Angiotensin II (AT1) Receptors and NADPH Oxidase Regulate Cl(−) Current Elicited by β1 Integrin Stretch in Rabbit Ventricular Myocytes

Direct stretch of β1 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(Cl,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 β1-integrin were applied to myocytes and pulled upward with an electromagnet while recording whole-cell anion current. Losartan (5 μM), 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 H(2)O(2) produced from superoxide generated by sarcolemmal NADPH oxidase. Diphenyleneiodonium (DPI, 60 μM), 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 μM). Moreover, exogenous H(2)O(2) (10, 100, and 500 μM), the eventual product of NADPH oxidase activity, also activated Cl(−) SAC in the absence of stretch, whereas catalase (1,000 U/ml), an H(2)O(2) scavenger, attenuated the response to stretch. Application of H(2)O(2) during NADPH oxidase inhibition by either DPI (60 μM) or AEBSF (0.5 mM) did not fully reactivate Cl(−) SAC, however. These results suggest that stretch of β1-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.