Michel M C, Kerker J, Branchek T A, Forray C
Department of Medicine, University of Essen, Germany.
Mol Pharmacol. 1993 Dec;44(6):1165-70.
We have determined the alkylating effects and affinity of chloroethylclonidine at alpha 1- and alpha 2-adrenoceptor subtypes in saturation and competition radioligand binding studies. Treatment with chloroethylclonidine (10 microM, for 30 min at 37 degrees, with subsequent washout) abolished [3H]prazosin binding to alpha 1B-adrenoceptors in rat spleen almost completely and reduced specific binding in rat kidney and cerebral cortex by a percentage comparable to the known alpha 1B-adrenoceptor content of these tissues. Chloroethylclonidine treatment also markedly reduced [3H]rauwolscine binding to human platelet and kidney membranes but did not affect [3H]rauwolscine binding to rat kidney. Similar chloroethylclonidine treatment (10 microM, 20 min at 37 degrees) reduced the number of detectable alpha 2-adrenoceptors in cell lines transfected with the alpha 2-C10 or alpha 2-C4 gene but not in those transfected with alpha 2-C2 adrenoceptors. In concentration-response experiments, higher chloroethylclonidine concentrations were required for inactivation of human platelet alpha 2A-adrenoceptors, compared with rat spleen alpha 1B-adrenoceptors, and a smaller maximal inactivation was achieved. The lack of inactivation of rat alpha 1A- and alpha 2B- and human alpha 2-C2-adrenoceptors was not due to a lack of chloroethylclonidine binding, because the affinity of chloroethylclonidine at these subtypes, as determined in competition binding experiments, was at least as high as the apparent affinity at the alkylated subtypes. alpha 2A-Adrenoceptor alkylation by chloroethylclonidine treatment was functionally relevant, because it significantly reduced alpha 2A-adrenoceptor-mediated Ca2+ elevations in HEL cells. We conclude that chloroethylclonidine binds to all major alpha-adrenoceptor subtypes and irreversibly inactivates not only alpha 1B-adrenoceptors but also alpha 2A- and alpha 2C-adrenoceptors, whereas alpha 1A- and alpha 2B-adrenoceptors are relatively resistant to its alkylating action, although they can bind chloroethylclonidine.
我们通过饱和及竞争放射性配体结合研究，确定了氯乙可乐定对α1和α2肾上腺素能受体亚型的烷基化作用及亲和力。用氯乙可乐定（10微摩尔，37℃处理30分钟，随后洗脱）处理，几乎完全消除了[3H]哌唑嗪与大鼠脾脏α1B肾上腺素能受体的结合，并使大鼠肾脏和大脑皮层中的特异性结合减少，减少的百分比与这些组织中已知的α1B肾上腺素能受体含量相当。氯乙可乐定处理还显著降低了[3H]育亨宾与人血小板及肾脏膜的结合，但不影响[3H]育亨宾与大鼠肾脏的结合。类似的氯乙可乐定处理（10微摩尔，37℃处理20分钟）减少了用α2 - C10或α2 - C4基因转染的细胞系中可检测到的α2肾上腺素能受体数量，但在用α2 - C2肾上腺素能受体转染的细胞系中未减少。在浓度 - 反应实验中，与大鼠脾脏α1B肾上腺素能受体相比，人血小板α2A肾上腺素能受体失活需要更高的氯乙可乐定浓度，且最大失活程度较小。大鼠α1A、α2B和人α2 - C2肾上腺素能受体未失活并非由于缺乏氯乙可乐定结合，因为在竞争结合实验中确定的氯乙可乐定对这些亚型的亲和力至少与对烷基化亚型的表观亲和力一样高。氯乙可乐定处理导致的α2A肾上腺素能受体烷基化在功能上是相关的，因为它显著降低了HEL细胞中α2A肾上腺素能受体介导的Ca2 +升高。我们得出结论，氯乙可乐定与所有主要的α肾上腺素能受体亚型结合，不仅不可逆地使α1B肾上腺素能受体失活，还能使α2A和α2C肾上腺素能受体失活，而α1A和α2B肾上腺素能受体对其烷基化作用相对抗性，尽管它们可以结合氯乙可乐定。
