Wu-Wong J R, Chiou W J, Magnuson S R, Opgenorth T J
Pharmaceutical Products Division, Abbott Laboratories, Illinois, USA.
J Pharmacol Exp Ther. 1995 Jul;274(1):499-507.
Endothelin (ET) receptor in human astrocytoma U373MG cells was characterized. ET-1, ET-3, sarafotoxin S6C, IRL1620, BQ788, Ro46-2005 and PD142893 inhibited specific [125I]ET-1 binding with Ki values of 0.03 0.06, 0.74, 5.01, 4.45, 2275 and 157 nM, respectively. ETA selective antagonists BQ123 and FR139317 at 1 microM did not block [125I]ET-1 binding. Reverse transcription-polymerase chain reaction confirmed the results from competition studies that U373 cells expressed predominantly ETB receptor. The Bmax and KD values of [125I]ET-1 binding were 0.15 pmol/1 x 10(6) cells and 0.23 nM. The molecular mass for the receptor was 45 kDa. ET-1 binding did not stimulate Ca+2 mobilization, phosphatidylinositol hydrolysis or arachidonic acid release, nor did it affect the intracellular cAMP or cGMP level. Interestingly, a majority of ET (> 80%) bound to the receptor was rapidly internalized, consistent with emerging evidence that a major function of ETB receptor is to clear ET. [125I]ET-1 binding was time-dependent and bound [125I]ET-1 was difficult to dissociate. In contrast, bound antagonists were much easier to dissociate. The results suggest that agonists and antagonists of the ET receptor exhibited different dissociation characteristics, with antagonist binding more reversible than agonist binding.
对人星形细胞瘤U373MG细胞中的内皮素（ET）受体进行了特性分析。ET-1、ET-3、萨拉毒素S6C、IRL1620、BQ788、Ro46-2005和PD142893抑制特异性[125I]ET-1结合，其Ki值分别为0.03、0.06、0.74、5.01、4.45、2275和157 nM。1 μM的ETA选择性拮抗剂BQ123和FR139317不阻断[125I]ET-1结合。逆转录-聚合酶链反应证实了竞争研究的结果，即U373细胞主要表达ETB受体。[125I]ET-1结合的Bmax和KD值分别为0.15 pmol/1×10(6)细胞和0.23 nM。该受体的分子量为45 kDa。ET-1结合不刺激Ca+2动员、磷脂酰肌醇水解或花生四烯酸释放，也不影响细胞内cAMP或cGMP水平。有趣的是，与受体结合的大部分ET（>80%）迅速内化，这与新出现的证据一致，即ETB受体的主要功能是清除ET。[125I]ET-1结合具有时间依赖性，且结合的[125I]ET-1难以解离。相比之下，结合的拮抗剂更容易解离。结果表明，ET受体的激动剂和拮抗剂表现出不同的解离特性，拮抗剂结合比激动剂结合更具可逆性。
