Department of Biology, Colorado State University-Pueblo, Pueblo, Colorado, United States of America.
PLoS One. 2014 Jan 16;9(1):e85957. doi: 10.1371/journal.pone.0085957. eCollection 2014.
In this study, a detailed characterization of Monocarboxylic Acid Transporter-1 (Mct1) in cytoplasmic vesicles of cultured rat brain microvascular endothelial cells shows them to be a diverse population of endosomes intrinsic to the regulation of the transporter by a brief 25 to 30 minute exposure to the membrane permeant cAMP analog, 8Br-cAMP. The vesicles are heterogeneous in size, mobility, internal pH, and co-localize with discreet markers of particular types of endosomes including early endosomes, clathrin coated vesicles, caveolar vesicles, trans-golgi, and lysosomes. The vesicular localization of Mct1 was not dependent on its N or C termini, however, the size and pH of Mct1 vesicles was increased by deletion of either terminus demonstrating a role for the termini in vesicular trafficking of Mct1. Using a novel BCECF-AM based assay developed in this study, 8Br-cAMP was shown to decrease the pH of Mct1 vesicles after 25 minutes. This result and method were confirmed in experiments with a ratiometric pH-sensitive EGFP-mCherry dual tagged Mct1 construct. Overall, the results indicate that cAMP signaling reduces the functionality of Mct1 in cerebrovascular endothelial cells by facilitating its entry into a highly dynamic vesicular trafficking pathway that appears to lead to the transporter's trafficking to autophagosomes and lysosomes.
在这项研究中，详细描述了培养的大鼠脑微血管内皮细胞细胞质小泡中的单羧酸转运蛋白 1（Mct1），表明其为内体的一个多样化群体，通过短暂暴露于膜通透性 cAMP 类似物 8Br-cAMP 25 至 30 分钟，可调节转运蛋白。这些小泡在大小、流动性、内部 pH 值方面存在异质性，并且与特定类型内体的离散标志物共定位，包括早期内体、网格蛋白包被小泡、小窝泡、反式高尔基体和溶酶体。Mct1 的小泡定位不依赖于其 N 或 C 末端，然而，通过删除任一末端，Mct1 小泡的大小和 pH 值增加，表明末端在 Mct1 小泡的运输中起作用。使用本研究中开发的一种新型 BCECF-AM 基于测定法，表明 8Br-cAMP 在 25 分钟后降低了 Mct1 小泡的 pH 值。该结果和方法在使用比率型 pH 敏感 EGFP-mCherry 双标记 Mct1 构建体的实验中得到了证实。总体而言，这些结果表明 cAMP 信号通过促进其进入高度动态的小泡运输途径，从而降低脑血管内皮细胞中 Mct1 的功能，该途径似乎导致转运蛋白向自噬体和溶酶体的运输。
