Serradeil-Le Gal C, Jouneaux C, Sanchez-Bueno A, Raufaste D, Roche B, Préaux A M, Maffrand J P, Cobbold P H, Hanoune J, Lotersztajn S
Sanofi Recherche, Biochimie Exploratoire, Toulouse, France.
J Clin Invest. 1991 Jan;87(1):133-8. doi: 10.1172/JCI114962.
High affinity binding sites for endothelin (ET) were identified on rat liver plasma membranes. Binding of 125I-ET-1 with its site was specific, saturable, and time dependent (kobs = 0.019 +/- 0.001 min-1), but dissociation of receptor-bound ligand was minimal. A single class of high affinity binding sites for 125I-ET-1 was identified with an apparent Kd of 32.4 +/- 9.8 pM and a Bmax of 1084 +/- 118 fmol/mg protein. ET-3 and big-ET-1 (1-38) (human) inhibited 125I-ET-1 binding with IC50 values of 1.85 +/- 1.03 nM and 43 +/- 6 nM, respectively. Aequorin measurements of cytosolic free Ca2+ in single, isolated rat hepatocytes showed that ET-1 at subnanomolar concentrations induced a series of repetitive, sustained Ca2+ transients. ET-1 had no effect on cAMP production. Finally, ET-1 caused a rapid and sustained stimulation of glycogenolysis in rat hepatocytes. A 1.8-fold maximal increase in glycogen phosphorylase alpha was observed at 1 pM ET-1, with an EC50 of 0.03 pM. Stimulation of the enzyme was specific for ET-1 since the order of potency of related peptides was similar to that in binding experiments (ET-1 greater than ET-3 greater than big ET-1). These data constitute the first demonstration of the presence of ET-1 binding sites in liver which is associated with a rise in cytosolic free Ca2+ and a potent glycogenolytic effect. We conclude that ET-1 behaves as a typical Ca2+ mobilizing hormone in liver.
在内质网膜上发现了内皮素（ET）的高亲和力结合位点。125I-ET-1与其位点的结合具有特异性、饱和性且依赖时间（观测速率常数kobs = 0.019 +/- 0.001 min-1），但受体结合配体的解离极少。确定了一类针对125I-ET-1的高亲和力结合位点，其表观解离常数Kd为32.4 +/- 9.8 pM，最大结合容量Bmax为1084 +/- 118 fmol/mg蛋白质。ET-3和大ET-1（1-38）（人）抑制125I-ET-1结合，其半数抑制浓度IC50值分别为1.85 +/- 1.03 nM和43 +/- 6 nM。对单个分离的大鼠肝细胞中胞质游离Ca2+的水母发光蛋白测量显示，亚纳摩尔浓度的ET-1可诱导一系列重复、持续的Ca2+瞬变。ET-1对环磷酸腺苷（cAMP）的产生没有影响。最后，ET-1可快速、持续地刺激大鼠肝细胞中的糖原分解。在1 pM ET-1时，糖原磷酸化酶α的最大增加倍数为1.8倍，半数有效浓度EC50为0.03 pM。该酶的刺激对ET-1具有特异性，因为相关肽的效力顺序与结合实验中的顺序相似（ET-1大于ET-3大于大ET-1）。这些数据首次证明肝脏中存在ET-1结合位点，其与胞质游离Ca2+升高及强大的糖原分解作用相关。我们得出结论，ET-1在肝脏中表现为典型的Ca2+动员激素。
