Endothelium-derived nitric oxide production is increased by ATP released from red blood cells incubated with hydroxyurea
Endothelium-derived nitric oxide production is increased by ATP released from red blood cells incubated with hydroxyurea
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DOI：
10.1016/j.niox.2014.02.003
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发表时间：
2014-04-30
影响因子：
3.9
通讯作者：
Spence, Dana M.
中科院分区：
文献类型：
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作者：
Lockwood, Sarah Y.;Erkal, Jayda L.;Spence, Dana M.
红细胞(RBC)在各种刺激下释放三磷酸腺苷(ATP)，包括流动诱导的变形。羟基尿素(HU)是一种已被证实的治疗镰状细胞病(SCD)的方法，已被认为可以改善血液流动。然而，导致血流改善的确切机制尚不完全。在这里，我们报告了人红细胞与HU的孵育增加了这些细胞的ATP释放，并且这种ATP能够刺激内皮细胞产生一氧化氮(NO)。将与HU孵育的红细胞在微流控装置中通过微米级的流道泵送。用荧光素-荧光素酶测定装置上的检测孔用聚碳酸酯多孔膜与流动通道隔开，测量红细胞的三磷酸腺苷的释放。用细胞外NO探针DAF-FM检测在聚碳酸酯膜上培养的牛动脉内皮细胞(BPAEC)释放的NO。经100 mU MHU孵育的人红细胞的ATP释放量是未加HU的对照样品的2.06+/-0.37倍(p=3)。当HU孵育的红细胞在一层bPAEC下流动时，bPAEC层释放的NO是对照组的1.34+/-0.10倍。一位P2Y受体的拮抗剂证实，这种额外增加30%的NO释放是由ATP介导的。此外，当红细胞与L-NAME共同孵育时，观察到内皮细胞产生的NO显著减少。对照实验表明，RBC产生的NO通过影响RBC产生的ATP而间接影响内皮细胞产生NO。(C)2014 Elsevier Inc.保留所有权利。
Red blood cells (RBCs) release adenosine triphosphate (ATP) in response to a variety of stimuli, including flow-induced deformation. Hydroxyurea (HU), a proven therapy for individuals with sickle cell disease (SCD), is known to improve blood flow. However, the exact mechanism leading to the improved blood flow is incomplete. Here, we report that the incubation of human RBCs with HU enhances ATP release from these cells and that this ATP is capable of stimulating nitric oxide (NO) production in an endothelium. RBCs incubated with HU were pumped through micron-size flow channels in a microfluidic device. The release of ATP from the RBCs was measured using the luciferin-luciferase assay in detection wells on the device that were separated from the flow channels by a porous polycarbonate membrane. NO released from a layer of bovine artery endothelial cells (bPAECs) cultured on the polycarbonate membrane was also measured using the extracellular NO probe DAF-FM. ATP release from human RBCs incubated with 100 mu M HU was observed to be 2.06 +/- 0.37-fold larger than control samples without HU (p= 3). When HU-incubated RBCs were flowed under a layer of bPAECs, NO released from the bPAEC layer was measured to be 1.34 +/- 0.10-fold higher than controls. An antagonist of the P2Y receptor established that this extra 30% increase in NO release is ATP mediated. Furthermore, when RBCs were incubated with L-NAME, a significant decrease in endothelium-derived NO production was observed. Control experiments suggest that RBC-generated NO indirectly affects endothelial NO production via its effects on RBC-derived ATP release. (C) 2014 Elsevier Inc. All rights reserved.