Receptors for bradykinin in intact cultured human fibroblasts. Identification and characterization by direct binding study.
Receptors for bradykinin in intact cultured human fibroblasts. Identification and characterization by direct binding study.
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完整培养的人成纤维细胞中缓激肽的受体。
DOI：
10.1172/jci111012
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
J. Moss
中科院分区：
文献类型：
--
作者：
A. Roscher;V. Manganiello;C. Jelsema;J. Moss
使用[2,3-脯氨酰-3,4-3H(N)]缓激肽作为放射性配体来表征培养的人成纤维细胞上的缓激肽受体。通过生物测定、高效液相色谱和离子交换色谱测定，在与完整成纤维细胞一起孵育期间，完整的[3H]缓激肽在37℃下比在4℃下损失得更快，并且很可能被降解。在 4 摄氏度（而非 37 摄氏度）时，缓激肽在 2 mM 杆菌肽存在下保持完整，但在大豆胰蛋白酶抑制剂或激肽酶 II 抑制剂 SQ-20881 存在下则不然。 4°C 下的特异性结合达到饱和，最大结合位点数量为 230 +/- 18 fmol/mg 蛋白质（平均值 +/- SE，n = 4），解离常数为 4.6 +/- 0.5 nM（平均值 +/- SE，n = 4）。线性 Scatchard 图、希尔系数接近 1 (0.95-1.06) 以及过量的缓激肽无法影响解离动力学，这与没有显着协同性的单组分结合系统一致。生理浓度的 Na+ 和 3-10 mM 的 Ca++ 或 Mg++ 使结合减少 25%。缓激肽类似物和无关肽竞争[3H]缓激肽结合的相对效力表明结合位点的特异性与B2型受体一致。这些肽取代 [3H] 缓激肽的效力与其释放前列环素的能力相关，前列环素通过其代谢物 6-酮-PGF1 α 确定。该系统是第一个对人类细胞上的缓激肽受体进行表征的系统，应该可用于研究缓激肽影响的生物过程的调节。
Bradykinin receptors on cultured human fibroblasts were characterized using [2,3-prolyl-3,4-3H(N)]bradykinin as radioligand. During incubation with intact fibroblasts, intact [3H]bradykinin was lost much more rapidly at 37 degrees than at 4 degrees C as determined by bioassay, high-performance liquid chromatography, and ion-exchange chromatography, and is likely to be degraded. At 4 degrees, but not at 37 degrees C, bradykinin remained intact in the presence of 2 mM bacitracin, but not in the presence of soybean trypsin inhibitor or SQ-20881, an inhibitor of kininase II. Specific binding at 4 degrees C was saturable with a maximum number of binding sites of 230 +/- 18 fmol/mg protein (mean +/- SE, n = 4) and a dissociation constant of 4.6 +/- 0.5 nM (mean +/- SE, n = 4). Linear Scatchard plots, Hill coefficients close to unity (0.95-1.06), and the failure of excess bradykinin to influence dissociation kinetics are consistent with a single component binding system with no significant cooperativity. Na+ at physiological concentrations and Ca++ or Mg++ at 3-10 mM reduced binding by 25%. The relative potencies of bradykinin analogues and unrelated peptides in competing for [3H]bradykinin binding indicated a specificity of the binding sites consistent with that of a B2 type receptor. Potencies of the peptides in displacing [3H]bradykinin correlated with their abilities to release prostacyclin, determined as its metabolite 6-keto-PGF1 alpha. This system, the first in which bradykinin receptors on human cells have been characterized, should prove useful for investigation of the regulation of bradykinin-influenced biological processes.
缓激肽刺激小鼠纤维肉瘤细胞中磷脂酰肌醇释放花生四烯酸。
DOI：
10.1016/s0090-6980(80)80045-1
发表时间：
1980
期刊：
Prostaglandins
影响因子：
--
作者：
Bell,RL;Baenziger,NL;Majerus,PW
通讯作者：
Majerus,PW