Brueggemann Lioubov I, Markun Daniel R, Henderson Kyle K, Cribbs Leanne L, Byron Kenneth L
Department of Pharmacology and Experimental Therapeutics, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA.
J Pharmacol Exp Ther. 2006 May;317(2):488-99. doi: 10.1124/jpet.105.095067. Epub 2006 Jan 13.
Capacitative Ca(2+) 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 Ca(2+) concentration (Ca(2+)) have demonstrated that depletion of intracellular Ca(2+) 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 I(SOC) 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 Gd(3+), 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 I(SOC) 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.
血管平滑肌细胞中的容量性钙内流（CCE）有助于血管活性激素的血管收缩和促有丝分裂作用。在A7r5大鼠主动脉平滑肌细胞中，胞质游离钙浓度（[Ca²⁺]i）的测量表明，细胞内钙库的耗竭会激活CCE。然而，关于血管收缩激素[精氨酸⁸] - 血管加压素（AVP）对该机制的调节，已发表的研究存在分歧。我们采用电生理方法来表征由钙库耗竭激活的膜电流[钙库操纵电流（I(SOC)）]。由于记录条件不同，此前尚未确定I(SOC)是否对应于使用fura - 2测量的CCE；负责CCE的通道蛋白也未被鉴定。在本研究中，发现I(SOC)的药理学特性，包括其对2 - 氨基乙氧基二苯硼烷、己烯雌酚或微摩尔浓度钆（Gd³⁺）阻断的敏感性，与这些药物在相同外部离子条件下使用fura - 2测量的对毒胡萝卜素或AVP激活的CCE的影响相似。在新鲜分离的大鼠肠系膜动脉平滑肌细胞（MASMC）中也测量了毒胡萝卜素刺激的I(SOC)。通过逆转录 - 聚合酶链反应和蛋白质免疫印迹法在A7r5细胞和MASMC中检测到非选择性阳离子通道瞬时受体电位（TRP）家族的成员TRPC1、TRPC4和TRPC6。在表达小干扰RNA（siRNA）的稳定A7r5细胞系中或通过用表达TRPC1反义核苷酸序列的腺病毒感染A7r5细胞，TRPC1表达降低。在表达TRPC1 siRNA和TRPC1反义的细胞中，毒胡萝卜素刺激的I(SOC)均降低，表明TRPC1通道参与I(SOC)/CCE途径。
