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首页> 外文期刊>American Journal of Physiology >Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells.
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Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells.

机译：Ehrlich腹水肿瘤细胞中肌球蛋白II耗尽的细胞质中NKCC1的收缩不敏感性。

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摘要

Protein phosphorylation/dephosphorylation and cytoskeletal reorganization regulate the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) during osmotic shrinkage; however, the mechanisms involved are unclear. We show that in cytoplasts, plasma membrane vesicles detached from Ehrlich ascites tumor cells (EATC) by cytochalasin treatment, NKCC1 activity evaluated as bumetanide-sensitive (86)Rb influx was increased compared with the basal level in intact cells yet could not be further increased by osmotic shrinkage. Accordingly, cytoplasts exhibited no regulatory volume increase after shrinkage. In cytoplasts, cortical F-actin organization was disrupted, and myosin II, which in shrunken EATC translocates to the cortical region, was absent. Moreover, NKCC1 activity was essentially insensitive to the myosin light chain kinase (MLCK) inhibitor ML-7, a potent blocker of shrinkage-induced NKCC1 activity in intact EATC. Cytoplast NKCC1 activity was potentiated by the Ser/Thr protein phosphatase inhibitor calyculin A, partially inhibited by the protein kinase A inhibitor H89, and blocked by the broad protein kinase inhibitor staurosporine. Cytoplasts exhibited increased protein levels of NKCC1, Ste20-related proline- and alanine-rich kinase (SPAK), and oxidative stress response kinase 1, yet they lacked the shrinkage-induced plasma membrane translocation of SPAK observed in intact cells. The basal phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in cytoplasts compared with intact cells, yet in contrast to the substantial activation in shrunken intact cells, p38 MAPK could not be further activated by shrinkage of the cytoplasts. Together these findings indicate that shrinkage activation of NKCC1 in EATC is dependent on the cortical F-actin network, myosin II, and MLCK.
机译：蛋白质磷酸化/去磷酸化和细胞骨架重组调节渗透收缩过程中的Na（+）-K（+）-2Cl（-）协同转运蛋白（NKCC1）;但是，涉及的机制尚不清楚。我们显示，在细胞质中，通过细胞松弛素处理从埃氏腹水肿瘤细胞（EATC）分离的质膜囊泡，与布美他尼敏感型（86）Rb涌入相比，NKCC1活性被评估为比完整细胞的基础水平更高，但无法进一步提高通过渗透收缩。因此，细胞质在收缩后没有显示出调节体积的增加。在细胞质中，皮质F-肌动蛋白的组织被破坏，而在收缩的EATC中易位至皮质区域的肌球蛋白II缺失。此外，NKCC1活性对肌球蛋白轻链激酶（MLCK）抑制剂ML-7基本上不敏感，后者是完整EATC中收缩诱导的NKCC1活性的有效阻断剂。胞质NKCC1活性被Ser / Thr蛋白磷酸酶抑制剂calyculin A增强，部分被蛋白激酶A抑制剂H89抑制，并被宽蛋白激酶抑制剂staurosporine阻断。细胞质显示出增加的NKCC1，Ste20相关的脯氨酸和丙氨酸富集激酶（SPAK）以及氧化应激反应激酶1的蛋白质水平，但它们缺乏在完整细胞中观察到的SPAK引起的收缩诱导的质膜易位。与完整细胞相比，p38丝裂原活化蛋白激酶（p38 MAPK）的基础磷酸化增加了，但是与收缩的完整细胞中的大量激活相反，p38 MAPK不能通过细胞质的收缩进一步激活。这些发现共同表明EATC中NKCC1的收缩活化取决于皮层F-肌动蛋白网络，肌球蛋白II和MLCK。

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