Mariela Puebla, Manuel F. Muñoz, Mauricio A. Lillo, Jorge E. Contreras, Xavier F. Figueroa
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Astrocytes were stimulated with glutamate or t-ACPD and NO-dependent changes in [Ca2+]i and ATP release were evaluated. In addition, the activity of Cx43 hemichannels, Panx-1 channels and CALHM1 channels was also analyzed. The expression of Cx43, Panx-1 and CALHM1 in astrocytes was confirmed by immunofluorescence analysis and both glutamate and t-ACPD induced NO-mediated activation of CALHM1 channels via direct S-nitrosylation, which was further confirmed by assessing CALHM1-mediated current using the two-electrode voltage clamp technique in Xenopus oocytes. Pharmacological blockade or siRNA-mediated inhibition of CALHM1 expression revealed that the opening of these channels provides a pathway for ATP release and the subsequent purinergic receptor-dependent activation of Cx43 hemichannels and Panx-1 channels, which further contributes to the astrocytic Ca2+ signaling. 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引用次数: 0
摘要
星形胶质细胞的 Ca2+ 信号在调节神经元功能方面发挥着核心作用。突触活动增加时释放的谷氨酸会激活代谢型谷氨酸受体(mGluR),从而触发星形胶质细胞中协调的 Ca2+ 信号。重要的是,星形胶质细胞表达依赖 Ca2+ 的一氧化氮(NO)合成酶 eNOS 和 nNOS,它们可能通过激活连接素 43(Cx43)半桥通道、pannexin-1(Panx-1)通道或 Ca2+ 平衡调节器 1(CALHM1)通道触发 Ca2+ 流入或 ATP 释放,从而促进 Ca2+ 信号。因此,我们旨在评估 NO 在大鼠大脑皮层星形胶质细胞原代培养物中参与由 mGluR 刺激引发的星形胶质细胞 Ca2+ 信号转导的情况。用谷氨酸或 t-ACPD 刺激星形胶质细胞,评估[Ca2+]i 和 ATP 释放的 NO 依赖性变化。此外,还分析了 Cx43 半通道、Panx-1 通道和 CALHM1 通道的活性。免疫荧光分析证实了 Cx43、Panx-1 和 CALHM1 在星形胶质细胞中的表达,谷氨酸和 t-ACPD 通过直接 S-亚硝基化诱导了 NO 介导的 CALHM1 通道活化。药理阻断或 siRNA 介导的 CALHM1 表达抑制显示,这些通道的开放为 ATP 释放以及随后嘌呤能受体依赖性激活 Cx43 半ich通道和 Panx-1 通道提供了途径,从而进一步促进了星形胶质细胞的 Ca2+ 信号转导。我们的研究结果表明,在体外星形胶质细胞中通过 NO 介导的 S-亚硝基化激活 CALHM1 通道对于谷氨酸引发的星形胶质细胞 Ca2+ 信号的产生至关重要。
Control of astrocytic Ca2+ signaling by nitric oxide-dependent S-nitrosylation of Ca2+ homeostasis modulator 1 channels
Astrocytes Ca2+ signaling play a central role in the modulation of neuronal function. Activation of metabotropic glutamate receptors (mGluR) by glutamate released during an increase in synaptic activity triggers coordinated Ca2+ signals in astrocytes. Importantly, astrocytes express the Ca2+-dependent nitric oxide (NO)-synthetizing enzymes eNOS and nNOS, which might contribute to the Ca2+ signals by triggering Ca2+ influx or ATP release through the activation of connexin 43 (Cx43) hemichannels, pannexin-1 (Panx-1) channels or Ca2+ homeostasis modulator 1 (CALHM1) channels. Hence, we aim to evaluate the participation of NO in the astrocytic Ca2+ signaling initiated by stimulation of mGluR in primary cultures of astrocytes from rat brain cortex. Astrocytes were stimulated with glutamate or t-ACPD and NO-dependent changes in [Ca2+]i and ATP release were evaluated. In addition, the activity of Cx43 hemichannels, Panx-1 channels and CALHM1 channels was also analyzed. The expression of Cx43, Panx-1 and CALHM1 in astrocytes was confirmed by immunofluorescence analysis and both glutamate and t-ACPD induced NO-mediated activation of CALHM1 channels via direct S-nitrosylation, which was further confirmed by assessing CALHM1-mediated current using the two-electrode voltage clamp technique in Xenopus oocytes. Pharmacological blockade or siRNA-mediated inhibition of CALHM1 expression revealed that the opening of these channels provides a pathway for ATP release and the subsequent purinergic receptor-dependent activation of Cx43 hemichannels and Panx-1 channels, which further contributes to the astrocytic Ca2+ signaling. Our findings demonstrate that activation of CALHM1 channels through NO-mediated S-nitrosylation in astrocytes in vitro is critical for the generation of glutamate-initiated astrocytic Ca2+ signaling.
期刊介绍:
Biological Research is an open access, peer-reviewed journal that encompasses diverse fields of experimental biology, such as biochemistry, bioinformatics, biotechnology, cell biology, cancer, chemical biology, developmental biology, evolutionary biology, genetics, genomics, immunology, marine biology, microbiology, molecular biology, neuroscience, plant biology, physiology, stem cell research, structural biology and systems biology.