ABSTRACT
Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain, working with wild-type enzyme, we developed an approach based on the combined use of N-domain-selective and C-domain-selective ACE inhibitors and fluorogenic substrates. With this approach, marked differences in substrate selectivity were revealed between rat, mouse and human somatic ACE. In particular, the fluorogenic substrate Mca-Ala-Ser-Asp-Lys-DpaOH was shown to be a strict N-domain-selective substrate of mouse ACE, whereas with rat ACE it displayed marked C-domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic substrate of ACE, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH. In support of these results, changes in amino-acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N-domain and C-domain depends on the ACE species. These results raise concerns about the interpretation of functional studies performed in animals using N-domain and C-domain substrate selectivity data derived only from human ACE.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Oligopeptides/pharmacology , Peptidyl-Dipeptidase A/chemistry , Phosphinic Acids/pharmacology , Animals , Binding Sites , Drug Combinations , Fluorescent Dyes , Humans , Mice , Mice, Inbred C57BL , Peptidyl-Dipeptidase A/metabolism , Rats , Rats, Inbred Lew , Structure-Activity Relationship , Substrate SpecificityABSTRACT
An alteration of extracellular matrix is involved in varicose veins. We have previously shown that collagen III production, but not its mRNA expression, is decreased in cultured smooth muscle cells (SMC) from varicose veins, involving an over-production of collagen I. In this study, the mechanisms involved in this collagen III reduction are explored. Steady state levels of collagen III mRNA and its ability to translate a protein were evaluated. Neither stability nor functionality of the alpha1(III) coding mRNA were affected in cells from varicose veins. Potential intracellular degradations of collagen III were investigated with inhibitors of intracellular proteases but the production was unaffected. The level of N-terminal propeptides of collagen III in the extracellular medium was determined and was similar in SMC from control and varicose veins. The stability of collagen III was determined by time-course experiments and a degradation of the protein was observed in cells from varicose veins. The production of collagen III was partially restored in cells from varicose veins in the presence of Marimastat, a matrix metalloproteinase (MMP) inhibitor. The mRNA expression and protein production of MMP3 were increased in cells from varicose veins. Fibronectin, a potential substrate of MMP3, was decreased in SMC from varicose veins. In conclusion, collagen III, and probably fibronectin, are degraded extracellularly in SMC from varicose veins by a mechanism involving MMPs, and maybe MMP3 by a direct or an indirect pathway. The degradation of collagen III and fibronectin may have repercussions for the mechanical properties of the venous wall.
