PKC‐dependent inhibition of CA2+‐dependent exocytosis from astrocytes
PKC‐dependent inhibition of CA2+‐dependent exocytosis from astrocytes
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DOI：
10.1002/glia.21083
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发表时间：
2011-01
期刊：
影响因子：
6.2
通讯作者：
K. Yasuda;M. Itakura;K. Aoyagi;T. Sugaya;Etsuko Nagata;H. Ihara;Masami Takahashi
中科院分区：
文献类型：
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作者：
K. Yasuda;M. Itakura;K. Aoyagi;T. Sugaya;Etsuko Nagata;H. Ihara;Masami Takahashi
星形胶质细胞通过依赖钙离子和可溶性N-乙基马来酰亚胺敏感因子结合蛋白受体(SNARE)的胞吐作用释放多种生物活性物质，但胶质细胞胞吐作用的调控机制尚不清楚。在本研究中，我们利用原代培养的星形胶质细胞和克隆性大鼠胶质瘤C6细胞，研究了蛋白激酶C(PKC)对胶质细胞胞吐的影响。质谱学和蛋白质印迹分析显示，佛波酯(PMA)可诱导培养的星形胶质细胞和C6细胞Ser95、Ser120和Ser160上23 kDa突触体相关蛋白(SNAP-23)的磷酸化。蛋白激酶C抑制剂双吲哚马来酰亚胺I(BIS)可抑制这些位点的磷酸化。相反，SNAP-23的Ser110在这些细胞中被结构性磷酸化，并以PKC依赖的方式去磷酸化。外源表达的人生长激素(HGH)聚集在培养的星形胶质细胞胞浆颗粒结构中，经ATP处理后其释放依赖于钙离子和陷阱。PMA可抑制ATP诱导的星形胶质细胞释放hGH，这种抑制作用可被BIS逆转。我们还观察到在离子霉素诱导的C6细胞释放hGH中，PMA依赖的抑制和BIS对这种抑制的减弱。这些结果表明，细胞内PKC的激活抑制了星形胶质细胞依赖于钙离子和SNARE的胞吐作用。©2010 Wiley-Liss公司。
Astrocytes release various bioactive substances via Ca2+‐ and soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE)‐dependent exocytosis; however the regulatory mechanisms of glial exocytosis are still poorly understood. In the present study, we investigated the effect of protein kinase C (PKC) on exocytosis in glial cells using primary cultured astrocytes and clonal rat glioma C6 cells. Mass spectrometry and Western blot analysis using phospho‐specific antibodies revealed that phorbol 12‐myristate 13‐acetate (PMA) treatment induced the phosphorylation of synaptosomal‐associated protein of 23 kDa (SNAP‐23) on Ser95, Ser120, and Ser160 in cultured astrocytes and C6 cells. Phosphorylation at these sites was suppressed by treatment with the PKC inhibitor, bisindolylmaleimide I (BIS). In contrast, Ser110 of SNAP‐23 was constitutively phosphorylated in these cells and was dephosphorylated in a PKC‐dependent manner. Exogenously expressed human growth hormone (hGH) accumulated in cytoplasmic granular structures in cultured astrocytes, and its release after ATP‐treatment was Ca2+‐ and SNARE‐dependent. PMA treatment suppressed the ATP‐induced hGH release from astrocytes and this inhibition was reversed by BIS. We also observed PMA‐dependent suppression and an attenuation of that suppression by BIS in ionomycin‐induced hGH release from C6 cells. These results suggest that intracellular activation of PKC suppresses Ca2+‐ and SNARE‐dependent exocytosis in astroglial cells. © 2010 Wiley‐Liss, Inc.