Adenylyl cyclase (AC) superactivation is thought to play an important role in opioid tolerance, dependence, and withdrawal. In the present study, we investigated the involvement of protein kinases in chronic ␦-opioid agonist-mediated AC superactivation in Chinese hamster ovary (CHO) cells stably expressing the human ␦-opioid receptor (hDOR/CHO). Maximal forskolin-stimulated cAMP formation in hDOR/CHO cells increased by 472 Ϯ 91, 399 Ϯ 2, and 433 Ϯ 73% after chronic treatment with the ␦-opioid-enkephalin, and deltorphin II, respectively. Concurrently, chronic SNC 80 (1 M, 4-h) treatment augmented 32 P incorporation into a 200-kDa protein immunoreactive with the ACV/VI antibody by 300 Ϯ 60% in hDOR/CHO cell lysates. The calmodulin antagonist calmidazolium significantly attenuated chronic deltorphin II-mediated AC superactivation. Tyrosine kinase (genistein) and protein kinase C (chelerythrine) inhibitors individually had minimal effect on chronic ␦-opioid agonist-mediated AC superactivation. Conversely, simultaneous treatment with both genistein and chelerythrine significantly attenuated AC superactivation. Because we showed previously that the Raf-1 inhibitor 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) attenuates AC superactivation, we hypothesize that parallel calmidazolium-, chelerythrine-, and genistein-sensitive pathways converge at Raf-1 to mediate AC superactivation by phosphorylating AC VI in hDOR/CHO cells.Chronic opioid receptor activation frequently leads to the sensitization of adenylyl cyclase to stimulators after the inhibitory agonist has been removed (AC superactivation). AC superactivation after chronic opioid agonist exposure is thought to contribute to the development of opioid tolerance, dependence, and withdrawal (Williams et al., 2001). A better understanding of the molecular mechanisms of chronic ␦-opioid agonist treatment-mediated AC superactivation should aid in the development of longer acting analgesics with fewer side effects.We have reported previously that in Chinese hamster ovary (CHO) cells stably expressing the human ␦-opioid receptor (hDOR/CHO), chronic ␦-opioid agonist treatment gives rise to AC superactivation (Malatynska et al., 1996). In addition, we also demonstrated that chronic ␦-opioid agonist treatment of the hDOR/CHO cells augments 32 P incorporation into proteins immunoreactive with an AC V/VI-specific antibody (Varga et al., 1999). The identity of protein kinase(s) involved in chronic ␦-opioid agonist-mediated phosphorylation in hDOR/CHO cells, and the role of the phosphorylation in AC superactivation, however, have not been investigated previously. In the present work, we studied the effect of protein kinase inhibitors on chronic ␦-opioid agonistmediated AC superactivation.Recent data (Tan et al., 2001) indicate the important role of the protein kinase p74Raf-1 in phosphorylation of adenylyl cyclase VI in transfected human embryonic kidney 293 cells. Moreover, Raf-1-mediated phosphorylation led to the sensitization of AC VI to s...

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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.

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Vasovagal reactions should be carefully evaluated. Additional studies may provide greater data in understanding and managing vasovagal reactions.

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