Quantification of intact plasma AGT consisting of oxidized and reduced conformations using a modified ELISA
Autor: | Ryousuke Satou, Hiroyuki Kobori, Akemi Katsurada, Kayoko Miyata, L. Gabriel Navar |
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Jazyk: | angličtina |
Rok vydání: | 2016 |
Předmět: |
0301 basic medicine
Male medicine.medical_specialty Physiology Reducing agent medicine.drug_class Angiotensinogen Renin-Substrate Enzyme-Linked Immunosorbent Assay 030204 cardiovascular system & hematology medicine.disease_cause Plasma renin activity Renin inhibitor Dithiothreitol Rats Sprague-Dawley Renin-Angiotensin System 03 medical and health sciences chemistry.chemical_compound Mice 0302 clinical medicine Internal medicine Renin–angiotensin system parasitic diseases medicine Animals Chemistry Disulfide bond Articles Rats 030104 developmental biology Endocrinology Oxidative stress |
Popis: | The pleiotropic actions of the renin-angiotensin system (RAS) depend on the availability of angiotensinogen (AGT) which generates angiotensin I (ANG I) when cleaved by renin. Thus, quantification of the intact AGT (iAGT) concentrations is important to evaluate the actual renin substrate available. The iAGT conformation exists as oxidized AGT (oxi-AGT) and reduced AGT (red-AGT) in a disulfide bond, and oxi-AGT has a higher affinity for renin, which may exacerbate RAS-associated diseases. Accordingly, we determined iAGT, oxi-AGT, and red-AGT levels in plasma from rats and mice. Blood samples were obtained by cardiac puncture and then immediately mixed with an inhibitor solution containing a renin inhibitor. Total AGT (tAGT) levels were measured by tAGT ELISA which detects both cleaved and iAGT. iAGT levels were determined by iAGT ELISA which was found to only detect red-AGT. Thus, it was necessary to treat samples with dithiothreitol, a reducing agent, to quantify total iAGT concentration. tAGT levels in rat and mouse plasma were 1,839 ± 139 and 1,082 ± 77 ng/ml, respectively. iAGT levels were 53% of tAGT in rat plasma but only 22% in mouse plasma, probably reflecting the greater plasma renin activity in mice. The ratios of oxi-AGT and red-AGT were ∼4:1 (rat) and 16:1 (mouse). Plasma iAGT consists of oxi-AGT and red-AGT, suggesting that oxidative stress can influence ANG I generation by the AGT conformation switch. Furthermore, the lower availability of plasma iAGT in mice suggests that it may serve as a limiting factor in ANG I formation in this species. |
Databáze: | OpenAIRE |
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