Abstract
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.
Highlights
Monocarboxylic acid transporter 1, Monocarboxylic Acid Transporter-1 (Mct1), is a ubiquitous transmembrane protein that facilitates proton coupled symport of important cellular energy substrates such as lactate and other monocarboxylates across plasma membranes [1,2]
Basic characteristics of Mct1 vesicles The major scientific objectives of this work were to provide a basic characterization of Mct1 vesicles in RBE4 brain microvascular endothelial cells, to elucidate how they are involved in the regulation of Mct1 function by cAMP, and to examine whether trafficking of Mct1 vesicles is dependent upon elements in the transporter’s cytoplasmic N or C termini
This study confirmed that Mct1 vesicles are not byproducts of a particular immunostaining process, or an artifact of our fusion protein’s expression
Summary
Monocarboxylic acid transporter 1, Mct, is a ubiquitous transmembrane protein that facilitates proton coupled symport of important cellular energy substrates such as lactate and other monocarboxylates across plasma membranes [1,2]. Mct facilitates an intercellular transport of lactic acid from astrocytes to neurons which is required for learning and memory [3], and it has important roles in brain cancer that point to it as a therapeutic target [4,5] Both of these involve a significant microvascular component that would likely involve acute cell signaling dependent Mct regulation, but this has not been well investigated in brain. It is important to understand basic mechanisms that regulate Mct function in cerebrovascular endothelial cells since they present specific targets for therapeutic drug development to treat brain diseases ranging from learning and memory disorders to stroke and cancer, and could enhance delivery of pharmaceuticals to brain
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