Effect of bestatin on angiotensin I-, II- and III-induced collagen gel contraction in cardiac fibroblasts

Abstract

Objective The purpose of this investigation was to determine whether the aminopeptidase inhibitor with broad specificity, bestatin, affects angiotensin I (Ang I)-, angiotensin II (Ang II)- or angiotensin III (Ang III)-stimulated collagen gel contraction in cardiac fibroblasts. Design and methods Cardiac fibroblasts (from normal male adult rats) were cultured to confluency in Dulbecco's modified Eagle's medium (DMEM) with 10% foetal bovine serum (FBS). These fibroblasts (100,000 cells) were then further incubated in a floating collagen gel lattice with the test products Ang I (1 µmol/L), Ang II (100 nmol/L), Ang III (100 nmol/L) and bestatin (100 µmol/L) for three days in DMEM without FBS. The area of the collagen gels embedded with cardiac fibroblasts was determined by a densitometric analysis. Aminopeptidase activity was estimated by spectrophotometric determination of the liberation of p-nitroaniline from alanine- or arginine-p-nitroanilide, Results p-nitroaniline from alanine- or arginine-p-nitroanilide. Results Ang I, II and III stimulated (p <0.05) collagen gel contraction by 30.4±4.8 (SEM)%, 27.1±3.1% and 15.4±3.6% respectively. Ang I- and II-induced stimulation of collagen gel contraction was of the same order but more pronounced (p<0.05) than Ang III-stimulated collagen gel contraction. The Ang I-, II- and III-stimulated collagen contraction was reduced by bestatin. Bestatin, however, did not affect basal collagen gel contraction in cardiac fibroblasts. Bestatin dose-dependently inhibited the hydrolysis of arginine- and alanine-p-nitroanilide in cardiac fibroblasts. When a neutralising antibody to transforming growth factor TGF-β 1 was added to the collagen gel simultaneously with the angiotensins, the stimulated collagen contraction was not affected. Beta-aminoproprionitrile, an inhibitor of lysyl oxidase, completely abolished basal as well as Ang I-, II- and III-stimulated collagen contraction in cardiac fibroblasts. Results Our data suggest that aminopeptidases are involved in the Ang I-, II- and III-induced stimulation of collagen contraction in cardiac fibroblasts.