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

To investigate the role of G-protein beta gamma subunits in delta -opiaid s ignal transduction, we have transfected Chinese hamster ovary (CHO) cells s tably expressing the human delta -opioid receptor (hDOR/CHO cells) with the G(alpha)-subunit of transducin-1 (hDOR/T1/CHO). Inhibition of forskolin-st imulated adenylyl cyclase and phospholipase Co (PLC beta) activation was me asured 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(alphai/o), the action of transducin was confirmed by measuring a si gnificant attenuation of (+)-4-[(alphaR)-alpha-((2S,5R)-4-Ally[-2,5-dimethy l-1 -piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)-mediated ma ximal inositol-1,4,5-trisphosphate formation in transducin-expressing cells of 59 +/- 12% compared with control cells. The acute inhibition of CAMP fo rmation was unchanged between control and transducin- expressing cells. We show that cells stably expressing the human delta -opioid receptor exhibite d a pertussis toxin-sensitive CAMP overshoot in response to chronic applica tion of SNC80. After 4 h of pretreatment and washout with 100 nM SNC80, max imal forskolin-stimulated CAMP formation in hDOR/CHO cells increased by 229 +/- 37% compared with buffer-treated cells. Expression of transducin in hD OR/CHO cells diminished this response: hDOR/T1/CHO cells showed no signific ant change in maximal forskolin-stimulated cAMP formation after pretreatmen t and washout. These data indicate that the expression of a-transducin scav enges 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 c AMP formation in hDOR/CHO cells.