EGFR Kinase Regulates Volume-sensitive Chloride Current Elicited by Integrin Stretch via PI-3K and NADPH Oxidase in Ventricular Myocytes

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 (I(Cl,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, I(Cl,swell).