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

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Abstract
We assessed whether hypoosmotic swelling of cardiac myocytes activates volume-sensitive Cl− current (ICl,swell) via the angiotensin II (AngII)-reactive oxygen species (ROS) signalling cascade. The AngII-ROS pathway previously was shown to elicit ICl,swell upon mechanical stretch of β1D integrin. Integrin stretch and osmotic swelling are, however, distinct stimuli. For example, blocking Src kinases stimulates swelling-induced but inhibits stretch-induced ICl,swell. ICl,swell was measured in rabbit ventricular myocytes by whole-cell voltage clamp. Swelling-induced ICl,swell was completely blocked by losartan and eprosartan, AngII type I receptor (AT1) antagonists. AT1 stimulation transactivates epidermal growth factor receptor (EGFR) kinase. Blockade of EGFR kinase with AG1478 abolished both ICl,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 ICl,swell. Ultimately, AngII signals via NADPH oxidase (NOX) and superoxide anion, •O2−. NOX inhibitors, diphenyleneiodonium, apocynin and gp91ds-tat, eliminated ICl,swell, whereas scramb-tat, an inactive gp91ds-tat analogue, was ineffective. •O2− rapidly dismutates to H2O2. Consistent with H2O2 being a downstream effector, catalase inhibited ICl,swell, and exogenous H2O2 overcame suppression of ICl,swell by AT1 receptor, EGFR kinase, and PI-3K blockers. H2O2-induced current was not blocked by osmotic shrinkage, however. Activation of ICl,swell by osmotic swelling is controlled by the AngII-ROS cascade, the same pathway previously implicated in ICl,swell activation by integrin stretch. This in part explains why ICl,swell is persistently activated in several models of cardiac disease.