Thrombin increases hyposmotic taurine efflux and accelerates ICI^sub swell^^sup -^ and RVD in 3T3 fibroblasts by a src-dependent EGFR transactivation

• Published in: Pflügers Archiv 2008-02, Vol.455 (5), p.859
• Main Authors: Vázquez-juárez, E, Ramos-mandujano, G, Lezama, R A, Cruz-rangel, S, Islas, L D, Pasantes-morales, H
• Format: Article
• Language: English
• ISSN: 0031-6768; 1432-2013
• DOI: 10.1007/s00424-007-0343-y

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 ^sup 3^H-taurine efflux (285%), accelerated the volume-sensitive Cl^sup -^ current (ICI^sub swell^^sup -^) and increased RVD rate. These effects were reduced (50-65%) by preventing the thrombin-induced intracellular Ca^sup 2+^ [Ca^sup 2+^]^sub i^ rise with EGTA-AM, or with the phospholipase C (PLC) blocker U73122. Ca^sup 2+^calmodulin (CaM) and calmodulin kinase II (CaMKII) also participate in this Ca^sup 2+^-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^sub swell^^sup -^ and RVD. Ca^sup 2+^- and src/EGFR-mediated pathways operate independently as shown by (1) the persistence of src and EGFR activation when [Ca^sup 2+^]^sub i^ rise is prevented and (2) the additive effect on taurine efflux, ICI^sub swell^^sup -^ or RVD by simultaneous inhibition of the two pathways, which essentially suppressed these events. PLC-Ca^sup 2+^- and src/EGFR-signaling pathways operate in the hyposmotic condition and because thrombin per se failed to increase taurine efflux and ICI^sub swell^^sup -^ 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 Ca^sup 2+^-dependent pathway.[PUBLICATION ABSTRACT]