Angiotensin II (AT1) receptors and NADPH oxidase regulate Cl- current elicited by β1 integrin stretch in rabbit ventricular myocytes

Directstretch of beta1 integrin activates an outwardly rectifying, tamoxifen-sensitive Cl- current (Cl- SAC) via focal adhesion kinase (FAK) and/or Src. The characteristics of Cl- SAC resemble those of the volume-sensitive Cl- current, I-CI.swell. Because myocyte stretch releases angiotensin II (AngII), which binds AT1 receptors (AT1R) and Stimulates FAK and Src in an autocrine-paracrine loop, we tested whether AT1R and their downstream signaling cascade participate in mechanotransduction. Paramagnetic beads coated with mAb for PI-integrin were applied to myocytes and pulled upward with an electromagnet while recording whole-cell anion current. Losartan (5 muM), an AT1R competitive antagonist, blocked Cl- SAC but did not significantly alter the background Cl- current in the absence of integrin stretch. AT1R signaling is mediated largely by H2O2 produced front superoxide generated by sarcolemmal NADPH oxidase. Diphenyleneiodonium (DPI, 60 muM), a potent NADPH oxidase inhibitor, rapidly and completely blocked both Cl- SAC elicited by stretch and the background Cl- current. A Structurally unrelated NADPH oxidase inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF, 0.5 and 2 mM), also rapidly and completely blocked Cl- SAC as well as a large fraction of the background Cl- current. With continuing integrin Stretch, Cl- SAC recovered upon washout of AEBSF (2 mM). In the absence of stretch, exogenous AngII (5 nM) activated an outwardly rectifying Cl- current that was rapidly and completely blocked by DPI (60 muM). Moreover, exogenous H2O2 (10, 100, and 500 muM), tire eventual product of NADPH oxidase activity, also activated Cl- SAC in the absence of Stretch, whereas catalase (1,000 U/ml), an H2O2 scavenger, attenuated the response to stretch. Application of H2O2 during NADPH oxidase inhibition by either DPI (60 muM) or AEBSF (0.5 mM) did not fully reactivate Cl- SAC, however. These results suggest that stretch of beta1-integrin in cardiac myocytes elicits Cl- SAC by, activating AT1R and NADPH oxidase and, thereby, producing reactive oxygen species. In addition, NADPH oxidase may be intimately coupled to the channel responsible for Cl- SAC, providing a second regulatory pathway.