Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, T2N 1N4, Canada.
Department of Biological Sciences, University of Alberta, Edmonton, AB, T6G 2E9, Canada.
Sci Rep. 2021 May 5;11(1):9621. doi: 10.1038/s41598-021-88957-3.
Glucocorticoids (GCs) are rapidly released in response to stress and play an important role in the physiological adjustments to re-establish homeostasis. The mode of action of GCs for stress coping is mediated largely by the steroid binding to the glucocorticoid receptor (GR), a ligand-bound transcription factor, and modulating the expression of target genes. However, GCs also exert rapid actions that are independent of transcriptional regulation by modulating second messenger signaling. However, a membrane-specific protein that transduces rapid GCs signal is yet to be characterized. Here, using freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and fura2 fluorescence microscopy, we report that stressed levels of cortisol rapidly stimulate the rise in cytosolic free calcium ([Ca]i). Pharmacological manipulations using specific extra- and intra-cellular calcium chelators, plasma membrane and endoplasmic reticulum channel blockers and receptors, indicated extracellular Ca entry is required for the cortisol-mediated rise in ([Ca]i). Particularly, the calcium release-activated calcium (CRAC) channel gating appears to be a key target for the rapid action of cortisol in the ([Ca]i) rise in trout hepatocytes. To test this further, we carried out in silico molecular docking studies using the Drosophila CRAC channel modulator 1 (ORAI1) protein, the pore forming subunit of CRAC channel that is highly conserved. The result predicts a putative binding site on CRAC for cortisol to modulate channel gating, suggesting a direct, as well as an indirect regulation (by other membrane receptors) of CRAC channel gating by cortisol. Altogether, CRAC channel may be a novel cortisol-gated Ca channel transducing rapid nongenomic signalling in hepatocytes during acute stress.
糖皮质激素（GCs）是对压力作出反应而迅速释放的，在重新建立体内平衡的生理调节中起着重要作用。GC 应对压力的作用模式主要通过类固醇与糖皮质激素受体（GR）结合来介导，GR 是一种配体结合的转录因子，调节靶基因的表达。然而，GC 也通过调节第二信使信号转导来发挥快速作用，而不依赖于转录调控。然而，一种介导快速 GC 信号的膜特异性蛋白尚未被描述。在这里，我们使用刚从虹鳟鱼（Oncorhynchus mykiss）中分离的肝细胞和 fura2 荧光显微镜，报告应激水平的皮质醇迅速刺激细胞质游离钙([Ca]i)的上升。使用特定的细胞外和细胞内钙螯合剂、质膜和内质网通道阻断剂和受体的药理学操作表明，皮质醇介导的([Ca]i)上升需要细胞外 Ca 进入。特别是，钙释放激活钙（CRAC）通道门控似乎是皮质醇在虹鳟鱼肝细胞中快速作用于([Ca]i)上升的关键靶标。为了进一步验证这一点，我们使用果蝇 CRAC 通道调节剂 1（ORAI1）蛋白（CRAC 通道的孔形成亚基，高度保守）进行了计算机分子对接研究。结果预测了 CRAC 上皮质醇调节通道门控的一个假定结合位点，表明 CRAC 通道门控受皮质醇的直接和间接调节（通过其他膜受体）。总之，CRAC 通道可能是一种新型的皮质醇门控 Ca 通道，在急性应激期间在肝细胞中快速传递非基因组信号。
