Neve K A, Molinoff P B
Mol Pharmacol. 1986 Aug;30(2):104-11.
The turnover of beta 1- and beta 2-adrenergic receptors was measured after both isoproterenol-induced down-regulation and irreversible blockade of receptors. Changes in the density of receptors were quantified using the radioligands 125I-iodopindolol and 125I-iodocyanopindolol. Treatment of intact L6 myoblasts or C6 glioma cells with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) inactivated beta-adrenergic receptors on membranes prepared from these cells. At a concentration of 100 microM EEDQ, more than 90% of beta 1- and beta 2-adrenergic receptors were inactivated within 2 hr of treatment. Recovery of beta-adrenergic receptors on intact cells after inactivation by EEDQ required more than 24 hr and was prevented by cycloheximide, an inhibitor of protein synthesis. The kinetics of recovery of the density of receptors were analyzed in terms of a model that allows estimation of the rate constants for receptor appearance in and disappearance from the membrane, assuming that the rate of appearance of receptors is constant and the rate of disappearance of receptors is proportional to the number of receptors. Beta 2-Adrenergic receptors on L6 myoblasts were incorporated into the membrane at a rate of 28 fmol/mg of protein/hr and had a half-life of 12.6 hr. On C6 glioma cells, Beta 1- and beta 2-adrenergic receptors appeared at rates of 13.3 and 6.6 fmol/mg of protein/hr, respectively, with half-lives of 9.4 and 6.4 hr. Recovery of receptors on C6 cells after isoproterenol-induced down-regulation was inhibited by cycloheximide. The rate of recovery of beta 1- and beta 2-adrenergic receptors was reduced after treatment with isoproterenol for 8 hr when compared to recovery after treatment with EEDQ. The major effect of treatment with isoproterenol was a persistent decrease in the rate of appearance of beta 1- and beta 2-adrenergic receptors (rate of synthesis and insertion into the membrane after treatment with isoproterenol = 4.0 fmol/mg of protein/hr). Since treatment with isoproterenol did not alter the rate of cell division or total protein synthesis, the isoproterenol-induced alteration was probably a specific effect on the rate of synthesis of beta-adrenergic receptors.
在异丙肾上腺素诱导的β1和β2肾上腺素能受体下调以及受体的不可逆阻断后，对其周转率进行了测定。使用放射性配体125I-碘吲哚洛尔和125I-碘氰吲哚洛尔对受体密度的变化进行定量。用N-乙氧羰基-2-乙氧基-1,2-二氢喹啉（EEDQ）处理完整的L6成肌细胞或C6胶质瘤细胞，可使从这些细胞制备的膜上的β肾上腺素能受体失活。在100μM EEDQ浓度下，处理2小时内超过90%的β1和β2肾上腺素能受体失活。EEDQ使完整细胞上的β肾上腺素能受体失活后，其恢复需要超过24小时，并且被蛋白质合成抑制剂环己酰亚胺所阻止。根据一个模型分析了受体密度恢复的动力学，该模型允许估计受体在膜上出现和消失的速率常数，假设受体出现的速率是恒定的，受体消失的速率与受体数量成正比。L6成肌细胞上的β2肾上腺素能受体以28 fmol/mg蛋白质/小时的速率整合到膜中，半衰期为12.6小时。在C6胶质瘤细胞上，β1和β2肾上腺素能受体出现的速率分别为13.3和6.6 fmol/mg蛋白质/小时，半衰期分别为9.4和6.4小时。异丙肾上腺素诱导下调后，C6细胞上受体的恢复受到环己酰亚胺的抑制。与EEDQ处理后的恢复相比，异丙肾上腺素处理8小时后β1和β2肾上腺素能受体的恢复速率降低。异丙肾上腺素处理的主要作用是β1和β2肾上腺素能受体出现速率的持续下降（异丙肾上腺素处理后合成并插入膜中的速率 = 4.0 fmol/mg蛋白质/小时）。由于异丙肾上腺素处理并未改变细胞分裂速率或总蛋白质合成，异丙肾上腺素诱导的改变可能是对β肾上腺素能受体合成速率的特异性作用。
