title: EGFR kinase regulates volume-sensitive chloride current elicited by integrin stretch via PI-3K and NADPH oxidase in ventricular myocytes.
abstract: Stretch of beta1 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 beta1 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 muM), an EGFR kinase blocker, inhibited 93 +/- 13% of Cl(-) SAC, and intracellular pretreatment with 1 muM 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 muM tamoxifen, an I(Cl,swell) blocker. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase. Wortmannin (500 nM) and LY294002 (100 microM), 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 beta1 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).
authors.affiliation: Department of Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, 23298, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Browe
authors.forename: Clive M
authors.initials: CM
authors.lastname: Baumgarten
mesh_terms: D000818:Animals; D048429:Cell Size; D002478:Cells, Cultured; D018118:Chloride Channels / Q000502:physiology*; D002713:Chlorine / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D005260:Female; D006352:Heart Ventricles / Q000378:metabolism; D019012:Integrin beta1 / Q000378:metabolism*; D015640:Ion Channel Gating; D008297:Male; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011817:Rabbits; D013314:Stress, Mechanical; D014882:Water-Electrolyte Balance / Q000502:physiology
chemical_list: D018118:Chloride Channels; D019012:Integrin beta1; D002713:Chlorine; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors