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Characterization and localization of Ac-SDKP receptor binding sites using ¹²⁵I-labeled Hpp-Aca-SDKP in rat cardiac fibroblasts

¹Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, Michigan; ²Department of Pharmacology, University of Sherbrooke, Sherbrooke, Canada; ³Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan

Submitted 19 July 2006 ; accepted in final form 2 October 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, ¹²⁵I-labeled 3-(p-hydroxyphenyl)-propionic acid (or desaminotyrosine) (Hpp)-Aca-SDKP, a biologically active analog of Ac-SDKP. ¹²⁵I-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). ¹²⁵I-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 µM). 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 µg/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 ¹²⁵I-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.

collagen synthesis; heart; autoradiography; receptor

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