Endothelin-1 Regulates Volume-Sensitive Chloride Current in Rabbit Atrial Myocytes via Reactive Oxygen Species from Mitochondria and NADPH Oxidase

Angiotensin II (AngII) signaling and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) are implicated in the activation of volume-sensitive Cl current (ICl,swell) by both beta1-integrin stretch and osmotic swelling. Because endothelin-1 (ET-1) is a potential downstream mediator of AngII and ET-1 blockade abrogates AngII-induced ROS generation, we studied how ET-1 signaling regulates ICl,swell. Under isosmotic conditions, ET-1 (10 nM) elicited an outwardly rectifying Cl current that was fully blocked by the highly selective ICl,swell inhibitor DCPIB (10 μM) and by osmotic shrinkage. Selective ETA (BQ-123, 1 μM) but not ETB blockade (BQ-788, 100 nM) fully suppressed ET-1-induced current. ET-1-induced ICl,swell also was abolished by inhibitors of EGFR kinase (AG1478, 10 μM) and PI-3K (LY294002, 20 μM; wortmannin, 500 nM), which also suppress stretch- and swelling-induced ICl,swell. ERK inhibitors (PD 98059, 10 μM; U0216, 1 μM) partially and fully blocked ET-1- and EGF- induced currents, respectively, but did not effect ICl,swell elicited by H2O2. ET-1 acts downstream from AngII. ETA blockade (BQ-123) abolished ICl,swell elicited by both AngII and osmotic swelling, whereas AT1 blockade (losartan, 5 μM) did not effect ET-1-induced ICl,swell. Both NOX and mitochondria are important sources of ROS in cardiomyocytes. Blocking NOX with apocynin (500 μM) or mitochondrial complex I with rotenone (10 μM) both completely suppressed ET-1-induced ICl,swell. In contrast, ICl,swell elicited by antimycin A (10 μM), which stimulates superoxide production by mitochondrial complex III, was insensitive to apocynin and the NOX fusion peptide inhibitor gp91ds-tat (500 nM). These data suggest that ET-1 and ETA receptors are required intermediates in AngII-, swelling-, and stretch-induced activation of ICl,swell. Moreover, enhancement of mitochondrial ROS production by ROS from NOX is likely to contribute to activation of ICl,swell by ET-1.