Thrombin increases hyposmotic taurine efflux and accelerates $$ {\text{ICI}}^{ - }_{{{\text{swell}}}} $$ 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 ( $$ {\text{ICI}}^{ - }_{{{\text{swell}}}} $$ ) 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, $$ {\text{ICI}}^{ - }_{{{\text{swell}}}} $$ 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, $$ {\text{ICI}}^{ - }_{{{\text{swell}}}} $$ 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 $$ {\text{ICI}}^{ - }_{{{\text{swell}}}} $$ 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. [ABSTRACT FROM AUTHOR]