Thrombin increases hyposmotic taurine efflux and accelerates ICI-swell and RVD in 3T3 fibroblasts by a src-dependent EGFR transactivation.
The present study in Swiss3T3 fibroblasts examines the effect of thrombin on hyposmolarity-induced osmolyte fluxes and RVD, and the contribution of the src/EGFR pathway. Thrombin (5 U/ml) added to a 30% hyposmotic medium markedly increased hyposmotic 3H-taurine efflux (285%), accelerated the volume-sensitive Cl- current (ICI-swell) and increased RVD rate. These effects were reduced (50-65%) by preventing the thrombin-induced intracellular Ca2+ [Ca2+]i rise with EGTA-AM, or with the phospholipase C (PLC) blocker U73122. Ca2+calmodulin (CaM) and calmodulin kinase II (CaMKII) also participate in this Ca2+-dependent pathway. Thrombin plus hyposmolarity increased src and EGFR phosphorylation, whose blockade by PP2 and AG1478, decreased by 30-50%, respectively, the thrombin effects on hyposmotic taurine efflux, ICI-swell and RVD. Ca2+- and src/EGFR-mediated pathways operate independently as shown by (1) the persistence of src and EGFR activation when [Ca2+]i rise is prevented and (2) the additive effect on taurine efflux, ICI-swell or RVD by simultaneous inhibition of the two pathways, which essentially suppressed these events. PLC-Ca2+- and src/EGFR-signaling pathways operate in the hyposmotic condition and because thrombin per se failed to increase taurine efflux and ICI-swell under isosmotic condition it seems that it is merely amplifying these previously activated mechanisms. The study shows that thrombin potentiates hyposmolarity-induced osmolyte fluxes and RVD by increasing src/EGFR-dependent signaling, in addition to the Ca2+-dependent pathway.
Vázquez-Juárez
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E
Departamento de Biofísica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, DF, Mexico.
Ramos-Mandujano
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G
Lezama
R A
RA
Cruz-Rangel
S
S
Islas
L D
LD
Pasantes-Morales
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H
D000818:Animals; D002118:Calcium / Q000378:metabolism; D048429:Cell Size; D018118:Chloride Channels / Q000502:physiology; D002712:Chlorides / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology; D006490:Hemostatics / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D009997:Osmotic Pressure / Q000187:drug effects; D018408:Patch-Clamp Techniques; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D011799:Quinazolines; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D041701:Swiss 3T3 Cells; D013654:Taurine / Q000493:pharmacokinetics*; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology; D014316:Tritium; D020032:Tyrphostins / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
C412373:AG 1879; D018118:Chloride Channels; D002712:Chlorides; D004791:Enzyme Inhibitors; D004963:Estrenes; D006490:Hemostatics; D010726:Phosphodiesterase Inhibitors; D011743:Pyrimidines; D011760:Pyrrolidinones; D011799:Quinazolines; D020032:Tyrphostins; D014316:Tritium; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C101044:RTKI cpd; D013654:Taurine; D004533:Egtazic Acid; C046770:EGTA acetoxymethyl ester; D066246:ErbB Receptors; D019061:src-Family Kinases; D013917:Thrombin; D002118:Calcium