title: Characterization and localization of Ac-SDKP receptor binding sites using 125I-labeled Hpp-Aca-SDKP in rat cardiac fibroblasts.
abstract: 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.
authors.affiliation: Division of Hypertension and Vascular Research, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202, USA. jzhuo1@hfhs.org
authors.forename: Jia L
authors.initials: JL
authors.lastname: Zhuo
authors.forename: Oscar A
authors.initials: OA
authors.lastname: Carretero
authors.forename: Hongmei
authors.initials: H
authors.lastname: Peng
authors.forename: Xiao C
authors.initials: XC
authors.lastname: Li
authors.forename: Domenico
authors.initials: D
authors.lastname: Regoli
authors.forename: Witold
authors.initials: W
authors.lastname: Neugebauer
authors.forename: Nour-Eddine
authors.initials: NE
authors.lastname: Rhaleb
mesh_terms: D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D001667:Binding, Competitive; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D007457:Iodine Radioisotopes / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008954:Models, Biological; D015394:Molecular Structure; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D017711:Nonlinear Dynamics; D009842:Oligopeptides / Q000378:metabolism* / Q000494:pharmacology; D011869:Radioligand Assay; D019275:Radiopharmaceuticals / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013997:Time Factors
chemical_list: D019332:Endothelin-1; C518122:Hpp-Aca-SDKP; D007457:Iodine Radioisotopes; D009842:Oligopeptides; D019275:Radiopharmaceuticals; D011956:Receptors, Cell Surface; D003094:Collagen; C058504:goralatide