A ligand-based approach to investigate the expression and function of angiotensin converting enzyme in intact human umbilical vein endothelial cells.
Angiotensin converting enzyme (ACE) is a drug target and an effective bradykinin (BK)-inactivating ectopeptidase. We exploited a recently described [(3)H]enalaprilat binding assay to quantify the full dynamic range of ACE expression in intact human umbilical vein endothelial cells (HUVECs) stimulated with known or novel modulators of ACE expression. Further, the affinities for ACE of a set of physiological substrates were determined using the same assay. BK has the highest affinity (K(i) 525 nM) among known substrates to displace [(3)H]enalaprilat binding from ACE. Tumor necrosis factor (TNF)-alpha repressed the expression of ACE in HUVECs while phorbol 12-myristate 13-acetate (PMA) upregulated it in 24h (approximately 12-fold dynamic range by [(3)H]enalaprilat binding, corroborated by ACE immunoblotting). Intermediate levels of ACE expression were seen in cells stimulated with both PMA and a cytokine. In contrast, high glucose, insulin or EGF failed to affect ACE expression. The effect of TNF-alpha was abated by etanercept, the IKK2 inhibitor TPCA-1, or a p38 inhibitor while that of PMA was reduced by inhibitors of PKC isoforms sensitive to phorbol esters and calcium. The short-term PKC- and MEK1-dependent increase of c-Fos expression was best correlated to PMA-induced ACE upregulation. The [(3)H]enalaprilat binding assay applied to HUVECs supports that ACE is a particularly active kininase and that endothelial ACE expression is dynamically and specifically regulated. This has potential importance in inflammatory diseases and diabetes.
Koumbadinga
Gérémy Abdull
GA
Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Québec, QC G1V 4G2, Canada.
Bawolak
Marie-Thérèse
MT
Marceau
Emilie
E
Adam
Albert
A
Gera
Lajos
L
Marceau
François
F
D000806:Angiotensin-Converting Enzyme Inhibitors / Q000378:metabolism; D001667:Binding, Competitive; D001920:Bradykinin / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D015773:Enalaprilat / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008024:Ligands; D007703:Peptidyl-Dipeptidase A / Q000096:biosynthesis* / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects
D000806:Angiotensin-Converting Enzyme Inhibitors; D008024:Ligands; D014409:Tumor Necrosis Factor-alpha; C016658:phorbolol myristate acetate; D010450:Endopeptidases; D007703:Peptidyl-Dipeptidase A; C032615:bradykinin kininase; D015773:Enalaprilat; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin