Regulation of monocarboxylic acid transporter-1 by cAMP dependent vesicular trafficking in brain microvascular endothelial cells.
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.
Department of Biology, Colorado State University-Pueblo, Pueblo, Colorado, United States of America.
Amy L
AL
Uhernik
Department of Biology, Colorado State University-Pueblo, Pueblo, Colorado, United States of America.
Lun
L
Li
Department of Biology, Colorado State University-Pueblo, Pueblo, Colorado, United States of America.
Nathan
N
LaVoy
Department of Biology, Colorado State University-Pueblo, Pueblo, Colorado, United States of America.
Micah J
MJ
Velasquez
Department of Biology, Colorado State University-Pueblo, Pueblo, Colorado, United States of America.
Jeffrey P
JP
Smith
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000098:blood supply*; D002460:Cell Line; D011992:Endosomes / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D055806:Microvessels / Q000166:cytology; D027501:Monocarboxylic Acid Transporters / Q000502:physiology*; D021381:Protein Transport; D051381:Rats; D015290:Second Messenger Systems; D027981:Symporters / Q000502:physiology*; D022161:Transport Vesicles / Q000378:metabolism*; D000099233:Monocarboxylate Transport Protein 1
D015124:8-Bromo Cyclic Adenosine Monophosphate; D027501:Monocarboxylic Acid Transporters; D027981:Symporters; D000099233:Monocarboxylate Transport Protein 1