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(Cl.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 (VO(4)(-3)). I(Cl.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 (EC(50) = 22.4 microM); 100 microM genistein stimulated I(Cl.vol) by 122.4 +/- 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 150 microM) and tamoxifen (20 microM), blockers of I(Cl.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 microM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited I(Cl.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(Cl.vol). In addition, the PTP inhibitor VO(4)(-3) (1 mM) reduced I(Cl.vol) by 53.5 +/- 4.5% (IC(50) = 249.6 microM). Pretreatment with VO(4)(-3) antagonized genistein-induced augmentation and A23- or A25-induced suppression of I(Cl.vol). Furthermore, the selective Src-family PTK inhibitor PP2 (5 microM) stimulated I(Cl.vol), mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 microM) reduced I(Cl.vol), mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO(4)(-3). The results suggest that I(Cl.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 I(Cl.vol), and multiple target proteins are likely to be involved.
Du
Xin-Ling
XL
Institute of Cardiovascular Science and Medicine/Department of Medicine, Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
Gao
Zhan
Z
Lau
Chu-Pak
CP
Chiu
Shui-Wah
SW
Tse
Hung-Fat
HF
Baumgarten
Clive M
CM
Li
Gui-Rong
GR
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000328:Adult; D000368:Aged; D018118:Chloride Channels / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D019833:Genistein / Q000494:pharmacology; D006325:Heart Atria / Q000166:cytology; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008875:Middle Aged; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D020032:Tyrphostins / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology; D014882:Water-Electrolyte Balance / Q000502:physiology; D019061:src-Family Kinases / Q000378:metabolism*
D018118:Chloride Channels; D002712:Chlorides; D004791:Enzyme Inhibitors; D020032:Tyrphostins; D014638:Vanadates; D019833:Genistein; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D019061:src-Family Kinases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid