Angiotensin II (AT1) receptors and NADPH oxidase regulate Cl- current elicited by beta1 integrin stretch in rabbit ventricular myocytes.
Direct stretch 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(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 beta1-integrin were applied to myocytes and pulled upward with an electromagnet while recording whole-cell anion current. Losartan (5 microM), 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 microM), 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 microM). Moreover, exogenous H(2)O(2) (10, 100, and 500 microM), 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 microM) 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.
Browe
David M
DM
Department of Physiology, Medical College of Virginia, Box 980551, Richmond, VA 23298-0551, USA.
Baumgarten
Clive M
CM
D000804:Angiotensin II / Q000378:metabolism* / Q000502:physiology; D000818:Animals; D018118:Chloride Channels / Q000378:metabolism / Q000502:physiology*; D004553:Electric Conductivity; D006352:Heart Ventricles / Q000166:cytology; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D019012:Integrin beta1 / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D019255:NADPH Oxidases / Q000378:metabolism* / Q000502:physiology*; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism; D011945:Receptors, Angiotensin / Q000378:metabolism* / Q000502:physiology*; D015398:Signal Transduction; D013314:Stress, Mechanical
D018118:Chloride Channels; D019012:Integrin beta1; D017382:Reactive Oxygen Species; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases