Gao Yadong, Zou Jinjing, Geng Shuang, Zheng Junwen, Yang Jiong
Department of Respiratory Medicine, Zhongnan Hospital, Wuhan University, Wuhan, 430071, China.
J Huazhong Univ Sci Technolog Med Sci. 2012 Jun;32(3):303-310. doi: 10.1007/s11596-012-0053-3. Epub 2012 Jun 9.
Store-operated Ca(2+) channels (SOCs) are plasma membrane Ca(2+) permeable channels activated by depletion of intracellular Ca(2+) store. Ca(2+) entry through SOCs is known as store-operated Ca(2+) entry (SOCE), which plays an important role in the functional regulation of airway smooth muscle cells (ASMCs). Protein kinase C (PKC) has been shown to have an activating or inhibiting effect on SOCE, depending on cell types and PKC isoforms that are involved. In ASMCs, the effect of PKC on SOCE has not been elucidated so far. In this study, the role of PKC in the activation of SOCE in rat ASMCs was examined by using Ca(2+) fluorescence imaging technique. The results showed that acute application of PKC activators PMA and PDBu did not affect SOCE induced by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin. The non-selective PKC inhibitor chelerythrine significantly inhibited thapsigargin- and bradykinin-induced SOCE. RT-PCR assay identified PKCα, δ and ɛ isoforms in rat ASMCs. PKCα-selective inhibitor Gö6976 and PKCɛ-inhibiting peptide Epsilon-V1-2 had no effect on SOCE; by contrast, PKCδ-selective inhibitor rottlerin attenuated SOCE dramatically, suggesting that PKCδ was the major PKC isoform involved in the activation of SOCE in ASMCs. Moreover, PKC down-regulation by extended exposure to high doses of PMA or PDBu also reduced SOCE, confirming the essential role of PKC in the activation of SOCE in ASMCs. In addition, PKC down-regulation did not influence the expression of stromal interaction molecule 1 (STIM1) and Orai1, two elementary molecules in the regulation and activation of SOCs. These results identified PKCδ as an essential PKC isoform involved in the activation of SOCE, and confirmed that PKC regulates the function of ASMCs in a SOCE-dependent manner.
储存性钙通道（SOCs）是一类质膜钙通透性通道，由细胞内钙库耗竭激活。通过SOCs的钙内流被称为储存性钙内流（SOCE），其在气道平滑肌细胞（ASMCs）的功能调节中起重要作用。蛋白激酶C（PKC）已被证明对SOCE有激活或抑制作用，这取决于所涉及的细胞类型和PKC亚型。在ASMCs中，PKC对SOCE的作用迄今尚未阐明。在本研究中，通过使用钙荧光成像技术研究了PKC在大鼠ASMCs中SOCE激活中的作用。结果表明，急性应用PKC激活剂佛波酯（PMA）和十四酰佛波醇乙酯（PDBu）不影响由肌浆网钙ATP酶（SERCA）抑制剂毒胡萝卜素诱导的SOCE。非选择性PKC抑制剂白屈菜红碱显著抑制毒胡萝卜素和缓激肽诱导的SOCE。逆转录-聚合酶链反应（RT-PCR）分析鉴定出大鼠ASMCs中有PKCα、δ和ɛ亚型。PKCα选择性抑制剂Gö6976和PKCɛ抑制肽Epsilon-V1-2对SOCE无影响；相反，PKCδ选择性抑制剂rottlerin显著减弱SOCE，表明PKCδ是参与ASMCs中SOCE激活的主要PKC亚型。此外，长时间暴露于高剂量PMA或PDBu导致的PKC下调也降低了SOCE，证实了PKC在ASMCs中SOCE激活中的重要作用。此外，PKC下调不影响基质相互作用分子1（STIM1）和Orai1的表达，这两个分子是SOCs调节和激活中的基本分子。这些结果确定PKCδ是参与SOCE激活的必需PKC亚型，并证实PKC以SOCE依赖的方式调节ASMCs的功能。
