Ebner Hannes L, Fiechtner Birgit, Pelster Bernd, Krumschnabel Gerhard
Institut für Zoologie und Limnologie, Leopold Franzens Universität Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria.
Biochim Biophys Acta. 2006 Jun;1760(6):941-50. doi: 10.1016/j.bbagen.2006.03.017. Epub 2006 Apr 19.
Activation of the extracellular signal-regulated MAP-kinase (ERK) by anisoosmotic conditions, the underlying signalling pathways, and the role of protein kinases in cell volume regulation were investigated in trout hepatocytes. While hyperosmolarity left phosphorylated ERK (pERK) levels unaffected, hypoosmolarity caused a significant increase of pERK within 2 min which peaked at around 30 min. Chelating extracellular Ca2+ to prevent the influx of Ca2+ associated with swelling reduced iso- and abolished hypoosmotic ERK activation. Similarly, inhibiting the ERK activator MEK, tyrosine kinases, or PKC inhibited the increase of pERK. In contrast, exposing cells to chelerytrine or staurosporine, PKC inhibitors of little specificity, increased pERK independently from osmotic conditions. Blocking PI3 kinase, application of 8-Br-cAMP, exposure to a P-receptor antagonist, and inhibition of p38 MAP-kinase had no effect on ERK activity. A significant reduction of regulatory volume decrease (RVD) after hypoosmotic swelling caused by MEK-inhibition and an even more pronounced reduction due to p38 inhibition indicates a role for MAP-kinases in volume regulation, but a lack of correlation between the impact of protein kinase inhibitors on pERK levels and on RVD suggests that ERK may merely modulate volume recovery. Immunocytochemical detection of pERK indicated cytoplasmic activation, but no nuclear accumulation within 30 min, supporting the notion that ERK exerts non-genomic effects. Overall, our data underscore the complexity of hypoosmotic ERK signalling and suggest a role of ERK and p38 in acute cell volume regulation.
在虹鳟肝细胞中，研究了等渗条件激活细胞外信号调节的丝裂原活化蛋白激酶（ERK）的情况、潜在的信号通路以及蛋白激酶在细胞体积调节中的作用。高渗状态下磷酸化ERK（pERK）水平未受影响，而低渗状态在2分钟内导致pERK显著增加，并在约30分钟时达到峰值。螯合细胞外Ca2+以防止与肿胀相关的Ca2+内流，可减少等渗状态下的ERK激活，并消除低渗状态下的ERK激活。同样，抑制ERK激活剂MEK、酪氨酸激酶或蛋白激酶C（PKC）可抑制pERK的增加。相比之下，将细胞暴露于白屈菜红碱或星形孢菌素（特异性较低的PKC抑制剂）中，可独立于渗透条件增加pERK。阻断磷脂酰肌醇-3激酶（PI3激酶）、应用8-溴环磷酸腺苷（8-Br-cAMP）、暴露于P受体拮抗剂以及抑制p38丝裂原活化蛋白激酶对ERK活性均无影响。MEK抑制导致低渗肿胀后调节性容积减小（RVD）显著降低，而p38抑制导致的降低更为明显，这表明丝裂原活化蛋白激酶在容积调节中发挥作用，但蛋白激酶抑制剂对pERK水平和RVD的影响之间缺乏相关性，提示ERK可能仅调节容积恢复。pERK的免疫细胞化学检测表明其在细胞质中被激活，但在30分钟内无核内积累，支持ERK发挥非基因组效应的观点。总体而言，我们的数据强调了低渗ERK信号传导的复杂性，并提示ERK和p38在急性细胞容积调节中的作用。
