title: Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays.
abstract: Angiotensin-converting enzyme 2 (ACE2) is a component of the renin-angiotensin system, and its expression and activity have been shown to be reduced in cardiovascular diseases. Enzymatic activity of ACE2 is commonly measured by hydrolysis of quenched fluorescent substrates in the absence or presence of an ACE2-specific inhibitor, such as the commercially available inhibitor DX600. Whereas recombinant human ACE2 is readily detected in mouse tissues using 1 μM DX600 at pH 7.5, the endogenous ACE2 activity in mouse tissues is barely detectable. We compared human, mouse, and rat ACE2 overexpressed in cell lines for their sensitivity to inhibition by DX600. ACE2 from all three species could be inhibited by DX600, but the half maximal inhibitory concentration (IC(50)) for human ACE2 was much lower (78-fold) than for rodent ACE2. Following optimization of pH, substrate concentration, and antagonist concentration, rat and mouse ACE2 expressed in a cell line could be accurately quantified with 10 μM DX600 (>95% inhibition) but not with 1 μM DX600 (<75% inhibition). Validation that the optimized method robustly quantifies ACE2 in mouse tissues (kidney, brain, heart, and plasma) was performed using wild-type and ACE2 knockout mice. This study provides a reliable method for measuring human, as well as endogenous ACE2 activity in rodents. Our data underscore the importance of validating the effect of DX600 on ACE2 from each particular species at the experimental conditions employed.
authors: Department of Pharmacology and Experimental Therapeutics and Cardiovascular Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
authors: Kim Brint
authors: KB
authors: Pedersen
authors: Srinivas
authors: S
authors: Sriramula
authors: Kavaljit H
authors: KH
authors: Chhabra
authors: Huijing
authors: H
authors: Xia
authors: Eric
authors: E
authors: Lazartigues
mesh_terms: D000085962:Angiotensin-Converting Enzyme 2; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000201:enzymology; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007668:Kidney / Q000187:drug effects / Q000201:enzymology; D007700:Kinetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009206:Myocardium / Q000201:enzymology; D010455:Peptides / Q000494:pharmacology*; D007703:Peptidyl-Dipeptidase A / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; C556151:DX600 peptide; D010455:Peptides; D007703:Peptidyl-Dipeptidase A; C000705307:ACE2 protein, human; C000705328:Ace2 protein, mouse; C000705329:Ace2 protein, rat; D000085962:Angiotensin-Converting Enzyme 2