Differential effects of tyrosine kinase inhibitors on volume-sensitive chloride current in human atrial myocytes: Evidence for dual regulation by Src and EGFR kinases

To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (I-CI.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). I-CI.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 muM); 100 muM genistein stimulated by 122.4 +/- 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4'-diisotliiocyanostilbene-2,2'-disulfonic acid, 150 muM) and tamoxifen (20 muM), blockers of I-CI.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 IT. In contrast to the stimulatory effects of genistein, 100 muM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited I-CI.vol by 38.2 +/- 4.9% and 40.9 +/- 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter I-CI.vol. In addition, the PTP inhibitor VO4-3 (1 mM) reduced I-CI.vol by 53.5 +/- 4.5% (IC50 = 249.6 muM). Pretreatment with VO4-3 antagonized genistein-induced augmentation and A23- or A25-induced suppression of I-CI.vol. Furthermore, the selective Src-family PTK inhibitor PP2 (5 muM) Stimulated mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 muM) reduced I-CI.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 I-CI.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 and multiple target proteins are likely to be involved.