Functional linkage of H+/peptide transporter PEPT2 and Na+/H+ exchanger in primary cultures of astrocytes from mouse cerebral cortex.
In our previous studies, we demonstrated that the high-affinity type peptide transporter PEPT2 is expressed in rat cerebral cortex using synaptosomal membrane study and that the uptake of dipeptide [14C]glycylsarcosine into synaptosomes was stimulated by an inwardly directed H+ gradient (Fujita et al., Brain Res. 972, 52-61, 2004). However, there is no information available for the driving force of PEPT2 function in the nervous system. In the present study, we investigated functional characteristics of PEPT2 mediated transport of Gly-Sar in primary cultured astrocytes from mouse cerebral cortex and examined the effects of Na+/H+ exchanger (NHE) inhibitor on Gly-Sar uptake in mouse astrocytes. In mouse astrocytes, extracellular H+ influenced only the maximal velocity (Vmax) of Gly-Sar uptake without affecting the apparent affinity (Kt). Interestingly, removal of Na+ from uptake buffer significantly reduced Gly-Sar uptake and Gly-Sar uptake was modulated by NHE inhibitors. The treatment of EIPA, an NHE inhibitor, altered the Vmax value of Gly-Sar uptake but had no effect on its Kt value. RT-PCR revealed that NHE1 and NHE2 mRNA are expressed in mouse cerebrocortical astrocytes. These results demonstrated that NHE activity is required to allow optimal uptake of dipeptides mediated by PEPT2 into the astrocytes. This study represents the first description of the functional co-operation of PEPT2 and NHE1 and/or NHE2 in cerebrocortical astrocytes.
Wada
Miyuki
M
Department of Biochemical Pharmacology, Kyoto Pharmaceutical University, Yamashina, Kyoto 607-8414, Japan.
Miyakawa
Sakiko
S
Shimada
Ayumi
A
Okada
Naoki
N
Yamamoto
Akira
A
Fujita
Takuya
T
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000502:physiology*; D015152:Blotting, Northern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D004151:Dipeptides / Q000493:pharmacokinetics; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry / Q000379:methods; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017923:Sodium-Hydrogen Exchangers / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects; D027981:Symporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25; D014316:Tritium / Q000493:pharmacokinetics
D004151:Dipeptides; D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C480499:Slc9c1 protein, mouse; C494209:Snap25 protein, mouse; D017923:Sodium-Hydrogen Exchangers; D027981:Symporters; D050825:Synaptosomal-Associated Protein 25; C093694:hydrogen-coupled oligopeptide transporter PepT2; D014316:Tritium; C004194:glycylsarcosine