Abstract

The rennin-angiotensin II system (RAS) and the insulin-PI3kinase signalling pathways cross-interact with important physiological and pathophysiological consequences for cells and the whole organism. Here, the effect of 24 h pre-incubation of EA.hy926 with two different concentrations of angiotensin II, on insulin-mediated activation of the PI3kinase-AKT-eNOS signalling was investigated. Quiescent EA.hy926 cells were treated with insulin (100 nM, 30 min) following 24 h pre-treatment with or without either 0.1 or 1 µM of angiotensin II. Cell lysates were immunoblotted for phospho AKT Ser-473, phospho eNOS Ser-1177 and normalized with β-actin. Homogenates of EA.hy926 treated with insulin in the presence or absence of 1 µM angiotensin II, were also subjected to nitric oxide synthase (NOS) activity assay using titrated arginine as substrate. To exclude cytotoxicity of the 1 µM angiotensin II concentration, Trypan blue cell viability assay as well as the microscopic examination of unstained and DAPI/Phalloidin stained EA.hy926 cells were undertaken. Insulin resulted in about 2 fold increase in phospho-eNOS Ser-1177 and 8 fold increase in phospho-AKT Ser-473 levels in treated compared to untreated cells. A 2 fold increase was observed in the NOS activity of insulin-treated and untreated cells preincubated with Ang II. 24 h pre-treatment with 0.1 µM Ang II did not significantly interfere with the responses to insulin but the 1 µM Ang II pre-treated EA.hy926 showed significant attenuated insulin induced phosphorylation of eNOS Ser-1177 and AKT Ser-473, alongside impaired NOS activity. The Ang II-treated cells showed normal nuclei and cytoskeletal architecture. Angiotensin II concentration-dependently regulated basal and insulin-mediated PI3Kinase-AKT-eNOS signalling in cultured endothelial cells.

Key words: 24 h pre-incubation, angiotensin II, regulates, insulin-PI3Kinase-AKT-eNOS signalling, concentration-dependently.