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Evidence for Dual Regulation by Src and EGFR Kinases
2 Cardiothoracic Unit, Grantham Hospital, Faculty of Medicine, University of Hong Kong, Hong Kong SAR, China
3 Department of Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298
Address correspondence to Dr. Gui-Rong Li, Laboratory Block, Faculty of Medicine Building, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong SAR, China. Fax: (852) 2855-9730; email: grli{at}hkucc.hku.hk
To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (ICl.vol) in human atrial myocytes and to identify the PTKs involved, we studied the effects of broad-spectrum and selective PTK inhibitors and the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (VO4-3). ICl.vol evoked by hyposmotic bath solution (0.6-times isosmotic, 0.6T) was enhanced by genistein, a broad-spectrum PTK inhibitor, in a concentration-dependent manner (EC50 = 22.4 µM); 100 µM genistein stimulated ICl.vol by 122.4 ± 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 150 µM) and tamoxifen (20 µM), blockers of ICl.vol. Moreover, the current augmented by genistein was volume dependent; it was abolished by hyperosmotic shrinkage in 1.4T, and genistein did not activate Cl- current in 1T. In contrast to the stimulatory effects of genistein, 100 µM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited ICl.vol by 38.2 ± 4.9% and 40.9 ± 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter ICl.vol. In addition, the PTP inhibitor VO4-3 (1 mM) reduced ICl.vol by 53.5 ± 4.5% (IC50 = 249.6 µM). Pretreatment with VO4-3 antagonized genistein-induced augmentation and A23- or A25-induced suppression of ICl.vol. Furthermore, the selective Src-family PTK inhibitor PP2 (5 µM) stimulated ICl.vol, mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 µM) reduced ICl.vol, mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO4-3. The results suggest that ICl.vol is regulated in part by the balance between PTK and PTP activity. Regulation is complex, however. Src and EGFR kinases, distinct soluble and receptor-mediated PTK families, have opposing effects on ICl.vol, and multiple target proteins are likely to be involved.
Key Words: cell volume • Src family kinases • EGFR kinase • protein tyrosine phosphatase • orthovanadate
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