Characterization and localization of Ac-SDKP receptor binding sites using I-labeled Hpp-Aca-SDKP in rat cardiac fibroblasts

Zhuo JL, Carretero OA, Peng H, Li XC, Regoli D, Neugebauer W, Rhaleb NE. Characterization and localization of Ac-SDKP receptor binding sites using I-labeled Hpp-Aca-SDKP in rat cardiac fibroblasts. Am J Physiol Heart Circ Physiol 292: H984–H993, 2007. First published October 6, 2006; doi:10.1152/ajpheart.00776.2006.—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, I-labeled 3-(p-hydroxyphenyl)-propionic acid (or desaminotyrosine) (Hpp)-Aca-SDKP, a biologically active analog of Ac-SDKP. I-labeled Hpp-Aca-SDKP bound to rat CFs and fractionated membranes with similar affinities and specificity in a concentrationand time-dependent fashion. Scatchard plot analyses revealed a single class of high-affinity Hpp-AcaSDKP binding sites (maximal binding: 1,704 198 fmol/mg protein; dissociation constant: 3.3 0.6 nM). 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 M). 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 g/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 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.