Extracellular ATP stimulates NO production in rat thick ascending limb
Extracellular ATP stimulates NO production in rat thick ascending limb
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DOI：
10.1161/01.hyp.0000197954.93874.ef
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发表时间：
2006-03-01
期刊：
影响因子：
8.3
通讯作者：
Garvin, JL
中科院分区：
文献类型：
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作者：
Silva, G;Beierwaltes, WH;Garvin, JL
由一氧化氮合酶(NOS)3产生的NO在粗大的升肢中扮演着调节盐分吸收的金字塔的角色。三磷酸腺苷通过一氧化氮合酶3诱导内皮细胞产生NO。我们推测细胞外的三磷酸腺苷通过P2受体激活粗大升肢中的一氧化氮合酶。为了验证这一点，我们使用NO选择性荧光染料DAF-2测量了大鼠髓质粗大上肢悬液中细胞内NO的产生。我们发现三磷酸腺苷以浓度依赖的方式增强DAF-2的荧光，在约200mU/L时达到饱和，EC_(50)为37mU/L。非选择性一氧化氮合酶抑制剂L-奥米伽-硝基-精氨酸甲酯(2 mmol/L；60+/-7对16+/-6个任意荧光单位；P<；0.02；n=5)可使DAF-2的荧光增强74%。在P2受体拮抗剂苏拉明存在下，三磷酸腺苷诱导的NO生成减少了%(101+/-11比37+/-5个任意荧光单位；P<；0.002；n=5)。用5‘-胞外核苷酸酶抑制剂ARL67156(30 mU·m o l/L)阻断三磷酸腺苷水解酶，可增强对三磷酸腺苷的反应，使EC_(50)移至0.8 mU·m o l/L。在ARL67156存在下，P_(2X)选择性激动剂β-亚甲基-5’-三磷酸的EC_(50)为4.8 mU/L，P_(2Y)选择性激动剂UTP的EC_(50)为40.4 m U/L，两者的最大反应相似。综上所述，这些数据表明，ATP主要通过激活P2X受体来刺激粗大升肢中NO的产生，并且ATP水解物可能调节NO的产生。
NO produced by NO synthase (NOS) 3 acts as an autacoid to regulate NaCl absorption in the thick ascending limb. ATP induces NO production by NOS 3 in endothelial cells. We hypothesized that extracellular ATP activates NOS in thick ascending limbs through P2 receptors. To test this, we measured intracellular NO production using the NO-selective fluorescent dye DAF-2 in suspensions of rat medullary thick ascending limbs. We found that ATP increased DAF-2 fluorescence in a concentration-dependent manner, reaching saturation at approximate to 200 mu mol/L with an EC50 of 37 mu mol/L. The increase was blunted by 74% by the nonselective NOS inhibitor L-omega-nitro-arginine-methyl-ester (2 mmol/L; 60 +/- 7 versus 16 +/- 6 arbitrary fluorescence units; P < 0.02; n = 5). In the presence of the P2 receptor antagonist suramin (300 mu mol/L), ATP-induced NO production was reduced by 64% (101 +/- 11 versus 37 +/- 5 arbitrary fluorescence units; P < 0.002; n = 5). Blocking ATP hydrolysis with a 5'-ectonucleotidase inhibitor, ARL67156 (30 mu mol/L) enhanced the response to ATP and shifted the EC50 to 0.8 mu mol/L. In the presence of ARL67156, the EC50 of the P2X-selective agonist beta,gamma-methylene-adenosine 5'-triphosphate was 4.8 mu mol/L and the EC50 for the P2Y- selective agonist UTP was 40.4 mu mol/L. The maximal responses for both agonists were similar. Taken together, these data indicate that ATP stimulates NO production in the thick ascending limb primarily through P2X receptor activation and that ATP hydrolysis may regulate NO production.