Cholesterol modulates the volume-regulated anion current in Ehrlich-Lettre ascites cells via effects on Rho and F-actin
Cholesterol modulates the volume-regulated anion current in Ehrlich-Lettre ascites cells via effects on Rho and F-actin
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DOI：
10.1152/ajpcell.00029.2006
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发表时间：
2006-10-01
影响因子：
5.5
通讯作者：
Pedersen, Stine F.
中科院分区：
文献类型：
--
作者：
Klausen, Thomas Kjaer;Hougaard, Charlotte;Pedersen, Stine F.
控制体积调节阴离子电流（VRAC）的机制尚未完全阐明。在这里，我们研究了细胞胆固醇对VRAC的调节，以及f -肌动蛋白、Rho、Rho激酶和磷脂酰肌醇-(4,5)-二磷酸[PtdIns(4,5)P-2]在这一过程中的潜在参与。在埃利希-莱氏腹水（ELA）细胞中，低渗肿胀激活了具有VRAC生物物理和药理学特征的电流。细胞胆固醇含量增加44%对f -肌动蛋白组织或VRAC活性没有可检测到的影响。细胞胆固醇含量降低47%，肿胀细胞中皮质和应激纤维相关的f -肌动蛋白含量增加。在适度（15%）降低细胞外渗透压后，胆固醇消耗增加了VRAC激活率和最大电流，但在严重（36%）降低细胞外渗透压后没有增加。使用拉runculin B （LB）破坏f -肌动蛋白可以阻止胆固醇消耗引起的最大VRAC电流的增加，而电流激活率不受LB的影响，但依赖于Rho激酶。Rho活性在轻度肿胀细胞中下降了20%，在严重肿胀细胞中下降了50%。在适度肿胀的细胞中，这种减少被胆固醇消耗所阻止，这也增加了等渗Rho活性。凝血酶，刺激Rho并引起肌动蛋白聚合，在适度肿胀的细胞中增强VRAC。在移液管中加入水溶性PtdIns(4,5)P-2类似物或PtdIns(4,5)P-2阻断抗体，或用新霉素隔离PtdIns(4,5)P-2，均不影响VRAC活性。这表明，在ELA细胞中，f -肌动蛋白和Rho-Rho激酶分别调节VRAC的大小和激活率，胆固醇消耗至少在一定程度上通过防止低张力引起的Rho活性降低和引发肌动蛋白聚合来增强VRAC。
The mechanisms controlling the volume-regulated anion current (VRAC) are incompletely elucidated. Here, we investigate the modulation of VRAC by cellular cholesterol and the potential involvement of F-actin, Rho, Rho kinase, and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P-2] in this process. In Ehrlich-Lettre ascites (ELA) cells, a current with biophysical and pharmacological properties characteristic of VRAC was activated by hypotonic swelling. A 44% increase in cellular cholesterol content had no detectable effects on F-actin organization or VRAC activity. A 47% reduction in cellular cholesterol content increased cortical and stress fiber-associated F-actin content in swollen cells. Cholesterol depletion increased VRAC activation rate and maximal current after a modest (15%), but not after a severe (36%) reduction in extracellular osmolarity. The cholesterol depletion-induced increase in maximal VRAC current was prevented by F-actin disruption using latrunculin B (LB), while the current activation rate was unaffected by LB, but dependent on Rho kinase. Rho activity was decreased by similar to 20% in modestly, and similar to 50% in severely swollen cells. In modestly swollen cells, this reduction was prevented by cholesterol depletion, which also increased isotonic Rho activity. Thrombin, which stimulates Rho and causes actin polymerization, potentiated VRAC in modestly swollen cells. VRAC activity was unaffected by inclusion of a water-soluble PtdIns(4,5)P-2 analogue or a PtdIns(4,5)P-2-blocking antibody in the pipette, or neomycin treatment to sequester PtdIns(4,5)P-2. It is suggested that in ELA cells, F-actin and Rho-Rho kinase modulate VRAC magnitude and activation rate, respectively, and that cholesterol depletion potentiates VRAC at least in part by preventing the hypotonicity-induced decrease in Rho activity and eliciting actin polymerization.