Abstract

To investigate the role of G-protein βγ subunits in δ-opioid signal transduction, we have transfected Chinese hamster ovary (CHO) cells stably expressing the human δ-opioid receptor (hDOR/CHO cells) with the G_α-subunit of transducin-1 (hDOR/T1/CHO). Inhibition of forskolin-stimulated adenylyl cyclase and phospholipase Cβ (PLCβ) activation was measured in each of these cell lines. Because PLCβ₃ activation in CHO cells has been shown to be mediated by free G_βγ subunits derived from G_αi/o, the action of transducin was confirmed by measuring a significant attenuation of (+)-4-[(αR)-α-((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 δ-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 α-transducin scavenges free G_βγsubunits and, furthermore, that free G_βγ subunits play a role in opioid-mediated PLCβ activation and adenylyl cyclase superactivation, but not acute inhibition of forskolin-stimulated cAMP formation in hDOR/CHO cells.