The peptide transporter PepT2 mediates the uptake of the glutathione precursor CysGly in astroglia-rich primary cultures

The intracellular content of glutathione in astroglia-rich primary cultures derived from the brains of newborn rats was used as an indicator for the ability of these cultures to utilize cysteinylglycine (CysGly) for glutathione synthesis. After a 24-h starvation period in the absence of glucose and amino acids, CysGly was able to substitute for cysteine plus glycine in the restoration of glutathione. Glutathione restoration from CysGly plus glutamate was only slightly affected by the dipeptides carnosine or serylglycine in a 200-fold excess. Captopril, a substrate of the peptide transporter PepT1, had almost no effect on glutathione restoration. In contrast, with increasing concentrations of alanylalanine or cefadroxil, known substrates of the peptide transporter PepT2, the amount of glutathione restored in the presence of CysGly and glutamate was strongly reduced. Cefadroxil in a 200-fold excess totally prevented the utilization of CysGly for glutathione restoration. The presence of mRNA for PepT2 in astroglia-rich primary cultures was demonstrated by application of RT-PCR. These results demonstrate that PepT2 is expressed in astroglia-rich primary cultures and that this transporter is highly likely to be responsible for the uptake of CysGly in these cultures.     

Cloning and expression of the gene for the Na+-coupled serine transporter from Escherichia coli and characteristics of the transporter

We cloned a gene (sstT) for the Na+/serine symporter from the chromosome of Escherichia coli by using a low-copy-number vector and sequenced it. According to the deduced amino acid sequence, the transporter (SstT) consists of 414 amino acid residues. Hydropathy analysis suggested that the SstT protein possesses 9, instead of 12, hydrophobic domains.     

Identification, cloning and characterization of a derepressible Na+-coupled phosphate transporter in Saccharomyces cerevisiae

Based on the high sequence homology between the yeast ORF YBR296c (accession number P38361 in the SWISS-PROT database) and the PHO4 gene of Neurospora crassa, which codes for a Na+/Pi cotransporter with twelve putative transmembrane domains, the YBR296c ORF was considered to be a promising candidate gene for a plasma membrane-bound phosphate transporter in Saccharomyces cerevisiae. Therefore, this gene, here designated PHO89, was cloned and a set of deletion mutants was constructed. We then studied their Pi uptake activity under different conditions. We show here that a transport activity displayed by PHO89 strains under alkaline conditions and in the presence of Na+ is absent in pho89 null mutants. Moreover, when the pH was lowered to pH 4.5 or when Na+ was omitted, this activity decreased significantly, reaching values close to those exhibited by the deltapho89 mutant. Studies of the acid phosphatase activity of these strains, as well as promoter sequence analysis, suggest that expression of the PHO89 gene is under the control of the PHO regulatory system. Northern analysis shows that this gene is only transcribed under conditions of Pi limitation. This is, to our knowledge, the first demonstration that the PHO89 gene codes for the Na+/Pi cotransporter previously characterized by kinetic studies, and represents the only Na+-coupled secondary anion transport system so far identified in S. cerevisiae. Pho89p has been shown to have an apparent Km of 0.5 microM and a pH optimum of 9.5, and is highly specific for Na+; activation of transport is maximal at a Na+ concentration of 25 mM.     

Regulation of the high-affinity H+/peptide cotransporter in renal LLC-PK1 cells

Di- and tripeptides and peptide mimetics such as beta-lactam antibiotics are efficiently reabsorbed from the tubular lumen by a high-affinity peptide transporter. We have recently identified and characterized this H+-coupled high-affinity peptide transport system in the porcine proximal tubular cell line LLC-PK1. Here we describe for the first time the regulation of the renal high-affinity peptide cotransporter at the cellular level. Uptake of 5 microM 3H-D-Phe-L-Ala into LLC-PK1 cells was significantly increased by lowering [Ca2+]in and decreased by increasing [Ca2+] in. Moreover, it was shown that the [Ca2+]in effects on peptide transport activity were dependent on Ca2+ entry from the extracellular site (e.g., via a store-regulated capacitative Ca2+ influx). Protein kinase C (PKC) was found to transmit the effects of [Ca2+]in on peptide transport. Although we demonstrate by pHin measurements that the PKC inhibitor staurosporine did decrease the transmembrane H+ gradient and consequently should have reduced the driving force for peptide uptake, the only effect on transport kinetics of 3H-D-Phe-L-Ala observed was a significant decrease in Km from 22.7+/-2.5 microM to 10.2+/-1.9 microM with no change in maximal velocity.     

H(+)-coupled alpha-methylaminoisobutyric acid transport in human intestinal Caco-2 cells

Numerous theoretical as well as pharmacological arguments lead to the assumption that anxiety and memory are two closely linked concepts. Nevertheless, the study of this relationship is full of complexities because neither memory nor anxiety are unitary phenomena. Indeed, the term memory covers a large number of concepts, and anxiety has been divided in two main classes, "state" and "trait" anxiety. Recently the neophobic responses exhibited by Balb/c mice confronted to the free exploratory paradigm have been proposed as a "trait anxiety" model while response exhibited in the light/dark choice procedure as a "state anxiety" one. The aim of this study was to further clarify the link between these two anxiety types and memory of emotional events assessed in the passive avoidance test. The relationship between the variables measured in these three tests were assessed by a principal component analysis that confirmed that the behavior recorded in the two anxiety tests does not reflect the same psychological state, and showed that emotional memory is linked to "state" but not "trait" anxiety.     

H+ permeation and pH regulation at a mammalian serotonin transporter

The rat serotonin transporter expressed in Xenopus oocytes displays an inward current in the absence of 5-HT when external pH is lowered to 6.5 or below. The new current differs from the leakage current described previously in two ways. (1) It is approximately 10-fold larger at pH 5 than the leakage current at pH 7.5 and reaches 1000 H+/sec per transporter at extremes of voltage and pH with no signs of saturation. (2) It is selective for H+ by reversal potential measurements. Similar H+-induced currents are also observed in several other ion-coupled transporters, including the GABA transporter, the dopamine transporter, and the Na+/glucose transporter. The high conductance and high selectivity of the H+-induced current suggest that protons may be conducted via a hydrogen-bonded chain (a "proton-wire mechanism") formed at least partially by side chains within the transporter. In addition, pH affects other conducting states of rat serotonin transporter. Acidic pH potentiates the 5-HT-induced, transport-associated current and inhibits the hyperpolarization-activated transient current. The dose-response relationships for these two effects suggest that two H+ binding sites, with pKa values close to 5.1 and close to 6.3, govern the potentiation of the 5-HT-induced current and the inhibition of the transient current, respectively. These results are important for developing structure-function models that explain permeation properties of neurotransmitter transporters.     

Structure, function, and molecular modeling approaches to the study of the intestinal dipeptide transporter PepT1

The proton-coupled intestinal dipeptide transporter, PepT1, has 707 amino acids, 12 putative transmembrane domains (TMD), and is of importance in the transport of nutritional di- and tripeptides and structurally related drugs, such as penicillins and cephalosporins. By using a combination of molecular modeling and site-directed mutagenesis, we have identified several key amino acid residues that effect catalytic transport properties of PepT1. Our molecular model of the transporter was examined by dividing it into four sections, parallel to the membrane, starting from the extracellular side. The molecular model revealed a putative transport channel and the approximate locations of several aromatic and charged amino acid residues that were selected as targets for mutagenesis. Wild type or mutagenized human PepT1 cDNA was transfected into human embryonic kidney (HEK293) cells, and the uptake of tritiated glycylsarcosine [3H]-(Gly-Sar) was measured. Michaelis-Menton analysis of the wild-type and mutated transporters revealed the following results for site-directed mutagenesis. Mutation of Tyr-12 or Arg-282 into alanine has only a very modest effect on Gly-Sar uptake. By contrast, mutation of Trp-294 or Glu-595 into alanine reduced Gly-Sar uptake by 80 and 95%, respectively, and mutation of Tyr-167 reduced Gly-Sar uptake to the level of mock-transfected cells. In addition, preliminary data from fluorescence microscopy following the expression of N-terminal-GFP-labeled PepT1Y167A in HEK cells indicates that the Y167A mutation was properly inserted into the plasma membrane but has a greatly reduced Vmax.     

Thyrotropin-releasing activity of histone H2A, H2B and peptide MB35

Histones possess multiple hormone-like activities. We studied the specificity and signal transduction pathways involved in the thyrotrophin (TSH)-releasing activity of histones H2A, H2B and peptide MB35, a H2A fragment, using perifused and incubated dispersed rat pituitary cells and measuring TSH release by a specific R1A. Histones released TSH in a dose- and time-dependent fashion while peptide MB35 behaved as a weaker secretagogue. Trifluoperazine and EGTA blocked histone-stimulated TSH release while forskolin and other cAMP enhancers did not. We conclude that the TSH-releasing activity of histones H2A and H2B is mediated by calcium- and diacylglycerol-associated pathways.     

Spectroscopic characterization of a high-affinity calmodulin-target peptide hybrid molecule

We describe the properties of a hybrid protein comprising the full length of the Xenopus laevis calmodulin sequence, followed by a pentapeptide linker (GGGGS), and residues 3-26 of M13, the calmodulin binding region of skeletal muscle myosin light chain kinase. The properties of the hybrid protein are compared with those of the complex formed between Drosophila calmodulin and a peptide corresponding to residues 1-18 of the M13 sequence. The addition of calcium to the hybrid protein produces pronounced changes in the near- and far-UV CD spectra, in the fluorescence emission spectrum of the single tryptophan residue at position 4 in the M13 sequence, and in the accessibility of this tryptophan residue to acrylamide quenching. These changes are consistent with the tryptophan residue being immobilized in a hydrophobic environment and with the hybrid protein adopting a more alpha-helical structure when calcium is bound. The increased alpha-helicity derives from changes in both the calmodulin and peptide regions of the hybrid protein. Changes in the circular dichroism and fluorescence properties of the hybrid protein as a function of the calcium to hybrid protein ratio are consistent with the fact that these changes parallel the cooperative binding of all four calcium ions. The hybrid protein shows greatly increased affinity (>250-fold) for calcium compared with calmodulin itself. Macroscopic calcium binding constants (K(1)-K(4)) were determined from calcium titrations performed in the presence of the calcium chelator Quin 2. Values for log(K(1)K(2)) and log(K(3)K(4)) were determined to be 15.4 +/- 0.2 and 15.59 +/- 0.22 (20 degrees C). The corresponding values for Drosophila calmodulin alone are 11.65 +/- 0.15 and 9.66 +/- 0.25. Consistent with this increased affinity for calcium stopped-flow kinetic studies suggest that the dissociation rate for the N-terminal calcium ions is reduced to at least 0.77 s(-1), compared with approximately 700 s(-1) for Drosophila calmodulin in the absence of peptide. This hybrid protein illustrates the principle whereby the binding of a peptide sequence covalently attached to calmodulin can enhance the average calcium affinity by more than 2 orders of magnitude. Conversely, the target sequence in the hybrid protein undergoes a calcium-induced conformational change to bind to the calmodulin in a conformation very similar to that of the corresponding dissociable target sequence binding to calmodulin, but with a greatly enhanced affinity due to its physical proximity to the binding site. This avoidance of the energetic penalty of dissociation may be a key contributory factor in determining the high affinity and specificity of the complex multiple interactions involved in recognition of biological targets by calmodulin.     

The predominant contribution of oligopeptide transporter PepT1 to intestinal absorption of beta-lactam antibiotics in the rat small intestine

Although recent evidence suggests that certain beta-lactam antibiotics are absorbed via a specific transport mechanism, its nature is unclear. To confirm whether peptide transport in the rat can be largely ascribed to the intestinal oligopeptide transporter PepT1, the transporter has been functionally characterized and its significance in the intestinal absorption of beta-lactam antibiotics was evaluated. For evaluation of transport activity complementary RNA (cRNA) of rat PepT1 was synthesized in-vitro and expressed in Xenopus laevis oocytes. cRNA induced uptake of several beta-lactam antibiotics and the dipeptide [14C]glycylsarcosine; this was specifically inhibited by various dipeptides and tripeptides but not by their constituent amino acids or by tetra- or pentapeptides. The transport activity of PepT1 for beta-lactam antibiotics correlated well with their in-vivo intestinal transport and absorption. Furthermore, mutual inhibitory effects on uptake were observed between glyclsarcosine and beta-lactam antibiotics. Hybrid depletion of the functional expression of rat PepT1 in oocytes injected with rat intestinal epithelial total mRNA was studied using an antisense oligonucleotide corresponding to the 5'-coding region of PepT1. In oocytes injected with rat mRNA pre-hybridized with the antisense oligonucleotide against rat PepT1, the uptake of [14C]glycylsarcosine was almost completely abolished, whereas its uptake was not influenced by a sense oligonucleotide for the same region of PepT1. Similarly, the uptake of beta-lactam antibiotics was also reduced by the antisense oligonucleotide against rat PepT1. These results demonstrate that the intestinal proton-coupled oligopeptide transporter PepT1 plays a predominant role in the carrier-mediated intestinal absorption of beta-lactam antibiotics and native oligopeptides in the rat.     

Protein kinase CK2: biphasic kinetics with peptide substrates

1. The human umbilical vein has been found to contract in response to bradykinin (BK) and desArg9BK. 2. The rank order of potency of agonists, in the presence of the B1 receptor antagonist Lys[Leu8]desArg9BK, is as follows: [Hyp3, Tyr(Me)8]BK (pD2 8.88) = [Hyp3]BK (pD2 8.86) = LysBK (pD2 8.81) > or = BK (pD2 8.60) > [Aib7]BK (pD2 6.38) > desArg9BK and LysdesArg9BK (inactive). 3. Hoe 140 (pA2 8.42) inhibits the effects of BK while other B2 receptor peptide antagonists are very weak and WIN 64338 is practically inactive. 4. Venoconstrictor responses to desArg9BK of fresh tissues increase with time during the in vitro incubation and reach a maximum after 4-6 h. The activity of Hoe 140 (pA2 5.48) is negligible against B1 receptor agonists. 5. When measured in the presence of the selective B2 receptor antagonist Hoe 140 (400 nM), the order of potency of kinin related peptides on the B1 receptor is Lys[desArg9]BK (pD2 8.60) > desArg9BK (pD2 6.69). BK, LysBK, [Hyp3]BK and other B2 receptor agonists are inactive. 6. The B1 receptor antagonist, Lys[Leu8]desArg9BK (pA2 7.99), inhibits the response of the human vein to B1 receptor agonists (LysdesArg9BK or desArg9BK), but do not alter the effect of BK. 7. The results summarized in this paper indicate that the human isolated umbilical vein is a sensitive preparation containing both B1 and B2 receptors. The human B2 receptor shows some similarity with that of the rabbit (at least for agonist potencies) and differs from the B2 receptor of the guinea-pig. Compared to the rabbit B1 receptor, the human B1 receptor shows low sensitivity to peptides that lack the N-terminal Lys.