Functional expression of the peptide transporter PEPT2 in the mammalian enteric nervous system.
The peptide transporter PEPT2 mediates transmembrane uptake of small peptides. So far, its expression has not been evidenced in the gastrointestinal tract. We have investigated peptide transport activity in the neuromuscular layers of the gastrointestinal tract by using the fluorescent tracer-dipeptide beta-Ala-Lys-Nepsilon-7-amino-4-methyl-coumarin-3-acetic acid (Ala-Lys-AMCA). Whole-mount preparations from mouse, rat, and guinea pig stomach and small and large intestine were incubated with Ala-Lys-AMCA in the presence or absence of the uptake-inhibitors L-histidine, D-phenylalanyl-L-alanine (D-Phe-Ala), glycyl-L-sarcosine (Gly-Sar), glycyl-L-glutamine (Gly-Gln), benzylpenicillin, and cefadroxil. Fluorescence microscopy revealed that Ala-Lys-AMCA specifically accumulated in both ganglionic layers of the enteric nervous system (ENS) in all regions and species studied. This could be inhibited by Gly-Sar, D-Phe-Ala, Gly-Gln, and cefadroxil, but not by free histidine and benzylpenicillin, indicating uptake via PEPT2. Accordingly, dipeptide uptake was completely abolished in PEPT2-deficient mice. Reverse transcriptase-polymerase chain reaction analysis detected a PEPT2-specific transcript in extracts from the ganglionic ENS layers of mouse small and large intestine, further proving that enteric dipeptide transport activity is specifically mediated via PEPT2. The cellular site of dipeptide uptake was immunohistochemically localized to enteric glial cells and tissue-resident macrophages. In addition, dipeptide uptake occurred in a neurochemically defined subset of neurons in the guinea pig ENS. Our results constitute the first functional evidence for dipeptide transport activity in the ENS. PEPT2-mediated dipeptide transport in enteric glia could contribute to the clearance of neuropeptides in the ENS. In addition, the fluorophore-coupled dipeptide uptake via PEPT2 is a novel vital marker for glial cells in the ENS.
Rühl
Anne
A
Department of Human Biology, Technical University of Munich, D-85350 Freising-Weihenstephan, Germany. ruehl@wzw.tum.de
Hoppe
Susanne
S
Frey
Isabelle
I
Daniel
Hannelore
H
Schemann
Michael
M
D000818:Animals; D030801:Animals, Genetically Modified; D000943:Antigens, Differentiation / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D015152:Blotting, Northern / Q000379:methods; D064026:Calbindins; D002452:Cell Count / Q000379:methods; D002795:Choline O-Acetyltransferase / Q000378:metabolism; D003374:Coumarins / Q000493:pharmacokinetics; D004151:Dipeptides / Q000493:pharmacokinetics / Q000494:pharmacology; D051959:ELAV Proteins; D000067778:ELAV-Like Protein 3; D017615:Enteric Nervous System / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006168:Guinea Pigs; D006639:Histidine / Q000494:pharmacology; D007150:Immunohistochemistry / Q000379:methods; D066298:In Vitro Techniques; D008322:Mammals; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D016601:RNA-Binding Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D009418:S100 Proteins / Q000378:metabolism; D051640:Small-Conductance Calcium-Activated Potassium Channels; D027981:Symporters / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
D000943:Antigens, Differentiation; D064026:Calbindins; D003374:Coumarins; D004151:Dipeptides; D051959:ELAV Proteins; D000067778:ELAV-Like Protein 3; C490262:Elavl3 protein, rat; D005904:Glial Fibrillary Acidic Protein; D009419:Nerve Tissue Proteins; D024681:Potassium Channels, Calcium-Activated; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D064030:S100 Calcium Binding Protein G; D009418:S100 Proteins; D051640:Small-Conductance Calcium-Activated Potassium Channels; D027981:Symporters; C434800:alanyl-lysyl-N(epsilon)-7-amino-4-methylcoumarin-3-acetic acid; C093694:hydrogen-coupled oligopeptide transporter PepT2; C036763:monocyte-macrophage differentiation antigen; C039788:glycylglutamine; C039552:phenylalanylalanine; D006639:Histidine; D002795:Choline O-Acetyltransferase; D010751:Phosphopyruvate Hydratase