PEPT2 is a high-affinity H⁺/dipeptide transporter expressed in kidney, brain, lung, and mammary gland. The physiological role of PEPT2 in kidney is to reabsorb small peptides generated by luminal peptidases. PEPT2 is also a transporter for peptide-like drugs such as penicillins and cephalosporins. We have conducted a haplotype analysis of 27 single nucleotide polymorphisms located in or near exons of the human gene encoding hPEPT2 (SLC15A2), using genotyping data from 247 genomic DNA samples from the Coriell collection. Our analysis reveals that hPEPT2 has a >6-kilobase sequence block with at least 10 abundant polymorphisms in almost complete linkage disequilibrium. As a result, only two main hPEPT2 variants exist (hPEPT2*1 and *2) with several phased amino acid substitutions, present in substantial frequencies in all ethnic groups tested. When expressed in Chinese hamster ovary cells, hPEPT2*1 and *2 displayed similar V_max values for glycyl-sarcosine (Gly-Sar), but they differed significantly in their K_m values (83 ± 16 and 233 ± 38 μM, respectively). Moreover, hPEPT2*1 and *2 differed in their pH sensitivity for H⁺/Gly-Sar transport. In addition, hPEPT2*1 and *2 generated varying levels of mRNA in nine heterozygous kidney tissue samples, including one allele expressing no detectable mRNA, suggesting the presence of cis-acting polymorphisms affecting transcription or mRNA processing. The results indicate that polymorphisms in the gene encoding hPEPT2 can alter substrate transport and therefore could affect drug disposition in vivo.

Two human di / tri-peptide transporters, hPepT1 and hPepT2 have been identified and functionally characterized. In the small intestine hPepT1 is exclusively expressed, whereas both PepT1 and PepT2 are expressed in the proximal tubule. The transport via di / tri-peptide transporters is proton-dependent, and the transporters thus belong to the Proton-dependent Oligopeptide Transporter (POT)-family. The transporters are not drug targets per se, however due to their uniquely broad substrate specificity; they have proved to be relevant drug targets at the level of drug transport. Drug molecules such as oral active β-lactam antibiotics, bestatin, prodrugs of aciclovir and ganciclovir have oral bioavailabilities, which largely are a result of their interaction with PepT1. In the last few years an increasing number of studies concerned with regulation of di / tri-peptide transporter capacity have appeared. Studies on receptor-mediated regulation has shown that both PepT1 and PepT2 is down-regulated by long-term exposure to epidermal growth factor (EGF) due to a decreased gene transcription. PepT1-mediated transport is upregulated by certain substrates and in response to fasting and starvation at the level of increased gene transcription. PepT1-mediated transport is up-regulated by short-term exposure to receptor agonists such as EGF, insulin, leptin, and clonidine, and down-regulated by VIP. Overall, the regulation of di / tri-peptide transport may be contributed to 1) changes in apical proton-motive force 2) recruitment of di / tri-peptide transporters from vesicular storages 3) changes in gene transcription / mRNA stability. The aim of the present review is to discuss physiological, patho-physiological and druginduced regulation of di / tri-peptide transporter mediated transport.

The human intestinal cell line Caco-2 was used as a model system to study the effects of epidermal growth factor (EGF) on peptide transport. EGF decreased apical-to-basolateral fluxes of [¹⁴C]glycylsarcosine ([¹⁴C]Gly-Sar) up to 50.2 ± 3.6% ( n = 6) of control values. Kinetic analysis of the fluxes showed that maximal flux ( V_max) of transepithelial transport decreased from 3.00 ± 0.17 nmol · cm⁻²· min⁻¹in control cells to 0.50 ± 0.07 nmol · cm⁻²· min⁻¹in cells treated with 5 ng/ml EGF ( n = 6, P < 0.01). The apparent Michaelis-Menten constant ( K_m) was 2.71 ± 0.31 mM ( n= 6) in control cells and 1.89 ± 0.28 mM ( n = 6, not significantly different from control) in EGF-treated cells. Similarly, apical uptake of [¹⁴C]Gly-Sar decreased in cells treated with EGF, with an ED₅₀value of 0.36 ± 0.06 ng/ml ( n = 6) EGF and a maximal inhibition of 80 ± 0.02% ( n = 6). V_maxdecreased from 2.61 ± 0.4 to 1.06 ± 0.1 nmol · cm⁻²· min⁻¹( n = 3, P < 0.05), whereas K_mremained constant. Basolateral Gly-Sar uptake showed no changes in V_maxor K_mafter EGF treatment ( n = 3). RT-PCR showed a decrease in hPepT1 mRNA (using glucose-6-phosphate dehydrogenase mRNA as control) in cells treated with EGF. Western blotting indicated a decrease in hPepT1 protein in cell lysates. We conclude that EGF treatment decreases Gly-Sar transport in Caco-2 cells by decreasing the number of peptide transporter molecules in the apical membrane.

The concentration of the excitotoxic amino acid, L‐glutamate, in brain interstitial fluid is tightly regulated by uptake transporters and metabolism in astrocytes and neurons. The aim of this study was to investigate the possible role of the blood‐brain barrier endothelium in brain L‐glutamate homeostasis. Transendothelial transport‐ and accumulation studies of ³H‐L‐glutamate, ³H‐L‐aspartate, and ³H‐D‐aspartate in an electrically tight bovine endothelial/rat astrocyte blood‐brain barrier coculture model were performed. After 6 days in culture, the endothelium displayed transendothelial resistance values of 1014 ± 70 Ω cm², and ¹⁴C‐D‐mannitol permeability values of 0.88 ± 0.13 × 10⁻⁶ cm s⁻¹. Unidirectional flux studies showed that L‐aspartate and L‐glutamate, but not D‐aspartate, displayed polarized transport in the brain‐to‐blood direction, however, all three amino acids accumulated in the cocultures when applied from the abluminal side. The transcellular transport kinetics were characterized with a K_m of 69 ± 15 μM and a J_max of 44 ± 3.1 pmol min⁻¹ cm⁻² for L‐aspartate and a K_m of 138 ± 49 μM and J_max of 28 ± 3.1 pmol min⁻¹ cm⁻² for L‐glutamate. The EAAT inhibitor, DL‐threo‐ß‐Benzyloxyaspartate, inhibited transendothelial brain‐to‐blood fluxes of L‐glutamate and L‐aspartate. Expression of EAAT‐1 (Slc1a3), −2 (Slc1a2), and −3 (Slc1a1) mRNA in the endothelial cells was confirmed by conventional PCR and localization of EAAT‐1 and −3 in endothelial cells was shown with immunofluorescence. Overall, the findings suggest that the blood‐brain barrier itself may participate in regulating brain L‐glutamate concentrations.