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


METHODS
Cardiac fibroblasts (from normal male adult rats) were cultured to confluency in Dulbeccos modified Eagles 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 micromol/L), Ang II (100 nmol/L), Ang III (100 nmol/L) and bestatin (100 micromol/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
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-b1 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.
Lijnen
Paul
P
Hypertension Unit, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. paul.lijnen@med.kuleuven.ac.be
Petrov
Victor
V
Diaz-Araya
Guillermo
G
Fagard
Robert
R
D000626:Aminopeptidases / Q000037:antagonists & inhibitors*; D000803:Angiotensin I / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology; D000805:Angiotensin III / Q000494:pharmacology; D000809:Angiotensins / Q000494:pharmacology*; D000813:Anilides / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D003094:Collagen / Q000187:drug effects* / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D005347:Fibroblasts / Q000378:metabolism*; D005782:Gels; D006868:Hydrolysis; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D008297:Male; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009574:Nitro Compounds / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013936:Thymidine / Q000378:metabolism
D000809:Angiotensins; D000813:Anilides; D005782:Gels; D009574:Nitro Compounds; D000804:Angiotensin II; D000805:Angiotensin III; D003094:Collagen; D004247:DNA; D000803:Angiotensin I; D000626:Aminopeptidases; D007930:Leucine; C012211:ubenimex; D013936:Thymidine