Functional expression of the peptide transporter PEPT2 in the mammalian enteric nervous system

Abstract

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.

Quantitative assessment of glial cells in the human and guinea pig enteric nervous system with an anti‐Sox8/9/10 antibody

“…The neuronal counts that we have obtained are largely in accordance with previously published data (Schneider et al, 2001;Rü hl et al, 2005), whereas a comparable EGC data set has not yet been made available. With respect to neuronal cell counts, there are some disparities in the MP for which we are reporting higher numbers in human and lower numbers in guinea pig preparations than were previously published (Young et al, 1993;Neunlist et al, 2003;Rü hl et al, 2005), which may be explained by variable definitions of ganglia and ganglionic borders. In the present study, we used rigid definitions for ganglionic borders depending on the presence of neuronal cell bodies.…”

Significance and Regional Dependency of Peptide Transporter (PEPT) 1 in the Intestinal Permeability of Glycylsarcosine: In Situ Single-Pass Perfusion Studies in Wild-Type andPept1Knockout Mice

“…It should also be appreciated that other POT family members are expressed in the intestine. For example, PEPT2 is found in glial cells and in tissue-resident macrophages of the enteric nervous system (Rühl et al, 2005). Moreover, the peptide/histidine transporters PHT1 and PHT2 have been found in intestinal tissue segments (Herrera-Ruiz et al, 2001), and immunohistochemical analyses have indicated that PHT1 is expressed in the villous epithelium of small intestine (Bhardwaj et al, 2006).…”

Effect of Dose Escalation on the In Vivo Oral Absorption and Disposition of Glycylsarcosine in Wild-Type andPept1Knockout Mice

“…For example, transcripts of PHT1 and PHT2 are found in human and rat intestinal tissue segments (Herrera-Ruiz et al, 2001), and immunohistochemical analysis indicates that PHT1 is expressed in the villous epithelium of human small intestine (Bhardwaj et al, 2006). Moreover, PEPT2 is expressed and functionally active in glial cells and tissue-resident macrophages in neuromuscular layers of the gastrointestinal tract (Rühl et al, 2005).…”