Pharmacological and electrophysiological characterization of store-operated currents and capacitative Ca2+ entry in vascular smooth muscle cells
Pharmacological and electrophysiological characterization of store-operated currents and capacitative Ca2+ entry in vascular smooth muscle cells
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DOI：
10.1124/jpet.105.095067
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发表时间：
2006-05-01
影响因子：
3.5
通讯作者：
Byron, KL
中科院分区：
文献类型：
--
作者：
Brueggemann, LI;Markun, DR;Byron, KL
血管平滑肌细胞的容性钙内流（CCE）参与血管活性激素的缩血管和促有丝分裂作用。在A7 r5大鼠主动脉平滑肌细胞中，细胞溶质游离Ca 2+浓度（[Ca 2 +]（i））的测量结果表明，细胞内Ca 2+储存的耗竭激活了CCE。然而，在已发表的研究中，关于血管收缩激素[Arg（8）]-加压素（AVP）对该机制的调节存在分歧。我们采用电生理学方法来表征由存储耗尽激活的膜电流[存储操作电流（I-SOC）]。由于不同的记录条件，它还没有以前确定是否ISOC对应于CCE使用Fura-2测量;也没有负责CCE的通道蛋白被确定。在本研究中，I-SOC的药理学特征，包括其对2-氨基乙氧基二苯基硼烷，己烯雌酚，或微摩尔Gd 3+封锁的敏感性，被发现平行的影响，这些药物毒胡萝卜素或AVP激活的CCE在相同的外部离子条件下使用呋喃-2测量。还在新鲜分离的大鼠肠系膜动脉平滑肌细胞（MASMC）中测定了毒胡萝卜素刺激的ISOC。非选择性阳离子通道的瞬时受体电位（TRP）家族成员，TRPC 1，TRPC 4和TRPC 6，通过逆转录-聚合酶链反应和Western blot在A7 r5细胞和MASMC中检测。在表达小干扰RNA（siRNA）的稳定A7 r5细胞系中或通过用表达TRPC 1反义核苷酸序列的腺病毒感染A7 r5细胞来降低TRPC 1表达。在TRPC 1 siRNA和TRPC 1反义表达细胞中，毒胡萝卜素刺激的I-SOC均减少，表明TRPC 1通道有助于I-SOC/CCE途径。
Capacitative Ca2+ entry (CCE) in vascular smooth muscle cells contributes to vasoconstrictor and mitogenic effects of vasoactive hormones. In A7r5 rat aortic smooth muscle cells, measurements of cytosolic free Ca2+ concentration ([Ca2+](i)) have demonstrated that depletion of intracellular Ca2+ stores activates CCE. However, there is disagreement in published studies regarding the regulation of this mechanism by the vasoconstrictor hormone [Arg(8)]-vasopressin (AVP). We have employed electrophysiological methods to characterize the membrane currents activated by store depletion [store-operated current (I-SOC)]. Because of different recording conditions, it has not been previously determined whether ISOC corresponds to CCE measured using fura-2; nor has the channel protein responsible for CCE been identified. In the present study, the pharmacological characteristics of I-SOC, including its sensitivity to blockade by 2-aminoethoxydiphenylborane, diethylstilbestrol, or micromolar Gd3+, were found to parallel the effects of these drugs on thapsigargin- or AVP-activated CCE measured under identical external ionic conditions using fura-2. Thapsigargin-stimulated ISOC was also measured in freshly isolated rat mesenteric artery smooth muscle cells (MASMC). Members of the transient receptor potential (TRP) family of nonselective cation channels, TRPC1, TRPC4, and TRPC6, were detected by reverse transcription-polymerase chain reaction and Western blot in both A7r5 cells and MASMC. TRPC1 expression was reduced in a stable A7r5 cell line expressing a small interfering RNA (siRNA) or by infection of A7r5 cells with an adenovirus expressing a TRPC1 antisense nucleotide sequence. Thapsigargin-stimulated I-SOC was reduced in both the TRPC1 siRNA- and TRPC1 antisense-expressing cells, suggesting that the TRPC1 channel contributes to the I-SOC/CCE pathway.