Antagonistic regulation of swelling-activated Cl- current in rabbit ventricle by Src and EGFR protein tyrosine kinases.
Regulation of swelling-activated Cl(-) current (I(Cl,swell)) is complex, and multiple signaling cascades are implicated. To determine whether protein tyrosine kinase (PTK) modulates I(Cl,swell) and to identify the PTK involved, we studied the effects of a broad-spectrum PTK inhibitor (genistein), selective inhibitors of Src (PP2, a pyrazolopyrimidine) and epidermal growth factor receptor (EGFR) kinase (PD-153035), and a protein tyrosine phosphatase (PTP) inhibitor (orthovanadate). I(Cl,swell) evoked by hyposmotic swelling was increased 181 +/- 17% by 100 microM genistein, and the genistein-induced current was blocked by the selective I(Cl,swell) blocker tamoxifen (10 microM). Block of Src with PP2 (10 microM) stimulated tamoxifen-sensitive I(Cl,swell) by 234 +/- 27%, mimicking genistein, whereas the inactive analog of PP2, PP3 (10 microM), had no effect. Moreover, block of PTP by orthovanadate (1 mM) inhibited I(Cl,swell) and prevented its stimulation by PP2. In contrast with block of Src, block of EGFR kinase with PD-153035 (20 nM) inhibited I(Cl,swell). Several lines of evidence argue that the PP2-stimulated current was I(Cl,swell): 1) the stimulation was volume dependent, 2) the current was blocked by tamoxifen, 3) the current outwardly rectified with both symmetrical and physiological Cl(-) gradients, and 4) the current reversed near the Cl(-) equilibrium potential. To rule out contributions of other currents, Cd(2+) (0.2 mM) and Ba(2+) (1 mM) were added to the bath. Surprisingly, Cd(2+) suppressed the decay of I(Cl,swell), and Cd(2+) plus Ba(2+) eliminated time-dependent currents between -100 and +100 mV. Nevertheless, these divalent ions did not eliminate I(Cl,swell) or prevent its stimulation by PP2. The results indicate that tyrosine phosphorylation controls I(Cl,swell), and regulation of I(Cl,swell) by the Src and EGFR kinase families of PTK is antagonistic.
Ren
Zuojun
Z
Dept. of Physiology, Box 980551, Medical College of Virginia, Virginia Commonwealth Univ., 1101 E. Marshall St., Richmond, VA 23298, USA.
Baumgarten
Clive M
CM
D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000502:physiology*; D019833:Genistein / Q000494:pharmacology; D006352:Heart Ventricles / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D011743:Pyrimidines / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D011817:Rabbits; D013629:Tamoxifen / Q000494:pharmacology; D016276:Ventricular Function; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C412373:AG 1879; D018118:Chloride Channels; D004791:Enzyme Inhibitors; D011743:Pyrimidines; D011799:Quinazolines; D002104:Cadmium; D013629:Tamoxifen; D019833:Genistein; D066246:ErbB Receptors; D019061:src-Family Kinases; C088860:4-((3-bromophenyl)amino)-6,7-dimethoxyquinazoline