Beskina Olga, Miller Anna, Mazzocco-Spezzia Amparo, Pulina Maria V, Golovina Vera A
Dept. of Physiology, Univ. of Maryland School of Medicine, 685 W. Baltimore St., HSF1, Rm. 565, Baltimore, MD 21201, USA.
Am J Physiol Cell Physiol. 2007 Sep;293(3):C1103-11. doi: 10.1152/ajpcell.00249.2007. Epub 2007 Aug 1.
Many neurodegenerative disorders are accompanied by chronic glial activation, which is characterized by the abundant production of proinflammatory cytokines, such as IL-1beta. IL-1beta disrupts Ca(2+) homeostasis and stimulates astrocyte reactivity. The mechanisms by which IL-1beta induces Ca(2+) dysregulation are not completely defined. Here, we examined how acute and chronic (24-48 h) treatment with IL-1beta affect Ca(2+) homeostasis in freshly dissociated and primary cultured mouse cortical astrocytes. Cytosolic free Ca(2+) concentration (Ca(2+)) was measured with fura-2 using digital imaging. An acute application of 10 ng/ml IL-1beta induced Ca(2+) mobilization from intracellular stores and activated store-operated Ca(2+) entry (SOCE) and receptor-operated Ca(2+) entry (ROCE) in both freshly dissociated and cultured actrocytes. Treatment of cultured astrocytes with IL-1beta for 24 and 48 h elevated resting Ca(2+), decreased Ca(2+) store content [associated with sarco(endo)plasmic reticulum Ca(2+)-ATPase 2b downregulation], and augmented ROCE. Based on evidence that receptor-operated, but not store-operated Ca(2+) channels are Ba(2+) permeable, Ba(2+) entry was used to distinguish receptor-operated Ca(2+) channels from store-operated Ca(2+) channels. ROCE was activated by the diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In the presence of extracellular Ba(2+), OAG-induced elevations of cytosolic Ba(2+) (fura-2 340-to-380-nm ratio) were significantly larger in astrocytes treated with IL-1beta. These changes in IL-1beta-treated astrocytes correlate with augmented expression of transient receptor potential cation channel (TRPC)6 protein, which likely mediates ROCE. Knockdown of the TRPC6 gene markedly reduced ROCE. The data suggest that IL-1beta-induced dysregulation of Ca(2+) homeostasis is the result of enhanced ROCE and TRPC6 expression. The disruption of Ca(2+) homeostasis appears to be an upstream component in the cascade of IL-1beta-activated pathways leading to neurodegeneration.
许多神经退行性疾病都伴有慢性胶质细胞激活，其特征是促炎细胞因子大量产生，如白细胞介素-1β（IL-1β）。IL-1β会破坏钙离子（Ca(2+)）稳态并刺激星形胶质细胞反应性。IL-1β诱导Ca(2+)失调的机制尚未完全明确。在此，我们研究了用IL-1β进行急性和慢性（24 - 48小时）处理如何影响新鲜分离和原代培养的小鼠皮质星形胶质细胞中的Ca(2+)稳态。使用fura-2通过数字成像测量胞质游离Ca(2+)浓度（Ca(2+)）。急性应用10 ng/ml IL-1β可诱导细胞内钙库释放Ca(2+)，并激活新鲜分离和培养的星形胶质细胞中的钙库操纵性Ca(2+)内流（SOCE）和受体操纵性Ca(2+)内流（ROCE）。用IL-1β处理培养的星形胶质细胞24小时和48小时会提高静息Ca(2+)，降低钙库含量（与肌浆网Ca(2+)-ATP酶2b下调有关），并增强ROCE。基于受体操纵性而非钙库操纵性Ca(2+)通道对钡离子（Ba(2+)）具有通透性这一证据，利用Ba(2+)内流来区分受体操纵性Ca(2+)通道和钙库操纵性Ca(2+)通道。ROCE由二酰基甘油类似物1-油酰基-2-乙酰基-sn-甘油（OAG）激活。在细胞外存在Ba(2+)的情况下，OAG诱导的胞质Ba(2+)升高（fura-2 340至380纳米比值）在用IL-1β处理的星形胶质细胞中显著更大。IL-1β处理的星形胶质细胞中的这些变化与瞬时受体电位阳离子通道（TRPC）6蛋白表达增加相关，TRPC6蛋白可能介导ROCE。敲低TRPC6基因可显著降低ROCE。数据表明，IL-1β诱导的Ca(2+)稳态失调是ROCE增强和TRPC6表达增加的结果。Ca(2+)稳态的破坏似乎是导致神经退行性变的IL-1β激活途径级联反应中的一个上游成分。
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