Kinetic Measurements of Di- and Tripeptide and Peptidomimetic Drug Transport in Different Kidney Regions Using the Fluorescent Membrane Potential-Sensitive Dye, DiS-C3-(3).

Tri- and dipeptides are transported in the kidney by PEPT1 and PEPT2 isoforms. The aim of this study was to investigate differences in transport kinetics between renal brush border (BBMV) and outer medulla (OMMV) membrane vesicles (where PEPT1 and PEPT2 are sequentially available) for a range of di- and tripeptides and peptidomimetic drugs. This was accomplished through the use of the potential-sensitive fluorescent dye 3,3'-dipropylthiacarbocyanine iodide [DiS-C3-(3)]. BBMV and OMMV were prepared from the rat kidney using standard techniques. The presence of PEPT1 in BBMV and PEPT2 in OMMV was confirmed using Western blotting. Fluorescence changes were measured when extravesicular medium at pH 6.6 containing 0-1 mM substrates was added to a cuvette containing vesicles pre-equilibrated at pH 7.4 and 2.71 μM DiS-C3-(3). An increase in fluorescence intensity occurred upon substrate addition reflecting the expected positive change in membrane potential difference. Of the range of substrates studied, OMMV manifested the highest affinity to cefadroxil and valacyclovir (K m 4.3 ± 1.2 and 11.7 ± 3.2 µM, respectively) compared to other substrates, whilst the BBMV showed a higher affinity to Gly-His (K m 15.4 ± 3.1 µM) compared to other substrates. In addition, OMMV showed higher affinity and capacity to Gly-Gln (K m 47.1 ± 9.8 µM, 55.5 ± 2.8 ΔF/s/mg protein) than BBMV (K m 78.1 ± 13.3 µM and 35.5 ± 1.7 ΔF/s/mg protein, respectively). In conclusion, this study successfully separated the expression of PEPT1 and PEPT2 into different vesicle preparations inferring their activity in different regions of the renal proximal tubule

Function and immunolocalization of overexpressed human intestinal H+/peptide cotransporter in adenovirus-transduced Caco-2 cells

Purpose. To determine the localization of the human intestinal H+/peptide cotransporter (hPepT1) and its function in intestinal epithelial cells after adenoviral transduction. Methods. Caco-2 cells grown on Transwell membrane filters were transduced with a recombinant replication-deficient adenovirus carrying the hPepT1 gene. The transport of Gly-Sar across both apical and basolateral membranes was measured after adenoviral transduction as a function of pH, temperature, inhibitors, and substrate concentration. The localization of hPepT1 was examined by immunocytochemistry using confocal laser scanning microscopy. Results. The apical-to-basolateral and basolateral-to-apical transport of Gly-Sar in Caco-2 cells after viral transduction was increased 3.3 and 3.5-fold, respectively. The similar magnitude of Gly-Sar permeability from either direction indicates involvement of identical transport pathways in both membranes. This was further confirmed by immunocytochemistry showing that hPepT1 was localized in the apical and basolateral membrane of Caco-2 cells after adenoviral transduction. In both directions, Gly-Sar transport was enhanced in the presence of a pH gradient. In addition, the basolateral-to-apical Gly-Sar transport was dependent on temperature, multiplicity of infection (MOI), and Gly-Sar concentration. It was inhibited in the presence of excess Gly-Pro and cephalexin. Conclusions. Caco-2 cell monolayers represent an appropriate model to study gene expression in intestinal epithelial cells. Transport characteristics of Gly-Sar from the basolateral to the apical side in adenovirus-transduced Caco-2 cells are in agreement with those from the apical to the basolateral side, indicating that hPepT1 is also expressed in the basolateral membrane and displays a similar level of transport enhancement after adenovirus mediated hPepT1 gene expression

Glycylsarcosine uptake in rabbit renal brush border membrane vesicles isolated from outer cortex or outer medulla: Evidence for heterogeneous distribution of oligopeptide transporters

Studies were initially performed in rabbit brush border membrane vesicles (BBMV) prepared from whole cortex plus outer medulla. In these studies using combined tissues, two distinct peptide/H+ transport systems were found for glycylsarcosine (GlySar) uptake, with one representing a low-affinity/high-capacity system (Vm1=974 pmol/mg/10 sec and Km1=4819 μM) and the other a high-affinity/low-capacity system (Vm2=220 pmol/mg/10 sec and Km2=96 μM). Thus, under linear conditions, the high-affinity transporter accounted for about 92% of the total transport of dipeptide. To better define the regional heterogeneity of peptide transporter activity in kidney, subsequent studies were performed in vesicles prepared from separately harvested outer cortical and outer medullary tissue. In BBMV studies prepared from outer cortex, two saturable components were revealed for GlySar transport (low-affinity/high-capacity transport system: Vm1=1921 pmol/mg/10 sec and Km1=11714 μM; high-affinity/low-capacity transport system: Vm2=143 pmol/mg/10 sec and Km2=138 μM). However, in BBMV studies prepared from outer medulla, only one saturable component was revealed for GlySar transport (high-affinity/low-capacity transport system: Vm2=168 pmol/mg/10 sec and Km2=230 μM). Overall, these studies support the contention that peptides are handled sequentially in kidney (ie, first by low-affinity transporter PEPT1, and then by high-affinity transporter PEPT2) and that PEPT2 is primarily responsible for the renal reabsorption of peptides and peptidomimetics

Transport systems for opioid peptides in mammalian tissues

Transmembrane transport of endogenous as well as synthetic opioid peptides is a critical determinant of pharmacokinetics and biologic efficacy of these peptides. This transport process influences the distribution of opioid peptides across the blood-brain barrier and their elimination from the body. A multitude of transport systems that recognize opioid peptides as substrates have been characterized at the functional level, and these transport systems are expressed differentially at different sites in the body. Many of these transport systems have been identified at the molecular level. These include the H+-coupled peptide transporters PEPT1 and PEPT2, the adenosine triphosphate-dependent efflux transporters P-glycoprotein and multidrug resistance-related protein 2, and several members of the organic anion-transporting polypeptide gene family. There are however many additional transport systems that are known to transport opioid peptides but their molecular identities still remain unknown