Immunohistochemical analysis of MCT1, MCT2 and MCT4 expression in rat plantaris muscle
Immunohistochemical analysis of MCT1, MCT2 and MCT4 expression in rat plantaris muscle
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DOI：
10.1113/jphysiol.2005.087411
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发表时间：
2005-08-15
影响因子：
5.5
通讯作者：
Brooks, GA
中科院分区：
文献类型：
--
作者：
Hashimoto, T;Masuda, S;Brooks, GA
我们解决了对单羧酸转运蛋白（MCT1、MCT2 和 MCT4）占据的肌细胞结构域进行组织学评估的需求。从乳酸穿梭假说的角度来看，我们假设 MCTI 在氧化纤维中高表达，而 MCT4 在高糖酵解纤维中高表达。此外，我们假设 MCTI 将在原纤维间以及肌膜下和肌膜细胞域检测到，而 MCT2 和 MCT4 丰度在肌膜处最为突出。为了检验这些假设，我们通过亲和素-生物素复合物 (ABC) 以及其他方法检查了大鼠跖肌中不同纤维类型（慢氧化，SO；快速氧化糖酵解，FOG；快速糖酵解，FG）中 MCTI、MCT2 和 MCT4 转运蛋白的细胞位置。使用跖肌是因为它是混合纤维骨骼肌。使用亲和纯化的抗体通过免疫组织化学和蛋白质印迹法评估 MCT、葡萄糖转运蛋白 (GLUT4) 蛋白和线粒体成分细胞色素氧化酶 (COX) 丰度。该染色方法特异且稳定，可对MCT表达进行半定量评估。此外，共焦激光扫描显微镜评估了 MCT 同工型定位。本研究的结果是：（1）MCTI位于肌膜和整个细胞内部的SO和FOG纤维中，其中存在线粒体网状结构； (2)相比之下，MCT4在FG和FOG纤维的肌膜结构域中高表达，而在SO纤维中低表达； (3) 共焦激光扫描显微镜表明，MCT I 和 COX 共定位于纤维间和肌膜下细胞域，而 MCT2 仅在氧化纤维的肌膜处微弱地检测到。 MCT 和相关蛋白质的定位是为了促进乳酸穿梭机的功能。
We addressed the need for histological assessment of myocellular domains occupied by monocarboxylate transporters (MCT1, MCT2 and MCT4). From the perspective of lactate shuttle hypotheses we posited that MCTI would be highly expressed in oxidative fibres, whereas MCT4 would be found in highly glycolytic fibres. Furthermore, we hypothesized that MCTI would be detected at interfibrillar as well as at subsarcolemmal and sarcolemmal cell domains, whereas MCT2 and MCT4 abundances would be most prominent at the sarcolemma. To test these hypotheses, we examined cellular locations of MCTI, MCT2 and MCT4 transporter proteins in different fibre types (slow oxidative, SO; fast oxidative glycolytic, FOG; fast glycolytic, FG) in rat plantaris muscles by the avidin-biotin complex (ABC) as well as other methods. The plantaris was used as it is a mixed fibre skeletal muscle. MCTs, glucose transporter (GLUT4) protein, and mitochondrial constituent cytochrome oxidase (COX) abundances were assessed by immunohistochemistry and Western blotting using affinity-purified antibodies. The staining method was specific and stable, which allowed for semiquantitative assessment of MCT expression. As well, confocal laser scanning microscopy assessed MCT isoform localizations. Findings of the present study were: (1) MCTI is located at the sarcolemma and throughout the cell interior in SO and FOG fibres where the mitochondrial reticulum was present; (2) in contrast, MCT4 was highly expressed in the sarcolemmal domain of FG and FOG fibres but poorly expressed in SO fibres; and (3) confocal laser-scanning microscopy demonstrated that MCT I and COX are co-localised at both interfibrillar and subsarcolemmal cell domains, whereas MCT2 is only faintly detected at the sarcolemma of oxidative fibres. MCTs and associated proteins are positioned to facilitate the function of the lactate shuttles.