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Sartans are very effective drugs for treatment of hypertension, heart failure, and other cardiovascular disorders. They antagonize the effects of angiotensin II at the AT(1) receptor and display p.o. bioavailability rates of 13 to 80%. Because some sartans sterically resemble dipeptide derivatives, we investigated whether they are transported by peptide transporters. We first assessed the effects of sartans on [(14)C]glycylsarcosine uptake into Caco-2 cells expressing H(+)/peptide transporter (PEPT) 1 and into SKPT cells expressing PEPT2. Losartan, irbesartan, valsartan, and eprosartan inhibited [glycine-1-(14)C]glycylsarcosine ([(14)C]Gly-Sar) uptake into Caco-2 cells in a competitive manner with K(i) values of 24, 230, 390, and >1000 microM. Losartan and valsartan also strongly inhibited the total transepithelial flux of [(14)C]Gly-Sar across Caco-2 cell monolayers. In SKPT cells, [(14)C]Gly-Sar uptake was inhibited with K(i) values of 2.2 microM (losartan), 65 microM (irbesartan), 260 microM (valsartan), and 490 microM (eprosartan). We determined by the two-electrode voltage-clamp technique whether the compounds elicited transport currents by PEPT1 or PEPT2 when expressed in Xenopus laevis oocytes. No currents were observed for any of the sartans, but the compounds strongly and reversibly inhibited peptide-induced currents. Uptake of valsartan, losartan, and cefadroxil was quantified in HeLa cells after heterologous expression of human PEPT1 (hPEPT1). In contrast to cefadroxil, no PEPT1-specific uptake of valsartan and losartan was found. We conclude that the sartans tested in this study display high-affinity interaction with PEPTs but are not transported themselves. However, they strongly inhibit hPEPT1-mediated uptake of dipeptides and cefadroxil.

Angiotensin-converting enzyme (ACE) inhibitors are often regarded as substrates for the H+/peptide transporters (PEPT)1 and PEPT2. Even though the conclusions drawn from published data are quite inconsistent, in most review articles PEPT1 is claimed to mediate the intestinal absorption of ACE inhibitors and thus to determine their oral availability. We systematically investigated the interaction of a series of ACE inhibitors with PEPT1 and PEPT2. First, we studied the effect of 14 ACE inhibitors including new drugs on the uptake of the dipeptide [14C]glycylsarcosine into human intestinal Caco-2 cells constitutively expressing PEPT1 and rat renal SKPT cells expressing PEPT2. In a second approach, the interaction of ACE inhibitors with heterologously expressed human PEPT1 and PEPT2 was determined. In both assay systems, zofenopril and fosinopril were found to have very high affinity for binding to peptide transporters. Medium to low affinity for transporter interaction was found for benazepril, quinapril, trandolapril, spirapril, cilazapril, ramipril, moexipril, quinaprilat, and perindopril. For enalapril, lisinopril, and captopril, very weak affinity or lack of interaction was found. Transport currents of PEPT1 and PEPT2 expressed in Xenopus laevis oocytes were recorded by the two-electrode voltage-clamp technique. Statistically significant, but very low currents were only observed for lisinopril, enalapril, quinapril, and benazepril at PEPT1 and for spirapril at PEPT2. For the other ACE inhibitors, electrogenic transport activity was extremely low or not measurable at all. The present results suggest that peptide transporters do not control intestinal absorption and renal reabsorption of ACE inhibitors.

The adaptation of the capacity of the intestinal peptide transporter PEPT1 to varying substrate concentrations may be important with respect to its role in providing bulk quantities of amino acids for growth, development, and other nutritional needs. In the present study, we describe a novel phenomenon of the regulation of PEPT1 in the Xenopus oocyte system. Using electrophysiological and immunofluorescence methods, we demonstrate that a prolonged substrate exposure of rabbit PEPT1 (rPEPT1) caused a retrieval of transporters from the membrane. Capacitance as a measure of membrane surface area was increased in parallel with the increase in rPEPT1-mediated transport currents with a slope of approximately 5% of basal surface per 100 nA. Exposure of oocytes to the model peptide Gly-l-Gln for 2 h resulted in a decrease in maximal transport currents with no change of membrane capacitance. However, exposure to substrate for 5 h decreased transport currents but also, in parallel, surface area by endocytotic removal of transporter proteins from the surface. The reduction of the surface expression of rPEPT1 was confirmed by presteady-state current measurements and immunofluorescent labeling of rPEPT1. A similar simultaneous decrease of current and surface area was also observed when endocytosis was stimulated by the activation of PKC. Cytochalasin D inhibited all changes evoked by either dipeptide or PKC stimulation, whereas the PKC-selective inhibitor bisindolylmaleimide only affected PKC-stimulated endocytotic processes but not substrate-dependent retrieval of rPEPT1. Coexpression experiments with human Na(+)-glucose transporter 1 (hSGLT1) revealed that substrate exposure selectively affected PEPT1 but not the activity of hSGLT1.

In this study we described the design, rational synthesis and functional characterization of a novel radiolabeled hydrolysis-resistant high-affinity substrate for H(+)/peptide cotransporters. L-4,4'-Biphenylalanyl-L-Proline (Bip-Pro) was synthesized according to standard procedures in peptide chemistry. The interaction of Bip-Pro with H(+)/peptide cotransporters was determined in intestinal Caco-2 cells constitutively expressing human H(+)/peptide cotransporter 1 (PEPT1) and in renal SKPT cells constitutively expressing rat H(+)/peptide cotransporter 2 (PEPT2). Bip-Pro inhibited the [(14)C]Gly-Sar uptake via PEPT1 and PEPT2 with exceptional high affinity (K(i) = 24 microm and 3.4 microm, respectively) in a competitive manner. By employing the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT1 or PEPT2 it was found that Bip-Pro was transported by both peptide transporters although to a much lower extent than the reference substrate, Gly-Gln. Bip-Pro remained intact to > 98% for at least 8 h when incubated with intact cell monolayers. Bip-[(3)H]Pro uptake into SKPT cells was linear for up to 30 min and pH dependent with a maximum at extracellular pH 6.0. Uptake was strongly inhibited, not only by unlabeled Bip-Pro but also by known peptide transporter substrates such as dipeptides, cefadroxil, Ala-4-nitroanilide and delta-aminolevulinic acid, but not by glycine. Bip-Pro uptake in SKPT cells was saturable with a Michaelis-Menten constant (K(t)) of 7.6 microm and a maximal velocity (V(max)) of 1.1 nmol x 30 min(-1) x mg of protein(-1). Hence, the uptake of Bip-Pro by PEPT2 is a high-affinity, low-capacity process in comparison to the uptake of Gly-Sar. We conclude that Bip-[(3)H]Pro is a valuable substrate for both mechanistic and structural studies of H(+)/peptide transporter proteins.

The human gene RSC1A1 codes for a 67-kDa protein named RS1 that mediates transcriptional and post-transcriptional regulation of Na(+)-D-glucose cotransporter SGLT1. The post-transcriptional regulation occurs at the trans-Golgi network (TGN). We identified two tripeptides in human RS1 (Gln-Cys-Pro (QCP) and Gln-Ser-Pro (QSP)) that induce posttranscriptional down-regulation of SGLT1 at the TGN leading to 40-50% reduction of SGLT1 in plasma membrane. For effective intracellular concentrations IC(50) values of 2.0 nM (QCP) and 0.16 nm (QSP) were estimated. Down-regulation of SGLT1 by tripeptides was attenuated by intracellular monosaccharides including non-metabolized methyl-alpha-D-glucopyranoside and 2-deoxyglucose. In small intestine post-transcriptional regulation of SGLT1 may contribute to glucose-dependent regulation of liver metabolism and intestinal mobility. QCP and QSP are transported by the H(+)-peptide cotransporter PepT1 that is colocated with SGLT1 in small intestinal enterocytes. Using coexpression of SGLT1 and PepT1 in Xenopus oocytes or polarized Caco-2 cells that contain both transporters we demonstrated that the tripeptides were effective when applied to the extracellular compartment. After a 1-h perfusion of intact rat small intestine with QSP, glucose absorption was reduced by 30%. The data indicate that orally applied tripeptides can be used to down-regulate small intestinal glucose absorption, e.g. in diabetes mellitus.

There is controversy as to whether intestinal absorption of glycosylated flavonoids, and particularly quercetin glycosides, involves their uptake in intact form via the human sodium-coupled glucose transporter hSGLT1. We here describe studies using Xenopus oocytes that express hSGLT1 and the two-electrode voltage clamp technique to determine the transport characteristics of a variety of flavonoids carrying glucose residues at different positions as well as of their aglycones (altogether 27 compounds). Neither quercetin, luteolin, apigenin, naringenin, pelarginidin, daidzein, or genistein, nor any of their glycosylated derivatives generated significant transport currents. However, the inward current evoked by 1 mM of the hSGLT1 substrate alpha-methyl-D-glucopyranoside was potently reduced by the simultaneous application of not only various flavonoid glycosides but also by some aglycones. The inhibitory potency remained unchanged when the attached glucose was replaced by galactose, suggesting that these residues may bind to SGLT1. Kinetic analysis by Dixon plots revealed inhibition of competitive type with high affinities, particularly when the glucose was attached to the position 4' of the aromatic ring of the flavonoids. The affinities became lower when the glucose was attached to a different position. Unexpectedly, the aglycone form of luteolin also inhibited the transport competitively with high affinity. These data show that hSGLT1 does not transport any of the flavonoids and seems therefore not involved in their intestinal absorption. However, not only glycosylated but also a few nonglycosylated flavonoids show a structure-dependent capability for effective inhibition of SGLT1.

Uptake of nutrients into cells is essential to life and occurs in all organisms at the expense of energy. Whereas in most prokaryotic and simple eukaryotic cells electrochemical transmembrane proton gradients provide the central driving force for nutrient uptake, in higher eukaryotes it is more frequently coupled to sodium movement along the transmembrane sodium gradient, occurs via uniport mechanisms driven by the substrate gradient only, or is linked to the countertransport of a similar organic solute. With the cloning of a large number of mammalian nutrient transport proteins, it became obvious that a few "archaic'' transporters that utilize a transmembrane proton gradient for nutrient transport into cells can still be found in mammals. The present review focuses on the electrogenic peptide transporters as the best studied examples of proton-dependent nutrient transporters in mammals and summarizes the most recent findings on their physiological importance. Taking peptide transport as a general phenomenon found in nature, we also include peptide transport mechanisms in bacteria, yeast, invertebrates, and lower vertebrates, which are not that often addressed in physiology journals.

Solute carrier 15 (SLC15) membrane proteins PEPT1 (SLC15A1) and PEPT2 (SLC15A2) have been described in great detail in mammals. In contrast, information in lower vertebrates is limited. We characterized the functional properties of a novel zebrafish peptide transporter orthologous to mammalian and avian PEPT2, described its gene (pept2) structure, and determined mRNA tissue distribution. An expressed sequence tag (EST) cDNA (Integrated Molecular Analysis of Gene Expression; IMAGE) corresponding to zebrafish pept2 was completed by inserting a stretch of 75 missing nucleotides in the coding sequence to obtain a 3,238-bp functional clone. The complete open reading frame (ORF) was 2,160 bp and encoded a 719-amino acid protein. Electrophysiological analysis after cRNA injection in Xenopus laevis oocytes suggested that zebrafish PEPT2 is a high-affinity/low-capacity transporter (K(0.5) for glycyl-L-glutamine approximately 18 microM at -120 mV and pH 7.5). Zebrafish pept2 gene was 19,435 kb, thus being the shortest vertebrate pept2 fully characterized so far. Also, zebrafish pept2 exhibited 23 exons and 22 introns, whereas human and rodent pept2 genes contain 22 exons and 21 introns only. Zebrafish pept2 mRNA was mainly detected in brain, kidney, gut, and, interestingly, otic vesicle, the embryonic structure that develops into the auditory/vestibular organ, homolog to the higher vertebrate inner ear, of the adult fish. Characterization of zebrafish pept2 will contribute to the investigation of peptide transporters using a well-established genetic model and will allow the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful marker to screen mutations that affect choroid plexus and inner ear development.

The proton-coupled amino acid transporter PAT1, cloned recently from brain and intestine, mediates the uphill transport of l- and d-proline, l-alanine, glycine, taurine, d-serine, GABA, and many other related compounds and drugs. Here we describe the novel finding that l-tryptophan and its derivatives tryptamine, 5-hydroxy-l-tryptophan, serotonin, and indole-3-propionic acid strongly inhibit H+-dependent l-[3H]proline uptake into Caco-2 cells with inhibition constants (K(i)) of 0.9 to 6.1 mM. Uptake of l-[3H]tryptophan into Caco-2 cells on the other hand was not inhibited by l-proline. Whereas PAT1 substrates produced significant changes in a membrane potential assay for electrogenic transport in Caco-2 cells, l-tryptophan, tryptamine, and 5-hydroxy-l-tryptophan failed to alter membrane voltage. When PAT1 was expressed in Xenopus laevis oocytes and analyzed by the two-electrode voltage clamp technique, glycine elicited high inward currents that were dependent on membrane potential but no currents were observed with l-tryptophan, tryptamine, 5-hydroxy-l-tryptophan, or serotonin. Although not transported electrogenically by PAT1, l-tryptophan and its derivatives inhibited glycine-evoked currents dose-dependently. We conclude that serotonin, l-tryptophan, and tryptamine bind to PAT1 with potencies similar to the prototype substrates, inhibit transport function but are not transported by this carrier protein. They may be considered as the carriers' naturally occurring inhibitors that may alter the transport function of PAT1.

PAT1 is a recently identified member of the PAT family of proton/amino acid co-transporters with predominant expression in the plasma membrane of enterocytes and in lysosomal membranes of neurons. Previous studies in Xenopus oocytes expressing PAT1 established proton/substrate co-transport associated with positive inward currents for a variety of small neutral amino acids. Here we provide a detailed analysis of the transport mode of the murine PAT1 in oocytes using the two-electrode voltage-clamp technique to measure steady-state and pre-steady-state currents. The GPC (giant patch clamp) technique and efflux studies were employed to characterize the reversed transport mode. Kinetic parameters [K(m) (Michaelis constant) and I(max) (maximum current)] for transport of various substrates revealed a dependence on membrane potential: hyperpolarization increases the substrate affinity and maximal transport velocity. Proton affinity for interaction with PAT1 is almost 100 nM, corresponding to a pH of 7.0 and is independent of substrate. Kinetic analysis revealed that binding of proton most likely occurs before substrate binding and that the proton and substrate are translocated in a simultaneous step. No evidence for a substrate-uncoupled proton shunt was observed. As shown by efflux studies and current measurements by the GPC technique, PAT1 allows bidirectional amino acid transport. Surprisingly, PAT1 exhibits no pre-steady-state currents in the absence of substrate, even at low temperatures, and therefore PAT1 takes an exceptional position among the ion-coupled co-transporters.

Recently, the PAT family of proton-dependent amino acid transporters has been identified as a novel class of mammalian amino acid symporters. PAT1 and PAT2 members mediate electrogenic uptake of small, neutral amino acids and derivatives by cotransport of protons. Analysis of the structural requirements for substrate recognition by PAT1 identified that a free amino group in a substrate is not essential for recognition. We therefore hypothesized that PAT1 and its ortholog PAT2 may also be able to recognize and transport the homologous short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate. We examined in Xenopus laevis oocytes whether the SCFAs interact with the transporter by employing flux studies, electrophysiology and intracellular pH recordings. SCFAs did not induce positive inward currents but inhibited glycine-induced transport currents. PAT-mediated uptake of radiolabeled proline was also dose-dependently reduced by SCFA and could be described by first order competition kinetics with apparent Ki-values for butyrate of 6.0 +/- 0.7 and 7.6 +/- 1.3 mM for PAT1 and PAT2, respectively. Acetate as well as propionate uptake was significantly enhanced in oocytes expressing PAT1 or PAT2. An electroneutral H+/SCFA symport mode was demonstrated by recording intracellular pH changes under voltage clamp conditions with rate constants for the initial intracellular acidification in the presence of SCFAs significantly increased in PAT-expressing oocytes. In conclusion, our data demonstrate that the PAT1 and PAT2 proteins are capable to transport selected SCFAs in an electroneutral and the homologous amino acids in an electrogenic mode and are therefore a paradigm for bifunctional solute carriers.

The PAT2 transporter has been shown to act as an electrogenic proton/amino acid symporter. The PAT2 cDNA has been cloned from various human, mouse and rat tissues and belongs to a group of four genes (pat1 to pat4) with PAT3 and PAT4 still resembling orphan transporters. The first immunolocalization studies demonstrated that the PAT2 protein is found in the murine central nervous system in neuronal cells with a proposed role in the intra and/or intercellular amino acid transport. Here we provide a detailed analysis of the transport mode and substrate specificity of the murine PAT2 transporter after expression in Xenopus laevis oocytes, by electrophysiological techniques and flux studies. The structural requirements to the PAT2 substrates - when considering both low and high affinity type substrates - are similar to those reported for the PAT1 protein with the essential features of a free carboxy group and a small side chain. For high affinity binding, however, PAT2 requires the amino group to be located in an alpha-position, tolerates only one methyl function attached to the amino group and is highly selective for the L-enantiomers. Electrophysiological analysis revealed pronounced effects of membrane potential on proton binding affinity, but substrate affinities and maximal transport currents only modestly respond to changes in membrane voltage. Whereas substrate affinity is dependent on extracellular pH, proton binding affinity to PAT2 is substrate-independent, favouring a sequential binding of proton followed by substrate. Maximal transport currents are substrate-dependent which suggests that the translocation of the loaded carrier to the internal side is the rate-limiting step.

Mammalian members of the SLC15 family are electrogenic transporters that utilize the proton-motive force for uphill transport of short chain peptides and peptido-mimetics into a variety of cells. The prototype transporters of this family are PEPT1 (SLC15A1) and PEPT2 (SLC15A2), which mediate the uptake of peptide substrates into intestinal and renal epithelial cells. More recently, other sites of functional expression of the two proteins have been identified such as bile duct epithelium (PEPT1), glia cells and epithelia of the choroid plexus, lung and mammary gland (PEPT2). Both proteins can transport essentially every possible di- and tripeptide regardless of the substrate's net charge, but operate stereoselectively. Based on peptide-like structures, various drugs and prodrugs are transported as well, allowing efficient intestinal absorption of the compounds via PEPT1. In kidney tubules both peptide transporters can mediate the renal reabsorption of the filtered compounds thus affecting their pharmacokinetics. Recently, two new peptide transporters, PHT1 (SLC15A4) and PHT2 (SLC15A3), were identified in mammals. They possess an overall amino acid identity with the PEPT-series of 20% to 25%. PHT1 and PHT2 were shown to transport free histidine and certain di- and tripeptides, but it is not yet clear whether they are located on the plasma membrane or represent lysosomal transporters for the proton-dependent export of histidine and dipeptides from lysosomal protein degradation into the cytosol.

The new member of the mammalian amino acid/auxin permease family, PAT2, has been cloned recently and represents an electrogenic proton/amino acid symporter. PAT2 and its paralog, PAT1/LYAAT-1, are transporters for small amino acids such as glycine, alanine, and proline. Our immunodetection studies revealed that the PAT2 protein is expressed in spinal cord and brain. It is found in neuronal cell bodies in the anterior horn in spinal cord and in brain stem, cerebellum, hippocampus, hypothalamus, rhinencephalon, cerebral cortex, and olfactory bulb in the brain. PAT2 is expressed in neurons positive for the N-methyl-d-aspartate subtype glutamate receptor subunit NR1. PAT2 is not found in lysosomes, unlike its paralog PAT1, but is present in the endoplasmic reticulum and recycling endosomes in neurons. PAT2 has a high external proton affinity causing half-maximal transport activation already at a pH of 8.3, suggesting that its activity is most likely not altered by physiological pH changes. Transport of amino acids by PAT2 activity is dependent on membrane potential and can occur bidirectionally; membrane depolarization causes net glycine outward currents. Our data suggest that PAT2 contributes to neuronal transport and sequestration of amino acids such as glycine, alanine, and/or proline, whereby the transport direction is dependent on the sum of the driving forces such as substrate concentration, pH gradient, and membrane potential.

We report the molecular and functional characterisation of a novel peptide transporter from zebrafish, orthologue to mammalian and avian PEPT1. Zebrafish PEPT1 is a low-affinity/high-capacity system. However, in contrast to higher vertebrate counterparts in which maximal transport activity is independent of extracellular pH, zebrafish PEPT1 maximal transport rates unexpectedly increase at alkaline extracellular pH. Zebrafish pept1 is highly expressed in the proximal intestine since day 4 post-fertilisation, thus preceding functional maturation of the gut, first feeding and complete yolk resorption. Zebrafish PEPT1 might help to understand the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, zebrafish pept1 can be a useful marker for screening mutations that affect gut regionalisation, differentiation and morphogenesis.

The PAT family of proton-dependent amino acid transporters has recently been identified at the molecular level. This paper describes the structural requirements in substrates for their interaction with the cloned murine intestinal proton/amino acid cotransporter (PAT1). By using the Xenopus laevis oocytes as an expression system and by combining the two-electron voltage clamp technique with radiotracer flux studies, it was demonstrated that the aliphatic side chain of L-alpha-amino acids substrates can consist maximally of only one CH2-unit for high affinity interaction with PAT1. With respect to the maximal separation between the amino and carboxyl groups, only two CH2-units, as in gamma-aminobutyric acid (GABA), are tolerated. PAT1 displays no or even a reversed stereoselectivity, tolerating serine and cystein only in the form of D-enantiomers. A methyl-substitution of the carboxyl group (e.g. O-methyl-glycine) markedly diminishes substrate affinity and transport rates, whereas methyl-substitutions at the amino group (e.g. sarcosine or betaine) have only minor effects on substrate interaction with the transporter binding site. Furthermore, it has been shown (by kinetic analyses of radiolabelled betaine influx and inhibition studies) that the endogenous PAT system of human Caco-2 cells has very similar transport characteristics to mouse PAT1. In summary, one has defined the structural requirements and limitations thet determine the substrate specificity of PAT1. A critical recognition criterion of PAT1 is the backbone charge separation distance and the side chain size, whereas substitutions on the amino group are well tolerated.

Mastoparan, a polypeptide known to activate heterotrimeric GTP-binding proteins, enhances the transport of Ca2+ and K+ across membranes. In the present study we investigated the influence of mastoparan on transepithelial resistance (TER) and on short circuit current (SCC) of the intestinal cell line T84. Mastoparan decreased the TER by 80% of baseline and induced a SCC of 8.34+/-1.38 microAcm(-2). The changes in paracellular conductance were estimated using the nystatin technique and showed that mastoparan increased the paracellular conductance 4-fold. Basolateral Cl(-)-free medium, or blockade of the basolateral Cl(-) uptake via the Na+/K+/2Cl(-) co-transporter with bumetanide, reduced SCC of T84 cells, but did not abolish the effect of mastoparan on the TER. Luminal addition of the Cl(-)-channel blocker DIDS or NPPB had no effect on the increase in SCC. In contrast, blocking the basolateral K(+)-channels by 2mM Ba2+ inhibited both the resistance decrease and elevation of the SCC, and further inhibited the mastoparan-induced increase in intracellular free Ca2. This indicates that mastoparan acts primarily via activating K+ channels with a secondary Cl(-) secretion and Ca2+ influx. Reduction of intracellular free Ca2+ did not alter the effect of mastoparan on TER. Stimulation with mastoparan led to a biphasic rearrangement of actin filaments and increased globular actin content in T84 cells. Depolymerization of actin filaments also correlated with inactivation of Rho-proteins, which are known regulators of the cytoskeleton. Mastoparan induced a 2-fold increase in GDI-complexed Rho. We conclude that mastoparan-induced changes in paracellular permeability are mediated via enhanced basolateral K+ conductance and Rho-protein inactivation. A secondary increase in intracellular Ca2+ or direct interaction of small GTPases with the cytoskeleton are likely mediators of the remodeling of the cytoskeleton with subsequent changes in paracellular permeability.

The effects of protein kinase A (PKA)-mediated and protein kinase C (PKC)-mediated stimulation on the tight junctions of the moderately tight Xenopus gallbladder epithelium have been investigated. Transepithelial impedance and DC voltage divider ratio measurements in Ussing-type chambers were used to calculate the cell membrane and tight junction resistances in the stimulated state. Under control conditions the TE resistance was used as a lowest estimate of tight junction resistance. Stimulation of PKA by forskolin and theophyllin as well as stimulation of PKC by phorbol dibutyrate lowered the TE resistance mainly via the reduction of the tight junctional resistance. PKA stimulation opened, in addition, an apical Cl- selective conductance. The paracellular pathway activated by PKA or PKC did not discriminate between small anions and cations. The effects of PKA stimulation could be blocked by the selective inhibition of PKA (with H89) or of PKC (with bisindolylmaleimide). By contrast the PKC-evoked effects were insensitive to H89, showing that the effects of PKA on the paracellular pathway were mediated by PKC.

The capability for electrogenic inward transport of substrates that carry different net charge is a phenomenon observed in a variety of membrane-solute transporters but is not yet understood. We employed the two-electrode voltage clamp technique combined with intracellular pH recordings and the giant patch technique to assess the selectivity for bidirectional transport and the underlying stoichiometries in proton to substrate flux coupling for electrogenic transfer of selected anionic, cationic, and neutral dipeptides by the intestinal peptide transporter PEPT1. Anionic dipeptides such as Gly-Asp and Asp-Gly are transported in their neutral and negatively charged forms with high and low affinities, respectively. The positive transport current obtained with monoanionic substrates results from the cotransport of two protons. Cationic dipeptides can be transported in neutral and positively charged form, resulting in an excess transport current as compared with neutral substrates. However, binding and transport of cationic dipeptides shows a pronounced selectivity for the position of charged side chains demonstrating that the binding domain of PEPT1 is asymmetric, both in its inward and outward facing conformation. The simultaneous presence of identically charged substrates on both membrane surfaces generates outward and, unexpectedly, enhanced inward transport currents probably by increasing the turnover rate.

We cloned two cDNAs encoding proton/amino acid cotransporters, designated as mPAT1 and mPAT2, from murine tissues. They were identified by sequence similarity to the amino acid/auxin permease family member of lower eukaryotes. We functionally characterized both transporters by flux studies and electrophysiology after expression in Xenopus laevis oocytes. Both mPAT1 and mPAT2 induced a pH-dependent electrogenic transport activity for small amino acids (glycine, alanine, and proline) that is altered by membrane potential. Direct evidence for amino acid/H(+)-symport was shown by intracellular acidification, and a flux coupling stoichiometry for proline/H(+)-symport of 1:1 was determined for both transporters. Besides small apolar L-amino acids, the transporters also recognize their D-enantiomers and selected amino acid derivatives such as gamma-aminobutyric acid. The mPAT1 transporter, the murine orthologue of the recently cloned rat LYAAT-1 transporter, can be considered as a low affinity system when compared with mPAT2. The mRNA of mPAT1 is highly expressed in small intestine, colon, kidney, and brain; the mPAT2-mRNA is mainly found in heart and lung. Phenotypically, the PAT1 transporter possesses the same functional characteristics as the previously described proton-dependent amino acid transport process in apical membranes of intestinal and renal epithelial cells.

In this study, we describe the rational synthesis and functional analysis of novel high affinity inhibitors for the mammalian peptide transporter PEPT2. Moreover, we demonstrate which structural properties convert a transported compound into a non-translocated inhibitor. Starting from Lys[Z(NO(2))]-Pro (where Z is benzyloxycarbonyl), which we recently identified as the first competitive high affinity inhibitor of the intestinal peptide transporter PEPT1, a series of different lysine-containing dipeptide derivatives was synthesized and studied for interaction with PEPT2 based on transport competition assays in Pichia pastoris yeast cells expressing PEPT2 heterologously and in renal SKPT cells expressing PEPT2. In addition, the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT2 was used to determine whether the compounds are transported electrogenically or block the uptake of dipeptides. Synthesis and functional analysis of Lys-Lys derivatives containing benzyloxycarbonyl or 4-nitrobenzyloxycarbonyl side chain protections provided a set of inhibitors that reversibly inhibited the uptake of dipeptides by PEPT2 with K(i) values as low as 10 +/- 1 nm. This is the highest affinity of a ligand of PEPT2 ever reported. Moreover, based on the structure-function relationship, we conclude that the spatial location of the side chain amino protecting group in a dipeptide containing a diaminocarbonic acid and its intramolecular distance from the Calpha atom are key factors for the transformation of a substrate into an inhibitor of PEPT2.

The peptide transporter PEPT2, expressed in a variety of tissues, including kidney, lung, and the central nervous system, mediates the uphill transport of di- and tripeptides, as well as a variety of peptidomimetic drugs. To identify the essential molecular features of substrates that determine affinity and transport by PEPT2, we synthesized a series of amino acid derivatives as well as modified dipeptides. Kinetic constants for the interaction of test compounds with PEPT2 were obtained in a competition assay using Pichia pastoris yeast cells expressing mammalian PEPT2. The two-electrode voltage-clamp technique in Xenopus laevis oocytes was used to assess the substrate's electrogenic transport properties. Whereas omega bar-amino fatty acids showed no affinity for PEPT2, the introduction of a single carbonyl group into the backbone increased both affinity and transport currents more than 30-fold. omega bar-amino fatty acids, at their amino or carboxyl group coupled to an alanine residue, allowed us to determine the importance of the spatial position of functional groups within the molecule. Affinity and transport function declined by elongating the omega bar-amino acid chain when located in the N-terminal position, whereas the elongation in the carboxyl terminal with an N-terminal alanine caused less pronounced effects. The results clearly establish that a free N terminus, a correctly positioned backbone carbonyl group, and a carboxylic group that is in a suitable distance from the intramolecular carbonyl function and the amino terminal head group are the main features for substrate recognition and transport by PEPT2. This information provides the framework for a rational design of peptidomimetic drugs for delivery via PEPT2.

1. The giant patch clamp technique in the inside-out configuration and the two-electrode voltage clamp technique were used to characterize the bidirectional transport properties of the proton-coupled peptide carrier PEPT1 expressed in Xenopus laevis oocytes. 2. The addition of the neutral dipeptide Gly-L-Gln to the cytoplasmic solution induced a net outward transport current in a membrane potential range between -80 and +60 mV, even in the absence of a pH gradient. 3. The concentration dependency of the outwardly directed transport currents followed Michaelis-Menten-type kinetics, with an apparent K0.5 of 3.28 mM (at pH 7.5 and +60 mV membrane potential). This apparent affinity is around fivefold lower than the apparent affinity measured for the inward transport mode (K0.5 of 0.70 mM (at pH 7.5 and -60 mV) under identical experimental conditions). 4. Apparent K0.5 values were strongly pH and potential dependent only on the external face for inward transport. The transport currents were potential dependent, but essentially pH independent for inward transport and only modestly altered by pH in the reverse direction. In addition to the membrane potential, the transmembrane substrate gradient acts as a driving force and contributes significantly to total transport currents. 5. The differences in apparent substrate affinity under identical experimental conditions suggest major differences in the conformation of the substrate binding pocket of PEPT1 when exposed to the external versus the internal face of the membrane. The lower affinity on the internal face allows the substrate to be released into the cytosolic compartment even in the absence of a proton-motive force. 6. Our study demonstrates for the first time that PEPT1 can transport dipeptides bidirectionally in an electrogenic and proton-coupled symport mode. When substrates are present on both sides of the membrane in sufficiently high concentrations, the direction and rate of transport are solely dependent on the membrane potential, and transport occurs symmetrically.

The existence of tight junctions between epithelial cells has been known for over 100 years, but their exact functions remained elusive until recently. The present paper summarizes the newest knowledge about morphology, molecular components and regulation of tight junctions. In the chapter about morphology the criteria for classification of epithelia as well as the importance of the "freeze fracture" electron-microscopic technique in forming our present ideas about the fine structure of tight junctions will be discussed. The two existing theories about the molecular composition of the resistance-forming barrier ("lipid-model" and "protein-model", resp.) and the important properties of the tight-junction-associated proteins characterized in the past years are discussed in the chapter about molecular components. Finally, in the last part of the paper the factors playing an important role in the regulation of tight junctions, including the cytoskeleton, the protein kinases A and C, the phospholipase C and two categories of the GTP-binding proteins are reviewed.

We here review the functional properties of the paracellular pathway of leaky epithelia such as gallbladder and renal proximal tubule. These epithelia are characterized by leaky terminal bars between adjacent cells which allow small ions, non-electrolytes and water to leak from lumen to interstitial fluid or back. In the past 10 years a great deal of information has been obtained about the properties of the misnamed 'tight' junctions in the terminal bars, by assuming that the overall permeation pattern reflected predominantly the junctional permeation properties. Although recent trans- and intraepithelial impedance analyses indicate that this assumption is not always justified (the contribution of the lateral intercellular space to the paracellular shunt resistance is not negligible, when the spaces are collapsed) it seems that the major conclusions are correct. The properties of the terminal junctions may thus be summarized as follows. (1) Large molecules such as horseradish peroxidase are not able to pass. (2) Passage of lipophilic substances is insignificant, as these substances permeate by the cellular route. (3) Depending on the tissue, ion permeation is either governed by channels with negative fixed charges, or positive fixed charges, or both. As inferred from ion selectivity patterns the channels of different epithelia are either wide and highly hydrated or narrow and poorly hydrated, thus allowing more or less water molecules to pass besides the ions. In narrow channels single-file diffusion may occur. (4) Besides the selective channels a free solution shunt seems to be present in some epithelia. (5) When applied in millimolar concentrations 2,4,6-triaminopyrimidinium and amiloride block negatively charged junctional channels. However these substances do not simply turn leaky epithelia into tight epithelia, because they have additional effects on the cell membranes. (6) As observed in cell cultures, formation of tight junctions requires connecting particles to be present on the cell surface--which seems to be controlled by the cytoskeleton-- and requires the presence of calcium ions as ligands. (7) Cellular control over paracellular permeability may be exerted through changes of intracellular calcium concentration.

A "minimal natriuretic and diuretic" dose (3.4--4.2 ng/min/kg) of prostaglandin E1 (PGE1) as previously estimated was infused into the aorta in anaesthetized rats. During the PGE1 infusion parameters for renal haemodynamics, Na+ and water excretion, intra- and peritubular pressures were studied ("electronic servo nulling device"). During the solvent period parameters for the infused left kidney did not differ from those on the control side. Diuresis and Na+ excretion were increased significantly due to PGE1 infusion in both series of investigations. Cortical blood flow (radio isotope labelled microsphere technique) and glomerular filtration (inulin clearance) did not change significantly. Oncotic pressure in the afferent arteriole was not affected by PGE1, whereas it was reduced significantly in the efferent arteriole (control, 22.9 +/- 1.7; PGE1, 19.2 +/- 0.9 mmHg). Transit times to the early and late distal tubules were not affected by PGE1. There were no changes in the hydrostatic pressure in the proximal tubule and efferent arteriole, whereas the peritubular capillary hydrostatic pressure was significantly increased (control, 9.6 +/- 0.3; PGE1, 11.2 +/- 0.2 mmHg). The present results indicate that PGE1 is capable of enhancing Na+ and water excretion without affecting RBF and GFR, and peritubular physical factors might play an auxiliary role in this effect.

Proximal transtubular potential difference (PD) was measured using the semi-microelectrode technique in control (C, n = 10) and unilaterally denervated (D, n = 10) rats. Acute renal sympathectomy resulted in a twofold and fourfold increase in diuresis and sodium excretion, respectively, with no change in GFR. PD (mean +/- S.E.M.) in the earliest accessible proximal convolutions (EPT) of group C was +/- 0.27 +/- 0.08 mV (n = 16), while in group D it was -0.16 +/- 0.07 mV (n = 18) (p less than 0.01). PD in mid (MPT) and late (LPT) proximal segments was unchanged by denervation (MPT: C = 0.94 +/- 0.05, n = 21; D = 0.98 +/- 0.04, n = ns. LPT: C = 1.04 +/- 0.11, n = 17; D = 0.95 +/- 0.06, n = 18 NS.). The shift to the negative of PD in EPT caused by denervation lends support to our earlier suggestion that active transport of solutes and organic substances is depressed by sympathectomy in the proximal tubule. The unaltered PD in MPT and LPT seems to indicate that passive forces promoting reabsorption in this part of the nephron are probably preserved.

The acetylcholine induced contralateral renorenal vasomotor reflex has been re-examined. In dogs under pentobarbital anaesthesia blood flow of the contralateral kidney as measured by electromagnetic flow probe did not change after an infusion or bolus injection of Ach. Basal sympathetic nervous activity of the contralateral kidney increased only slightly and these changes correlated with the decrease of arterial blood pressure. In conclusion, the renorenal vasomotor reflex could not be evoked under the present experimental conditions.