Iron uptake in Pseudomonas aeruginosa mediated by N-(2,3-dihydroxybenzoyl)-L-serine and 2,3-dihydroxybenzoic acid

Abstract Pseudomonas aeruginosa is known to have an inducible uptake system for the enterobacterial siderophore enterobactin. In this work we have examined iron transport mediated by the biosynthetic precursor 2,3-dihydroxybenzoic acid and N -(2,3-dihydroxybenzoyl)- l -serine, a breakdown product of enterobactin. Iron complexed with 2,3-dihydroxybenzoyl-L-serine was transported into P. aeruginosa IA1 via a transport system which is energy-dependent and iron-repressible. The rate of transport was not altered by growing the cells in the presence of either pyoverdin or pyochelin, which have been shown previously to induce transport via that system. Growth of the cells in the presence of enterobactin did cause an increase in the rate of transport, indicating that the complex can be transported by the inducible enterobactin uptake system, but also that a separate system must exist. In contrast, transport of iron complexed with 2,3-dihydroxybenzoic acid was neither iron-repressible nor strongly energy-dependent, from which we conclude that there must be a novel mode of transport not characteristic of iron-siderophore transport systems.     

Characterization of basal and methylxanthine-stimulated Ca2+ transport in abalone spermatozoa

Methylxanthines, such as 1-methyl-3-isobutylxanthine (MIX) and theophylline, stimulate abalone sperm 45Ca2+ uptake in a time- and concentration-dependent manner. MIX is the most potent compound tested, and the ability of these compounds to alter 45Ca2+ uptake resides with methyl or isobutyl substitution of the xanthine nucleus at multiple sites. Methylxanthine-stimulated 45Ca2+ uptake does not occur as a secondary consequence of cyclic nucleotide phosphodiesterase inhibition, and added cyclic nucleotides are also without effect. The dramatic elevation of intracellular cAMP concentrations and induction of the acrosome reaction of sperm incubated with methylxanthines in the presence of Ca2+ is mediated by a primary effect of methylxanthines on Ca2+ transport. Basal 45Ca2+ uptake occurs through a verapamil-insensitive site that obeys the properties of a simple diffusion-mediated process. MIX-stimulated 45Ca2+ uptake occurs through a carrier-mediated transport site that has low affinity for Ca2+ (Km = 19.9 mM) and is verapamil sensitive. 45Ca2+ uptake through both basal and MIX-stimulated sites is enhanced by low extracellular Na+ concentrations (less than or equal to 15 mM) and is not affected by either extracellular Mg2+ or K+ X 45Ca2+ uptake through both sites is pH sensitive, but this sensitivity is different for each site. These data suggest that methylxanthines can affect sperm function via primary effects on Ca2+ transport, which occur through a specific carrier-mediated site(s). It is possible that many of the previously described effects of methylxanthines on sperm function are mediated via such changes in Ca2+ conductance.     

Vanadate uptake in Neurospora crassa occurs via phosphate transport system II.

Vanadate, a potent inhibitor of plasma membrane ATPases, is taken up by Neurospora crassa only when cells are growing in alkaline medium and starving for phosphate. The appearance of a vanadate uptake system (Km = 8.2 microM; Vmax = 0.15 mmol/min per liter of cell water) occurs under the same conditions required for derepression of a high-affinity phosphate transport system. Phosphate is a competitive inhibitor of vanadate uptake, and vanadate is a competitive inhibitor of phosphate uptake. Furthermore, mutant strains which are either partially constitutive or non-derepressible for the high-affinity phosphate transport system are also partially constitutive or non-derepressible for vanadate uptake. These data indicate that vanadate enters the cell via phosphate transport system II.     

Nateglinide uptake by a ceftibuten transporter in the rat kidney brush-border membrane

Nateglinide, a novel oral hypoglycemic agent, possesses a carbonyl group and a peptide-type bond in its structure. We previously reported that nateglinide transport occurs via a single system that may be identical to the ceftibuten/H(+) cotransport system by the rat small intestine. We speculated that the absorption system present on the intestinal epithelium may be similar to that found on the renal tubular epithelium. The aim of this study was to characterize the transporters on the apical side of the kidney that may contribute to the reabsorption of ceftibuten and nateglinide. The uptake of nateglinide by rat renal brush-border membranes is associated with an H(+)-coupled transport system. Ceftibuten competitively inhibited H(+)-dependent nateglinide uptake. In contrast, Gly-Sar, cephradine and cephalexin had no effect on nateglinide uptake. Nateglinide competitively inhibited H(+)-driven transporter-mediated ceftibuten uptake. We conclude that nateglinide transport occurs via a single system that is H(+)-dependent and may be identical to the ceftibuten/H(+) cotransport system.     

ON THE UPTAKE OF INOSITOL BY RAT BRAIN SYNAPTOSOMES

The uptake of inositol by rat brain synaptosomes occurs via an unsaturable process that even at substrate concentrations as low as 1 μM is unable to achieve a concentration gradient indicative of active transport. Dinitrophenol, ouabain and cytochalasin B did not affect uptake of the cyclitol. The data indicate that inositol uptake by rat synaptosomes occurs by diffusion or by a system with an affinity so low it is difficult to discern. The low capacity, saturable inositol uptake system observed in rabbit brain slices may reflect a species difference or uptake by elements of the slice other than neuronal membranes.     

Effect of 2,4 dinitrophenol and ouabain on the ability of cesium ions to substitute for intracellular potassium ions in isolated and perfused turtle heart (author's transl)]

1. It has been shown that most of the intracellular potassium of turtle heart can be replaced by cesium. 2. The uptake of cesium is reduced by the presence of either DNP 5.10(-5) M or ouabain 10(-6) M in the external medium. The presence of 10(-5) M ouabain markedly inhibits the uptake of cesium. 3. These results lead to the conclusion that the intracellular accumulation of cesium occurs via an active inward transport system wich operates with the simultaneous efflux of sodium.     

Cadmium transport in isolated enterocytes of freshwater rainbow trout: interactions with zinc and iron, effects of complexation with cysteine, and an ATPase-coupled efflux

The present study investigated the mechanisms of intestinal cadmium (Cd) uptake and efflux, using isolated enterocytes of freshwater rainbow trout (Oncorhynchus mykiss) as the experimental model. The apical uptake of free Cd(2+) in the enterocytes was a saturable and high-affinity transport process. Both zinc (Zn(2+)) and iron (Fe(2+)) inhibited cellular Cd(2+) uptake through a competitive interaction, suggesting that Cd(2+) enters enterocytes via both Zn(2+) (e.g., ZIP8) and Fe(2+) (e.g., DMT1) transport pathways. Cellular Cd(2+) uptake increased in the presence of HCO(3)(-), which resembled the function of mammalian ZIP8. Cellular Cd(2+) uptake was unaffected by Ca(2+), indicating that Cd(2+) does not compete with Ca(2+) for apical uptake. Interestingly, Cd uptake was influenced by the presence of l-cysteine, and under the exposure condition where Cd(Cys)(+) was the predominant Cd species, cellular Cd uptake rate increased with the increased concentration of Cd(Cys)(+). The kinetic analysis indicated that the uptake of Cd(Cys)(+) occurs through a low capacity transport mechanism relative to that of free Cd(2+). In addition, Cd efflux from the enterocytes decreased in the presence of an ATPase inhibitor (orthovanadate), suggesting the existence of an ATPase-coupled extrusion process. Overall, our findings provide new mechanistic insights into the intestinal Cd transport in freshwater fish.     

Identification of a ryanodine receptor in rat heart mitochondria

Recent studies have shown that, in a wide variety of cells, mitochondria respond dynamically to physiological changes in cytosolic Ca(2+) concentrations ([Ca(2+)](c)). Mitochondrial Ca(2+) uptake occurs via a ruthenium red-sensitive calcium uniporter and a rapid mode of Ca(2+) uptake. Surprisingly, the molecular identity of these Ca(2+) transport proteins is still unknown. Using electron microscopy and Western blotting, we identified a ryanodine receptor in the inner mitochondrial membrane with a molecular mass of approximately 600 kDa in mitochondria isolated from the rat heart. [(3)H]Ryanodine binds to this mitochondrial ryanodine receptor with high affinity. This binding is modulated by Ca(2+) but not caffeine and is inhibited by Mg(2+) and ruthenium red in the assay medium. In the presence of ryanodine, Ca(2+) uptake into isolated heart mitochondria is suppressed. In addition, ryanodine inhibited mitochondrial swelling induced by Ca(2+) overload. This swelling effect was not observed when Ca(2+) was applied to the cytosolic fraction containing sarcoplasmic reticulum. These results are the first to identify a mitochondrial Ca(2+) transport protein that has characteristics similar to the ryanodine receptor. This mitochondrial ryanodine receptor is likely to play an essential role in the dynamic uptake of Ca(2+) into mitochondria during Ca(2+) oscillations.     

Silicon reduces sodium uptake in rice (Oryza sativa L.) in saline conditions and this is accounted for by a reduction in the transpirational bypass flow

Rice is relatively sensitive to salinity and is classified as a silicon accumulator. There have been reports that silicon can reduce sodium uptake in crop grasses in saline conditions, but the mechanism by which silicon might alleviate salinity damage is unclear. We report on the effects of silicon on growth, gas exchange and sodium uptake in rice genotypes differing in salt tolerance. In non-saline media there were no effects of supplementary silicate upon shoot fresh or dry weight or upon root dry weight, indicating that the standard culture solution was not formally deficient with respect to silicon. Plants grown with supplementary silicate had slightly, but significantly, shorter leaves than plants grown in a standard culture solution. Salinity reduced growth and photosynthetic gas exchange. Silicate supplementation partly overcame the reduction in growth and net photosynthesis caused by salt. This amelioration was correlated with a reduction in sodium uptake. Silicate supplementation increased the stomatal conductance of salt-treated plants, showing that silicate was not acting to reduce sodium uptake via a reduction in the transpiration rate. Silicate reduced both sodium transport and the transport of the apoplastic tracer trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS). This implies that the mode of action of silicate was by partial blockage of the transpirational bypass flow, the pathway by which a large proportion of the uptake of sodium in rice occurs. Mechanisms by which silicate might reduce the transpirational bypass flow directly are discussed.     

5-fluorotryptophan resistant mutants affecting the A and L transport systems in the mouse L cell line A9

Tryptophan transport has been examined in A9 and in mutants resistant to 5-fluorotryptophan (5-FT). Evidence indicates that in A9 cells two systems are present for tryptophan transport, which are analogous to the A and L systems found in Ehrlich ascites cells differing, however, in terms of amino acid specificity. Tryptophan uptake via the L system, a high affinity, low capacity system, is Na⁺ independent and occurs by a counter transport mechanism, while uptake via the A system, a low affinity, high capacity system, is Na⁺ dependent. Alanine, arginine, lysine, proline, asparagine, and aspartate (listed in order of decreasing inhibitory effect) inhibit tryptophan uptake via the A system from approximately 80-50% while having no inhibitory effect on the L system. In addition, glutamine which inhibits tryptophan uptake by 80% via the L system only inhibits to the extent of 20% via the A system. Previous kinetic studies of 5FT resistant clone FT^r37 indicated system A was altered while the analysis of the effects of the mutation on system L was inconclusive. However, in these studies Na⁺ independent uptake was not altered in FT^r 37 indicating system L was not affected. Amino acid competition studies confirmed this observation and suggested that a change in the specificity of system A had occurred in FT^r 37. The amino acid competition studies in FT^r 23, indicated that the specificities of both systems differed from A9. The possibility that this change may be due to a single mutational event is discussed.     

Nateglinide uptake by a ceftibuten transporter in the rat kidney brush-border membrane

Nateglinide, a novel oral hypoglycemic agent, possesses a carbonyl group and a peptide-type bond in its structure. We previously reported that nateglinide transport occurs via a single system that may be identical to the ceftibuten/H⁺ cotransport system by the rat small intestine. We speculated that the absorption system present on the intestinal epithelium may be similar to that found on the renal tubular epithelium. The aim of this study was to characterize the transporters on the apical side of the kidney that may contribute to the reabsorption of ceftibuten and nateglinide. The uptake of nateglinide by rat renal brush-border membranes is associated with an H⁺-coupled transport system. Ceftibuten competitively inhibited H⁺-dependent nateglinide uptake. In contrast, Gly-Sar, cephradine and cephalexin had no effect on nateglinide uptake. Nateglinide competitively inhibited H⁺-driven transporter-mediated ceftibuten uptake. We conclude that nateglinide transport occurs via a single system that is H⁺-dependent and may be identical to the ceftibuten/H⁺ cotransport system.     

Mechanism of Na(+)-dependent citrate transport in Klebsiella pneumoniae.

Citrate transport via CitS of Klebsiella pneumoniae has been shown to depend on the presence of Na+. This transport system has been expressed in Escherichia coli, and uptake of citrate in E. coli membrane vesicles via this uptake system was found to be an electrogenic process, although the pH gradient is the main driving force for citrate uptake (M. E. van der Rest, R. M. Siewe, T. Abee, E. Schwartz, D. Oesterhelt, and W. N. Konings, J. Biol. Chem. 267:8971-8976, 1992). Analysis of the affinity constants for the different citrate species at different pH values of the medium indicates that H-citrate2- is the transported species. Since the electrical potential across the membrane is a driving force for citrate transport, this indicates that transport occurs in symport with at least three monovalent cations. Citrate efflux is stimulated by Na+ concentrations of up to 5 mM but inhibited by higher Na+ concentrations. Citrate exchange, however, is stimulated by all Na+ concentrations, indicating sequential events in which Na+ binds before citrate for translocation followed by a release of Na+ after release of citrate. CitS has, at pH 6.0 and in the presence of 5 mM citrate on both sides of the membrane, an apparent affinity (K(app)) for Na+ of 200 microM. The Na+/citrate stoichiometry was found to be 1. It is postulated that H-citrate2- is transported via CitS in symport with one Na+ and at least two H+ ions.     

Existence of two parallel mechanisms for glucose uptake in heterotrophic plant cells

The implied existence of two mechanisms for glucose uptake into heterotrophic plant cells was investigated using the fluorescent glucose derivative 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), two membrane impermeable fluorescent markers (3000 mol. wt. dextran-Texas Red (d-TR) and Alexa-488), hexose carrier and endocytic inhibitors (phloridzin and wortmannin-A, respectively), and fluorescent and confocal microscopy. Both phloridzin and wortmannin-A significantly reduced the uptake of 2-NBDG into sycamore cultured cells, which was confirmed by fluorescent microscopy. Phloridzin prevented 2-NBDG uptake exclusively into the cytosol, whereas the wortmannin-A effect was more general, with 2-NBDG uptake into the vacuole being the more affected. Simultaneous incubation of cells in the membrane-impermeable fluorescent probes Alexa-488 and d-TR for 24 h resulted in co-localization of the labelling in the central vacuole and other endosomal compartments. Cytoplasts, cells devoid of vacuoles, were instrumental in demonstrating the transport of 2-NBDG by separate uptake mechanisms. In cytoplasts incubated simultaneously in 2-NBDG and d-TR for 2 h, a green fluorescent cytosol was indicative of transport of hexoses across the plasmalemma, while the co-localization of 2-NBDG and d-TR in internal vesicles demonstrated transport via an endocytic system. The absence of vesicles when cytoplasts were pre-incubated in wortmannin-A authenticated the endocytic vesicular nature of the co-shared 2-NBDG and d-TR fluorescent structures. In summary, uptake of 2-NBDG occurs by two separate mechanisms: (i) a plasmalemma-bound carrier-mediated system that facilitates 2-NBDG transport into the cytosol, and (ii) an endocytic system that transports most of 2-NBDG directly into the vacuole.     

Alanine metabolism, transport, and cycling in the brain

Brain glutamate/glutamine cycling is incomplete without return of ammonia to glial cells. Previous studies suggest that alanine is an important carrier for ammonia transfer. In this study, we investigated alanine transport and metabolism in Guinea pig brain cortical tissue slices and prisms, in primary cultures of neurons and astrocytes, and in synaptosomes. Alanine uptake into astrocytes was largely mediated by system L isoform LAT2, whereas alanine uptake into neurons was mediated by Na⁺-dependent transporters with properties similar to system B⁰ isoform B⁰AT2. To investigate the role of alanine transport in metabolism, its uptake was inhibited in cortical tissue slices under depolarizing conditions using the system L transport inhibitors 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and cycloleucine (1-aminocyclopentanecarboxylic acid; cLeu). The results indicated that alanine cycling occurs subsequent to glutamate/glutamine cycling and that a significant proportion of cycling occurs via amino acid transport system L. Our results show that system L isoform LAT2 is critical for alanine uptake into astrocytes. However, alanine does not provide any significant carbon for energy or neurotransmitter metabolism under the conditions studied.     

Potassium secretion by the cortical collecting tubule

The isolated perfused rabbit cortical collecting tubule has been shown to actively transport K from bath to lumen. The first step in this process is active uptake of K across the basolateral membrane via and Na:K exchange pump as evidenced by: 1) basolateral localization and Na:K exchange properties of the ouabain-sensitive Na,K-ATPase, 2) ouabain sensitivity of the Na and K fluxes, 3) interdependence of the Na and K fluxes, and 4) ouabain-sensitivity of 42K uptake into the cell across the basolateral membrane. At the luminal border, a significant K permeability of the apical cell membrane has been identified using electrophysiological techniques. This K permeability is insensitive to the diuretic amiloride, and, thus, differs from the pathway for Na entry, which is highly amiloride sensitive. A significant K permeability of the paracellular pathway is not apparent. It is concluded that K secretion by the rabbit cortical collecting tubule occurs via a two-step process: active uptake of K across the basolateral membrane via the Na:K exchange pump, followed by passive efflux of K across the apical membrane via an amiloride-insensitive K conductive pathway.     

Ruthenium red-insensitive Ca2+ uptake and release by mitochondria

Calcium uptake by rat liver mitochondria driven by an artificial pH gradient is ruthenium red insensitive, electrically neutral, and inhibited by the local anesthetic, nupercaine. This pH-driven Ca2+ transport is also inhibited by NH3, Pi, and acetate. Direct measurements of Pi indicate it is not translocated with Ca2+ during pH-driven Ca2+ uptake. Calcium is therefore not transported by a Ca2+-Pi symport mechanism. Ruthenium red-insensitive Ca2+ efflux is similar in its inhibition by nupercaine and its kinetics, and is also electroneutral. This suggests that the Ca2+ uptake described here occurs via reversal of the principal pathway of mitochondrial Ca2+ release. From the available data, pH-driven Ca2+ uptake (and presumably Ca2+ efflux) is hypothesized to occur by Ca2+ symport with unidentified anions. Protons may move counter to Ca2+ or reversibly dissociate from cotransported anions, which therefore couples Ca2+ transport to the pH gradient.     

Transport of nonmetabolizable opines by Agrobacterium tumefaciens.

We have examined the uptake of [14C]octopine and [14C]nopaline by Agrobacterium tumefaciens strains containing the C58 chromosomal background in medium suitable for the induction of vir genes. All strains tested could transport both of these opines, regardless of the presence or type of Ti plasmid (octopine or nopaline) present in the bacterium. The transport of these opines required active cellular metabolism. Nonradioactive octopine, nopaline, and arginine competed effectively with [14C]octopine and [14C]nopaline for transport into A. tumefaciens A136, suggesting that the transport of these opines occurs via an arginine transport pathway not encoded by the Ti plasmid.     

Characterization of tryptophan transport in human placental brush-border membrane vesicles.

The characteristics of tryptophan uptake in isolated human placental brush-border membrane vesicles were investigated. Tryptophan uptake in these vesicles was predominantly Na+-independent. Uptake of tryptophan as measured with short incubations occurred exclusively by a carrier-mediated process, but significant binding of this amino acid to the membrane vesicles was observed with longer incubations. The carrier-mediated system obeyed Michaelis-Menten kinetics, with an apparent affinity constant of 12.7 +/- 1.0 microM and a maximal velocity of 91 +/- 5 pmol/15 s per mg of protein. The kinetic constants were similar in the presence and absence of a Na+ gradient. Competition experiments showed that tryptophan uptake was effectively inhibited by many neutral amino acids except proline, hydroxyproline and 2-(methylamino)isobutyric acid. The inhibitory amino acids included aromatic amino acids as well as other system-1-specific amino acids (system 1 refers to the classical L system, according to the most recent nomenclature of amino acid transport systems). The transport system showed very low affinity for D-isomers, was not affected by phloretin or glucose but was inhibited by p-azidophenylalanine and N-ethylmaleimide. The uptake rates were only minimally affected by change in pH over the range 4.5-8.0. Tryptophan uptake markedly responded to trans-stimulation, and the amino acids capable of causing trans-stimulation included all amino acids with system-1-specificity. The patterns of inhibition of uptake of tryptophan and leucine by various amino acids were very similar. We conclude that system t, which is specific for aromatic amino acids, is absent from human placenta and that tryptophan transport in this tissue occurs via system 1, which has very broad specificity.     

Tryptophan and iodothyronine transport interactions in HepG2 human hepatoma cells

This study identifies interactions between transport of the aromatic amino acid l-tryptophan (Trp) and thyroid hormones (TH) in HepG2 human hepatoma cells. The major portion of Trp uptake in HepG2 cells occurs via the NEM-sensitive amino acid transport System L2 (consistent with hepatic LAT3 expression), with a smaller aromatic-AA selective System T (MCT10) component. LAT3 and MCT10 mRNA were both detected in HepG2 cells. Uptake of TH does not involve System L2, but a significant portion of T₃ uptake is mediated by System T, alongside a taurocholate-sensitive organic anion transporter. T₄ uptake into HepG2 cells appears to be mediated principally by organic anion/monocarboxylate transporters, with smaller contributions by System T and receptor-mediated endocytosis. TH–Trp transport interactions in liver cells centre on System T which, due to a perivenous localisation alongside deiodinase 1, may impact on hepatic T₃ generation and release.