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Effect of bestatin on angiotensin I-, II- and III-induced collagen gel contraction in cardiac fibroblastsHypertension and Cardiovascular Rehabilitation Unit, Department of Molecular and Cardiovascular Research, Katholieke Universiteit Leuven (KULeuven), Leuven, Belgium, paul.lijnen{at}med.kuleuven.ac.be
Hypertension and Cardiovascular Rehabilitation Unit, Department of Molecular and Cardiovascular Research, Katholieke Universiteit Leuven (KULeuven), Leuven, Belgium
Department of Chemical Pharmacology and Toxicology, Universidad de Chile, Santiago, Chile
Hypertension and Cardiovascular Rehabilitation Unit, Department of Molecular and Cardiovascular Research, Katholieke Universiteit Leuven (KULeuven), Leuven, Belgium 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.
Key Words: collagen • cardiac fibroblasts • contraction • aminopeptidase inhibition
Journal of Renin-Angiotensin-Aldosterone System, Vol. 5, No. 4,
183-188 (2004) |
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