L-Carnitine transport in mouse renal and intestinal brush-border and basolateral membrane vesicles.

We characterized the uptake of carnitine in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, isolated from mouse kidney and intestine. In kidney, carnitine uptake was Na(+)-dependent, showed a definite overshoot and was saturable for both membranes, but for intestine, it was Na(+)-dependent only in BLM. The uptake was temperature-dependent in BLM of both kidney and intestine. The BBM transporter in kidney had a high affinity for carnitine: apparent K(m)=18.7 microM; V(max)=7.85 pmol/mg protein/s. In kidney BLM, similar characteristics were obtained: apparent K(m)=11.5 microM and V(max)=3.76 pmol/mg protein/s. The carnitine uptake by both membranes was not affected within the physiological pH 6.5-8.5. Tetraethylammonium, verapamil, valproate and pyrilamine significantly inhibited the carnitine uptake by BBM but not by BLM. By Western blot analysis, the OCTN2 (a Na(+)-dependent high-affinity carnitine transporter) was localized in the kidney BBM, and not in BLM. Strong OCTN2 expression was observed in kidney and skeletal muscle, with no expression in intestine in accordance with our functional study. We conclude that different polarized carnitine transporters exist in kidney BBM and BLM. L-Carnitine uptake by mouse renal BBM vesicles involves a carrier-mediated system that is Na(+)-dependent and is inhibited significantly by specific drugs. The BBM transporter is likely to be OCTN2 as indicated by a strong reactivity with the anti-OCTN2 polyclonal antibody.     

Expression of OCTN2 and OCTN3 in the apical membrane of rat renal cortex and medulla

Immunological assays and transport measurements in apical membrane vesicles revealed that the apical membrane of rat kidney cortex and medulla presents OCTN2 and OCTN3 proteins and transports L ‐[³H]‐carnitine in a Na⁺‐dependent and ‐independent manner. OCTN2 mediates the Na⁺/L ‐carnitine transport activity measured in medulla because (i) the transport showed the same characteristics as the cortical Na⁺/L ‐carnitine transporter and (ii) the medulla expressed OCTN2 mRNA and protein. The Na⁺‐independent L ‐carnitine transport activity appears to be mediated by both OCTN2 and OCTN3 since: (i) Na⁺‐independent L ‐carnitine uptake was inhibited by both, anti‐OCTN2 and anti‐OCTN3 antibodies, (ii) kinetics studies revealed the involvement of a high‐ and a low‐affinity transport systems, and (iii) Western and immunohistochemistry studies revealed that OCTN3 protein is located at the apical membrane of the kidney epithelia. The Na⁺‐independent L ‐carnitine uptake exhibited trans‐stimulation by intravesicular L ‐carnitine or betaine. This trans‐stimulation was inhibited by anti‐OCTN3 antibody, but not by anti‐OCTN2 antibody, indicating that OCTN3 can function as an L ‐carnitine/organic compound exchanger. This is the first report showing a functional apical OCTN2 in the renal medulla and a functional apical OCTN3 in both renal cortex and medulla. J. Cell. Physiol. 223: 451–459, 2010. © 2010 Wiley‐Liss, Inc.     

L-Carnitine transport in human placental brush-border membranes is mediated by the sodium-dependent organic cation transporter OCTN2

Maternofetal transport of L-carnitine, a molecule that shuttles long-chain fatty acids to the mitochondria for oxidation, is thought to be important in preparing the fetus for its lipid-rich postnatal milk diet. Using brush-border membrane (BBM) vesicles from human term placentas, we showed that L-carnitine uptake was sodium and temperature dependent, showed high affinity for carnitine (apparent K_m = 11.09 ± 1.32 µM; V_max = 41.75 ± 0.94 pmol·mg protein^–1·min^–1), and was unchanged over the pH range from 5.5 to 8.5. L-Carnitine uptake was inhibited in BBM vesicles by valproate, verapamil, tetraethylammonium, and pyrilamine and by structural analogs of L-carnitine, including D-carnitine, acetyl-D,L-carnitine, and propionyl-, butyryl-, octanoyl-, isovaleryl-, and palmitoyl-L-carnitine. Western blot analysis revealed that OCTN2, a high-affinity, Na⁺-dependent carnitine transporter, was present in placental BBM but not in isolated basal plasma membrane vesicles. The reported properties of OCTN2 resemble those observed for L-carnitine uptake in placental BBM vesicles, suggesting that OCTN2 may mediate most maternofetal carnitine transport in humans. membrane transport; valproate; maternofetal; xenobiotics; acylcarnitine     

Expression and localization of organic cation/carnitine transporter OCTN2 in Caco-2 cells

l-Carnitine is derived both from dietary sources and biosynthesis. Dietary carnitine is absorbed in the small intestine and then distributed to other organs. Previous studies using Caco-2 cells demonstrated that the transport of l-carnitine in the intestine involves a carrier-mediated system. The purpose of this study was to determine whether the uptake of l-carnitine in Caco-2 cells is mediated by the recently identified organic cation/carnitine transporter (OCTN2). Kinetics of l-[(3)H]carnitine uptake were investigated with or without specific inhibitors. l-Carnitine uptake in mature cells was sodium dependent and linear with time. K(m) and V(max) values for saturable uptake were 14.07 +/- 1.70 micro M and 26.3 +/- 0.80 pmol. mg protein(-1). 6 min(-1), respectively. l-carnitine uptake was inhibited (P < 0.05-0.01) by valproate and other organic cations. Anti-OCTN2 antibodies recognized a protein in the brush-border membrane (BBM) of Caco-2 cells with an apparent molecular mass of 60 kDa. The OCTN2 expression was confirmed by double immunostaining. Our results demonstrate that l-carnitine uptake in differentiated Caco-2 cells is primarily mediated by OCTN2, located on the BBM.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Uptake of selenate and selenite by isolated intestinal brush border membrane vesicles from pig,sheep, and rat

Selenate and selenite uptakes by isolated intestinal brush border membrane vesicles (BBMV) from pig, sheep, and rat were investigated. Selenate uptake into jejunal and ileal, but not duodenal, BBMV from pig was stimulated by an inwardly directed transmembrane Na⁺ gradient (Na _out ⁺ >Na _in ⁺ ). Selenate transport into rat ileal and sheep jejunal BBMV was also enhanced in the presence of a Na⁺ gradient. Unlike selenate uptake, selenite uptake was not Na⁺ dependent, neither in pig small intestine nor in sheep jejunum and rat ileum. Uptake of selenate represented real uptake into the vesicular lumen, whereas selenite uptake was a result of an extensive binding of⁷⁵Se to the membranes. Thiosulfate at a 250-fold concentration of selenate completely inhibited Na⁺-dependent selenate uptake into pig jejunal BBMV. Furthermore, Na⁺-dependent sulfate uptake was totally inhibited in the presence of a 250-fold selenate concentration. The results clearly show that selenate transport across the BBM of pig jejunum and ileum, sheep jejunum, and rat ileum is partially energized by a transmembrane Na⁺ gradient. Moreover, it is concluded from the results that there exists a common transport mechanism for sulfate and selenate in the BBM. The extensive binding of⁷⁵Se from⁷⁵Se-labeled selenite to the membranes could be from a spontaneous reaction of selenite with membrane-associated SH groups.     

Reconstitution into liposomes and functional characterization of the carnitine transporter from renal cell plasma membrane

The carnitine transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalysed a first-order antiport reaction (carnitine/carnitine or carnitine/substrate) stimulated by external, not internal, Na⁺, with a positive cooperativity. Na⁺ was co-transported with carnitine. Optimal activity was found between pH 5.5 and pH 6.0. The sulfhydryl reagents MTSES, MTSET and mercurials strongly inhibited the transport. Substrate analogues inhibited the transport; the most effective were acylcarnitines and betaine, followed by dimethylglicine, tetraethylammonium and arginine. Besides carnitine, only acylcarnitines and betaine were efficiently translocated. The Km for carnitine on the external and internal side of the transporter was 0.08 and 1.2 mM, respectively. The transporter is asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. The reconstituted carnitine transporter corresponds, very probably, to the OCTN2 protein.     

Membrane vesicles from brown adipose tissue: A tool for the study of amino acid transport. The case of L-alanine

A density gradient method is used to isolate membrane vesicles from brown adipose tissue. These respond to changes in osmolarity and show the classical overshoot pattern when L-alanine uptake is assayed. Transport is shown to be effected by two components: a linear (K_d=0.498 min⁻¹) and Na⁺-dependent saturable component (K_m=2.3 mM) and a V_max=19.9 pmol/μg protein·min). This pattern is similar to that shown by cells isolated from brown adipose tissue.     

Na+-dependent,electroneutral l-ascorbate transport across brush border membrane vesicles from guinea pig small intestine

In brush border vesicles from guinea pig small intestine l-ascorbate transport is Na⁺-dependent and electroneutral (in the presence of Na⁺, as shown by its lack of response to either positive or negative Δψ across the membrane).l-Ascorbate transporter has the kinetic characteristics of a mobile carrier (K_m for l-ascorbate, 0.3 mM). d-Isoascorbate (erythorbate) seems to be another, but poorer, substrate of the same transporter.l-Ascorbate transport is subjected to heterologous inhibition by d-glucose.     

4-Azidophlorizin,a high affinity probe and photoaffinity label for the glucose transporter in brush border membranes

A new phlorizin derivative ($\text{2′-O-(β-}\text{d}\text{-}\text{glucopyranosyl}\text{)-4-}\text{azidophloretin}$, 4-azidophlorizin) has been synthesized and its affinity for the d-glucose, Na⁺ co-transport system in brush border vesicles from intestinal and renal membranes has been compared with that of phlorizin. The extent of the reversible interaction of the ligand with the transporter in dim light has been evaluated from three separate measurements: (1) K′_i, the constant for fully-competitive inhibition of (Na⁺, Δψ)-dependent d-glucose uptake, (2) K′_d, the dissociation constant of 4-azido[³H]phlorizin binding in the presence of an NaSCN inward gradient, and (3) K″_i, the constant for fully-competitive inhibition of the specific ((Na⁺, Δψ)-dependent, d-glucose protectable) high-affinity [³H]phlorizin binding. In experiments with vesicles derived from rat kidney, all three constants (K′_i, K′_d and K″_i) were essentially equal and ranged between 3.2 and 5.2 μM, that is, the azide derivative has almost the same affinity for this transporter as phlorizin itself. On the other hand, compared to phlorizin, the 4-azidophlorizin has a lower affinity for the transporter in vesicles prepared from rabbit; its K′_i values are some 15–20-times larger than those determined with rat membranes. However, the affinity of the azide for the sugar transporter in membranes from either the intestine or kidney of the same animal species (rabbit or rat) was essentially the same. In spite of the lower affinity for the transporter in either membrane system from the rabbit, results described elsewhere (Hosang, M., Gibbs, E.M., Diedrich, D.F. and Semenza, G. (1981) FEBS Lett., 130, 244–248) indicate that 4-azidophlorizin is an effective photoaffinity label in this species also. Photolysis of the azide yields a reactive intermediate which reacts with a 72 kDa protein in rabbit intestine brush borders. Covalent labeling of this protein occurred under conditions which suggests that it is (a component of) the glucose transporter.     

Molecular and functional characterization of choline transporter in rat renal tubule epithelial NRK-52E cells

Homeostatic regulation of the plasma choline concentration depends on the effective functioning of a choline transporter in the kidney. However, the nature of the choline transport system in the kidney is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake in the rat renal tubule epithelial cell line NRK-52E. Choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (K_m) of 16.5 μM and a maximal velocity (V_max) of 133.9 pmol/mg protein/min. The V_max value of choline uptake was strongly enhanced in the absence of Na⁺ without any change in K_m values. The increase in choline uptake under Na⁺-free conditions was inhibited by Na⁺/H⁺ exchanger (NHE) inhibitors. Choline uptake was inhibited by the choline uptake inhibitor hemicholinium-3 (HC-3) and organic cations, and was decreased by acidification of the extracellular medium and by intracellular alkalinization. Collapse of the plasma membrane H⁺ electrochemical gradient by a protonophore inhibited choline uptake. NRK-52E cells mainly express mRNA for choline transporter-like proteins (CTL1 and CTL2), and NHE1 and NHE8. CTL1 protein was recognized in both plasma membrane and mitochondria. CTL2 protein was mainly expressed in mitochondria. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in NRK-52E cells and is responsible for choline uptake. This choline transport system uses a directed H⁺ gradient as a driving force, and its transport functions in co-operation with NHE8. Furthermore, the presence of CTL2 in mitochondria provides a potential site for the control of choline oxidation.     

Angiotensin II directly increases rabbit renal brush-border membrane sodium transport: Presence of local signal transduction system

Summary In the present study, we have examined the direct actions of angiotensin II (AII) in rabbit renal brush border membrane (BBM) where binding sites for AII exist. Addition of AII (10^–11–10^–7 m) was found to stimulate²²Na^– uptake by the isolated BBM vesicles directly. AII did not affect the Na⁺-dependent BBM glucose uptake, and the effect of AII on BBM²²Na⁺ uptake was inhibited by amiloride, suggesting the involvement of Na⁺/H⁺ exchange mechanism. BBM proton permeability as assessed by acridine orange quenching was not affected by AII, indicating the direct effect of AII on Na⁺/H⁺ antiport system.In search of the signal transduction mechanism, it was found that AII activated BBM phospholipase A₂ (PLA) and that BBM contains a 42-kDa guanine nucleotide-binding regulatory protein (G-protein) that underwent pertussis toxin (PTX)-catalyzed ADP-ribosylation. Addition of GTP potentiated, while GDP-ßS or PTX abolished, the effects of AII on BBM PLA and²²Na⁺ uptake, suggesting the involvement of G-protein in AII's actions. On the other hand, inhibition of PLA by mepacrine prevented AII's effect on BBM²²Na⁺ uptake, and activation of PLA by mellitin or addition of arachidonic acid similarly enhanced BBM²²Na⁺ uptake, suggesting the role of PLA activation in mediating AII's effect on BBM²²Na⁺ uptake.In summary, results of the present study show a direct stimulatory effect of AII on BBM Na⁺/H⁺ antiport system, and suggest the presence of a local signal transduction system involving G-protein mediated PLA activation.     

Thyroid hormones increase Na+–Pi co-transport activity in intestinal brush border membrane: Role of membrane lipid composition and fluidity

In the present study, we documented the promising role of thyroid hormones status in animals in modulation of Na⁺–P_i transport activity in intestinal brush border membrane vesicles (BBMV) which was accompanied with alterations in BBM lipid composition and fluidity. Augmentation of net P_i balance in hyperthyroid (Hyper-T) rats was fraternized with accretion of P_i transport across BBMV isolated from intestine of Hyper-T rats as compared to hypothyroid (Hypo-T) and euthyroid (Eu-T) rats while Na⁺–P_i transport across BBMV was decreased in Hypo-T rats relative to Eu-T rats. Increment in Na⁺–P_i transport in intestinal BBMV isolated from Hyper-T rats was manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport system. Furthermore, BBMV lipid composition profile in intestinal BBM from Hyper-T was altered to that of Hypo-T rats and Eu-T rats. The molar ratio of cholesterol/phospholipids was higher in intestinal BBM from Hypo-T rats. Fluorescence anistropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the intestinal BBM of Hyper-T rats and decreased in Hypo-T rats as compared to Eu-T rats which corroborated with the alteration in membrane fluidity in response to thyroid hormone status of animals. Therefore, thyroid hormone mediated change in membrane fluidity might play an important role in modulating Na⁺–P_i transport activity of intestinal BBM. (Mol Cell Biochem 278: 195–202, 2005)     

Wide tolerance to amino acids substitutions in the OCTN1 ergothioneine transporter

BackgroundOrganic cation transporters transfer solutes with a positive charge across the plasma membrane. The novel organic cation transporter 1 (OCTN1) and 2 (OCTN2) transport ergothioneine and carnitine, respectively. Mutations in the SLC22A5 gene encoding OCTN2 cause primary carnitine deficiency, a recessive disorders resulting in low carnitine levels and defective fatty acid oxidation. Variations in the SLC22A4 gene encoding OCTN1 are associated with rheumatoid arthritis and Crohn disease.MethodsHere we evaluate the functional properties of the OCTN1 transporter using chimeric transporters constructed by fusing different portion of the OCTN1 and OCTN2 cDNAs. Their relative abundance and subcellular distribution was evaluated through western blot analysis and confocal microscopy.ResultsSubstitutions of the C-terminal portion of OCTN1 with the correspondent residues of OCTN2 generated chimeric OCTN transporters more active than wild-type OCTN1 in transporting ergothioneine. Additional single amino acid substitutions introduced in chimeric OCTN transporters further increased ergothioneine transport activity. Kinetic analysis indicated that increased transport activity was due to an increased V_max, with modest changes in K_m toward ergothioneine.ConclusionsOur results indicate that the OCTN1 transporter is tolerant to extensive amino acid substitutions. This is in sharp contrast to the OCTN2 carnitine transporter that has been selected for high functional activity through evolution, with almost all substitutions reducing carnitine transport activity.General significanceThe widespread tolerance of OCTN1 to amino acid substitutions suggests that the corresponding SLC22A4 gene may have derived from a recent duplication of the SLC22A5 gene and might not yet have a defined physiological role.     

Transport of carnosine by mouse intestinal brush-border membrane vesicles

The characteristics of carnosine (β-alanyl-l-histidine) transport have been studied using purified brush-border membrane vesicles from mouse small intestine. Uptake curves did not exhibit any overshoot phenomena, and were similar under Na⁺, K⁺ or choline⁺ gradient conditions (extravesicular > intravesicular). However, uptake of histidine showed an overshoot phenomenon in the presence of a Na⁺-gradient. There was no detectable hydrolysis of carnosine during 15 min of incubation with membrane vesicles under conditions used for transport experiments. Analysis of intravesicular contents further showed the complete absence of the constituent free amino acids of carnosine, and indicates that intact carnosine is transported. Studies on the effect of concentration on peptide uptake revealed that transport occurred by a saturable process conforming to Michaelis-Menten kinetics with a K_m of 9.6 ± 1.4 mM and a V_max of 2.9 ± 0.2 nmol / mg protein per 0.4 min. Uptake of carnosine was inhibited by both di- and tripeptides with a maximum inhibition of 68% by glycyl-l-leucyltyrosine. These results clearly demonstrate that carnosine is transported intact by a carrier-mediated, Na⁺-independent process.     

Sodium translocation by the iminoglycinuria associated imino transporter (SLC6A20)

The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na⁺ and Cl^?-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na⁺-binding sites and a putative Cl^?-binding site. Mutation of all residues in the two proposed Na⁺-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na⁺-binding site 1) and mutants S20A, S20G, S20G/G405S (Na⁺-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na⁺-binding site 1 caused a decrease of the Na⁺-K_0.5, whereas mutations of site 2 increased the K_0.5. Mutation of Na⁺-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates ³⁶Cl^? demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na⁺/1Cl^?-proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria.     

Ca2+ dependency of Na+ transport by rabbit renal brush border membrane

Summary Intracellular Ca²⁺ has been suggested to play an important role in the regulation of epithelial Na⁺ transport. Previous studies showed that preincubation of toad urinary bladder, a tight epithelium, in Ca²⁺-free medium enhanced Na⁺ uptake by the subsequently isolated apical membrane vesicles, suggesting the downregulation of Na⁺ entry across the apical membrane by intracellular Ca²⁺. In the present study, we have examined the effect of Ca²⁺-free preincubation on apical membrane Na⁺ transport in a leaky epithelium, i.e., brush border membrane (BBM) of rabbit renal proximal tubule. In contrast to toad urinary bladder, it was found that BBM vesicles derived from proximal tubules incubated in 1mm Ca²⁺ medium exhibited higher Na⁺ uptake than those derived from proximal tubules incubated in Ca²⁺-free EGTA medium. Such effect of Ca²⁺ in the preincubation medium was temperature dependent and could not be replaced by another divalent cation, Ba²⁺ (1mm). Ca²⁺ in the preincubation medium did not affect Na⁺-dependent BBM glucose uptake, and its effect on BBM Na⁺ uptake was pH gradient dependent and amiloride (10^–3 m) sensitive, suggesting the involvement of Na⁺/H⁺ antiport system. Addition of verapamil (10^–4 m) to 1mm Ca²⁺ preincubation medium abolished while ionomycin (10^–6 m) potentiated the effect of Ca²⁺ to increase BBM Na⁺ uptake, suggesting that the effect of Ca²⁺ in the preincubation medium is likely to be mediated by Ca²⁺-dependent cellular pathways and not due to a direct effect of extracellular Ca²⁺ on BBM. Neither the proximal tubule content of cAMP nor the inhibitory effect of 8, bromo-cAMP (0.1mm) on BBM Na⁺ uptake was affected by the presence of Ca²⁺ in the preincubation medium, suggesting that Ca²⁺ in the preincubation medium did not increase BBM Na⁺ uptake by removing the inhibitory effect of cAMP. Addition of calmodulin inhibitor, trifluoperazine (10^–4 m) to 1mm Ca²⁺ preincubation medium did not prevent the increase in BBM Na⁺ uptake. The effect of Ca²⁺ was, however, abolished when protein kinase C in the proximal tubule was downregulated by prolonged (24 hr) incubation with phorbol 12-myristate 13-acetate (10^–6 m). In summary, these results show the Ca²⁺ dependency of Na⁺ transport by renal BBM, possibly through stimulation of Na⁺/H⁺ exchanger by protein kinase C.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

Estrogen regulation and ion dependence of taurine uptake by MCF-7 human breast cancer cells

It has been reported that estrogen receptor-positive MCF-7 cells express TauT, a Na⁺-dependent taurine transporter. However, there is a paucity of information relating to the characteristics of taurine transport in this human breast cancer cell line. Therefore, we have examined the characteristics and regulation of taurine uptake by MCF-7 cells. Taurine uptake by MCF-7 cells showed an absolute dependence upon extracellular Na⁺. Although taurine uptake was reduced in Cl^- free medium a significant portion of taurine uptake persisted in the presence of NO₃ ^-. Taurine uptake by MCF-7 cells was inhibited by extracellular β-alanine but not by L-alanine or L-leucine. 17β-estadiol increased taurine uptake by MCF-7 cells: the V_max of influx was increased without affecting the K_m. The effect of 17β-estradiol on taurine uptake by MCF-7 cells was dependent upon the presence of extracellular Na⁺. In contrast, 17β-estradiol had no significant effect on the kinetic parameters of taurine uptake by estrogen receptor-negative MDA-MB-231 cells. It appears that estrogen regulates taurine uptake by MCF-7 cells via TauT. In addition, Na⁺-dependent taurine uptake may not be strictly dependent upon extracellular Cl^-.     

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.