Converging protein kinase pathways mediate adenylyl cyclase superactivation upon chronic delta-opioid agonist treatment.
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 delta-opioid agonist-mediated AC superactivation in Chinese hamster ovary (CHO) cells stably expressing the human delta-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 delta-opioid agonists (+)-4-[(alphaR)-alpha-((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 micro M, 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 delta-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.
Varga
Eva V
EV
Department of Pharmacology, College of Medicine, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA.
Rubenzik
Marc K
MK
Stropova
Dagmar
D
Sugiyama
Masano
M
Grife
Vanessa
V
Hruby
Victor J
VJ
Rice
Kenner C
KC
Roeske
William R
WR
Yamamura
Henry I
HI
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001549:Benzamides / Q000494:pharmacology*; D016466:CHO Cells; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006801:Humans; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D017465:Receptors, Opioid, delta / Q000819:agonists* / Q000235:genetics; D014162:Transfection
D001549:Benzamides; D010879:Piperazines; D017465:Receptors, Opioid, delta; C088464:4-(alpha-(4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl)-N,N-diethylbenzamide; D005576:Colforsin; D000242:Cyclic AMP; D011494:Protein Kinases; D000262:Adenylyl Cyclases