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Vol. 60, Issue 5, 1076-1082, November 2001
-Transducin in Chinese Hamster Ovary Cells
Stably Transfected with the Human 
-Opioid Receptor Attenuates
Chronic Opioid Agonist-Induced Adenylyl Cyclase Superactivation
Departments of Pharmacology (M.R., E.V., D.S., W.R.R., H.I.Y.),
Medicine (W.R.R.), and Biochemistry, Psychiatry, and the Program in
Neuroscience (H.I.Y.), College of Medicine, University of Arizona
Health Sciences Center, Tucson, Arizona
To investigate the role of G-protein 
subunits in
-opioid signal transduction, we have transfected Chinese hamster
ovary (CHO) cells stably expressing the human -opioid receptor
(hDOR/CHO cells) with the G
-subunit of transducin-1
(hDOR/T1/CHO). Inhibition of forskolin-stimulated adenylyl cyclase and
phospholipase C (PLC) activation was measured in each of these
cell lines. Because PLC3 activation in CHO cells has
been shown to be mediated by free G
subunits derived
from Gi/o, the action of transducin was confirmed by
measuring a significant attenuation of
(+)-4-[(
R)--((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 -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 -transducin scavenges free G
subunits and, furthermore, that free G subunits play
a role in opioid-mediated PLC activation and adenylyl cyclase
superactivation, but not acute inhibition of forskolin-stimulated cAMP
formation in hDOR/CHO cells.
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