Converging protein kinase pathways mediate adenylyl cyclase superactivation upon chronic δ-opioid agonist treatment
Converging protein kinase pathways mediate adenylyl cyclase superactivation upon chronic δ-opioid agonist treatment
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DOI：
10.1124/jpet.103.049643
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发表时间：
2003-07-01
影响因子：
3.5
通讯作者：
Yamamura, HI
中科院分区：
文献类型：
--
作者：
Varga, EV;Rubenzik, MK;Yamamura, HI
腺苷酸环化酶（AC）的超激活被认为在阿片耐受、依赖和戒断中起重要作用。在本研究中，我们研究了蛋白激酶参与慢性δ阿片激动剂介导的AC超激活在中国仓鼠卵巢（CHO）细胞稳定表达人类δ阿片受体（hDOR/CHO）。在用δ-阿片激动剂（+）-4-[（α R）-α-（（2S，5 R）-4-烯丙基-2，5-二甲基-1-哌嗪基）-3-甲氧基-苄基]-N，N-二乙基苯甲酰胺（SNC 80），[D-Pen（2），D-Pen（5）]-脑啡肽和deltorphin II。同时，在hDOR/CHO细胞裂解物中，慢性SNC 80（1 μ M，4小时）处理使P-32掺入到与ACV/VI抗体免疫反应的200 kDa蛋白质中增加了300 +/- 60%。钙调素拮抗剂calmidazolium显着衰减慢性deltorphin II介导的AC超激活。酪氨酸激酶（染料木黄酮）和蛋白激酶C（白屈菜红碱）抑制剂单独对慢性δ阿片激动剂介导的AC超激活的影响最小。相反，同时治疗染料木黄酮和白屈菜红碱显着衰减AC超激活。因为我们先前表明Raf-1抑制剂3-（3，5-二溴-4-羟基苯亚甲基-5-碘-1，3-二氢-吲哚-2-酮（GW 5074）减弱AC超活化，所以我们假设平行的钙咪唑鎓、白屈菜红碱和染料木黄酮敏感性通路会聚在Raf-1处，通过磷酸化hDOR/CHO细胞中的AC VI来介导AC超活化。
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-Pen(2),D-Pen(5)]-enkephalin, and deltorphin II, respectively. Concurrently, chronic SNC 80 (1 muM, 4-h) treatment augmented P-32 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.