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首页> 外文期刊>Bioscience Reports >Dinucleotide polyphosphates contribute to purinergic signalling via inhibition of adenylate kinase activity
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Dinucleotide polyphosphates contribute to purinergic signalling via inhibition of adenylate kinase activity

机译：二核苷酸多磷酸通过抑制腺苷酸激酶活性促进嘌呤能传递信号

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摘要

Dinucleoside polyphosphates are well described as direct vasoconstrictors and as mediators with strong proliferative properties, however, less is known about their effects on nucleotide-converting pathways. Therefore, the present study investigates the effects of Ap4A (diadenosine tetraphosphate), Up4A (uridine adenosine tetraphosphate) and Ap5A (diadenosine pentaphosphate) and the non-selective P2 antagonist suramin on human serum and endothelial nucleotide-converting enzymes. Human serum and HUVECs (human umbilical vein endothelial cells) were pretreated with various concentrations of dinucleotide polyphosphates and suramin. Adenylate kinase and NDP kinase activities were then quantified radiochemically by TLC analysis of the ATP-induced conversion of [3H]AMP and [3H]ADP into [3H]ADP/ATP and [3H]ATP respectively. Endothelial NTPDase (nucleoside triphosphate diphosphohydrolase) activity was additionally determined using [3H]ADP and [3H]ATP as preferred substrates. Dinucleoside polyphosphates and suramin have an inhibitory effect on the serum adenylate kinase [plC50 values (-log IC50): Ap4A, 4.67 + 0.03; Up4A, 3.70 + 0.10; Ap5A, 6.31 + 0.03; suramin, 3.74 + 0.07], as well as on endothelial adenylate kinase (plC50 values: Ap4A, 4.17 + 0.07; Up4A, 2.94 + 0.02; Ap5A, 5.97 ± 0.04; suramin, 4.23 + 0.07), but no significant effects on serum NDP kinase, emphasizing the selectivity of these inhibitors. Furthermore, Ap4A, Up4A, Ap5Aand suramin progressively inhibited the rates of [3H]ADP (plC50 values: Ap4A, 3.38 + 0.09; Up4A, 2.78±0.06; Ap5A, 4.42 + 0.11; suramin, 4.10 ±0.07) and [3H]ATP (plC50 values: Ap4A, 3.06 ±0.06; Ap5A, 3.05 ±0.12; suramin, 4.14 ±0.05) hydrolyses by cultured HUVECs. Up4A has no significant effect on the endothelial NTPDase activity. Although the half-lives for Ap4A, Up4A and Ap5A in serum are comparable with the incubation times of the assays used in the present study, secondary effects of the dinucleotide metabolites are not prominent for these inhibitory effects, since the concentration of metabolites formed are relatively insignificant compared with the 800 umol/l ATP added as a phosphate donor in the adenylate kinase and NDP kinase assays. This comparative competitive study suggests that Ap4A and Ap5A contribute to the purinergic responses via inhibition of adenylate-kinase-mediated conversion of endogenous ADP. whereas Up4A most likely mediates its vasoregulatory effects via direct binding-mediated mechanisms.
机译：二磷酸核苷多磷酸被很好地描述为直接的血管收缩剂和具有强增殖特性的介体，但是，人们对它们对核苷酸转化途径的影响知之甚少。因此，本研究研究了Ap4A（四磷酸腺苷四磷酸），Up4A（尿苷四磷酸腺苷）和Ap5A（五磷酸腺苷五磷酸）以及非选择性P2拮抗剂苏拉明对人血清和内皮核苷酸转化酶的影响。用各种浓度的二核苷酸多磷酸盐和苏拉明预处理人血清和HUVEC（人脐静脉内皮细胞）。然后通过ATP诱导的[3H] AMP和[3H] ADP分别转化为[3H] ADP / ATP和[3H] ATP的TLC分析，通过放射化学定量腺苷酸激酶和NDP激酶的活性。还使用[3H] ADP和[3H] ATP作为优选底物测定了内皮NTPDase（核苷三磷酸二磷酸水解酶）活性。双核苷多磷酸盐和苏拉明对血清腺苷酸激酶具有抑制作用[pIC50值（-log IC50）：Ap4A，4.67 + 0.03; Up4A，3.70 + 0.10; Ap5A，6.31 + 0.03;苏拉明，3.74 + 0.07]，以及内皮腺苷酸激酶（pIC50值：Ap4A，4.17 + 0.07; Up4A，2.94 + 0.02; Ap5A，5.97±0.04;苏拉明，4.23 + 0.07），但对血清NDP无明显影响激酶，强调这些抑制剂的选择性。此外，Ap4A，Up4A，Ap5A和苏拉明逐渐抑制[3H] ADP的发生率（plC50值：Ap4A，3.38 + 0.09; Up4A，2.78±0.06; Ap5A，4.42 + 0.11;苏拉明，4.10±0.07）和[3H] ATP （pIC50值：Ap4A，3.06±0.06; Ap5A，3.05±0.12;苏拉明，4.14±0.05）通过培养的HUVEC水解。 Up4A对内皮NTPDase活性无明显影响。尽管血清中Ap4A，Up4A和Ap5A的半衰期与本研究中测定的孵育时间相当，但是二核苷酸代谢物的次级作用对于这些抑制作用并不突出，因为形成的代谢物浓度相对较高与在腺苷酸激酶和NDP激酶测定中作为磷酸盐供体添加的800 umol / l ATP相比，微不足道。这项比较竞争性研究表明，Ap4A和Ap5A通过抑制腺苷酸激酶介导的内源性ADP转化而促进了嘌呤能反应。而Up4A最有可能通过直接结合介导的机制来介导其血管调节作用。

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