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

Abstract

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, ICl,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 H2O2 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 H2O2 (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 H2O2 scavenger, attenuated the response to stretch. Application of H2O2 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.

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance

“…20,28 In addition, signaling pathways contributing to VRAC activity include phosphatidyl-inositol-3-phosphate kinase (PI3K), NADH-dependent oxidases and reactive oxygen species (ROS) as well as tyrosine kinases. [29][30][31][32][33][34][35][36] Recently it was demonstrated that knockdown of integrin b 1 reduces RVD in adherent Ehrlich Lettre ascites cells (ELA) cells 37 and as integrin b 1 stretch in Figure 1. VRAC characteristics.…”

“…9D), (iv) sensitivity to membrane potential (VSOAC inhibited by depolarization / VRAC shows outward rectification and varying degree of inactivation at positive potentials 7 ), and (v) regulation via integrin b 1 , i.e., VSOAC is unaffected by integrin b 1 knockdown 37 whereas VRAC is activated by integrin b 1 stretch. 34,38,92 Variation in stoichiometry of LRRC8 components and their regulation / localization to subdomain in the plasma membrane could explain differences between VSOAC and VRAC activities (see below).…”

Enhanced Angiotensin II-Induced Cardiac and Aortic Remodeling in ACE2 Knockout Mice

“…A study of the effects of Ang II in adult rats showed myocyte hypertrophy and midmyocardial interstitial fibrosis (Grobe et al, 2007). Ang II mediated cardiac remodeling is facilitated through myocardial AT 1 receptors and subsequent intracellular signaling (Browe & Baumgarten, 2004;de Gasparo et al, 2000).…”

Oxidative stress and oxidant signaling in obstructive sleep apnea and associated cardiovascular diseases

“…Wright et al [92] showed that prolyl hydroxylation is the oxygen-sensing mechanism in cardiac myocytes, which in turn regulates HIF-1, heme oxygenase-1, and GLUT1. While the role of NADPH oxidase in O 2 sensing in cardiac myocytes has not been reported, this enzyme has been shown to be expressed and play a role in the development of cardiac hypertrophy and in the transition to heart failure [93][94][95][96][97][98][99][100]. The concept that the heart directly senses changes in oxygen tension has recently been supported by results from our laboratory that 20 min of intermittent hypoxia can elicit significant changes in gene expression and protein modifications in mouse hearts [101].…”