Refsnes M, Sandnes D, Christoffersen T
Eur J Biochem. 1987 Mar 16;163(3):457-66. doi: 10.1111/j.1432-1033.1987.tb10891.x.
The role of beta-adrenoceptor regulation in the mechanisms controlling beta-adrenergic responsiveness in hepatocytes was explored, using primary monolayer cultures. When plated in vitro, these cells gradually acquire a strong catecholamine-sensitive adenylate cyclase activity and an enhanced ability to bind the beta-adrenoceptor ligand [125I]iodocyanopindolol (125ICYP). Examination of the time course showed that the increase in the number of 125ICYP binding sites was detectable within 1-2 h of culturing and slightly preceded the elevation of isoproterenol-responsive activity. Then the responsiveness rose steeply and between about 5-24 h it closely followed the increase in beta-receptor binding. Addition of isoproterenol (10 microM) to cells after 20 h of culturing caused a rapid homologous desensitization of the adenylate cyclase (50% after about 5 min). This was paralleled by a down-regulation of beta-adrenoceptors measured both in membrane particles and in total cell lysates. Removal of isoproterenol led to a resensitization of the adenylate cyclase, which was rapid and protein-synthesis-independent after a brief (10-min) desensitization, or slow and cycloheximide-sensitive after prolonged (4-h) exposure to the agonist. In both cases an up-regulation of the 125ICYP binding paralleled the recovery from refractoriness. In contrast, no concurring changes in 125ICYP binding were measured when the beta-adrenoceptor-linked adenylate cyclase activity was enhanced by pretreatment with pertussin toxin (islet-activating protein, IAP) or was desensitized by exposure of the cells to glucagon or 8-bromo-cAMP; however, these modulations of the adenylate cyclase were nonselective, since the pretreatments with IAP, glucagon or 8-bromo-cAMP affected both isoproterenol-sensitive and glucagon-sensitive activities. The results suggest that, in hepatocytes, regulation at the beta-adrenoceptor level is a major determinant for both short-term and long-term selective changes of the beta-adrenergic responsiveness.
利用原代单层培养技术，探讨了β-肾上腺素能受体调节在控制肝细胞β-肾上腺素能反应性机制中的作用。当在体外培养时，这些细胞逐渐获得强烈的儿茶酚胺敏感性腺苷酸环化酶活性以及增强的结合β-肾上腺素能受体配体[125I]碘氰吲哚洛尔（125ICYP）的能力。对时间进程的研究表明，培养1 - 2小时内可检测到125ICYP结合位点数量增加，且略早于异丙肾上腺素反应性活性的升高。然后反应性急剧上升，在约5 - 24小时之间，它紧密跟随β受体结合的增加。培养20小时后向细胞中加入异丙肾上腺素（10μM）导致腺苷酸环化酶迅速发生同源脱敏（约5分钟后50%）。这与在膜颗粒和全细胞裂解物中测量的β-肾上腺素能受体下调同时发生。去除异丙肾上腺素导致腺苷酸环化酶重新敏感化，短暂（10分钟）脱敏后快速且不依赖蛋白质合成，或长时间（4小时）暴露于激动剂后缓慢且对环己酰亚胺敏感。在这两种情况下，125ICYP结合的上调与从不应性恢复平行。相反，当用百日咳毒素（胰岛激活蛋白，IAP）预处理增强β-肾上腺素能受体连接的腺苷酸环化酶活性，或通过使细胞暴露于胰高血糖素或8-溴-cAMP使其脱敏时，未检测到125ICYP结合的同时变化；然而，这些腺苷酸环化酶的调节是非选择性的，因为用IAP、胰高血糖素或8-溴-cAMP预处理会影响异丙肾上腺素敏感性和胰高血糖素敏感性活性。结果表明，在肝细胞中，β-肾上腺素能受体水平的调节是β-肾上腺素能反应性短期和长期选择性变化的主要决定因素。
