Bhalla Shaifali, Ciaccio Natalie, Wang Zaijie Jim, Gulati Anil
Department of Biopharmaceutical Sciences (M/C 865), The University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612, USA.
Exp Biol Med (Maywood). 2006 Jun;231(6):1152-6.
Long-term use of morphine in pain management leads to adverse effects, such as development of antinociceptive tolerance. We have previously shown the involvement of central endothelin (ET) mechanisms in morphine analgesia and development of tolerance in vivo. The present study was conducted to investigate the in vitro mechanism of interaction of the ET(A) receptor antagonist, BMS182874, and morphine during acute and chronic morphine tolerance in SH-SY5Y cells. SH-SY5Y cells were exposed to acute and chronic treatment with vehicle, morphine, ET-1, BMS182874, or morphine plus BMS182874. Activation of G-protein-coupled receptors in SH-SY5Y cells was determined using [35S]GTPgammaS binding assays. Acute morphine treatment produced a concentration-dependent increase in GTP binding. Median effective concentration (EC50) values were significantly decreased after acute morphine treament, suggesting sensitization of opioid receptors. Chronic morphine treatment produced a lower maximal response of GTP binding compared with both control (vehicle treated) and acute morphine treatment, indicating uncoupling of G-proteins. Acute and chronic exposure of cells to ET-1 did not affect changes in ET-1-induced GTP binding. BMS182874 treatment alone (acute or chronic) did not produce G-protein activation. However, in cells chronically cotreated with 10 microM morphine and 1 microM BMS182874, morphine-induced GTP stimulation was significantly higher than control (vehicle treated). The EC50 value after control treatment was 414 nM, and was significantly increased in chronically morphine-treated cells (>1000 nM ). However, the EC50 value in cells receiving a chronic treatment of BMS182874 and 63 nM morphine was significantly reduced compared with control (vehicle treated) and chronic morphine treatment. ET(A) antagonists significantly enhance the coupling of G-protein to opioid receptors. Therefore, we propose that restoration of morphine antinociception by ET(A) antagonists in morphine-tolerant animals is likely via a G-protein mediated mechanism.
长期使用吗啡进行疼痛管理会导致不良反应，如抗伤害感受性耐受的产生。我们之前已经表明，中枢内皮素（ET）机制参与了吗啡镇痛及体内耐受的形成。本研究旨在探讨ET(A)受体拮抗剂BMS182874与吗啡在SH-SY5Y细胞急性和慢性吗啡耐受过程中的体外相互作用机制。将SH-SY5Y细胞分别用溶剂、吗啡、ET-1、BMS182874或吗啡加BMS182874进行急性和慢性处理。使用[35S]GTPγS结合试验测定SH-SY5Y细胞中G蛋白偶联受体的激活情况。急性吗啡处理使GTP结合呈浓度依赖性增加。急性吗啡处理后，半数有效浓度（EC50）值显著降低，提示阿片受体致敏。与对照组（溶剂处理组）和急性吗啡处理组相比，慢性吗啡处理使GTP结合的最大反应降低，表明G蛋白解偶联。细胞急性和慢性暴露于ET-1均不影响ET-1诱导的GTP结合变化。单独使用BMS182874处理（急性或慢性）不会产生G蛋白激活。然而，在长期用10μM吗啡和1μM BMS182874共同处理的细胞中，吗啡诱导的GTP刺激显著高于对照组（溶剂处理组）。对照组处理后的EC50值为414 nM，在慢性吗啡处理的细胞中显著升高（>1000 nM）。然而，与对照组（溶剂处理组）和慢性吗啡处理组相比，接受BMS182874和63 nM吗啡慢性处理的细胞中的EC50值显著降低。ET(A)拮抗剂显著增强G蛋白与阿片受体的偶联。因此，我们提出ET(A)拮抗剂在吗啡耐受动物中恢复吗啡抗伤害感受作用可能是通过G蛋白介导的机制实现的。
