Feoktistov I, Murray J J, Biaggioni I
Division of Clinical Pharmacology, Vanderbilt University, Nashville, Tennessee 37232.
Mol Pharmacol. 1994 Jun;45(6):1160-7.
Human erythroleukemia (HEL) cells express megakaryocyte/platelet membrane markers and thus have been used as a model for studying platelet membrane receptors and their coupling to cell signaling pathways. Our previous studies, however, indicated that platelets and HEL cells possess different subtypes of adenosine A2 receptors. Furthermore, we now report that, whereas adenosine inhibits intracellular Ca2+ increases in platelets, it potentiates the rise in intracellular Ca2+ produced by thrombin, prostaglandin E1, thapsigargin, and the calcium ionophore A23187 in HEL cells. Stable adenosine analogs potentiated intracellular Ca2+ increases with a rank order of potencies of 5'-N-ethylcarboxamidoadenosine (NECA) > (R)-(-)-N6-(2-phenylisopropyl)adenosine (R-PIA) >> CGS 21680, suggesting that this effect is mediated by A2b receptors. EC50 values for NECA and R-PIA were 0.8 and 42 microM, respectively. NECA (100 microM) potentiated by 2-3-fold the increase in intracellular Ca2+ produced by 0.3 unit/ml thrombin. This effect was mimicked by cholera toxin and was shared by other Gs-coupled receptors, such as those activated by the prostacyclin analog iloprost and prostaglandin E1, indicating the involvement of Gs proteins. Adenosine analogs also increased intracellular cAMP with the same rank order of potencies. The membrane-permeable analog 8-bromo-cAMP, however, had no effect on intracellular Ca2+ levels, indicating that the potentiation of intracellular Ca2+ increases and the activation of adenylate cyclase are parallel but independent events. The increase in intracellular Ca2+ produced by adenosine is due not to an increase in phosphoinositide hydrolysis but, rather, to an increase in calcium influx, and it is lost if cells are studied in the absence of extracellular Ca2+. We conclude, therefore, that adenosine A2b receptors in HEL cells are coupled to Gs proteins and their activation leads to stimulation of adenylate cyclase and, independently, to potentiation of the rise in intracellular Ca2+. We speculate that A2b receptors in HEL cells activate a calcium channel through a cholera toxin-sensitive mechanism that requires an initial increase in intracellular Ca2+.
人红白血病（HEL）细胞表达巨核细胞/血小板膜标志物，因此已被用作研究血小板膜受体及其与细胞信号通路偶联的模型。然而，我们之前的研究表明，血小板和HEL细胞具有不同亚型的腺苷A2受体。此外，我们现在报告，虽然腺苷抑制血小板内细胞内Ca2+的增加，但它能增强凝血酶、前列腺素E1、毒胡萝卜素和钙离子载体A23187在HEL细胞中产生的细胞内Ca2+的升高。稳定的腺苷类似物增强细胞内Ca2+升高的效力顺序为5'-N-乙基羧酰胺腺苷（NECA）>（R）-（-）-N6-（2-苯异丙基）腺苷（R-PIA）>> CGS 21680，表明这种效应是由A2b受体介导的。NECA和R-PIA的EC50值分别为0.8和42 microM。NECA（100 microM）使0.3单位/毫升凝血酶产生的细胞内Ca2+增加增强2-3倍。这种效应被霍乱毒素模拟，并且被其他Gs偶联受体共享，例如前列环素类似物伊洛前列素和前列腺素E1激活的受体，表明Gs蛋白参与其中。腺苷类似物也以相同的效力顺序增加细胞内cAMP。然而，膜通透性类似物8-溴-cAMP对细胞内Ca2+水平没有影响，表明细胞内Ca2+增加的增强和腺苷酸环化酶的激活是平行但独立的事件。腺苷产生的细胞内Ca2+增加不是由于磷酸肌醇水解增加，而是由于钙内流增加，如果在没有细胞外Ca2+的情况下研究细胞，这种增加就会消失。因此，我们得出结论，HEL细胞中的腺苷A2b受体与Gs蛋白偶联，它们的激活导致腺苷酸环化酶的刺激，并且独立地导致细胞内Ca2+升高的增强。我们推测，HEL细胞中的A2b受体通过一种霍乱毒素敏感机制激活钙通道，该机制需要细胞内Ca2+的初始增加。
