Zhuo Jia L, Carretero Oscar A, Peng Hongmei, Li Xiao C, Regoli Domenico, Neugebauer Witold, Rhaleb Nour-Eddine
Division of Hypertension and Vascular Research, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202, USA.
Am J Physiol Heart Circ Physiol. 2007 Feb;292(2):H984-93. doi: 10.1152/ajpheart.00776.2006. Epub 2006 Oct 6.
We have shown that the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibited endothelin-1 (ET-1)-induced cell proliferation and collagen synthesis in cultured rat cardiac fibroblasts (CFs) and reduced left ventricle collagen deposition in rats with aldosterone (salt)- and ANG II-induced hypertension. However, it is not known whether these effects are mediated by receptor binding sites specific for Ac-SDKP. We hypothesized that Ac-SDKP exerts antifibrotic effects by binding to specific receptor sites in cultured rat CFs, which mediate the inhibitory effects of Ac-SDKP on ET-1-stimulated collagen synthesis. Ac-SDKP binding sites in rat CFs and hearts were characterized by a specific radioligand, (125)I-labeled 3-(p-hydroxyphenyl)-propionic acid (or desaminotyrosine) (Hpp)-Aca-SDKP, a biologically active analog of Ac-SDKP. (125)I-labeled Hpp-Aca-SDKP bound to rat CFs and fractionated membranes with similar affinities and specificity in a concentration- and time-dependent fashion. Scatchard plot analyses revealed a single class of high-affinity Hpp-Aca-SDKP binding sites (maximal binding: 1,704 +/- 198 fmol/mg protein; dissociation constant: 3.3 +/- 0.6 nM). (125)I-labeled Hpp-Aca-SDKP binding in CFs was displaced by unlabeled native peptide Ac-SDKP (inhibition constant: 0.69 +/- 0.15 nM) and the analog Hpp-Aca-SDKP (inhibition constant: 10.4 +/- 0.2 nM) but not the unrelated peptide ANG II or ET-1 (10 microM). In vitro, both Ac-SDKP and Hpp-Aca-SDKP inhibited ET-1-stimulated collagen synthesis in CFs in a dose-dependent fashion, reaching a maximal effect at 1 nM (control: 7.5 +/- 0.4, ET-1: 19.9 +/- 1.2, ET-1+SDKP: 7.7 +/- 0.4, ET-1+Hpp-Aca-SDKP: 9.7 +/- 0.1 microg/mg protein; P < 0.001). Ac-SDKP also significantly attenuated ET-1-induced increases in intracellular calcium and MAPK ERK1/2 phosphorylation in CFs. In the rat heart, in vitro autoradiography revealed specific (125)I-labeled Hpp-Aca-SDKP binding throughout the myocardium, primarily interstitially. We believe that these results demonstrate for the first time that Hpp-Aca-SDKP is a functional ligand specific for Ac-SDKP receptor binding sites and that both Ac-SDKP and Hpp-Aca-SDKP exert antifibrotic effects by binding to Ac-SDKP receptors in rat CFs.
我们已经表明，四肽N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸（Ac-SDKP）可抑制内皮素-1（ET-1）诱导的培养大鼠心脏成纤维细胞（CFs）增殖和胶原蛋白合成，并减少醛固酮（盐）和血管紧张素II诱导的高血压大鼠左心室胶原蛋白沉积。然而，尚不清楚这些作用是否由Ac-SDKP特异性受体结合位点介导。我们推测，Ac-SDKP通过与培养的大鼠CFs中的特定受体位点结合发挥抗纤维化作用，该受体位点介导Ac-SDKP对ET-1刺激的胶原蛋白合成的抑制作用。大鼠CFs和心脏中的Ac-SDKP结合位点用特异性放射性配体（125）I标记的3-（对羟基苯基）-丙酸（或脱氨酪氨酸）（Hpp）-Aca-SDKP进行表征，Hpp-Aca-SDKP是Ac-SDKP的生物活性类似物。（125）I标记的Hpp-Aca-SDKP以浓度和时间依赖性方式与大鼠CFs和分级分离的膜以相似的亲和力和特异性结合。Scatchard图分析显示一类高亲和力的Hpp-Aca-SDKP结合位点（最大结合量：1,704±198 fmol/mg蛋白质；解离常数：3.3±0.6 nM）。CFs中（125）I标记的Hpp-Aca-SDKP结合被未标记的天然肽Ac-SDKP（抑制常数：0.69±0.15 nM）和类似物Hpp-Aca-SDKP（抑制常数：10.4±0.2 nM）取代，但不被无关肽血管紧张素II或ET-1（10μM）取代。在体外，Ac-SDKP和Hpp-Aca-SDKP均以剂量依赖性方式抑制CFs中ET-1刺激的胶原蛋白合成，在1 nM时达到最大效应（对照：7.5±0.4，ET-1：19.9±1.2，ET-1 + SDKP：7.7±0.4，ET-1 + Hpp-Aca-SDKP：9.7±0.1μg/mg蛋白质；P <0.001）。Ac-SDKP还显著减弱了ET-1诱导的CFs细胞内钙增加和MAPK ERK1/2磷酸化。在大鼠心脏中，体外放射自显影显示整个心肌中存在特异性（125）I标记的Hpp-Aca-SDKP结合，主要在间质中。我们认为这些结果首次证明Hpp-Aca-SDKP是Ac-SDKP受体结合位点的功能性配体，并且Ac-SDKP和Hpp-Aca-SDKP均通过与大鼠CFs中的Ac-SDKP受体结合发挥抗纤维化作用。
