Metabolic constraints of swelling-activated glutamate release in astrocytes and their implication for ischemic tissue damage.
Metabolic constraints of swelling-activated glutamate release in astrocytes and their implication for ischemic tissue damage.
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星形胶质细胞中肿胀激活的谷氨酸释放的代谢限制及其对缺血性组织损伤的影响。
DOI：
10.1111/jnc.14711
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发表时间：
2019
影响因子：
4.7
通讯作者：
Mongin,AlexanderA
中科院分区：
文献类型：
--
作者：
Wilson,CorinneS;Bach,MartinD;Ashkavand,Zahra;Norman,KennethR;Martino,Nina;Adam,AlejandroP;Mongin,AlexanderA
容量调节阴离子通道（VRAC）是一种谷氨酸渗透性通道，可被生理和病理性细胞肿胀激活，并促进缺血性脑损伤。然而，由于VRAC开放需要胞质ATP，因此尚不清楚其活性是否以及如何在代谢受损的CNS中维持。在本研究中，我们使用培养的星形胶质细胞-在中风中表现出明显肿胀的细胞类型-来模拟代谢应激和基因表达的变化如何影响缺血和缺血后大脑中的VRAC功能。用化学抑制剂修饰原代大鼠星形胶质细胞的代谢状态，并使用荧光素酶ATP测定和海马分析仪进行检查。用放射性示踪剂D-[3 H]天冬氨酸定量肿胀激活的谷氨酸释放。通过RNAi敲低必需亚基富含亮氨酸重复序列的8A（LRRC 8A），验证了VRAC对肿胀激活的谷氨酸外排的特异性贡献;使用qRT-PCR测量VRAC组分的表达水平。使用这种方法，我们发现用糖酵解阻断剂2-脱氧-D-葡萄糖和线粒体毒物氰化钠进行完全代谢抑制可使星形胶质细胞ATP水平降低> 90%，并消除肿胀细胞的谷氨酸释放（通过VRAC）。当只有线粒体呼吸被氰化物或鱼藤酮抑制时，细胞内ATP水平和VRAC活性在很大程度上得以保留。通过提供线粒体底物丙酮酸盐和/或谷氨酰胺来抑制糖酵解导致ATP水平和VRAC活性的部分恢复。出乎意料的是，当ATP耗竭细胞暴露于极端细胞肿胀（介质渗透压降低≥ 50%）时，VRAC的代谢阻断被覆盖。24小时缺氧适应导致VRAC组分LRRC 8A的表达水平中度降低，但VRAC活性无显著变化。总体而言，我们的研究结果表明，（i）星形胶质细胞VRAC活性和代谢可以通过低水平的葡萄糖维持，（ii）ATP水平降低的抑制作用和细胞肿胀的刺激作用是控制缺血性脑中VRAC活性的两个主要因素。
Volume‐regulated anion channel (VRAC) is a glutamate‐permeable channel that is activated by physiological and pathological cell swelling and promotes ischemic brain damage. However, because VRAC opening requires cytosolic ATP, it is not clear if and how its activity is sustained in the metabolically compromised CNS. In the present study, we used cultured astrocytes – the cell type which shows prominent swelling in stroke – to model how metabolic stress and changes in gene expression may impact VRAC function in the ischemic and post‐ischemic brain. The metabolic state of primary rat astrocytes was modified with chemical inhibitors and examined using luciferin–luciferase ATP assays and a Seahorse analyzer. Swelling‐activated glutamate release was quantified with the radiotracer D‐[3H]aspartate. The specific contribution of VRAC to swelling‐activated glutamate efflux was validated by RNAi knockdown of the essential subunit, leucine‐rich repeat‐containing 8A (LRRC8A); expression levels of VRAC components were measured with qRT‐PCR. Using this methodology, we found that complete metabolic inhibition with the glycolysis blocker 2‐deoxy‐D‐glucose and the mitochondrial poison sodium cyanide reduced astrocytic ATP levels by > 90% and abolished glutamate release from swollen cells (via VRAC). When only mitochondrial respiration was inhibited by cyanide or rotenone, the intracellular ATP levels and VRAC activity were largely preserved. Bypassing glycolysis by providing the mitochondrial substrates pyruvate and/or glutamine led to partial recovery of ATP levels and VRAC activity. Unexpectedly, the metabolic block of VRAC was overridden when ATP‐depleted cells were exposed to extreme cell swelling (≥ 50% reduction in medium osmolarity). Twenty‐four hour anoxic adaptation caused a moderate reduction in the expression levels of the VRAC component LRRC8A, but no significant changes in VRAC activity. Overall, our findings suggest that (i) astrocytic VRAC activity and metabolism can be sustained by low levels of glucose and (ii) the inhibitory influence of diminishing ATP levels and the stimulatory effect of cellular swelling are the two major factors that govern VRAC activity in the ischemic brain.