Vázquez-Juárez E, Ramos-Mandujano G, Lezama R A, Cruz-Rangel S, Islas L D, Pasantes-Morales H
Departamento de Biofísica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, DF, Mexico.
Pflugers Arch. 2008 Feb;455(5):859-72. doi: 10.1007/s00424-007-0343-y. Epub 2007 Sep 27.
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.
本研究在瑞士3T3成纤维细胞中检测了凝血酶对低渗诱导的渗透溶质通量和调节性容积减小（RVD）的影响，以及src/表皮生长因子受体（EGFR）信号通路的作用。向30%低渗培养基中添加凝血酶（5 U/ml）可显著增加低渗状态下3H-牛磺酸外流（增加285%），加速容积敏感性氯离子电流（ICI-swell）并提高RVD速率。通过用乙二醇双四乙酸乙酰甲酯（EGTA-AM）或磷脂酶C（PLC）阻断剂U73122阻止凝血酶诱导的细胞内钙离子（[Ca2+]i）升高，这些效应降低了50%-65%。钙离子-钙调蛋白（CaM）和钙调蛋白激酶II（CaMKII）也参与了这一依赖钙离子的信号通路。凝血酶加低渗状态可增加src和EGFR的磷酸化，用PP2和AG1478阻断它们，可分别使凝血酶对低渗牛磺酸外流、ICI-swell和RVD的作用降低30%-50%。钙离子和src/EGFR介导的信号通路独立发挥作用，这表现为：（1）当[Ca2+]i升高被阻止时src和EGFR仍持续激活；（2）同时抑制这两条信号通路时，对牛磺酸外流、ICI-swell或RVD有累加效应，这基本抑制了这些过程。PLC-钙离子和src/EGFR信号通路在低渗状态下起作用，并且由于凝血酶本身在等渗状态下不能增加牛磺酸外流和ICI-swell，因此似乎它只是在放大这些先前已激活的机制。该研究表明，除了依赖钙离子的信号通路外，凝血酶还通过增加src/EGFR依赖的信号转导来增强低渗诱导的渗透溶质通量和RVD。
