Tao P L, Lue W M, Lee C R, Chang L R
Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan, R.O.C.
Chin J Physiol. 1995;38(3):193-9.
Alteration in ligand-receptor interaction during chronic drug treatment has been suggested as a possible mechanism underlying opioid tolerance. However, our previous studies found that chronic PL017 (a selective mu-opioid agonist) treatment of adult animals resulted in down regulation of mu opioid receptor levels only after 5 days of PL017 treatment although tolerance had significantly developed after 3 days of PL017 treatment. Since G protein seems to be involved in regulation of opioid receptors, we suspect that opioid receptor-G protein interaction may be altered after chronic PL017 treatment before down-regulation of opioid receptors occurrs. Our investigation proceeded first, by measuring the ability of Gpp(NH)p to alter mu-opioid agonist: [3H]DAMGO binding; and second, by measuring the opioid agonist-stimulated GTPase activity before and after chronic PL017 treatment for 1 or 3 days when tolerance has developed but without down-regulation. We found that after 1 day and 3 days of PL017 treatment, rats produced 1.9 and 7.4 fold degree of tolerance. In receptor binding assay, we found the Bmax values did not show significant difference before and after chronic PL017 treatment. On the other hand, 10 microM Gpp(NH)p (a stable GTP analogue) significantly increased the Kd of the control midbrain by 2.59 +/- 0.21 fold but only increased the Kd by 1.92 +/- 0.11 fold after 3 days of PL017 treatment. Furthermore, the EC50 and maximal effect of DAMGO on stimulating low Km GTPase activity for control midbrain are 1.2 +/- 0.3 10(-8) M and 21.7 +/- 0.6%, respectively; in the experimental group, after 3 days PL017 treatment, the EC50 has increased to 7.3 +/- 2.7 x 10(-8) M and maximal stimulation decreased to 16.6 +/- 1.1%. The present findings indicate that after 3 days chronic PL017 treatment: (1) The effect of Gpp(NH)p on the affinity of mu-opioid receptor and DAMGO has been diminished. (2) The effect of DAMGO on stimulating low Km GTPase activity of G protein has been decreased. Therefore, it seems that the interaction between opioid receptor and G protein has been altered after chronic PL017 treatment. This phenomenum happens before down-regulation, and it may be one of the mechanisms for opioid tolerance.
慢性药物治疗期间配体-受体相互作用的改变被认为是阿片类药物耐受性潜在的一种可能机制。然而，我们之前的研究发现，对成年动物进行慢性PL017（一种选择性μ-阿片受体激动剂）治疗时，仅在PL017治疗5天后μ-阿片受体水平才出现下调，尽管在PL017治疗3天后耐受性已显著形成。由于G蛋白似乎参与阿片受体的调节，我们怀疑在阿片受体下调发生之前，慢性PL017治疗后阿片受体-G蛋白相互作用可能已发生改变。我们的研究首先通过测量Gpp(NH)p改变μ-阿片受体激动剂：[3H]DAMGO结合的能力来进行；其次，在慢性PL017治疗1天或3天后耐受性已形成但未出现下调时，测量阿片受体激动剂刺激的GTP酶活性。我们发现，PL017治疗1天和3天后，大鼠产生的耐受性分别为1.9倍和7.4倍。在受体结合试验中，我们发现慢性PL017治疗前后Bmax值未显示出显著差异。另一方面，10μM Gpp(NH)p（一种稳定的GTP类似物）使对照中脑的Kd显著增加2.59±0.21倍，但在PL017治疗3天后仅使Kd增加1.92±0.11倍。此外，DAMGO刺激对照中脑低Km GTP酶活性的EC50和最大效应分别为1.2±0.3×10(-8)M和21.7±0.6%；在实验组中，PL017治疗3天后，EC50已增加至7.3±2.7×10(-8)M，最大刺激降至16.6±1.1%。目前的研究结果表明，慢性PL017治疗3天后：（1）Gpp(NH)p对μ-阿片受体和DAMGO亲和力的影响减弱。（2）DAMGO刺激G蛋白低Km GTP酶活性的作用降低。因此，慢性PL017治疗后阿片受体与G蛋白之间的相互作用似乎已发生改变。这种现象发生在下调之前，可能是阿片类药物耐受性的机制之一。
