Abstract
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 δ-opioid agonist-mediated AC superactivation in Chinese hamster ovary (CHO) cells stably expressing the human δ-opioid receptor (hDOR/CHO). Maximal forskolin-stimulated cAMP formation in hDOR/CHO cells increased by 472 ± 91, 399 ± 2, and 433 ± 73% after chronic treatment with the δ-opioid agonists (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl benzamide (SNC 80), [d-Pen2,d-Pen5]-enkephalin, and deltorphin II, respectively. Concurrently, chronic SNC 80 (1 μM, 4-h) treatment augmented 32P incorporation into a 200-kDa protein immunoreactive with the ACV/VI antibody by 300 ± 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 δ-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.
Footnotes
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This work was supported in part by grants from the National Institutes of Health and the Arizona Disease Control Research Commission.
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DOI: 10.1124/jpet.103.049643.
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ABBREVIATIONS: AC, adenylyl cyclase; CHO, Chinese hamster ovary; hDOR, human δ-opioid receptor; GW5074, 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one; MAPK, mitogen-activated protein kinase; SNC 80, (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl benzamide; DPDPE, [d-Pen2-d-Pen5]-enkephalin; IMDM: Iscove's modified Dulbecco's medium; PAGE, polyacrylamide gel electrophoresis; ANOVA, analysis of variance; PKC, protein kinase C; LMMP, longitudinal muscle myenteric plexus; H-89, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinoline sulfonamide, 2HCl; IP1, inositol phosphate-1.
- Received January 27, 2003.
- Accepted March 20, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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Converging Protein Kinase Pathways Mediate Adenylyl Cyclase Superactivation upon Chronic δ-Opioid Agonist Treatment
