Clifford E E, Parker K, Humphreys B D, Kertesy S B, Dubyak G R
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH, USA.
Blood. 1998 May 1;91(9):3172-81.
Extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) activate multiple types of P2-nucleotide receptors expressed in platelets or leukocytes. Electrophysiological and biochemical studies have indicated expression of the P2X1 receptor, an ATP-gated cation channel, in human and rat platelets, rat basophilic leukemia (RBL) cells, and phorbol myristate acetate (PMA)-differentiated HL-60 myeloid cells. Although these findings suggest that P2X1 receptors are present in both blood leukocytes and blood platelets, the relative levels of P2X1 receptor expression and function in human blood leukocytes and platelets have not been directly characterized. On the basis of both immunoblot analysis and functional assays of P2X1 receptor-mediated ionic fluxes, we report that there is significant expression of P2X1 receptors in human platelets, but not in neutrophils, monocytes, or blood lymphocytes. Thus, unlike platelets and myeloid progenitor cell lines, fully differentiated human blood leukocytes do not express functionally significant numbers of P2X1 receptors, suggesting the downregulation of P2X1 receptor gene expression during the differentiation of phagocytic leukocytes. By contrast, P2X1 receptor expression is strongly maintained during megakaryocytic differentiation and platelet release. Immunoblot analysis indicated that the platelet P2X1 receptor migrates as an approximately 60-kD protein during SDS-electrophoresis under reducing or nonreducing conditions. Treatment of platelet membranes with endoglycosidase-F causes the P2X1 receptor band to migrate as a 46-kD protein, verifying the highly glycosylated nature of the mature receptor protein. Additional studies of nucleotide-induced changes in Ca2+ influx/mobilization demonstrated that the platelet P2X1 receptors are pharmacologically distinct from the well-characterized ADP receptors of these cells. This finding suggests a unique role for these ATP-gated ion channels during hemostasis or thrombosis.
细胞外三磷酸腺苷（ATP）和二磷酸腺苷（ADP）可激活血小板或白细胞中表达的多种类型的P2 - 核苷酸受体。电生理和生化研究表明，ATP门控阳离子通道P2X1受体在人和大鼠血小板、大鼠嗜碱性白血病（RBL）细胞以及佛波酯（PMA）分化的HL - 60髓样细胞中表达。尽管这些发现表明P2X1受体存在于血液白细胞和血小板中，但人血液白细胞和血小板中P2X1受体表达和功能的相对水平尚未得到直接表征。基于P2X1受体介导的离子通量的免疫印迹分析和功能测定，我们报告人血小板中存在大量P2X1受体表达，而中性粒细胞、单核细胞或血液淋巴细胞中则没有。因此，与血小板和髓样祖细胞系不同，完全分化的人血液白细胞不表达功能上显著数量的P2X1受体，这表明吞噬性白细胞分化过程中P2X1受体基因表达下调。相比之下，P2X1受体表达在巨核细胞分化和血小板释放过程中强烈维持。免疫印迹分析表明，在还原或非还原条件下进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - 电泳）时，血小板P2X1受体迁移为约60 kDa的蛋白质。用内切糖苷酶 - F处理血小板膜会使P2X1受体条带迁移为46 kDa的蛋白质，证实了成熟受体蛋白的高度糖基化性质。对核苷酸诱导的Ca2+内流/动员变化的进一步研究表明，血小板P2X1受体在药理学上与这些细胞中已充分表征的ADP受体不同。这一发现表明这些ATP门控离子通道在止血或血栓形成过程中具有独特作用。
