Transporter-Mediated Uptake of 2-Chloro- and 2-Hydroxybenzoate by Pseudomonas huttiensis Strain D1

We investigated the mechanisms of uptake of 2-chlorobenzoate (2-CBa) and 2-hydroxybenzoate (2-HBa) by Pseudomonas huttiensis strain D1. Uptake was monitored by assaying intracellular accumulation of 2-[UL-ring-¹⁴C]CBa and 2-[UL-ring-¹⁴C]HBa. Uptake of 2-CBa showed substrate saturation kinetics with an apparent K_m of 12.7 ± 2.6 μM and a maximum velocity (V_max) of 9.76 ± 0.78 nmol min⁻¹ mg of protein⁻¹. Enhanced rates of uptake were induced by growth on 2-CBa and 2-HBa, but not by growth on benzoate or 2,5-di-CBa. Intracellular accumulations of 2-CBa and 2-HBa were 109- and 42-fold greater, respectively, than the extracellular concentrations of these substrates and were indicative of uptake mediated by a transporter rather than driven by substrate catabolism (“metabolic drag”). Results of competitor screening tests indicated that the substrate range of the transporter did not include other o-halobenzoates that serve as growth substrates for strain D1 and for which the metabolism was initiated by the same dioxygenase as 2-CBa and 2-HBa. This suggested that multiple mechanisms for substrate uptake were coupled to the same catabolic enzyme. The preponderance of evidence from tests with metabolic inhibitors and artificial electrochemical gradients suggested that 2-CBa uptake was driven by ATP hydrolysis. If so, the 2-CBa transporter would be the first of the ATP binding cassette type implicated in uptake of haloaromatic acids.     

Mechanism of energization of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation [DMP+] into an acrA strain of Escherichia coli.

The mechanism of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) into cells and vesicles of the acrA strain AS-1 of Escherichia coli was examined. Uptake was energized by substrate oxidation and discharged by uncouplers. Uptake was enhanced by the presence of tetraphenylphosphonium cation, tetraphenylboron anion and tributyltin chloride, which may inhibit the efflux system for DMP+. Uptake was inhibited by 5-methoxyindole-2-carboxylic acid (MIC). By the use of ionophores with right-side-out vesicles loaded with monovalent cations it was shown that DMP+ uptake could be driven both by the establishment of a membrane potential across the vesicle membrane and by a H+/DMP+ antiport system. Attempts to demonstrate the latter mechanism in everted membrane vesicles were unsuccessful.     

Uptake of the cephalosporin, cephalexin, by a dipeptide transport carrier in the human intestinal cell line, Caco-2

The transport of the orally absorbed cephalosporin, cephalexin, was examined in the human epithelial cell line, Caco-2 that possesses intestinal enterocyte-like properties when cultured. In sodium-free buffer, the cells accumulated 1 mM D-[9-14C]cephalexin against a concentration gradient and obtained a distribution ratio of 3.5 within 180 min. Drug uptake was maximal when the extracellular pH was 6.0. Uptake was reduced by metabolic inhibitors and by protonophores indicating that uptake was energy- and proton-dependent. Kinetic analysis of the concentration dependence of the rate of cephalexin uptake showed that a non-saturable component (Kd of 0.18 +/- 0.01 nmol/min per mg protein per mM) and a transport system with a Km of 7.5 +/- 2.8 mM and a Vmax of 6.5 +/- 0.9 nmol/min per mg protein were responsible for drug uptake. Uptake was competitively inhibited by dipeptides. The transport carrier exhibited stereospecificity for the L-isomer of cephalexin. Drug uptake was not affected by the presence of amino acids, organic anions, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid or 4,4'-diisothiocyano-2,2'-disulfonic stilbene. Therefore, Caco-2 cells take up cephalexin by a proton-dependent dipeptide transport carrier that closely resembles the transporter present in the intestine. Caco-2 cells represent a cellular model for future studies of the dipeptide transporter.     

mhpT Encodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12

Escherichia coli K-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome, mhpT was proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated that mhpT is essential for 3HPP catabolism in E. coli K-12 W3110 at pH 8.2. Uptake assays with ¹⁴C-labeled substrates demonstrated that strain W3110 and strain W3110ΔmhpT containing recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110ΔmhpT containing the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital for E. coli K-12 W3110 growth on this substrate under basic conditions.     

Biphenyl uptake by psychrotolerant Pseudomonas sp. strain Cam-1 and mesophilic Burkholderia sp. strain LB400

We investigated the uptake of biphenyl by the psychrotolerant, polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp. strain Cam-1 and the mesophilic PCB-degrader, Burkholderia sp. strain LB400. The effects of growth substrates, metabolic inhibitors, and temperature on [14C]biphenyl uptake were studied. Biphenyl uptake by both strains was induced by growth on biphenyl, and was inhibited by dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), which are metabolic uncouplers. The Vmax and Km for biphenyl uptake by Cam-1 at 22 degrees C were 5.4 +/- 1.7 nmol x min(-1) x (mg of cell protein)(-1) and 83.1 +/- 15.9 micromol x L(-1), respectively. The Vmax and Km for biphenyl uptake by LB400 at 22 degrees C were 3.2 +/- 0.3 nmol x min(-1) x (mg of cell protein(-1)) and 51.5 +/- 9.6 micromol x L(-1), respectively. At 15 degrees C, the maximum rate for biphenyl uptake by Cam-1 and LB400 was 3.1 +/- 0.3 nmol x min(-1) x (mg of cell protein)(-1) and 0.89 +/- 0.1 nmol x min(-1) x (mg of cell protein)(-1), respectively. Thus, the maximum rate for biphenyl uptake by Cam-1 at 15 degrees C was more than 3 times higher than that for LB400.     

Cytidine is a novel substrate for wild-type concentrative nucleoside transporter 2

Nucleoside transporter (NT) plays key roles in the physiology of nucleosides and the pharmacology of its analogues in mammals. We previously cloned Na+/nucleoside cotransporter CNT2 from mouse M5076 ovarian sarcoma cells, the peptide encoded by it differing from that by the previously reported mouse CNT2 in five substitutions, and observed that the transporter can take up cytidine, like CNT1 and CNT3. In the present study, we examined which of the two aforementioned CNT2 is the normal one, and whether or not cytidine is transported via the previously reported CNT2. The peptide encoded by CNT2 derived from mouse intestine, liver, spleen, and ovary was identical to that previously reported. The uptake of [3H]cytidine, but not [3H]thymidine, by Cos-7 cells transfected with CNT2 cDNA obtained from mouse intestine was much greater than that by mock cells, as in the case of [3H]uridine, a typical substrate of NT. [3H]Cytidine and [3H]uridine were taken up via CNT2, in temperature-, extracellular Na+-, and substrate concentration-dependent manners. The uptake of [3H]cytidine and [3H]uridine mediated by CNT2 was significantly inhibited by the variety of nucleosides used in this study, except for thymidine, and inhibition of the [3H]uridine uptake by cytidine was competitive. The [3H]uridine uptake via CNT2 was significantly decreased by the addition of cytarabin or gemcitabine, antimetabolites of cytidine analogue. These results indicated that the previously reported mouse CNT2 is the wild-type one, and cytidine is transported mediated by the same recognition site on the CNT2 with uridine, and furthermore, cytidine analogues may be substrates for the transporter.     

Energization of phenylalanine transport and energy-dependent transhydrogenase by ATP in cytochrome-deficient Escherichia coli K12

$\text{E. coli}$ SASX76 does not form cytochromes unless supplemented with 5-aminolevulinic acid. Uptake of [¹⁴C]phenylalanine into cytochrome-deficient cells of this mutant was energized by glucose but not by endogenous substrates or D-lactate with or with or without fumarate. In contrast, uptake of this amino acid was supported in cytochrome-containing cells of this strain by oxidation of D-lactate or endogenous substrates. It is concluded that ATP can energize phenylalanine transport in cytochrome-deficient cells. Cytochrome-deficient cells lacked eńergy-dependent transhydrogenase activity driven by oxidation of NADH but ATP-driven transhydrogenation was unimpaired. Both transhydrogenase activities were present in cytochrome-containing cells.     

Methylammonium uptake by Rhizobium sp. strain 32H1

We present evidence that methylammonium is transported into cowpea Rhizobium sp. strain 32H1 cells by a membrane carrier whose natural substrate is ammonium. After growth in low (0.2%) oxygen, which is necessary for nitrogen fixation by these cells, respiring rhizobial cells took up [14C]methylammonium to high intracellular levels. Cells grown in atmospheric (21%) oxygen did not take up methylammonium. Uptake (transport plus metabolism) was maximal in cells harvested in the early stationary phase of batch culture and had a distinct pH optimum of 6.5 to 7.0. Uptake was inhibited by metabolic poisons that dissipate the proton motive force or inhibit ATP synthesis. Inhibition of uptake by ammonium and the counterflow phenomenon indicated that ammonium and methylammonium share a transport carrier. Of the methylammonium taken up, about 15% was accumulated to intracellular levels 20 times higher than those in the medium; most of the methylammonium was metabolized to gamma-N-methylglutamine.     

A highly selective, high-affinity transporter for uracil in Trypanosoma brucei brucei: evidence for proton-dependent transport.

The presence of an uptake mechanism for uracil in procyclic forms of the protozoan parasite Trypanosoma brucei brucei was investigated. Uptake of [3H]uracil at 22 degrees C was rapid and saturable and appeared to be mediated by a single high-affinity transporter, designated U1, with an apparent Km of 0.46 +/- 0.09 microM and a Vmax of 0.65 +/- 0.08 pmol x (10(7) cells)(-1) x s(-1). [3H]Uracil uptake was not inhibited by a broad range of purine and pyrimidine nucleosides and nucleobases (concentrations up to 1 mM), with the exception of uridine, which acted as an apparent weak inhibitor (Ki value of 48 +/- 15 microM). Similarly, most chemical analogues of uracil, such as 5-chlorouracil, 3-deazauracil, and 2-thiouracil, had little or no affinity for the U1 carrier. Only 5-fluorouracil was found to be a relatively potent inhibitor of uracil uptake (Ki = 3.2 +/- 0.4 microM). Transport of uracil was independent of extracellular sodium and potassium gradients, as replacement of NaCl in the assay buffer by N-methyl-D-glucamine, KCl, LiCl, CsCl, or RbCl did not affect initial rates of transport. However, the proton ionophore carbonyl cyanide chlorophenylhydrazone inhibited up to 70% of [3H]uracil flux. These data show that uracil uptake in T. b. brucei procyclics is mediated by a single high-affinity transporter with high substrate selectivity and are consistent with a nucleobase-H+-symporter model for this carrier.     

The organic cation transporters rOCT1 and hOCT2 are inhibited by cGMP

The electrogenic cation transporters OCT1 and OCT2 in the basolateral membrane of renal proximal tubules mediate the first step during secretion of organic cations. Previously we demonstrated stimulation and change of selectivity for rat OCT1 (rOCT1) by protein kinase C. Here we investigated the effect of cGMP on cation transport by rOCT1 or human OCT2 (hOCT2) after expression in human embryonic kidney cells (HEK293) or oocytes of Xenopus laevis. In HEK293 cells, uptake was measured by microfluorimetry using the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ) as substrate, whereas uptake into Xenopus laevis oocytes was measured with radioactively labelled cations. In addition, ASP +-induced depolarizations of membrane voltages (Vm) were measured in HEK293 cells using the slow whole-cell patch-clamp method. Incubation of rOCT1-expressing HEK293 cells for 10 min with 100 mM 8-Br-cGMP reduced initial ASP + uptake by maximally 78% with an IC50 value of 24 +/- 16 mM. This effect was not abolished by the specific PKG inhibitor KT5823, indicating that a cGMP-dependent kinase is not involved. An inhibition of ASP + uptake by rOCT1 in HEK293 cells was also obtained when the cells were incubated for 10 min with 100 mM cGMP, whereas no effect was obtained when cGMP was given together with ASP +. ASP + (100 mM)-induced depolarizations of Vm were reduced in the presence of 8-Br-cGMP (100 mM) by 44 +/- 11% (n = 6). Since it could be demonstrated that [3H]cGMP is taken up by an endogeneous cyanine863-inhibitable transporter, the effect of cGMP is probably mediated from inside the cell. Uptake measurements with [14C]tetraethylammonium and [3H]2-methyl-4-phenylpyridinium in Xenopus laevis oocytes expressing rOCT1 performed in the absence and presence of 8-Br-cGMP showed that cGMP does not interact directly with the transporter. The data suggest that the inhibition mediated by cGMP observed in HEK293 cells occurs most likely via a mammalian cGMP-binding protein that interacts with OCT1-2 transporters.     

Characterization of berberine transport into Coptis japonica cells and the involvement of ABC protein

Cultured Coptis japonica cells are able to take up berberine, a benzylisoquinoline alkaloid, from the medium and transport it exclusively into the vacuoles. Uptake activity depends on the growth phase of the cultured cells whereas the culture medium had no effect on uptake. Treatment with several inhibitors suggested that berberine uptake depended on the ATP level. Some inhibitors of P-glycoprotein, an ABC transporter involved in multiple drug resistance in cancer cells, strongly inhibited berberine uptake, whereas a specific inhibitor for glutathione biosynthesis and vacuolar ATPase, bafilomycin A1, had little effect. Vanadate-induced ATP trap experiments to detect ABC proteins expressed in C. japonica cells showed that three membrane proteins of between 120 and 150 kDa were photolabelled with 8-azido-[alpha-32P] ATP. Two revealed the same photoaffinity-labelling pattern as P-glycoprotein, and the interaction of these proteins with berberine was also demonstrated. These results suggest that ABC proteins of the MDR-type are involved in the uptake of berberine from the medium.     

Biogenesis of mitochondria. A requirement for mitochondrial protein synthesis for the formation of a normal adenine nucleotide transporter in yeast mitochondria

1. Parameters of ATP uptake by fully functional Saccharomyces cerevisiae mitochondria, including kinetic constants, binding constants and sensitivity to atractylate, closely resemble those of mammalian mitochondria. Scatchard plots of atractylate-sensitive adenine nucleotide binding indicate two distinct sites of high affinity (binding constant, K(D)'=1mum), and low affinity (binding constant, K(D)'=20mum) in the ratio 1:3. Uptake has high Arrhenius activation energies (+35 and +57kJ/mol), above and below a transition temperature of 11 degrees C. Atractylate-insensitive ATP uptake is apparently not saturable and has a low Arrhenius activation energy (6kJ/mol), suggesting a non-specific binding process. 2. Kinetic and binding constants for ATP uptake are not significantly changed in catabolite-repressed or anaerobic mitochondrial structures. 3. Inhibition of the mitochondrial protein-synthesizing system by growth of cells in the presence of erythromycin, or loss of mitochondrial DNA by mutation profoundly alters the adenine nucleotide transporter. ATP uptake becomes completely insensitive to atractylate, and the high-affinity binding site is lost. However, the adenine nucleotide transporter does not appear to be totally eliminated, as a moderate amount of saturable low-affinity ATP binding remains. 4. It is concluded that products of the mitochondrial protein-synthesizing system, probably coded by mitochondrial DNA, are required for the normal function of the adenine nucleotide transporter.     

Defective P2Y purinergic receptor function: A possible novel mechanism for impaired glucose transport

Extracellular ATP is an ubiquitous mediator that regulates several cellular functions via specific P2 plasma membrane receptors (P2Rs), for which a role in modulating intracellular glucose metabolism has been recently suggested. We have investigated glucose uptake in response to P2Rs stimulation in fibroblasts from type 2 diabetic (T2D) patients and control subjects. P2Rs expression was evaluated by RT-PCR; intracellular calcium release by fluorometry; glucose transporter (GLUT1) translocation by immunoblotting and chemiluminescence; glucose uptake was measured with 2-deoxy-D-[1-(3)H]glucose (2-DOG) and ATP by luminometry. Cells from T2D patients, in contrast to those from healthy controls, showed no increase in glucose uptake after ATP stimulation; extracellular ATP caused, however, a similar GLUT1 recruitment to the plasma membrane in both groups. P2Rs expression did not differ between fibroblasts from diabetic and healthy subjects, but while plasma membrane depolarization, a P2X-mediated response was similar in both groups, no evident intracellular calcium increase was detectable in the cells from the former group. The calcium response in fibroblasts from diabetics was restored by co-incubation with apyrase or hexokinase, suggesting that P2YRs in those cells were normally expressed but chronically desensitised. In support to this finding, fibroblasts from T2D subjects secreted a two-fold larger amount of ATP compared to controls. Pre-treatment with apyrase or hexokinase also restored ATP stimulated glucose uptake in fibroblasts from diabetic subjects. These results suggest that extracellular ATP plays a role in the modulation of glucose transport via GLUT1, and that the P2Y-dependent GLUT1 activation is deficient in fibroblasts from T2D individuals. Our observations may point to additional therapeutic targets for improving glucose utilization in diabetes.     

Functional characterization of the Saccharomyces cerevisiae ABC-transporter Yor1p overexpressed in plasma membranes

Yor1p, a Saccharomyces cerevisiae plasma membrane ABC-transporter, is associated to oligomycin resistance and to rhodamine B transport. Here, by using the overexpressing strain Superyor [A. Decottignies, A.M. Grant, J.W. Nichols, H. de Wet, D.B. McIntosh, A. Goffeau, ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p, J. Biol. Chem. 273 (1998) 12612-12622], we show that Yor1p also confers resistance to rhodamine 6G and to doxorubicin. In addition, Yor1p protects cells, although weakly, against tetracycline, verapamil, eosin Y and ethidium bromide. The basal ATPase activity of the overexpressed form of Yor1p was studied in membrane preparations. This activity is quenched upon addition of micromolar amounts of vanadate. Vmax and Km values of approximately 0.8 s(-1) and 50+/-8 microM are measured. Mutations of essential residues in the nucleotide binding domain 2 reduces the activity to that measured with a Deltayor1 strain. ATP hydrolysis is strongly inhibited by the addition of potential substrates of the transporter. Covalent reaction of 8-azido-[alpha-(32)P]ATP with Yor1p is not sensitive to the presence of excess oligomycin. Thus, competition of the drug with ATP binding is unlikely. Finally, we inspect possible hypotheses accounting for substrate inhibition, rather than stimulation, of ATP hydrolysis by the membrane preparation.     

Regulation of dopamine transporter function and cell surface expression by D3 dopamine receptors

D(3) dopamine receptors are expressed by dopamine neurons and are implicated in the modulation of presynaptic dopamine neurotransmission. The mechanisms underlying this modulation remain ill defined. The dopamine transporter, which terminates dopamine transmission via reuptake of released neurotransmitter, is regulated by receptor- and second messenger-linked signaling pathways. Whether D3 receptors regulate dopamine transporter function is unknown. We addressed this issue using a fluorescent imaging technique that permits real time quantification of dopamine transporter function in living single cells. Accumulation of the fluorescent dopamine transporter substrate trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium (ASP(+)) in human embryonic kidney cells expressing human dopamine transporter was saturable and temperature-dependent. In cells co-expressing dopamine transporter and D3 receptors, the D2/D3 agonist quinpirole produced a rapid, concentration-dependent, and pertussis toxin-sensitive increase of ASP(+) uptake. Similar agonist effects were observed in Neuro2A cells and replicated in human embryonic kidney cells using a radioligand uptake assay in which binding to and activation of D3 receptors by [(3)H]dopamine was prevented. D3 receptor stimulation activated phosphoinositide 3-kinase and MAPK. Inhibition of either kinase prevented the quinpirole-induced increase in uptake. D3 receptor activation differentially affected dopamine transporter function and subcellular distribution depending on the duration of agonist exposure. Biotinylation experiments revealed that the rapid increase of uptake was associated with increased cell surface and decreased intracellular expression and increased dopamine transporter exocytosis. In contrast, prolonged agonist exposure reduced uptake and transporter cell surface expression. These results demonstrate that D3 receptors regulate dopamine transporter function and identify a novel mechanism by which D3 receptors regulate extracellular dopamine concentrations.     

Study on the effect of hypergravity stress on L-glutamate uptake by rat brain nerve endings

Using rat brain synaptosomes, we have investigated the effect of hypergravity on the kinetic parameters of Na(+)-dependent, high-affinity L-glutamate transport activity. The time-course of L-[14C]-glutamate uptake and dependence of L-[14C]-glutamate uptake velocity on glutamate concentrations were analyzed. K(m) and Vmax of this process have been determined. The hypergravity stress was created by centrifugation of rats for 1 hour at 10 g. We observed no differences in K(m) values between the control rats (10.7 +/- 2.5 microM) and animals exposed to hypergravity (6.7 +/- 1.5 microM). The similarity of this parameter for the two studied groups of animals showed that affinity of glutamate transporter to substrate was not sensitive to hypergravity stress. In contrast, the maximal velocity of glutamate uptake changed in hypergravity conditions. Vmax reduced from 12.5 +/- +/- 3.2 nmol/min per 1 mg of protein (control group) to 5.6 +/- 0.9 nmol/min per 1 mg of protein (animals, exposed to hypergravity stress). The possible mechanisms of attenuation of the glutamate transporter activity without modifying K(m) of glutamate uptake were discussed.     

Sodium-dependent and inhibitor-insensitive uptake of adenosine by mouse peritoneal exudate cells

[8-3H]Adenosine uptake in mouse peritoneal exudate cells, harvested following i.p. challenge with Complete Freund's Adjuvant from BALB/c mice, was found to be insensitive to common nucleoside transport inhibitors such as dilazep or 6-[(4-nitrobenzyl)mercapto]purine ribonucleoside and to require sodium ion, being inactive when sodium was replaced by lithium or potassium. These findings also applied to the adherent (macrophages) and nonadherent (polymorphonuclear cells) cell fractions prepared from the peritoneal cell mixture. Uptake was inhibited by several nucleosides including deoxyadenosine, inosine, uridine, thymidine and, to a lesser extent, by the adenosine analog tubercidin, while adenine, fructose, glucose and ribose were without effect. Uptake [8-3H]adenosine was fully matched by rapid intracellular phosphorylation to AMP, ADP and ATP. Inosine was a substrate for the transporter, but tubercidin was not. The system clearly is distinct from carrier-mediated, nonconcentrative transport and has similarities to concentrative, sodium-dependent nucleoside transporters described in other cell types.     

Synthesis and characterization of a new and radiolabeled high-affinity substrate for H+/peptide cotransporters

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.     

Molecular mechanisms of organic cation transport in OCT2-expressing Xenopus oocytes

The molecular mechanisms of organic cation transport by rat OCT2 was examined in the Xenopus oocyte expression system. When extracellular Na+ ions were replaced with K+ ions, uptake of tetraethylammonium (TEA) by OCT2-expressing oocytes was decreased, suggesting that TEA uptake by OCT2 is dependent on membrane potential. Kinetic analysis revealed that the decreased TEA uptake by ion substitution was caused at least in part by decreased substrate affinity. Acidification of extracellular buffer resulted in decreased uptake of TEA, whereas TEA efflux from OCT1- and OCT2-expressing oocytes was not stimulated by inward proton gradient, in consistent with basolateral organic cation transport in the kidney. Inhibition of TEA uptake by various organic cations revealed that apparent substrate spectrum of OCT2 was similar with that of OCT1. However, the affinity of procainamide to OCT1 was higher than that to OCT2. Uptake of 1-methyl-4-phenylpyridinium was stimulated by OCT2 as well as OCT1, but uptake of levofloxacin, a zwitterion, was not stimulated by both OCTs. These results suggest that OCT2 is a multispecific organic cation transporter with the characteristics comparable to those of the basolateral organic cation transporter in the kidney.