Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland 21224, USA.
authors.forename: Heng
authors.initials: H
authors.lastname: Xu
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Richard B
authors.initials: RB
authors.lastname: Rothman
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000701:Analgesics, Opioid / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004361:Drug Tolerance; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D009020:Morphine / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D020710:RGS Proteins / Q000096:biosynthesis* / Q000235:genetics*; D017450:Receptors, Opioid, mu / Q000187:drug effects* / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D000701:Analgesics, Opioid; D016376:Oligonucleotides, Antisense; D020710:RGS Proteins; D017450:Receptors, Opioid, mu; C116925:regulator of g-protein signaling 9; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009020:Morphine; D000242:Cyclic AMP; D000262:Adenylyl Cyclases