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首页> 外文期刊>Life sciences >Cyclic AMP increases bradykinin receptor binding affinity in human endothelial cells
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Cyclic AMP increases bradykinin receptor binding affinity in human endothelial cells

机译：环状AMP增加人内皮细胞中缓激肽受体的结合亲和力

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We demonstrated bradykinin receptors in human endothelial cells and studied whether bradykinin receptors might be regulated by cyclic AMP. Messenger RNA for bradykinin B-1 and B-2 receptors was detected with real-time PCR and B-2 receptor protein was confirmed by immunoblotting. Saturation binding experiments with increasing concentrations of I-125-[Tyr(8)]-bradykinin (25-700 pM) were made to determine maximal binding capacity and dissociation constant. However, saturation binding experiments suggested one class of binding sites, maximal binding capacity of 39.3 +/- 1.3 fmol/mg protein and dissociation constant of 352 27 pM. Competition studies with bradykinin B-1 and B-2 receptor antagonists showed that binding was competed by a B-1 antagonist, and when internalization was inhibited with hypertonic buffer,, by both B-1 and B-2 antagonists. Stimulating cells with dibutyryl-cAMP, cholera toxin and forskolin for 24 h increased I-125-[Tyr(8)]-bradykinin (90 pM) binding with approximately 50%. Saturation binding expenments with dibutyryl-cAMP stimulated cells showed, that the dissociation constant was altered from 352 +/- 27 pM in non-stimulated cells, to 203 +/- 18 pM (P < 0.001) in stimulated cells, while maximal binding capacity remained unchanged: Binding was competed similarly by the B-1 antagonist in stimulated and control cells. These results suggest, that the dibutyryl-cAMP stimulated increase in I-125-[Tyr8]-bradykinin binding is probably due to increased B, receptor affinity with no change in receptor capacity. In conclusion, bradykinin B-1 and B-2 receptor mRNA was shown in human, endothelial cells. Binding studies suggest that bradykinin receptors are competable with bradykinin antagonists. Adenylate cyclase activators probably increase bradykinin B-1 receptor affinity, without changing, capacity; and thus increase bradykinin binding. (C) 2004 Elsevier Inc. All rights reserved.
机译：我们证明了人类内皮细胞中的缓激肽受体，并研究了缓激肽受体是否可能受环状AMP的调节。通过实时PCR检测缓激肽B-1和B-2受体的信使RNA，并通过免疫印迹确认B-2受体蛋白。进行了增加浓度的I-125- [Tyr（8）]-缓激肽（25-700 pM）的饱和结合实验，以确定最大结合容量和解离常数。然而，饱和结合实验提示了一类结合位点，最大结合能力为39.3 +/- 1.3 fmol / mg蛋白，解离常数为352 27 pM。缓激肽B-1和B-2受体拮抗剂的竞争研究表明，结合由B-1拮抗剂竞争，当高渗缓冲液抑制内在化时，B-1和B-2拮抗剂均会竞争。用二丁酰-cAMP，霍乱毒素和毛喉素刺激细胞24小时可增加I-125- [Tyr（8）]-缓激肽（90 pM）的结合率，约为50％。与二丁酰-cAMP刺激的细胞的饱和结合实验表明，解离常数从未刺激的细胞中的352 +/- 27 pM更改为刺激的细胞中的203 +/- 18 pM（P <0.001），同时具有最大的结合能力保持不变：B-1拮抗剂在刺激和对照细胞中的结合竞争相似。这些结果表明，二丁酰基-cAMP刺激的I-125- [Tyr8]-缓激肽结合增加可能是由于B受体亲和力增加而受体容量没有改变。总之，在人的内皮细胞中显示缓激肽B-1和B-2受体mRNA。结合研究表明，缓激肽受体可与缓激肽拮抗剂竞争。腺苷酸环化酶激活剂可能会增加缓激肽B-1受体的亲和力，而不会改变容量。从而增加缓激肽的结合。 （C）2004 Elsevier Inc.保留所有权利。

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