Abstract P117: MECHANISMS OF SIGNALING PATHWAYS TRIGGERED BY ANGIOTENSIN I CONVERTING ENZYME IN CHO ECA CELLS TREATED WITH CAPTOPRIL

Abstract

Renin-Angiotensin System (RAS) is a hormonal system associated with hydroelectrolytic homeostasis and blood pressure control. Angiotensin II (Ang II) is the main and most potent biologically active product in the system, which is produced by the action of Angiotensin Converting Enzyme (ACE) on Ang I. Dysregulation of RAS is an important factor in the development and progression of cardiovascular/renal diseases and diabetes. Experiments and clinical practice have shown that the use of an ACE inhibitor (ACEi) is an effective treatment to attenuate hypertension and diabetic renal damage. In addition, recently the use of ACEi has also been associated with the activation of signaling pathways thus suggesting that ACE can act as a signal transductional molecule. The purpose of this study is to demonstrate the proteins that are differentially regulated when CHO-ECA cells are treated with the ACE inhibitor, captopril. The CHO-ACE cells were cultured in DMEM-SILAC medium in different media: Medium with light chain lysine (LV), medium with heavy chain lysine (P), and control (CT), with DMEM-High Glucose medium. At the 75% confluency, the cells were deprived of serum and then stimulated with captopril (1μM) or vehicle for 2 and 5 minutes (min). CHO-ACE cells were also cultured for the assay analysis of the Kinase Array. ACE activity was determined spectrofluorimetrically using ZPhe-His-Leu as substrate. With the SILAC medium, it was not possible to detect the differently modulated proteins by captopril. The kinase array demonstrated that some signaling pathways were not modulated after Captopril (eg, MSK1/2 and CREB). Other kinases were positively modulated after 2 min stimulation (eg, AMPKa1, HSP27, Akt 1/2/3 T308) and 5 min stimulation (STATa/b, HSP60, b-Catenin) with Captopril. Our screening also has shown that some kinases were negatively modulated following Captopril (eg, p53, PLC-γ1, Pyk2, eNOS). ACE activity in cell lysate group was inexpressive when treated with captopril. Our results expanded the repertoire of kinases modulated by the binding of Captopril to ACE, as well as, the role of ACE as a receptor and the modulation of signaling pathways triggered by this binding, which may imply in the treatment of hypertension.