Neurokinin 1 Receptor Mediates Membrane Blebbing in HEK293 Cells through a Rho/Rho-associated Coiled-coil Kinase-dependent Mechanism
Neurokinin 1 Receptor Mediates Membrane Blebbing in HEK293 Cells through a Rho/Rho-associated Coiled-coil Kinase-dependent Mechanism
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DOI：
10.1074/jbc.m808825200
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发表时间：
2009-04-03
影响因子：
4.8
通讯作者：
Tuluc, Florin
中科院分区：
文献类型：
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作者：
Meshki, John;Douglas, Steven D.;Tuluc, Florin
我们研究了神经激肽1受体(NK1R)激动剂对转染NK1R受体的HEK293细胞的影响。NK1R受体介导剧烈的形状变化，包括膜皮质的收缩导致膜泡的形成。我们发现细胞形状的变化与在细胞单层中测量的电阻抗的变化相关。用NK1受体拮抗剂阿普匹林和L-73060预先孵育后，可阻止其形态和阻抗的改变。虽然气泡的形成通常预示着细胞的死亡，但我们发现NK1R介导的细胞气泡与细胞凋亡无关。与阻断Rho的C3转移酶的细胞通透性衍生物或与Rho相关的卷曲盘状激酶抑制剂Y27632预先孵育可完全阻止NK1R诱导的形状和阻抗变化。肌球蛋白轻链激酶抑制剂ML-9也能完全抑制起泡。此外，磷脂酶C抑制剂U73,122不干扰P物质(SP)对细胞形态和细胞阻抗的影响，但可完全阻断SP诱导的细胞内钙升高，表明泡化是一个不依赖于细胞内钙升高的过程。由于非选择性蛋白激酶C抑制剂GF109203X不干扰SP诱导的效应，因此泡沫化是一个不依赖于蛋白激酶C的过程。基于这些结果，我们首次提供了NK1R受体-配体相互作用可以导致细胞不依赖于凋亡的泡泡的证据，并且这一过程是由Rho/Rho相关的螺旋卷曲激酶通路介导的。
We have investigated the effect of neurokinin 1 receptor (NK1R) agonists on HEK293 cells transfected with the NK1R receptor. The NK1R receptor mediates dramatic shape changes that include contractions of the membrane cortex resulting in membrane bleb formation. We have found that the cell shape changes correlate with changes in electrical impedance measured in cellular monolayers. The shape and impedance changes were prevented after preincubation with NK1R antagonists aprepitant and L-73060. Although bleb formation usually heralds apoptotic cell death, we have found that NK1R-mediated cellular blebbing does not associate with apoptosis. Preincubation with a cell-permeable derivative of C3 transferase that blocks Rho or with the Rho-associated coiled-coil kinase inhibitor Y27632 completely prevented NK1R-induced shape and impedance changes. Blebbing was also completely inhibited by ML-9, a myosin light chain kinase inhibitor. Furthermore, the phospholipase C inhibitor U73,122 did not interfere with the effect of Substance P(SP) on cellular morphology and cellular impedance but completely blocked SP-induced intracellular calcium increase, indicating that the blebbing is a process independent of intracellular calcium elevations. Blebbing is a protein kinase C-independent process, since the nonselective protein kinase C inhibitor GF109203X did not interfere with SP-induced effects. Based on these results, we provide the first evidence that NK1R receptor-ligand interaction can cause apoptosis-independent cellular blebbing and that this process is mediated by the Rho/Rho-associated coiled-coil kinase pathway.