Search

Acute encephalopathy (AE) can be a manifestation of an acute porphyric attack. Clinical data were studied in 32 patients during AE with or without polyneuropathy (PNP) together with 12 subjects with PNP but no AE, and 17 with dysautonomia solely. Brain neuroimaging was done in 20 attacks during AE, and PEPT2 polymorphisms were studied in 56 subjects, 24 with AE. AE manifested as a triad of seizures, confusion and/or blurred vision. Symptoms lasting 1–5 days manifested 3–19 days from the onset of an attack. 55% of these patients had acute PNP independent of AE. Posterior reversible encephalopathy syndrome (PRES) was detected in 42% of the attacks. These patients were severely affected and hyponatremic (88%). Reversible segmental vasoconstriction was rare. There was no statistical difference in hypertension or urinary excretion of porphyrin precursors among the patients with or without AE. In 94% of the attacks with AE, liver transaminases were elevated significantly (1.5 to fivefold, P = 0.034) compared to a normal level in 87% of the attacks with dysautonomia, or in 25% of patients with PNP solely. PEPT2*2/2 haplotype was less common among patients with AE than without (8.3% vs. 25.8%, P = 0.159) and in patients with PNP than without (9.5% vs. 22.9%, P = 0.207), suggesting a minor role, if any, in acute neurotoxicity. In contrast, PEPT2*2/2 haplotype was commoner among patients with chronic kidney disease (P = 0.192). Acute endothelial dysfunction in porphyric encephalopathy could be explained by a combination of abrupt hypertension, SIADH, and acute metabolic and inflammatory factors of hepatic origin. [ABSTRACT FROM AUTHOR]

Peptide transporter 2 (PepT2) transports short peptides from the blood into bovine mammary epithelial cells (BMEC) to stimulate milk protein synthesis. Despite the fact that the effect of PepT2 is acknowledged in BMEC, little is known about its regulation. This study was completed to investigate the role of mammalian target of the rapamycin (mTOR) signaling in regulating the expression and function of PepT2 in BMEC. The regulation of PepT2 by mTOR in BMEC was studied in vitro using peptide transport assay, gene silencing, Western blot. The membrane expression of PepT2 and the uptake of β-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (β-Ala-Lys-AMCA), a model dipeptide, in BMEC were reduced by rapamycin (a mTOR inhibitor) and silencing of either mTOR complex 1 (mTORC1) or mTOR complex 2 (mTORC2), stimulated by DEP domain-containing mTOR-interacting protein (DEPTOR, endogenous inhibitor of mTORC1 and mTORC2) silencing. The trafficking of PepT2 to the membrane and the uptake of β-Ala-Lys-AMCA was promoted by neuronal precursor cell-expressed developmentally down-regulated 4 isoform 2 (Nedd4-2) silencing. The effects of knockdown of mTORC1, but not mTORC2, on cell membrane expression and transport activity of PepT2 was abolished by Nedd4-2 silencing. With immunofluorescence staining, PepT2 was identified to be interacting with Nedd4-2. The Nedd4-2 expression and the interaction between PepT2 and Nedd4-2 was increased through mTORC1 knockdown, indicating an increased ubiquitination of PepT2. The results revealed that mTORC1 can regulate the expression and function of PepT2 through Nedd4-2 in BMEC.

Peptide transporter 2 (PepT2) in mammals plays essential roles in the reabsorption and conservation of peptide-bound amino acids in the kidney and in maintaining neuropeptide homeostasis in the brain. It is also of significant medical and pharmacological significance in the absorption and disposing of peptide-like drugs, including angiotensin-converting enzyme inhibitors, β-lactam antibiotics and antiviral prodrugs. Understanding the structure, function and regulation of PepT2 is of emerging interest in nutrition, medical and pharmacological research. In this review, we provide a comprehensive overview of the structure, substrate preferences and localization of PepT2 in mammals. As PepT2 is expressed in various organs, its function in the liver, kidney, brain, heart, lung and mammary gland has also been addressed. Finally, the regulatory factors that affect the expression and function of PepT2, such as transcriptional activation and posttranslational modification, are also discussed. [ABSTRACT FROM AUTHOR]

l-Carnosine (β-alanyl-l-histidine) is a well-known antioxidant and neuroprotector in various models on animals and cell cultures. However, while there is a plethora of data demonstrating its efficiency as a neuroprotector, there is a distinct lack of data regarding the mechanism of its take up by neurons. According to literature, cultures of rat astrocytes, SKPT cells and rat choroid plexus epithelial cells take up carnosine via the H+-coupled PEPT2 membrane transporter. We've assessed the effectiveness and mechanism of carnosine transport, and its stability in primary rat cortical culture neurons. We demonstrated that neurons take up carnosine via active transport with Km = 119 μM and a maximum velocity of 0.289 nmol/mg (prot)/min. Passive transport speed constituted 0.21∙10–4 nmol/mg (prot)/min (with 119 μM concentration in the medium)—significantly less than active transport speed. However, carnosine concentrations over 12.5 mM led to passive transport speed becoming greater than active transport speed. Using PEPT2 inhibitor zofenopril, we demonstrated that PEPT2-dependent transport is one of the main modes of carnosine take up by neurons. Our experiments demonstrated that incubation with carnosine does not affect PEPT2 amount present in culture. At the same time, after removing carnosine from the medium, its elimination speed by culture cells reached 0.035 nmol/mg (prot)/min, which led to a decrease in carnosine quantity to control levels in culture within 1 h. Thus, carnosine is taken up by neurons with an effectiveness comparable to that of other PEPT2 substrates, but its elimination rate suggests that for effective use as a neuroprotector it's necessary to either maintain a high concentration in brain tissue, or increase the effectiveness of glial cell synthesis of endogenous carnosine and its shuttling into neurons, or use more stable chemical modifications of carnosine. [ABSTRACT FROM AUTHOR]

Protein post-translational modification by the Small Ubiquitin-like MOdifier (SUMO) on lysine residues is a reversible process highly important for transcription and protein stability. In the kidney, SUMOylation appears to be important for the cellular response to aldosterone. Therefore, in this study, we generated a SUMOylation profile of the aldosterone-sensitive kidney distal convoluted tubule (DCT) as a basis for understanding SUMOylation events in this cell type. Using mass spectrometry-based proteomics, 1037 SUMO1 and 552 SUMO2 sites, corresponding to 546 SUMO1 and 356 SUMO2 proteins, were identified from a modified mouse kidney DCT cell line (mpkDCT). SUMOylation of the renal hydrogen-coupled oligopeptide and drug co-transporter (Pept2) at one site (K139) was found to be highly regulated by aldosterone. Using immunolabelling of mouse kidney sections Pept2 was localized to DCT cells in vivo. Aldosterone stimulation of mpkDCT cell lines expressing wild-type Pept2 or mutant K139R-Pept2, post-transcriptionally increased Pept2 expression up to four-fold. Aldosterone decreased wild-type Pept2 abundance in the apical membrane domain of mpkDCT cells, but this response was absent in K139R-Pept2 expressing cells. In summary, we have generated a SUMOylation landscape of the mouse DCT and determined that SUMOylation plays an important role in the physiological regulation of Pept2 trafficking by aldosterone.

The objectives of this study were clone oligopeptide transport carrier PepT2 and compare its abundance in kidney tissues of yak and cattle in response to different dietary nitrogen levels. Four adult female yaks were enrolled as donor animals for profiling the molecular characteristics and expression specificity of yak PepT2 (yPepT2), and twenty-four castrated males of each of two genotypes, yak (Bos grunniens) and indigenous cattle (Bos taurus) were used to explore PepT2 mRNA expression in kidney tissue in different nitrogen (N) levels (10.3, 19.5, 28.5 or 37.6 g N/kg dry matter; DM). Our results showed that the yPepT2 coding sequence region contains 2190 bp, and encodes a putative protein of 729 amino acids (AA) residues. The yPepT2 AA sequence identified eight putative extracellular N-glycosylation sites (Asn7, Asn80, Asn373, Asn435, Asn472, Asn508, Asn528, Asn587) and eight intracellular putative protein kinase C sites (Ser34, Ser264, Ser274, Ser376, Ser442, Ser586, Ser640, Ser724). The yPepT2 AA sequence was 98 and 94% identical to PepT2 from zebu cattle (Bos indicus) and sheep (Ovis aries), respectively. The relative PepT2 expression in kidney tissue for yak was greater than of indigenous cattle in the10.3 and 28.5 N/kg DM diet, but it was lower for yaks in 37.6 N/kg DM diet. These implied that relative PepT2 mRNA expression was higher in yak kidney than that in indigenous cattle at lower dietary N supplies, but more research on PepT2 will be required to determine the renal regulatory mechanisms.

Peptide transporter 2 (PepT2) in mammals plays essential roles in the reabsorption and conservation of peptide-bound amino acids in the kidney and in maintaining neuropeptide homeostasis in the brain. It is also of significant medical and pharmacological significance in the absorption and disposing of peptide-like drugs, including angiotensin-converting enzyme inhibitors, β-lactam antibiotics and antiviral prodrugs. Understanding the structure, function and regulation of PepT2 is of emerging interest in nutrition, medical and pharmacological research. In this review, we provide a comprehensive overview of the structure, substrate preferences and localization of PepT2 in mammals. As PepT2 is expressed in various organs, its function in the liver, kidney, brain, heart, lung and mammary gland has also been addressed. Finally, the regulatory factors that affect the expression and function of PepT2, such as transcriptional activation and posttranslational modification, are also discussed.

Fish larvae differ greatly from the adult form in their morphology and organ functionality. The functionality of the gastrointestinal tract depends on the expression of various pumps, transporters, and channels responsible for feed digestion and nutrients absorption. During the larval period, the gastrointestinal tract develops from a simple closed tube, into its complex form with differentiated segments, crypts and villi, as found in the adult. In this study, we characterized the expression of three peptide transporters (PepT1a, PepT1b, and PepT2) in the gastrointestinal tract of Mozambique tilapia (Oreochromis mossambicus) larvae along 12 days of development, from pre-hatching to the completion of yolk sac absorption. Gene expression analysis revealed differential and complimentary time-dependent expression of the PepT1 variants and PepT2 along the larval development period. Immunofluorescence analysis showed differential protein localization of the three peptide transporters (PepTs) along the gastrointestinal tract, in a similar pattern to the adult. In addition, PepT1a was localized in mucosal cells in the larvae esophagus, in much higher abundance than in the adults. The results of this study demonstrate specialization of intestinal sections and absorbance potential of the enterocytes prior to the onset of active exogenous feeding, thus pointing to an uncharacterized function and role of the gastrointestinal tract and its transporters during the larval period.

Fish larvae differ greatly from the adult form in their morphology and organ functionality. The functionality of the gastrointestinal tract depends on the expression of various pumps, transporters, and channels responsible for feed digestion and nutrients absorption. During the larval period, the gastrointestinal tract develops from a simple closed tube, into its complex form with differentiated segments, crypts and villi, as found in the adult. In this study, we characterized the expression of three peptide transporters (PepT1a, PepT1b, and PepT2) in the gastrointestinal tract of Mozambique tilapia (Oreochromis mossambicus) larvae along 12 days of development, from pre-hatching to the completion of yolk sac absorption. Gene expression analysis revealed differential and complimentary time-dependent expression of the PepT1 variants and PepT2 along the larval development period. Immunofluorescence analysis showed differential protein localization of the three peptide transporters (PepTs) along the gastrointestinal tract, in a similar pattern to the adult. In addition, PepT1a was localized in mucosal cells in the larvae esophagus, in much higher abundance than in the adults. The results of this study demonstrate specialization of intestinal sections and absorbance potential of the enterocytes prior to the onset of active exogenous feeding, thus pointing to an uncharacterized function and role of the gastrointestinal tract and its transporters during the larval period. [ABSTRACT FROM AUTHOR]

Renal PEPT1 and PEPT2 cotransporters play an important role in the balance of circulating body oligopeptides and selected peptidomimetic drugs. We aim to comprehensively characterise age-related changes of the renal PEPT cotransporters at the gene, protein, and functional level. Brush border membrane vesicles (BBMV) and outer medulla membrane vesicles (OMMV) were isolated from the kidneys of young, middle-aged and old rats. The protein expression of PEPT1 was not only increased in BBMV from old rats, but PEPT1 also appeared in OMMV from middle-aged and old rats. SLC15A1 gene expression in the renal cortex increased in middle-aged group. PEPT2 protein expression was not only increased with ageing, but PEPT2 also was found in BBMV from middle-aged and old groups. SLC15A2 gene expression in the renal outer medulla increased in the old group. These changes in the expressions and localisations of PEPT1 and PEPT2 could explain the changes to transport activity in BBMV and OMMV. These findings provide novel insights that would be useful for maintaining protein nutrition and optimising the delivery of some peptidomimetic drugs in elderly individuals. [ABSTRACT FROM AUTHOR]

Background/Aims: β-Klotho, a transmembrane protein expressed in several tissues including the brain and the kidney, is critically important for inhibition of 1,25(OH)2D3 formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. Soluble klotho is a β-glucuronidase participating in the regulation of several ion channels and carriers. The present study explored the effect of β-Klotho protein on the peptide transporters PEPT1 and PEPT2. Methods: cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes and glycine-glycine (2 mM)-induced inward current (IGly) taken as measure of glycine-glycine transport. Measurements were made without or with prior 24 h treatment with soluble β-Klotho protein (30 ng/ml) in the absence and presence of β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM). Ussing chamber experiments were employed to determine electrogenic peptide transport across intestinal epithelia of klotho deficient (kl-/-) and corresponding wild type (kl+/+) mice. Results: IGly was observed in PEPT1 and in PEPT2 expressing oocytes but not in water injected oocytes. In both, PEPT1 and PEPT2 expressing oocytes IGly was significantly decreased by treatment with soluble β-Klotho protein. As shown for PEPT1, β-klotho protein decreased significantly the maximal transport rate without significantly modifying the affinity of the carrier. The effect of β-Klotho on PEPT1 was reversed by DSAL. Intestinal IGly was significantly larger in kl-/- than in kl+/+ mice. Conclusion: β-Klotho participates in the regulation of the peptide transporters PEPT1 and PEPT2.

Tilapiine species, widely distributed across habitats with diverse water salinities, are important to aquaculture as well as a laboratory model. The effects of water salinity on two tilapia species, that differ in their salinity tolerance, was evaluated. Oreochromis niloticus reared in brackish-water, showed a significant decrease in growth and feed efficiency, whereas O. mossambicus reared in seawater did not show any significant changes. The expression and activity of Na + /K + -ATPase (NKA), V-type H + -ATPase (VHA) and carbonic anhydrase (CA), as well as expression levels of genes encoding two HCO 3 − and three peptide transporters ( nbc1 , slc26a6 , slc15a1a , slc15a1b and slc15a2 ) were measured in three intestinal sections of these two species, grown in freshwater and brackish/sea-water. Overall, the spatial distribution along the intestine of the genes examined in this study was similar between the two species, with the exception of tcaIV . The salinity response, on the other hand, varied greatly between these species. In O. mossambicus , there was a salinity-dependent increased expression of most of the examined genes (except slc26a6 and slc15a2 ), while in O. niloticus the expression of most genes did not change, or even decreased ( tcaIV , nbc1 and slc15a1b ). This study highlighted differences in the intestinal response to salinity acclimation between closely- related species that differ in their salinity tolerance. O. mossambicus , which has a high salinity tolerance, showed expression patterns and responses similar to marine species, and differed from the low-salinity-tolerance O. niloticus , which showed a response that differed from the accepted models, that are based on marine and diadromous fishes. [ABSTRACT FROM AUTHOR]

The peptide transporter (PepT) systems are well-known for their importance to protein absorption in all vertebrate species. These symporters use H+ gradient at the apical membrane of the intestinal epithelial cells to mediate the absorption of small peptides. In fish, the intestine is a multifunctional organ, involved in osmoregulation, acid-base regulation, and nutrient absorption. Therefore, we expected environmental stimuli to affect peptide absorption. We examined the effect of three environmental factors; salinity, pH and feeding, on the expression, activity and localization of three PepT transporters (PepT1a, PepT1b, PepT2) along the intestine of the Mozambique tilapia (Oreochromis mossambicus). Quantitative real time PCR (qPCR) analysis demonstrated that the two PepT1 variants are typical to the proximal intestinal section while PepT2 is typical to the distal intestinal sections. Immunofluorescence analysis with custom-made antibodies supported the qPCR results, localized both transporters on the apical membrane of enterocytes and provided the first evidence for the participation of PepT2 in nutrient absorption. This first description of segment-specific expression and localization points to a complementary role of the different peptide transporters, corresponding to the changes in nutrient availability along the intestine. Both gene expression and absorption activity assays showed that an increase in water salinity shifted the localization of the PepT genes transcription and activity down along the intestinal tract. Additionally, an unexpected pH effect was found on the absorption of small peptides, with increased activity at higher pH levels. This work emphasizes the relationships between different functions of the fish intestine and how they are affected by environmental conditions.

The peptide transporter (PepT) systems are well-known for their importance to protein absorption in all vertebrate species. These symporters use H+ gradient at the apical membrane of the intestinal epithelial cells to mediate the absorption of small peptides. In fish, the intestine is a multifunctional organ, involved in osmoregulation, acid-base regulation, and nutrient absorption. Therefore, we expected environmental stimuli to affect peptide absorption. We examined the effect of three environmental factors; salinity, pH and feeding, on the expression, activity and localization of three PepT transporters (PepT1a, PepT1b, PepT2) along the intestine of the Mozambique tilapia (Oreochromis mossambicus). Quantitative real time PCR (qPCR) analysis demonstrated that the two PepT1 variants are typical to the proximal intestinal section while PepT2 is typical to the distal intestinal sections. Immunofluorescence analysis with custom-made antibodies supported the qPCR results, localized both transporters on the apical membrane of enterocytes and provided the first evidence for the participation of PepT2 in nutrient absorption. This first description of segment-specific expression and localization points to a complementary role of the different peptide transporters, corresponding to the changes in nutrient availability along the intestine. Both gene expression and absorption activity assays showed that an increase in water salinity shifted the localization of the PepT genes transcription and activity down along the intestinal tract. Additionally, an unexpected pH effect was found on the absorption of small peptides, with increased activity at higher pH levels. This work emphasizes the relationships between different functions of the fish intestine and how they are affected by environmental conditions. [ABSTRACT FROM AUTHOR]

Understanding how the nervous system functions requires mapping synaptic connections between neurons. Several methods are available for imaging neurons connected by chemical synapses, but few enable marking neurons connected by electrical synapses. Here, we demonstrate that a peptide transporter, Pept2, can be used for this purpose. Pept2 transports a gap junction-permeable fluorophore-coupled dipeptide, beta-alanine-lysine-N-7-amino-4-methyl coumarin-3-acid (βALA). Cre-dependent expression of pept2 in specific neurons followed by incubation in βALA labeled electrically-coupled synaptic partners. Using this method, we analyze light-dependent modulation of electrical connectivity among retinal horizontal cells.

Background/Aims: β-Klotho, a transmembrane protein expressed in several tissues including the brain and the kidney, is critically important for inhibition of 1,25(OH)2D3 formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. Soluble klotho is a β-glucuronidase participating in the regulation of several ion channels and carriers. The present study explored the effect of β-Klotho protein on the peptide transporters PEPT1 and PEPT2. Methods: cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes and glycine-glycine (2 mM)-induced inward current (IGly) taken as measure of glycine-glycine transport. Measurements were made without or with prior 24 h treatment with soluble β-Klotho protein (30 ng/ml) in the absence and presence of β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM). Ussing chamber experiments were employed to determine electrogenic peptide transport across intestinal epithelia of klotho deficient (kl-/-) and corresponding wild type (kl+/+) mice. Results: IGly was observed in PEPT1 and in PEPT2 expressing oocytes but not in water injected oocytes. In both, PEPT1 and PEPT2 expressing oocytes IGly was significantly decreased by treatment with soluble β-Klotho protein. As shown for PEPT1, β-klotho protein decreased significantly the maximal transport rate without significantly modifying the affinity of the carrier. The effect of β-Klotho on PEPT1 was reversed by DSAL. Intestinal IGly was significantly larger in kl-/- than in kl+/+ mice. Conclusion: β-Klotho participates in the regulation of the peptide transporters PEPT1 and PEPT2. [ABSTRACT FROM AUTHOR]

Elasmobranchs are considered to be top marine predators, and in general play important roles in the transfer of energy within marine ecosystems. Despite this, little is known regarding the physiological processes of digestion and nutrient absorption in these fishes. One topic that is particularly understudied is the process of nutrient uptake across the elasmobranch gastrointestinal tract. Given their carnivorous diet, the present study sought to expand knowledge on dietary nutrient uptake in elasmobranchs by focusing on the uptake of products of protein digestion. To accomplish this, a full-length cDNA encoding peptide transporter 1 (PepT1), a protein previously identified within the brush border membrane of vertebrates that is responsible for the translocation of peptides released during digestion by luminal and membrane-bound proteases, was isolated from the bonnethead shark ( Sphyrna tiburo). A cDNA encoding the related peptide transporter PepT2 was also isolated from S. tiburo using the same methodology. The presence of PepT1 was then localized in multiple components of the bonnethead digestive tract (esophagus, stomach, duodenum, intestine, rectum, and pancreas) using immunohistochemistry. Vesicle studies were used to identify the apparent affinity of PepT1 and to quantify the rate of dipeptide uptake by its H-dependent cotransporter properties. The results of this study provide insight into the properties of peptide uptake within the bonnethead gut, and can facilitate future work on physiological regulation of protein metabolism and absorption including how these processes may vary in elasmobranchs that exhibit different feeding strategies. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine absolute protein expression levels of transporters in rat choroid plexus, that is, the blood-cerebrospinal fluid barrier, and to compare them with the levels in the human choroid plexus. Plasma membrane fractions were prepared from pooled, freshly isolated choroid plexuses of 30 male Wistar rats and from frozen choroid plexus of one male human donor. Protein expression levels of 54 rat and 121 human molecules were measured, using a quantitative targeted absolute proteomics technique. In rat, oatp1a5 showed the most abundant protein expression (30.3 fmol/μg protein), and its expression level was 3.1-, 4.5-, 5.5-, 8.4-, 9.0-, 9.9-, 22-, 91-, and 95-fold greater than those of glut1, oatp1c1, mrp1, mct1, oat3, pept2, mrp4, bcrp, and mdr1a, respectively. OATP1A2 (a possible homolog of rat oatp1a5), OATP1C1 and PEPT2 were not detected in human choroid plexus. MRP1, OAT3, and MRP4 showed 4.0-, 1.8-, and 1.7-fold smaller expression levels in human than rat, respectively. MATE1 was detected in human, but not rat, and its expression level (8.61 fmol/μg protein) was the highest among the xenobiotic transporters examined in human choroid plexus. These findings should be useful for understanding rat blood-cerebrospinal fluid barrier function and its differences from that in human. [ABSTRACT FROM AUTHOR]

Oligopeptide transporters serve important functions in nutrition and pharmacology. In particular, these transporters help maintain the homeostasis of peptides. The peptide-transporter PEPT2 is a high-affinity and low-capacity type oligopeptide transporter from the proton-coupled oligopeptide transporter family. PEPT2 has recently received attention because of its potential application in targeted drug delivery. PEPT2 is widely distributed in kidney, central nervous system, and lung of organisms. In general, all dipeptides, tripeptides, and peptide-like drugs such as β-lactam antibiotics and angiotensin-converting enzyme inhibitors could be mediated and transported as a substrate of PEPT2. The design of many extant drugs and prodrugs is based on the substrate structure of PEPT2 to accelerate absorption via peptide transporters. Thus, this paper summarizes the substrate features of PEPT2 to promote the rational design of drugs and prodrugs that target peptide transporters. [ABSTRACT FROM AUTHOR]

This study evaluated the developmental gene and protein expression of proton-coupled oligopeptide transporters ( POTs: peptide transporter, PepT1 and PepT2; peptide-histidine transporter, PhT1 and PhT2) in different regions of rodent brain, and the age-dependent uptake of a POT substrate, glycylsarcosine (GlySar), in brain slices. Slices were obtained from cerebral cortex, cerebellum and hippocampus of wildtype and PepT2 null mice, and from rats at different ages. Gene and protein expression were determined by real-time PCR and immunoblot analyses. Brain slice uptakes of radiolabeled glycylsarcosine were determined in the absence and presence of excess unlabeled glycylsarcosine or l-histidine, the latter being an inhibitor of PhT1/2 but not PepT1/2. As PepT2 and PhT1 transcripts were abundantly expressed in all three regions of mouse brain, little to no expression was observed for PepT1 and PhT2. PhT1 protein was present in brain regions of adult but not neonatal mice and expression levels increased with age in rats. Glycylsarcosine uptake, inhibition and transporter dominance did not show regional brain or species differences. However, there were clear age-related differences in functional activity, with PepT2 dominating in neonatal mice and rats, and PhT1 dominating in adult rodents. These developmental changes may markedly impact the neural activity of both endogenous and exogenous (drug) peptides/mimetics. [ABSTRACT FROM AUTHOR]

We investigated whether entecavir is a substrate of the oligopeptide transporter 2 (PEPT2) and whether reabsorption of entecavir is mediated by PEPT2 as well as what is the contribution of PEPT2 to entecavir reabsorption during urinary excretion. Entecavir uptake in transfected cells and rat kidney slices, changes in urine entecavir concentrations following isolated kidney perfusion and in vivo entecavir plasma and urine concentrations were determined with LC–MS/MS. In hPEPT2-HELA cells, entecavir uptake was significantly higher compared to vector-HELA cells and was sharply inhibited by Gly-sar and JBP485, and there were two distinct transport systems. The Km and Vmax of entecavir were 427 (μM) and 1.60 (nmol/mg protein/30s) (low-affinity, high-velocity system) and 24.0 (μM) and 0.296 (nmol/mg protein/30s) (high-affinity, low-velocity system). In rat kidney slices, uptake of entecavir was not markedly inhibited by Gly-sar. In isolated kidney perfusion experiments, entecavir cumulative urinary excretion was statistically significant at 45 and 60min. CLR(4°C), CLR(37°C Control) and CLR(37°C Experiment) were 12.6, 27.6 and 36 (ml/min/kg), respectively. CLTS and TR rate (for PEPT2) were 25.3 and 9.4 (ml/min/kg). In vivo, the cumulative urinary excretion of entecavir had statistical significance at 3 and 4h with CLR(Control) and CLR(Experiment) values of 31 and 42 (ml/min/kg), respectively. The CLTS and TR rate (for PEPT2) were 32 and 11.6 (ml/min/kg), respectively. The present study demonstrated that entecavir is a substrate of PEPT2. Moreover, reabsorption of entecavir is mediated by PEPT2, and 25% of urinary entecavir is reabsorbed by PEPT2. [ABSTRACT FROM AUTHOR]

Abstract: Aims: The purpose of this study was to clarify the expression and function of peptide transporter 2 (PEPT2) in primary cultured alveolar type II epithelial cells and in transdifferentiated type I-like cells. Main methods: Real-time PCR analysis, uptake study of [3H]Gly-Sar, and immunostaining were performed in alveolar epithelial cells. Key findings: The expression of PEPT2 mRNA in type II cells isolated from rat lungs was highest at day 0, and decreased rapidly during culture of the cells. In accordance with this change, PEPT2 activity estimated as cefadroxil-sensitive [3H]Gly-Sar uptake also decreased along with transdifferentiation. The expression of PEPT2 protein in type II cells was confirmed by immunostaining and Western blot analysis. The uptake of [3H]Gly-Sar in type II cells was time- and pH-dependent. In contrast, minimal time-dependence and no pH-dependence of [3H]Gly-Sar uptake were observed in type I-like cells. The maximal [3H]Gly-Sar uptake was observed at pH6.0, and the uptake decreased at higher pHs in type II cells. The uptake of [3H]Gly-Sar in type II cells was inhibited by cefadroxil in a concentration-dependent manner, the IC50 value being 4.3μM. On the other hand, no significant inhibition by cefadroxil was observed in type I-like cells. In addition, [3H]Gly-Sar uptake in type II cells was saturable, the Km value being 72.0μM. Significance: PEPT2 is functionally expressed in alveolar type II epithelial cells, but the expression decreases along with transdifferentiation, and PEPT2 would be almost completely lost in type I cells. [Copyright &y& Elsevier]

Objective: Pulmonary fibrosis is a common pathological phenomena in lung cancer patients after chemotherapy or radiotherapy, which is a key factor hindering to transport ion of high concentrated drug to the lung tissue, peptide transporter has become targets of the rational design of peptides and peptide drug. The purpose of the study is to investigate the expression of PEPT2 mRNA in the lung of rats with bleomycin (BLM)-induced pulmonary fibrosis. Methods: Fifty healthy adult Spragne-Dawley rats were randomized into five groups, the rats in BLM 7d, 14d and 28d groups were treated with a single instillation of 5 mg/kg of BLM, to induced pulmonary fibrosis models. On days 7, 14 and 28, the animals were killed by exsanguination respectively. Normal saline (NS) group were treated by NS, on days 14, the animals were killed by exsanguinations. Control group were untreated. The lung samples were processed for light microscopy and determined the hydroxyproline (HYP) concentration. The expression of PEPT2 mRNA were measured by RT-PCR. PEPT2 cDNA fragments were tested by dideoxy chain termination. Results: Compared with control and NS group, HYP levels increased on day 7 of BLM group, but there was no statistical significant difference ( P > 0.05). HYP levels markedly increased on days 14 and 28 of BLM group, there was statistical significant difference ( P < 0.01). The morphological study showed that collagenous fiber proliferated on days 14 and 28 of BLM group, especially on day 28, formed pulmonary fibrosis. There were no significant changes of pulmonary PEPT2 mRNA expression at different groups ( P > 0.05). Conclusion: The pulmonary fibrosis models of SD rats can be induced by a single instillation of 5 mg/kg of bleomycin on 28d. There were no significant changes of PEPT2 mRNA expression in the lung of rats with bleomycin-induced pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Peptide transporters 1 and 2 (PepTl and PepT2) and peptide/histidine transporter 1 (PHT1) are all members of the proton-coupled oligopeptide transporter family, which are important for the trans-port of amino acids in peptide form. The PepTl acts as a low-affinity/high-capacity transporter and PepT2 as a high-affinity/low-capacity transporter for di- and tri-peptides. The PHT1 transports di- and tri-peptides as well as histidine. The objective of this study was to pro-file PepTl, PepT2, and PHT1 mRNA expression in the proventriculus, duodenum, jejunum, ileum, ceca, large intestine, brain, heart, bursa of Fabricius, lung, kidney, and liver in layer chicks on embryonic d 18 and 20 and d 1, 3, 7, 10, and 14 posthatch. Absolute quantification real-time PCR was used to measure gene expression. Expression of PepTl was greatest in the duodenum, jejunum, and ileum. Expression of PepTl increased in the duodenum, jejunum, and ileum from late embryonic stages to posthatch and in the large intestine from late embryonic stages to d 10 posthatch. In the ceca, PepTl expression increased from embryonic d 20 to d 1 post-hatch and then decreased. Expression of PepT2 was greatest in the brain and kidney. Expression of PepT2 increased from d 10 to 14 in the bursa of Fabricius and decreased in the proventriculus, duodenum, jejunum, and liver from late embryonic stages to posthatch. In the small intestine and liver, PepT2 may function to transport di- and tri-peptides during embryogenesis. The PHT1 was expressed in all tissues analyzed. Ex-pression of PHT1 increased in the jejunum, large intes-tine, brain, and liver posthatch and decreased in the proventriculus from embryonic stages to posthatch. A tissue x age interaction was observed for all genes. The uptake of peptides in the developing chick is regulated by peptide transporters that are expressed in a tissue-and development-specific manner. [ABSTRACT FROM AUTHOR]

Abstract: Aim: To investigate the effect of key tissue culture conditions on cell growth, gene expression and functional uptake of peptide and organic cation transporter substrates in the human nasal epithelium (HNE). Methods: HNE were cultured on different growth surfaces (polystyrene plastic, collagen film, and hydrated collagen gel) and were maintained with three popular nasal tissue culture media supplements [DMEM/F12 supplemented with Ultroser® G (2%), FBS (10%) and NuSerum® (10%)], respectively. The expression of gene transcripts for organic cation and peptide transporters were screened using qPCR and substrate uptake studies. Results: Cell growth surface (polystyrene plastic surface, dried collagen film and hydrated collagen gel) did not significantly alter gene expression levels. However, Ultroser® G and FBS caused significant increase in PEPT1, PEPT2, PHT1, OCT3, and OCTN1 levels (≅2–5-fold for FBS and 2–8-fold for Ultroser® G). In terms of the degree to which the supplements affected gene expression, the following observations were made: effect on OCTN1>PEPT2>OCT3>PHT1>PEPT1. Functional uptake of organic cation (4-Di-1-ASP) and peptide [β-Ala-Lys (AMCA)] transporter substrates was significantly lower in cells cultured with NuSerum® compared to Ultroser® G and FBS cultured cells (p >0.05). Conclusions: Tissue culture media had a major effect on SLC gene expression levels of the human nasal epithelium in primary culture. Ultroser® G was identified as the most efficient culture supplement in maintaining SLC transporter expression under most culture conditions, whereas FBS appears to be an economical choice. We do not recommend the use of NuSerum® as a supplement for growing HNE for transport studies involving SLC transporters. [Copyright &y& Elsevier]

Abstract: The purpose of this study was to construct stably transfected HeLa cells with human peptide transporters (hPEPT1/hPEPT2) and to identify the function of the transfected cells using the substrate JBP485 (a dipeptide) and a typical substrate for PEPTs, glycylsarcosine (Gly-Sar). An efficient and rapid method was established for the preparation and transformation of competent cells of Escherichia coli. After extraction and purification, hPEPT1/hPEPT2-pcDNA3 was transfected into HeLa cells by the liposome transfection method, respectively. HeLa-hPEPT1/hPEPT2 cells were selected by measuring the protein expression and the uptake activities of JBP485 and Gly-Sar. A simple and rapid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of JBP485 and Gly-Sar in biological samples. The Michaelis–Menten constant (K m) values of Gly-Sar uptake by the hPEPT1 and hPEPT2-expressing transfectants were 1.03mM and 0.0965mM, respectively, and the K m values of JBP485 uptake were 1.33mM for PEPT1 and 0.144mM for PEPT2. The uptake of Gly-Sar was significantly inhibited by JBP485 with a K i value of 8.11mM (for PEPT1) and 1.05mM (for PEPT2). Maximal uptake of Gly-Sar were detected at pH 5.8 (for PEPT1) and pH 6.5 (for PEPT2), suggesting that both HeLa-hPEPT1 and HeLa-hPEPT2 were H+ dependent transporters. Stably transfected HeLa-hPEPT1/HeLa-hPEPT2 cells were constructed successfully, and the functions of hPEPT1/hPEPT2 were identified using their substrates, JBP485 and Gly-Sar. The transfected cells with transporters were used to investigate drug–drug interactions (DDIs) between JBP485 and other substrates (cephalexin or lisinopril) of PEPT1 and PEPT2. [Copyright &y& Elsevier]

Abstract: Common carp (Cyprinus carpio) of average body mass 0.07±0.02g were fed three formulated diets: wheat gluten protein-based diet supplemented with Lys–Gly dipeptide (PP), wheat gluten protein-based diet supplemented with free lysine and glycine (AA), and a wheat gluten protein-based control diet without lysine supplementation (CON), frozen zooplankton (Z) (restricted diet), and a commercial starter food Aglo Norse (AN). After 4weeks of experimental feeding, fish fed AN diet showed the highest body mass and length. Significantly lower mass occurred in groups fed PP, AA, CON, and Z. Fish fed CON diet showed the lowest intestinal folds and the highest number of mucous cells. Fish fed PP diet showed a significantly higher number of gastrin/cholecystokinin (CCK) positive cells. The diameter of lipid vacuoles in hepatocyte cytoplasm of fish fed formulated diets (PP, AA and CON) was significantly higher than in fish fed zooplankton (Z) and the commercial diet (AN). Hepatocytes of fish fed AA and CON showed a higher nucleus proliferation rate than in the other experimental groups. The quantitative analysis of the number of proliferating cell nuclear antigen (PCNA) and caspase-3(rabbit polyclonal antibody CPP-32)-positive cells showed that the highest proliferation rate was accompanied by the high apoptosis in the intestine of fish fed AA and CON. After 4weeks of experimental feeding the highest relative expression of PepT1 gene was observed in fish fed PP diet, while the lowest expression occurred in fish fed CON. Feeding carp plant protein-based diet supplemented with Lys–Gly dipeptide (PP) had a beneficial influence on fish growth and metabolism in the digestive tract as compared to fish fed control diet without lysine supplementation (CON). [Copyright &y& Elsevier]

Abstract: Neuropeptide inactivation is generally thought to occur via peptidase-mediated degradation. However, a recent study found increased analgesia after L-kyotorphin (L-Tyr-L-Arg; L-KTP) administration in mice lacking an oligopeptide transporter, PEPT2. The current study examines the role of PEPT2 in L-KTP uptake by astrocytes and compares it to astrocytic L-KTP degradation. L-[3H]KTP uptake was measured in primary cultures of neonatal astrocytes from rats and from Pept2 +/+ and Pept2 −/− mice. Uptake was further characterized using potential inhibitors. L-[3H]KTP degradation was examined in primary astrocyte cultures from Pept2 −/− mice by following the formation of L-[3H]tyrosine. The uptake of L-[3H]KTP in both rat and Pept2 +/+ mouse neonatal astrocytes was inhibited by known PEPT2 inhibitors. L-[3H]KTP uptake was also reduced in Pept2 −/− astrocytes as compared to those from Pept2 +/+ mice. Kinetic analysis indicated the presence of a high affinity (K m ∼50μM) transporter for L-[3H]KTP, identified as Pept2, and a low affinity transporter (K m ∼3–4mM), inhibited by amastatin, bestatin and tyrosine. Astrocytes also degraded L-KTP through a low affinity peptidase (K m ∼2mM). Astrocytic clearance of L-KTP occurs via both peptidase activity and transport. These processes occur at similar rates and may be linked. This supports the contention that oligopeptide transport may have an impact on the extracellular clearance (and potentially activity) of certain neuropeptides. [Copyright &y& Elsevier]

The bioactive dipeptide derivative anserine (β-alanyl-1- N-methyl-l-histidine) is absorbed from the human diet in intact form at the intestinal epithelium. The purpose of this study was to investigate whether anserine is a substrate of the H+/peptide cotransporters 1 and 2 (PEPT1 and PEPT2). We first assessed the effects of anserine on [14C]glycylsarcosine ([14C]Gly-Sar) uptake into Caco-2 cells expressing human PEPT1 and into spontaneous hypertensive rat kidney proximal tubule (SKPT) cells expressing rat PEPT2. Anserine inhibited [14C]Gly-Sar uptake with Ki values of 1.55 mm (Caco-2) and 0.033 mm (SKPT). In HeLa cells transfected with pcDNA3-hPEPT1 or pcDNA3-hPEPT2, Ki values of 0.65 mm (hPEPT1) and 0.18 mm (hPEPT2) were obtained. We conclude from these data that anserine is recognized by PEPT1 and PEPT2. Carnosine also inhibited [14C]Gly-Sar uptake. Using the two-electrode, voltage-clamp technique at Xenopus laevis oocytes, strong hPEPT1-specific inward transport currents were recorded for Gly-Sar, anserine and carnosine, but not for glycine. We conclude that anserine and carnosine interact with the human intestinal peptide transporter and are transported by hPEPT1 in an active, electrogenic H+ symport. As PEPT1 is the predominant transport system for di- and tripeptides at the intestinal epithelium, this transporter is most probably responsible for the intestinal absorption of anserine after food intake. In addition, anserine might be useful for the design of new substrates of peptide transporters, such as prodrugs, that can be administered orally. [ABSTRACT FROM AUTHOR]

Abstract: Low-level lead exposure during early childhood has long been associated with altered neurocognitive development and diminished cognitive functions. Over nine thousand U.S. industrial facilities annually emit significant amounts of lead, creating exposure risk particularly for minority children. The mechanisms by which low-level lead exerts neurotoxic effects are poorly understood. Once absorbed, the only intervention is source removal, thus primary prevention is key. Genetic biomarkers could provide an efficient means of identifying children at greatest risk. Common functional variants of genes that alter lead''s neurotoxic potential have been identified and include delta-aminolevulinic acid dehydratase (ALAD2) and peptide transporter 2 (PEPT2*2). These polymorphisms have not been examined previously in Hispanic minority samples, or with regard to lowest level lead exposure. In 116 children of Mexican-American/Hispanic descent residing in zip codes previously designated as “high risk” for lead exposure (mean age=8.1, S.D.=1.9), blood lead level was measured at three time points over a 3-month period and averaged. DNA extraction was completed using buccal swab samples. The frequencies of the ALAD2 and PEPT2*2 polymorphisms observed in this sample closely approximated those previously reported for Anglo, European and Asian samples. As compared to children heterozygous for the PEPT2*2 polymorphism, and without the PEPT2*2 polymorphism, the geometric mean blood lead level of children homozygous for the PEPT2*2 polymorphism was significantly higher. In contrast to past studies, mean blood lead level of children heterozygous and homozygous for the ALAD2 polymorphism in this sample did not differ from that of children without the ALAD2 polymorphism. Higher blood lead burden in children with the PEPT2*2 mutation may suggest that this common genetic variant is a biomarker of increased vulnerability to the neurotoxic effects of lowest level lead exposure. [Copyright &y& Elsevier]

l-Kyotorphin (l-KTP), an endogenous analgesic neuropeptide, is a substrate for aminopeptidases and a proton-coupled oligopeptide transporter, PEPT2. This study examined the CSF efflux, antinociceptive response, and hydrolysis kinetics in brain ofl-KTP and its synthetic diastereomerd-kyotorphin (d-KTP) in wild-type and Pept2 null mice. CSF clearance ofl-KTP was slower in Pept2 null mice than in wild-type animals, and this difference was reflected in greaterl-KTP-induced analgesia in Pept2 null mice. Moreover, dose-response analyses showed that the ED50 ofl-KTP in Pept2-deficient animals was one-fifth of the value observed in Pept2-competent animals (4 vs. 21 nmol for null vs. wild-type mice, respectively). In contrast, the ED50 ofd-KTP was very similar between the two genotypes (9–10 nmol). Likewise, there was little difference between genotypes in slope factor or baseline effects ofl-KTP andd-KTP. The enhanced antinociceptive response tol-KTP in Pept2 null mice could not be explained by differences in neuropeptide degradation as Vmax and Km values did not differ between genotypes. Our results demonstrate that PEPT2 can significantly impact the analgesic response to an endogenous neuropeptide by altering CSF (and presumably brain interstitial fluid) concentrations and that it may influence the disposition and response to exogenous peptide/mimetic substrates. [ABSTRACT FROM AUTHOR]

1. The peptide transporters belong to the peptide transporter (PTR) family and serve as integral membrane proteins for the cellular uptake of di- and tripeptides in the organism. By their ability also to transport peptidomimetics and other substrates with therapeutic activities or precursors of pharmacologically active agents, they are of considerable importance in pharmacology. 2. PEPT1 is the low-affinity, high-capacity transporter and is mainly expressed in the small intestine, whereas PEPT2 is the high-affinity, low-capacity transporter and has a broader distribution in the organism. 3. Targeted mouse models have revealed PEPT2 to be the dominant transporter for the reabsorption of di- and tripeptides and its pharmacological substrates in the organism, and for the removal of these substrates from the cerebrospinal fluid. Moreover, the peptide transporters undergo physiological and pharmacological regulation and, of great interest, are present in disease states where PEPT1 exhibits ectopic expression in colonic inflammation. 4. The paper reviews the structural characteristics of the peptide transporters, the structural requirements for substrates, the distribution of the peptide transporters in the organism, and finally their regulation in the organism in healthy and pathological situations. [ABSTRACT FROM AUTHOR]

Abstract: The H+/peptide cotransporters PEPT1 and PEPT2 have gained considerable interest in pharmaceutical sciences as routes for drug delivery. It is, therefore, of interest to develop uncommon artificial substrates for the two carriers. This study was initiated to investigate the binding affinity of 2-aminothiazole-4-acetic acid (ATAA) conjugates with amino acids to PEPT1 and PEPT2. The 2-aminothiazole-4-acetic acid derivatives have been synthesised and tested for their affinity to PEPT1 and PEPT2. The K i values were compared with in silico predicted values from CoMSIA models. C-terminal ATAA-Xaa conjugates proved to be low to medium inhibitors of the [14C]Gly-Sar uptake at both carrier systems whereas N-terminal Xaa-ATAA conjugates exhibited medium to high affinity. A promising candidate for further functionalisation is Val-ATAA which shows extraordinary high affinity to PEPT1. [Copyright &y& Elsevier]

The transporters PEPT1 and PEPT2 accept a broad spectrum of substrates including small, naturally occurring peptides and peptidomimetic drugs. This study aimed to investigate for the first time whether these transporters are expressed and active in isolated cardiomyocytes. PEPT1/PEPT2 expression in rat kidney (positive control), guinea pig kidney and cardiomyocytes were investigated by reverse transcription polymerase chain reaction. l-Glycyl- l-[14C]sarcosine (Gly-sar) uptake was characterised using freshly isolated suspensions of adult male guinea pig cardiomyocytes. PEPT2-specific primers recognised mRNA of appropriate size and sequence in cardiomyocytes and kidney, whilst PEPT1 was expressed in the kidney only. The initial uptake (30 s) of 200 μM Gly-sar was dependent on extracellular pH with a maximum at pH 6.0 (237.8 ± 12.2 pmol/μl) and a minimum at pH 8.0 (72.1 ± 13.4 pmol/μl, n = 6 ± SE, p < 0.01, T test). The K m and V max of Gly-sar uptake at pH 6.0 were 495.5 ± 69.6μM and 1470.5 ± 69.6 pmol μl−1 min−1. The addition of 10 mM fosinopril, cefadroxil, carnosine, cyclacillin or a variety of l-amino acid containing dipeptides/tripeptides significantly reduced Gly-sar uptake. Gly-sar uptake was not affected by 10 mM d-ala- d-ala, glycine or sarcosine. These results support the presence of a functional dipeptide transporter in isolated cardiomyocytes, with accompanying expression of PEPT2. [ABSTRACT FROM AUTHOR]

Abstract: 5-Aminolevulinic acid (ALA) and carnosine have important physiological and pathophysiological roles in the CNS. Both are substrates for the proton-coupled oligopeptide transporter PEPT2. The purpose of the current study was to determine the importance of PEPT2 in the uptake of ALA and carnosine in rat and mouse (PEPT2+/+ and PEPT2−/−) cultured neonatal astrocytes. Although neonatal astrocytes are known to express PEPT2, its quantitative importance in the transport of these compounds is not known. [14C]ALA uptake in neonatal rat astrocytes was inhibited by dipeptides, an α-amino containing cephalosporin (which is a PEPT2 substrate) but was not affected by a non-amino containing cephalosporin (which is not a PEPT2 substrate). Uptake was pH sensitive as expected from a proton-coupled transporter and was saturable (V max =715±29 pmol/mg/min, K m =606±14 μM). [3H]Carnosine uptake in neonatal rat astrocytes was inhibited by dipeptides but not by histidine (a substrate for the peptide/histidine transporters PHT1 and PHT2) and also showed saturable transport (V max =447±23 pmol/mg/min, K m =43±5.5 μM). Neonatal astrocytes from PEPT2−/− mice had a 62% reduction in [14C]ALA uptake and a 92% reduction in [3H]carnosine uptake compared to PEPT2+/+ mice. These results demonstrate that PEPT2 is the primary transporter responsible for the astrocytic uptake of ALA and carnosine. [Copyright &y& Elsevier]

The proton-coupled uptake of di- and tri-peptides is the major route of dietary nitrogen absorption in the intestine and of reabsorption of filtered protein in the kidney. In addition, the transporters involved, PepT1 (SLC15a1) and PepT2 (SLC15a2), are responsible for the uptake and tissue distribution of a wide range of pharmaceutically important compounds, including beta-lactam antibiotics, angiotensin-converting enzyme inhibitors, anti-cancer and anti-viral drugs. PepT1 and PepT2 are large proteins, with over 700 amino acids, and to date there are no reports of their crystal structures, nor of those of related proteins from lower organisms. Therefore there is virtually no information about the protein 3-D structure, although computer-based approaches have been used to both model the transmembrane domain (TM) layout and to produce a substrate binding template. These models will be discussed, and a new one proposed from homology modeling rabbit PepT1 to the recently crystallized bacterial transporters LacY and GlpT. Understanding the mechanism by which PepT1 and PepT2 bind and transport their substrates is of great interest to researchers, both in academia and in the pharmaceutical industries. [ABSTRACT FROM AUTHOR]

Abstract: Endogenous porphyrin accumulation after administration of 5-aminolevulinic acid is employed in photodynamic therapy of tumours. Due to its low membrane permeability, esterified 5-aminolevulinic acid derivatives less hydrophilic than the parental compound are under investigation. Knowledge of the mechanisms of 5-aminolevulinic acid derivatives uptake into target cells is essential to understand and improve photodynamic therapy and useful in the design of new derivatives with better affinity and with higher selectivity for tumour cells in specific tissues. The aim of this work was to assess the interaction of 5-aminolevulinic acid derivatives with the intestinal PEPT1 and renal transporter PEPT2 expressed in Pichia pastoris yeasts. We found that Undecanoyl, Hexyl, Methyl and 2-(hydroxymethyl)tetrahydropyranyl 5-aminolevulinic acid esters and the dendron 3m-ALA inhibited 14C-5-aminolevulinic acid uptake by PEPT2. However, only the Undecanoyl ester inhibited 5-aminolevulinic acid uptake by PEPT1. We have also found through a new developed colorimetric method, that Hexyl and 2-(hydroxymethyl)tetrahydropyranyl 5-aminolevulinic acid esters display more affinity than 5-aminolevulinic acid for PEPT2 whereas none of the compounds surpass 5-aminolevulinic acid affinity for PEPT1. In addition, the Undecanoyl ester binds with high affinity to the membranes of PEPT2 and PEPT1-expressing yeasts and to the control yeasts. The main finding of this work was that some derivatives have the potential to improve 5-aminolevulinic acid-based photodynamic therapy by increased efficiency of transport into cells expressing PEPT2 such as kidney, mammary gland, brain or lung whereas in tissues expressing exclusively PEPT1 the parent 5-aminolevulinic acid remains the compound of choice. [Copyright &y& Elsevier]

The proton-coupled peptide transporter PEPT2 (SLC15A2) mediates the high-affinity low-capacity transport of small peptides as well as various oral peptide-like drugs in the kidney. In contrast to its well-characterized transport properties, there is less information available on its regulatory mechanism, although the interaction of PEPT2 to the PDZ (PSD-95, DglA, and ZO-1)-domain protein PDZK1 has been preliminarily reported. To examine whether PDZK1 is a physiological partner of PEPT2 in kidneys, we started from a yeast two-hybrid screen of a human kidney cDNA library with the C-terminus of PEPT2 (PEPT2 C-terminus (PEPT2-CT)) as bait. We could identify PDZK1 as one of the positive clones. This interaction requires the PDZ motif of PEPT2-CT detected by a yeast two-hybrid assay, in vitro binding assay and co-immunoprecipitation. The binding affinities of second and third PDZ domains of PDZK1 to PEPT2-CT were measured by surface plasmon resonance. Co-immunoprecipitation using human kidney membrane fraction and localization of PEPT2 in renal apical proximal tubules revealed the physiological meaning of this interaction in kidneys. Furthermore, we clarified the mechanism of enhanced glycylsarcosine (Gly-Sar) transport activity in PEPT2-expressing HEK293 cells after the PDZK1 coexpression. This augmentation was accompanied by a significant increase in the Vmax of Gly-Sar transport via PEPT2 and it was also associated with the increased surface expression level of PEPT2. These results indicate that the PEPT2–PDZK1 interaction thus plays a physiologically important role in both oligopeptide handling as well as peptide-like drug transport in the human kidney.Kidney International (2006) 70, 275–282. doi:10.1038/sj.ki.5001522; published online 31 May 2006 [ABSTRACT FROM AUTHOR]

The kidney is a target organ for thyroid hormone action and a variety of renal transport processes are altered in response to impaired thyroid functions. To investigate the effect of thyroid hormone on the expression of the renal proximal tubular high-affinity-type H+-peptide cotransporter (PEPT2) in rats, hypothyroidism was induced in animals by administration of methimazole (0.05%) via drinking water. After 7 weeks of treatment, hypothyroidism was confirmed by determining serum free T and free T concentrations. Northern blotting was used to examine the expression of PEPT2 mRNA in kidney tissues from hypothyroid rats compared to control rats. Hypothyroidism resulted in an increased level of total renal PEPT2 mRNA (121.1±3.3% vs. control 100±2.8%; p=0.008). The mRNA results were confirmed by immuno-blotting, which demonstrated significantly increased protein levels (162% vs. control 100%; p<0.01). Immunohistochemistry also revealed increased PEPT2 protein levels in the proximal tubules of treated compared to non-treated rats. In summary, PEPT2 is the first proximal tubule transporter protein that shows increased expression in states of hypothyreosis. As PEPT2 reabsorbs filtered di- and tripeptides and peptide-like drugs, the present findings may have important implications in nutritional amino acid homeostasis and for drug dynamics in states of altered thyroid function. [ABSTRACT FROM AUTHOR]

Abstract: 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 (V max) of Gly-Sar uptake without affecting the apparent affinity (K t). 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 V max value of Gly-Sar uptake but had no effect on its K t 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. [Copyright &y& Elsevier]

In addition to being the main source of cerebrospinal fluid (CSF) secretion, the choroid plexuses are involved in the supply and distribution of peptides to brain, the removal of toxic metabolites, the excretion of xenobiotics, and the delivery of drugs as an alternative route to the blood–brain barrier (BBB). The discovery of proton-coupled oligopeptide transporters in choroid plexus has generated considerable interest regarding their physiologic role at the blood–cerebrospinal fluid interface and their potential for peptide/antagonist pharmacotherapy in the central nervous system. Many of the same factors that affect the disposition of naturally occurring peptides in brain will also affect the disposition of exogenously delivered peptide or peptidomimetic drugs. Therefore, this review addresses three main areas: (1) choroid plexus structure, physiology, and barrier function in relation to peptide transport; (2) polypeptide transport and secretion mechanisms into cerebrospinal fluid; and (3) molecular physiology, expression, and functional activity of proton-coupled oligopeptide transporters in choroid plexus. [Copyright &y& Elsevier]

We evaluated the functional consequences of genetic variations in human H+/peptide cotransporter 2 (hPEPT2, SLC15A2) resulting in the amino acid changes Arg57His (R57H) and Pro409Ser (P409S). The transport activity of variant R57H was completely abolished, whereas that of variant P409S was comparable with that of wild-type hPEPT2 at pH 5.0–8.0. R57H variant protein was detected in the crude membranes of transiently expressed HEK293 cells by Western blot analysis. The expression of the R57H variant at the plasma membrane was confirmed by indirect immunofluorescence in Xenopus oocytes, suggesting that the loss of transport function of hPEPT2 R57H was not due to a change in membrane protein expression. This is the first demonstration of a functional impairment of the SLC15A family induced by a single nucleotide polymorphism. [Copyright &y& Elsevier]

In this report, we studied the functional characteristics of a brain peptide transporter using synaptosomes prepared from rat cerebral cortex. Crude synaptosomes (P2 fraction) were prepared from cerebral cortices in male Wistar rats. Uptake of [14C]glycylsarcosine (Gly–Sar), a substrate for H+/oligopeptide transporters PEPT1 and PEPT2, and [3H]histidine, a substrate for peptide/histidine transporters PHT1 and PHT2, was measured at 37 °C by a rapid filtration technique. The uptake of [14C]Gly–Sar into synaptosomes was stimulated by an inwardly directed H+-gradient. The uptake system exhibited a Michaelis–Menten constant (Kt) of 110±20 μM for Gly–Sar. This value is comparable to the Kt value for Gly–Sar uptake via the high-affinity H+/peptide transporter PEPT2. The H+-dependent uptake of [14C]Gly–Sar into synaptosomes was inhibited by di- and tripeptides and β-lactam antibiotics, but was unaffected by amino acids glycine and histidine. In particular, kyotorphin (Tyr–Arg) completely inhibited Gly–Sar uptake with the Ki value of 29±14 μM. These uptake properties of the brain peptide transporter (i.e., the Kt value for Gly–Sar uptake and the Ki value of kyotorphin for Gly–Sar uptake) are very similar to those of PEPT2. RT-PCR and Western blotting analyses revealed that PEPT2 is actually expressed in the cerebral cortex in rat. These results indicate that a H+-coupled high affinity peptide transport system is functionally expressed in the cerebral cortex and that this transport system is identical to PEPT2. [Copyright &y& Elsevier]

Purpose. To determine the expression and functional activity of proton-coupled oligopeptide transporters (POT) in retinal pigment epithelial (RPE) cells. Methods. RT-PCR was used to probe the presence of POT mRNA in freshly isolated bovine RPE (BRPE) and human RPE (HRPE) cells, a human RPE cell line (ARPE-19), and human and bovine neural retina. [[sup14]C]GlySar uptake was used to characterize POT activity in cultured ARPE-19 cells and freshly isolated BRPE cell sheet suspensions. Results. PHT1 mRNA was expressed in BRPE, HRPE, ARPE-19, and bovine and human neural retina. In contrast, PEPT2 and PHT2 were expressed only in bovine and human retina, and PEPT1 could not be detected. GlySar exhibited a linear uptake over 6 h at pH values of 6.0 and 7.4, with greater uptake at pH 7.4 (p Purpose. To determine the expression and functional activity of proton-coupled oligopeptide transporters (POT) in retinal pigment epithelial (RPE) cells. Methods. RT-PCR was used to probe the presence of POT mRNA in freshly isolated bovine RPE (BRPE) and human RPE (HRPE) cells, a human RPE cell line (ARPE-19), and human and bovine neural retina. [[sup14]C]GlySar uptake was used to characterize POT activity in cultured ARPE-19 cells and freshly isolated BRPE cell sheet suspensions. Results. PHT1 mRNA was expressed in BRPE, HRPE, ARPE-19, and bovine and human neural retina. In contrast, PEPT2 and PHT2 were expressed only in bovine and human retina, and PEPT1 could not be detected. GlySar exhibited a linear uptake over 6 h at pH values of 6.0 and 7.4, with greater uptake at pH 7.4 (p < 0.01). GlySar uptake did not exhibit saturability (5-2000 μM) and was unchanged when studied in the presence of 1 mM L-histidine. In contrast, GlySar uptake was significantly decreased when studied at 4°C or in the presence of endocytic inhibitors at 37°C (p < 0.01). [ABSTRACT FROM AUTHOR]

Purpose. The peptide transporter PEPT2 was recently shown to be functionally active in rat choroid plexus, suggesting that it may play a role in neuropeptide homeostasis in the cerebrospinal fluid. This study, therefore, examined the role of PEPT2 in mediating neuropeptide uptake into choroid plexus. Methods. Whole-tissue rat choroid plexus uptake studies were performed on GlySar in the absence and presence of neuropeptides and on carnosine. Results. The neuropeptides NAAG, CysGly, GlyGln, kyotorphin, and carnosine inhibited the uptake of radiolabeled GlySar at 1.0 mM concentrations. In contrast, TRH, [D-Arg2]-kyotorphin, glutathione, and homocarnosine did not inhibit GlySar uptake. Kyotorphin, an analgesic, was a competitive inhibitor of GlySar with a Ki of 8.0 μM. The direct uptake of carnosine was also shown to be mediated by PEPT2 in isolated choroid plexus (Km = 39.3 μM; Vmax = 73.9 pmol/mg/min). Radiolabeled carnosine uptake was inhibited by 1.0 mM concentrations of GlySar or carnosine but not homocarnosine, L-histidine, or β-alanine. Conclusions. These findings indicate that PEPT2 mediates the uptake of a diverse group of neuropeptides in choroid plexus, and suggests a role for PEPT2 in the regulation of neuropeptides, peptide fragments, and peptidomimetics in cerebrospinal fluid. [ABSTRACT FROM AUTHOR]