Institute of Veterinary Physiology, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Germany
Institute of Veterinary Physiology, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Germany.
Am J Physiol Regul Integr Comp Physiol. 2014 Dec 15;307(12):R1428-37. doi: 10.1152/ajpregu.00408.2013. Epub 2014 Oct 15.
In the intact rumen epithelium, isoforms 1 and 4 of the monocarboxylate transporter (MCT1 and MCT4) are thought to play key roles in mediating transcellular and intracellular permeation of short-chain fatty acids and their metabolites and in maintaining intracellular pH. We examined whether both MCT1 and MCT4 are expressed at mRNA and protein levels in ovine ruminal epithelial cells (REC) maintained in primary culture and whether they are regulated by peroxisome proliferator-activated receptor-α (PPARα). Because both transporters have been characterized to function coupled to protons, the influence of PPARα on the recovery of intracellular pH after l-lactate exposure was evaluated by spectrofluorometry. MCT1 and MCT4 were detected using immunocytochemistry both at the cell margins and intracellularly in cultured REC. To test regulation by PPARα, cells were exposed to WY 14.643, a selective ligand of PPARα, for 48 h. The subsequent qPCR analysis resulted in a dose-dependent upregulation of MCT1 and PPARα target genes, whereas response of MCT4 was not uniform. Protein expression of MCT1 and MCT4 quantified by Western blot analysis was not altered by WY 14.643 treatment. l-Lactate-dependent proton export was blocked almost completely by pHMB, a specific inhibitor of MCT1 and MCT4. However, l-lactate-dependent, pHMB-inhibited proton export in WY 14.643-treated cells was not significantly altered compared with cells not treated with WY 14.643. These data suggest that PPARα is particularly regulating MCT1 but not MCT4 expression. Extent of lactate-coupled proton export indicates that MCT1 is already working on a high level even under unstimulated conditions.
在完整的瘤胃上皮中，单羧酸转运蛋白（MCT1 和 MCT4）的同工型 1 和 4 被认为在介导短链脂肪酸及其代谢物的细胞间和细胞内渗透以及维持细胞内 pH 方面发挥关键作用。我们研究了在原代培养的绵羊瘤胃上皮细胞（REC）中，MCT1 和 MCT4 是否在 mRNA 和蛋白质水平上表达，以及它们是否受过氧化物酶体增殖物激活受体-α（PPARα）调节。由于这两种转运蛋白都被证明与质子耦联发挥作用，因此通过荧光光谱法评估了 PPARα 对 l-乳酸暴露后细胞内 pH 恢复的影响。使用免疫细胞化学在培养的 REC 的细胞边缘和细胞内检测到 MCT1 和 MCT4。为了测试 PPARα 的调节作用，将细胞暴露于 WY 14.643 48 小时，这是 PPARα 的选择性配体。随后的 qPCR 分析导致 MCT1 和 PPARα 靶基因的剂量依赖性上调，而 MCT4 的反应并不一致。通过 Western blot 分析定量的 MCT1 和 MCT4 蛋白表达未受 WY 14.643 处理的影响。MCT1 和 MCT4 的特异性抑制剂 pHMB 几乎完全阻断了 l-乳酸依赖性质子外排。然而，与未用 WY 14.643 处理的细胞相比，WY 14.643 处理的细胞中 l-乳酸依赖性、pHMB 抑制的质子外排没有明显改变。这些数据表明，PPARα 特别调节 MCT1 而不是 MCT4 的表达。乳酸偶联质子外排的程度表明，即使在未刺激的情况下，MCT1 已经在高水平上工作。
