Hydrogen sulfide inhibited L-type calcium channels (CaV1.2) via up-regulation of the channel sulfhydration in vascular smooth muscle cells
Hydrogen sulfide inhibited L-type calcium channels (CaV1.2) via up-regulation of the channel sulfhydration in vascular smooth muscle cells
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硫化氢通过上调血管平滑肌细胞中的通道硫化作用来抑制 L 型钙通道 (CaV1.2)。
DOI：
10.1016/j.ejphar.2019.172455
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发表时间：
2019-09-05
影响因子：
5
通讯作者：
Li, Shengnan
中科院分区：
文献类型：
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作者：
Dai, Li;Qian, Yuanyuan;Li, Shengnan
硫化氢 (H2S) 通过调节离子通道对心血管系统产生不同的影响。本研究旨在确定H2S是否影响血管平滑肌细胞（VSMC）中的L型钙（Ca2+）通道以及随后的信号通路。在这里，使用全细胞膜片钳技术，在 A7r5 细胞系中，CaV1.2 L 型 Ca2+ 电流 (I-Ca,I- (L)) 被硫氢化钠（NaHS，H2S 供体）抑制。然后，通过流式细胞术，NaHS 显着降低 Bayk8644 刺激的 CaV1.2-HEK293 细胞中的细胞内 Ca2+ 浓度 ([Ca2+](i))。然而，通过共聚焦显微镜观察，NaHS并不影响兰尼碱诱导的[Ca2+](i)升高，排除了其对细胞内Ca2+释放的影响。下面，通过埃尔曼试验测定L型Ca 2+ 通道的硫酸化。结果表明，NaHS 减少了游离硫氢基的数量，氧化剂硫氢基改性剂二酰胺 (DM) 进一步增强了游离硫氢基的数量，而还原剂硫氢基改性剂二硫苏糖醇 (DTT) 则显着抵消了这种影响。 DTT 还部分逆转 CaV1.2-HEK293 细胞中 NaHS 还原的 [Ca2+](i)。此外，NaHS 不会改变 CaV1.2 的表达。此外，NaHS 以浓度和时间依赖性方式增加 VSMC 中 PKC 和 ERK 的磷酸化。伊拉地平 (Isradipine) 是 L 型 Ca2+ 通道特异性阻断剂，可进一步增加 H2S 诱导的 PKC 和 ERK 磷酸化，显示出与 H2S 的相加效应。因此，我们的结果表明，H2S 减少了 I-Ca、I-L 和 [Ca2+](i)，从而影响下游 PKC/ERK 通路，这可能是通过调节 VSMC 中 L 型 Ca2+ 通道的硫酸化作用来实现的。
Hydrogen sulfide (H2S) exerts different effects on the cardiovascular system by modulating ion channels. The present study was to ascertain whether H2S affects L-type calcium (Ca2+) channels in vascular smooth muscle cells (VSMCs) and the subsequent signaling pathways. Here, CaV1.2 L-type Ca2+ currents (I-Ca,I- (L)) were inhibited by sodium hydrosulfide (NaHS, an H2S donor) in A7r5 cell lines using the whole-cell patch-clamp technique. Then NaHS significantly reduced intracellular Ca2+ concentration ([Ca2+](i)) in Bayk8644-stimulated CaV1.2-HEK293 cells by using flow cytometry. However, NaHS did not affect the ryanodine-induced elevation of [Ca2+](i) by means of confocal microscopy, ruling out its influence on the intracellular Ca2+ release. In the following, the sulfhydration of L-type Ca2+ channels was determined by Ellman's Test. The results showed that NaHS decreased the number of free sulfhydryls, which was further strengthened by the oxidant sulfhydryl modifier diamide (DM) and significantly counteracted by the reductant sulfhydryl modifier dithiothreitol (DTT). DTT also partly reversed the NaHS-reduced [Ca2+](i) in CaV1.2-HEK293 cells. Additionally, NaHS did not change CaV1.2 expression. Furthermore, NaHS increased phosphorylation of PKC and ERK in both a concentration- and a time-dependent manner in VSMCs. Isradipine, L-type Ca2+ channel specific blocker, further increased H2S-induced phosphorylation of PKC and ERK, showing an additive effect with H2S. Therefore, our results suggest that H2S reduced I-Ca,I- L & [Ca2+](i) and hence influenced the downstream PKC/ERK pathway, which was likely through regulating the sulfhydration of L-type Ca2+ channels in VSMCs.