Mahaut-Smith M P, Ennion S J, Rolf M G, Evans R J
Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG.
Br J Pharmacol. 2000 Sep;131(1):108-14. doi: 10.1038/sj.bjp.0703517.
ADP, an important agonist in thrombosis and haemostasis, has been reported to activate platelets via three receptors, P2X(1), P2Y(1) and P2T(AC). Given the low potency of ADP at P2X(1) receptors and recognized contamination of commercial samples of adenosine nucleotides, we have re-examined the activation of P2X(1) receptors by ADP following HPLC and enzymatic purification. Native P2X(1) receptor currents in megakaryocytes were activated by alpha, beta-meATP (10 microM) and commercial samples of ADP (10 microM), but not by purified ADP (10 - 100 microM). Purified ADP (up to 1 mM) was also inactive at recombinant human P2X(1) receptors expressed in Xenopus oocytes. Purification did not modify the ability of ADP to activate P2Y receptors coupled to Ca(2+) mobilization in rat megakaryocytes. In human platelets, P2X(1) and P2Y receptor-mediated Ca(2+) responses were distinguished by their different kinetics at 13 degrees C. In 1 mM Ca(2+) saline, alpha,beta-meATP (10 microM) and commercial ADP (40 microM) activated a rapid Ca(2+) increase (lag time < or =0.5 s) through the activation of P2X(1) receptors. Hexokinase treatment of ADP shifted the lag time by approximately 2 s, indicating loss of the P2X(1) receptor-mediated response. A revised scheme is proposed for physiological activation of P2 receptors in human platelets. ATP stimulates P2X(1) receptors, whereas ADP is a selective agonist at metabotropic (P2Y(1) and P2T(AC)) receptors.
ADP是血栓形成和止血过程中的一种重要激动剂，据报道它可通过三种受体P2X(1)、P2Y(1)和P2T(AC)激活血小板。鉴于ADP对P2X(1)受体的作用效力较低以及腺苷核苷酸商业样品中存在公认的污染问题，我们在经过高效液相色谱（HPLC）和酶纯化后，重新研究了ADP对P2X(1)受体的激活情况。巨核细胞中的天然P2X(1)受体电流可被α,β-甲硫基ATP（10微摩尔）和ADP商业样品（10微摩尔）激活，但不能被纯化的ADP（10 - 100微摩尔）激活。纯化后的ADP（浓度高达1毫摩尔）对非洲爪蟾卵母细胞中表达的重组人P2X(1)受体也无活性。纯化过程并未改变ADP激活大鼠巨核细胞中与Ca(2+)动员偶联的P2Y受体的能力。在人血小板中，P2X(1)和P2Y受体介导的[Ca(2+)]i反应在13摄氏度时因其不同的动力学特性而得以区分。在1毫摩尔Ca(2+)盐溶液中，α,β-甲硫基ATP（10微摩尔）和商业ADP（40微摩尔）通过激活P2X(1)受体激活了快速的[Ca(2+)]i升高（延迟时间≤0.5秒）。用己糖激酶处理ADP可使延迟时间延长约2秒，表明P2X(1)受体介导的反应丧失。本文提出了一种关于人血小板中P2受体生理激活的修订方案。ATP刺激P2X(1)受体，而ADP是代谢型（P2Y(1)和P2T(AC)）受体的选择性激动剂。
