Expression of alpha-transducin in Chinese hamster ovary cells stably transfected with the human delta-opioid receptor attenuates chronic opioid agonist-induced adenylyl cyclase superactivation

• Published in: Molecular pharmacology Vol. 60; no. 5; pp. 1076 - 1082
• Main Authors: Rubenzik, M, Varga, E, Stropova, D, Roeske, W R, Yamamura, H I
• Format: Journal Article
• Language: English
• Published: United States 01.11.2001
• Subjects: Adenylate Cyclase Toxin; Adenylyl Cyclases - metabolism; Animals; Benzamides - pharmacology; CHO Cells; Colforsin - pharmacology; Cricetinae; Cyclic AMP - metabolism; Drug Interactions; Enzyme Activation - drug effects; Gene Expression; Humans; Inositol 1,4,5-Trisphosphate - metabolism; Pertussis Toxin; Piperazines - pharmacology; Receptors, Opioid, delta - genetics; Receptors, Opioid, delta - metabolism; Transducin - biosynthesis; Transducin - metabolism; Transfection; Virulence Factors, Bordetella - pharmacology
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.60.5.1076

To investigate the role of G-protein beta gamma subunits in delta-opioid signal transduction, we have transfected Chinese hamster ovary (CHO) cells stably expressing the human delta-opioid receptor (hDOR/CHO cells) with the G(alpha)-subunit of transducin-1 (hDOR/T1/CHO). Inhibition of forskolin-stimulated adenylyl cyclase and phospholipase C beta (PLC beta) activation was measured in each of these cell lines. Because PLC beta(3) activation in CHO cells has been shown to be mediated by free G(beta gamma) subunits derived from G(alpha i/o), the action of transducin was confirmed by measuring a significant attenuation of (+)-4-[(alpha R)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)-mediated maximal inositol-1,4,5-trisphosphate formation in transducin-expressing cells of 59 +/- 12% compared with control cells. The acute inhibition of cAMP formation was unchanged between control and transducin-expressing cells. We show that cells stably expressing the human delta-opioid receptor exhibited a pertussis toxin-sensitive cAMP overshoot in response to chronic application of SNC80. After 4 h of pretreatment and washout with 100 nM SNC80, maximal forskolin-stimulated cAMP formation in hDOR/CHO cells increased by 229 +/- 37% compared with buffer-treated cells. Expression of transducin in hDOR/CHO cells diminished this response: hDOR/T1/CHO cells showed no significant change in maximal forskolin-stimulated cAMP formation after pretreatment and washout. These data indicate that the expression of alpha-transducin scavenges free G(beta gamma) subunits and, furthermore, that free G(beta gamma) subunits play a role in opioid-mediated PLC beta activation and adenylyl cyclase superactivation, but not acute inhibition of forskolin-stimulated cAMP formation in hDOR/CHO cells.