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Localization and function of the brain excitatory amino acid transporter type 1 in cardiac mitochondria. J Mol Cell Cardiol 2004 Jul;37(1):33-41

Date

07/10/2004

Pubmed ID

15242733

DOI

10.1016/j.yjmcc.2004.04.008

Scopus ID

2-s2.0-3142617875   31 Citations

Abstract

Glutamate is the only amino acid extracted by healthy myocardium in net amounts, with uptake further increased during hypoxic or ischemic conditions. Glutamate supplementation provides cardioprotection from hypoxic and reperfusion injury through several metabolic pathways that depend upon adequate transport of glutamate into the mitochondria. Glutamate transport across the inner mitochondrial membrane is a key component of the malate/aspartate shuttle. Glutamate transport in the brain has been well characterized since the discovery of the excitatory amino acid transporter (EAAT) family. We hypothesize that a protein similar to EAAT1 found in brain may function as a glutamate transporter in cardiac mitochondria. Rat heart total RNA was screened by reverse transcriptase-polymerase chain reaction with an array of primer pairs derived from the rat brain EAAT1 cDNA sequence, yielding a 3786-bp cDNA comprising a 1638-bp open reading frame identical to rat brain EAAT1 with flanking 5'- and 3'-untranslated regions. Northern blot analysis confirmed a 4-kb mRNA product in rat heart and brain, with greater abundance in brain. A protein of the predicted approximate 60-kD size was recognized in myocardial lysates by an anti-EAAT1 polyclonal antibody produced against an amino-terminal peptide from human EAAT1. The protein enriched in rat heart mitochondria by immunoblot, co-localized with the mitochondrial protein cytochrome c by immunohistochemistry, and further localized to the inner mitochondrial membrane upon digitonin fractionation of the mitochondria. In myocytes overexpressing EAAT1, activity of the malate/aspartate shuttle increased by 33% compared to non-transfected cells (P = 0.004). These data indicate that EAAT1 is expressed in myocardial mitochondria, and functions in the malate/aspartate shuttle, suggesting a role for EAAT1 in myocardial glutamate metabolism.

Author List

Ralphe JC, Segar JL, Schutte BC, Scholz TD

Author

Jeffrey L. Segar MD Professor in the Pediatrics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Adenoviridae
Animals
Aspartic Acid
Blotting, Northern
Brain
Cells, Cultured
Coloring Agents
Cytochromes c
DNA, Complementary
Digitonin
Excitatory Amino Acid Transporter 1
Genetic Vectors
Glutamic Acid
Hypoxia
Immunoblotting
Immunohistochemistry
Malates
Microscopy, Fluorescence
Mitochondria
Mitochondria, Heart
Myocardium
Open Reading Frames
RNA
RNA, Messenger
Rats
Rats, Inbred WKY
Rats, Sprague-Dawley
Reperfusion Injury
Reverse Transcriptase Polymerase Chain Reaction
Subcellular Fractions
Tetrazolium Salts
Thiazoles
Transfection
jenkins-FCD Prod-486 e3098984f26de787f5ecab75090d0a28e7f4f7c0