Regulation of selling-activated Cl- current by angiotensin II signalling and NADPH oxidase in rabbit ventricle

Aims We assessed whether hypoosmotic swelling of cardiac myocytes activates volume-sensitive Cl- current (I-Cl,I-swell) via the angiotensin II (AngII)-reactive oxygen species (ROS) signalling cascade. The AngII-ROS pathway previously was shown to elicit I-Cl,I-swell upon mechanical stretch of beta(1D) integrin. Integrin stretch and osmotic swelling are, however, distinct stimuli. For example, blocking Src kinases stimulates swelling-induced but inhibits stretch-induced I-Cl,I-swell. Methods and results I-Cl,I-swell was measured in rabbit ventricular myocytes by whole-cell voltage clamp. Swelling-induced I-Cl,I-swell was completely blocked by losartan and eprosartan, AngII type I receptor (AT(1)) antagonists. AT(1) stimulation transactivates epidermal growth factor receptor (EGFR) kinase. Blockade of EGFR kinase with AG1478 abolished both I-Cl,I-swell and AngII-induced Cl- current, whereas exogenous EGF evoked a Cl- current that was suppressed by osmotic shrinkage. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase, and PI-3K inhibitors LY294002 and wortmannin blocked I-Cl,I-swell. Ultimately, AngII signals via NADPH oxidase (NOX) and superoxide anion, center dot O-2(-). NOX inhibitors, diphenyleneiodonium, apocynin and gp91ds-tat, eliminated I-Cl,I-swell, whereas scramb-tat, an inactive gp91ds-tat analogue, was ineffective. center dot O-2(-) rapidly dismutates to H2O2. Consistent with H2O2 being a downstream effector, catalase inhibited I-Cl,I-swell, and exogenous H2O2 overcame suppression of I-Cl,I-swell by AT(1) receptor, EGFR kinase, and PI-3K blockers. H2O2-induced current was not blocked by osmotic shrinkage, however. Conclusion Activation of I-Cl,I-swell by osmotic swelling is controlled by the AngII-ROS cascade, the same pathway previously implicated in I-Cl,I-swell activation by integrin stretch. This in part explains why I-Cl,I-swell is persistently activated in several models of cardiac disease.