A novel glutamate transporter blocker, LL-TBOA, attenuates ischaemic injury in the isolated, perfused rat heart despite low transporter levels.
A novel glutamate transporter blocker, LL-TBOA, attenuates ischaemic injury in the isolated, perfused rat heart despite low transporter levels.
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一种新型谷氨酸转运蛋白阻断剂 LL-TBOA，尽管转运蛋白水平较低，但可以减轻离体灌注大鼠心脏的缺血性损伤。
DOI：
10.1093/ejcts/ezt487
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Valen G.
中科院分区：
文献类型：
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作者：
Martinov V;Dehnes Y;Holmseth S;Shimamoto K;Danbolt NC;Valen G.
心肌缺血时谷氨酸的丢失可能加重心内直视手术中的缺血-再灌注损伤。这可能是由于兴奋性氨基酸转运蛋白（EAAT）的逆转。然而，这种转运蛋白在心肌细胞中的表达是模糊的，缺乏定量数据。我们的目的是研究是否EAAT在大鼠心脏表达，并研究是否在心脏缺血期间的转运操作的阻断可能是beneficial. METHODS我们使用TaqMan实时PCR和免疫隔离，然后通过蛋白质印迹明确识别EAAT亚型在大鼠心脏。我们使用了一种新的高亲和力的非转运竞争性抑制剂，命名为LL-TBOA [（2S，3S）-3-（3-（6-（6-（2-（2-（2-（2-（2-氨基乙氧基）乙氧基）-乙氧基）乙氧基）乙酰氨基）己酰胺基）-己酰胺基）-5-甲基-N-（2-（2-（2-（2-（2-氨基乙氧基）乙氧基）-乙氧基）乙酰氨基）-N-（2-（2-（2-（2-（2-氨基乙氧基）乙氧基）-己酰胺基）-N-（2-（2-（2-（2-（2-氨基乙氧基）-乙氧基）-乙酰氨基）己酰胺基）-N-（2-（2-（2-（2-氨基乙氧基）-己酰胺基）-N-（2-（2-（2-氨基乙氧基）-己酰胺基）-N-（2-（2-（2-氨基乙氧基）-己酰胺基）-N-（2-（2-（2-氨基乙氧基）-己酰胺基）-N-（2-（2-氨基乙氧基）-己酰胺基）-N-（2-（2-氨基乙氧基（4-（三氟甲基）苯甲酰氨基）苄氧基）天冬氨酸]，阻断EAAT介导的离体大鼠心脏缺血和再灌注过程中的转运。EAAT心脏表达EAAT亚型1和3，而亚型2和4未检测到。分离心脏并用1.6 µ M LL-TBOA灌注5 min，然后诱导全身缺血30 min和再灌注60 min（n = 8）。对照心脏用溶剂二甲基亚砜3.5 mM（n = 7）或无预处理（n = 8）灌注。通过氯化三苯基四氮唑（TTC）染色评估细胞大小。LL-TBOA使梗死面积从33 ± 14%减少到20 ± 5%（平均值± SD）（P = 0.015）。二甲亚砜单独给药无影响（35 ± 2%）。再灌注心律失常减少LL-TBOA（P = 0.009），但不是由二甲亚砜单独。CONCLUSION大鼠心脏表达EAAT 1和EAAT 3，但mRNA水平，分别为1.25和200倍低于大脑。加入LL-TBOA对缺血再灌注损伤具有有益作用。
OBJECTIVESLoss of glutamate from cardiomyocytes during ischaemia may aggravate ischaemia-reperfusion injury in open heart surgery. This may be due to reversal of excitatory amino acid transporters (EAATs). However, the expression of such transporters in cardiomyocytes is ambiguous and quantitative data are lacking. Our objective was to study whether EAATs were expressed in the rat heart and to study whether blocking of transporter operation during cardiac ischaemia could be beneficial.METHODSWe used TaqMan real-time PCR and immunoisolation followed by western blotting to unequivocally identify EAAT subtypes in rat hearts. We used a novel high-affinity non-transportable competitive inhibitor, named LL-TBOA [(2S,3S)-3-(3-(6-(6-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)-ethoxy)ethoxy) acetamido)hexanamido)- hexanamido)-5-(4-(trifluoromethyl)benzamido)benzyloxy) aspartic acid], to block EAAT-mediated transport during global ischaemia and reperfusion of isolated rat hearts.RESULTSRat hearts expressed EAAT subtypes 1 and 3, while subtypes 2 and 4 were not detected. Hearts were isolated and perfused with 1.6 µM LL-TBOA for 5 min before 30 min of induced global ischaemia and 60 min of reperfusion (n= 8). Control hearts were perfused either with the solvent dimethylsulfoxide 3.5 mM (n= 7) or with no pretreatment (n= 8). Infarct size was evaluated by triphenyl tetrazolium chloride (TTC) staining. LL-TBOA reduced infarct size from 33 ± 14 to 20 ± 5% (mean ± SD) (P= 0.015). Dimethylsulfoxide alone had no effect (35 ± 2%). Reperfusion arrhythmias were reduced by LL-TBOA (P= 0.009), but not by dimethylsulfoxide alone.CONCLUSIONRat hearts express EAAT1 and EAAT3, but the mRNA levels are, respectively, ∼25 and 200 times lower than in the brain. Addition of LL-TBOA has a beneficial effect against ischaemia-reperfusion injury.