Gosse M E, Frey E A, Cote T E
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Mol Endocrinol. 1989 Feb;3(2):315-24. doi: 10.1210/mend-3-2-315.
In this study, the influence of the inhibitory mu-opioid receptor on the potencies of 5'-guanosine alpha-thiotriphosphate (GTP gamma S) and GDP at the inhibitory GTP-binding protein (Gi) were investigated in an adenylyl cyclase system. It was hoped that a receptor-mediated change in the potency of either GTP gamma S or GDP in affecting adenylyl cyclase activity may elucidate how a receptor alters cyclase activity via its G-protein. In an adenylyl cyclase system employing 5'-adenylyl imidodiphosphate as substrate, GTP gamma S, a nonhydrolyzable analog of GTP, inhibited forskolin-stimulated adenylyl cyclase activity in the absence of morphine; morphine failed to significantly affect the apparent potency of GTP gamma S. GDP blocked the GTP gamma S-induced inhibition of adenylyl cyclase; morphine profoundly diminished the ability of GDP to block the inhibitory effect of GTP gamma S. The IC50 values of GTP gamma S were 0.02 +/- 0.01, 0.18 +/- 0.04, and 2.2 +/- 0.5 microM in the absence of other drugs, in the presence of a combination of 100 microM GDP and morphine, and in the presence of 100 microM GDP, respectively. GDP blocked the inhibitory effect of GTP gamma S (0.3 microM) in a concentration-dependent manner; the EC50 for GDP was 16 +/- 2.6 microM in the absence of morphine and 170 +/- 32 microM in the presence of morphine. Exposure of 7315c cells to pertussis toxin for 3 h resulted in a small decrease in the potency of GTP gamma S in inhibiting cyclase. However, the relative potency of GDP in blocking the GTP gamma S-mediated inhibition of cyclase was increased: the EC50 values of GDP were 11 +/- 4 and 0.81 +/- 0.2 microM in untreated and pertussis toxin-treated membranes, respectively. In untreated membranes, there was a brief lag in the GTP gamma S-induced inhibition of adenylyl cyclase; morphine diminished this lag. In membranes treated with pertussis toxin, there was an exaggerated lag in the onset of GTP gamma S inhibition of adenylyl cyclase activity; morphine could no longer affect this lag. Thus, uncoupling the mu-opioid receptor from Gi appeared to increase the affinity of Gi for GDP. These data suggest that the effect of an inhibitory receptor is to decrease the affinity of Gi for GDP by virtue of its interaction with the carboxy-terminal region of Gi alpha.(ABSTRACT TRUNCATED AT 400 WORDS)
在本研究中，我们在腺苷酸环化酶系统中研究了抑制性μ-阿片受体对5'-鸟苷α-硫代三磷酸（GTPγS）和GDP在抑制性GTP结合蛋白（Gi）上活性的影响。人们希望，GTPγS或GDP影响腺苷酸环化酶活性的效力发生受体介导的变化，可能会阐明受体如何通过其G蛋白改变环化酶活性。在以5'-腺苷酰亚胺二磷酸为底物的腺苷酸环化酶系统中，GTPγS是一种不可水解的GTP类似物，在没有吗啡的情况下可抑制福斯高林刺激的腺苷酸环化酶活性；吗啡未能显著影响GTPγS的表观效力。GDP可阻断GTPγS诱导的腺苷酸环化酶抑制作用；吗啡显著降低了GDP阻断GTPγS抑制作用的能力。在没有其他药物时、在100μM GDP和吗啡联合存在时以及在100μM GDP存在时，GTPγS的IC50值分别为0.02±0.01、0.18±0.04和2.2±0.5μM。GDP以浓度依赖性方式阻断GTPγS（0.3μM）的抑制作用；在没有吗啡时，GDP的EC50为16±2.6μM，在有吗啡时为170±32μM。将7315c细胞暴露于百日咳毒素3小时，导致GTPγS抑制环化酶的效力略有下降。然而，GDP阻断GTPγS介导的环化酶抑制作用的相对效力增加：在未处理和经百日咳毒素处理的膜中，GDP的EC50值分别为11±4和0.81±0.2μM。在未处理的膜中，GTPγS诱导的腺苷酸环化酶抑制作用有短暂延迟；吗啡减少了这种延迟。在用百日咳毒素处理的膜中，GTPγS抑制腺苷酸环化酶活性的起始延迟更为明显；吗啡不再能影响这种延迟。因此，使μ-阿片受体与Gi解偶联似乎增加了Gi对GDP的亲和力。这些数据表明，抑制性受体的作用是通过与Giα的羧基末端区域相互作用来降低Gi对GDP的亲和力。（摘要截选至400字）
