EGFR kinase regulates volume-sensitive chloride current elicited by integrin stretch via PI-3K and NADPH oxidase in ventricular myocytes

Abstract

Stretch of β1 integrins activates an outwardly rectifying, tamoxifen-sensitive Cl- current (Cl- SAC) via AT1 receptors, NADPH oxidase, and reactive oxygen species, and Cl- SAC resembles the volume-sensitive Cl- current (ICl,swell). Epidermal growth factor receptor (EGFR) kinase undergoes transactivation upon stretch, integrin engagement, and AT1 receptor activation and, in turn, stimulates NADPH oxidase. Therefore, we tested whether Cl- SAC is regulated by EGFR kinase signaling and is volume sensitive. Paramagnetic beads coated with mAb for β1 integrin were attached to myocytes and pulled with an electromagnet. Stretch activated a Cl- SAC that was 1.13 ± 0.10 pA/pF at +40 mV. AG1478 (10 μM), an EGFR kinase blocker, inhibited 93 ± 13% of Cl- SAC, and intracellular pretreatment with 1 μM AG1478 markedly suppressed Cl- SAC activation. EGF (3.3 nM) directly activated an outwardly rectifying Cl- current (0.81 ± 0.05 pA/pF at +40 mV) that was fully blocked by 10 μM tamoxifen, an I Cl,swell blocker. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase. Wortmannin (500 nM) and LY294002 (100 μM), blockers of PI-3K, inhibited Cl- SAC by 67 ± 6% and 91 ± 25% respectively, and the EGF-induced Cl- current also was fully blocked by LY294002. Furthermore, gp91ds-tat (500 nM), a cell-permeable, chimeric peptide that specifically blocks NADPH oxidase assembly, profoundly inhibited the EGF-induced Cl- current. Inactive permeant and active impermeant control peptides had no effect. Myocyte shrinkage with hyperosmotic bathing media inhibited the Cl- SAC and EGF-induced Cl- current by 88 ± 9% and 127 ± 11%, respectively. These results suggest that β1 integrin stretch activates Cl- SAC via EGFR, PI-3K, and NADPH oxidase, and that both the Cl- SAC and the EGF-induced Cl - currents are likely to be the volume-sensitive Cl- current, ICl,swell. © The Rockefeller University Press.