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C A Wagner

Publications and source records attributed to C A Wagner.

At least 37 records · Page 2Linked to original sources
PubMed2000

Inhibition of Jurkat-T-lymphocyte Na+/H+-exchanger by CD95(Fas/Apo-1)-receptor stimulation.

Mitogenic factors are known to stimulate the Na+/H+-exchanger (NHE), leading to cytosolic alkalinization and/or cell swelling. Conversely, a hallmark of apoptosis is cell shrinkage and CD95-induced apoptosis has been reported to be paralleled by cytosolic acidification. To assess whether the CD95-receptor regulates NHE activity in Jurkat T-lymphocytes, we performed conventional BCECF fluorescence measurements and SNARF flow cytometric analysis (FACS). The recoveries from acidifications following application of butyrate or a NH3 pulse were both abolished by a specific NHE-inhibitor, HOE694, indicating that they fully depend on NHE activity. Thus they were taken as a measure of NHE activity. CD95-receptor stimulation caused a cytosolic acidification and blunted the recovery from acidification following application of butyrate or a NH3 pulse. Moreover, the NHE-dependent alkalinization following osmotic cell shrinkage was almost abolished by CD95-receptor stimulation. As apparent from the effect of osmotic cell shrinkage, inhibition of the NHE by CD95-receptor stimulation was absent in Lck56-deficient J-CaM1.6 cells and restored by retransfection of J-CaM1.6-cells with Lck56. CD95-receptor stimulation led within 4 h to a decrease of cellular ATP which could contribute to NHE inhibition. Treatment of Jurkat cells with the NHE inhibitor HOE694 accelerated CD95-induced DNA fragmentation. In conclusion, CD95-receptor stimulation inhibits NHE activity through a mechanism that depends directly or indirectly on the activation of the Src-like kinase Lck56. This effect contributes to CD95-induced cytosolic acidification, DNA fragmentation and cell shrinkage.

Adenosine Triphosphate↗
PubMed2000

Expression of cell volume-regulated kinase h-sgk in pancreatic tissue.

Transcript levels of the human serine/threonine kinase h-sgk have been found to be highest in pancreas. In the present study, localization and regulation of h-sgk transcription in pancreatic tissue were elucidated. As was apparent from radioactive in situ hybridization, most pancreatic acinar cells expressed high levels of h-sgk mRNA. h-sgk mRNA-positive cells were also found in ductal epithelia but not in pancreatic islets. In biopsy specimens from patients with pancreatitis, h-sgk mRNA levels were decreased in acinar cells but abundant in numerous mononuclear interstitial cells within areas of pancreatic necrosis and fibrosis. As shown by Northern blotting, h-sgk transcription in DAN-G pancreatic tumor cells is upregulated by osmotic cell shrinkage, serum, phorbol esters (phorbol 12,13-didecanoate), and Ca(2+) ionophore A-23187 and decreased by staurosporine and cAMP. In conclusion, h-sgk transcription is regulated not only by cell volume but also by serum, protein kinase C stimulation, cAMP, and increase of intracellular Ca(2+) activity. The kinase may participate not only in normal function of exocrine pancreas but also in fibrosing pancreatitis.

Acute Disease↗
PubMed2000

Functional and pharmacological characterization of human Na(+)-carnitine cotransporter hOCTN2.

L-Carnitine is essential for the translocation of acyl-carnitine into the mitochondria for beta-oxidation of long-chain fatty acids. It is taken up into the cells by the recently cloned Na(+)-driven carnitine organic cation transporter OCTN2. Here we expressed hOCTN2 in Xenopus laevis oocytes and investigated with two-electrode voltage- clamp and flux measurements its functional and pharmacological properties as a Na(+)-carnitine cotransporter. L-carnitine transport was electrogenic. The L-carnitine-induced currents were voltage and Na(+) dependent, with half-maximal currents at 0.3 +/- 0.1 mM Na(+) at -60 mV. Furthermore, L-carnitine-induced currents were pH dependent, decreasing with acidification. In contrast to other members of the organic cation transporter family, hOCTN2 functions as a Na(+)-coupled carnitine transporter. Carnitine transport was stereoselective, with an apparent Michaelis-Menten constant (K(m)) of 4.8 +/- 0.3 microM for L-carnitine and 98.3 +/- 38.0 microM for D-carnitine. The substrate specificity of hOCTN2 differs from rOCT-1 and hOCT-2 as hOCTN2 showed only small currents with classic OCT substrates such as choline or tetraethylammonium; by contrast hOCTN2 mediated transport of betaine. hOCTN2 was inhibited by several drugs known to induce secondary carnitine deficiency. Most potent blockers were the antibiotic emetine and the ion channel blockers quinidine and verapamil. The apparent IC(50) for emetine was 4.2 +/- 1.2 microM. The anticonvulsant valproic acid did not induce a significant inhibition of carnitine transport, pointing to a different mode of action. In summary, hOCTN2 mediates electrogenic Na(+)-dependent stereoselective high-affinity transport of L-carnitine and Na(+). hOCTN2 displays transport properties distinct from other members of the OCT family and is directly inhibited by several substances known to induce systemic carnitine deficiency.

Amino Acids↗
PubMed2000

The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins.

The oocytes of the South African clawed frog X. laevis are widely used for the expression of heterologous proteins. The functional characterization of membrane proteins in particular has significantly profited from the use of this expression system. Heterologous cRNA can easily be injected and protein expression and function be studied with several techniques. This review will give a short overview into the variety of methods applicable. They span from different electrophysiological methods such as two electrode voltage clamp, patch clamp and ion-selective electrodes over cytochemistry to protein biochemistry. In spite of the wide usage of Xenopus oocytes, caution should be taken interpreting the results of protein expression. Heterologous proteins may either interact with endogenous proteins, the background of endogenous protein function may be relatively high, or altered protein behaviour may occur due to differences of the ambient temperature or altered cellular environment.

Animals↗
PubMed2000

The shrinkage-activated Na(+) conductance of rat hepatocytes and its possible correlation to rENaC.

At moderate cell shrinkage, activation of Na(+) channels is the most prominent mechanism of regulatory cell volume increase in rat hepatocytes. The amiloride sensitivity of these channels suggests a relation to the family of epithelial Na(+) channels (ENaCs). The present study was performed to determine the pharmacological profile of shrinkage-activated Na(+) channels and to test for ENaC expression in primary cultures of rat hepatocytes; in addition, the influence of the cell volume regulated serine/threonine kinase hSGK on activity and pharmacological profile of rENaC was examined in Xenopus oocytes. Conventional electrophysiology in hepatocytes reveals that the shrinkage-activated Na(+) channel is inhibited by amiloride and EIPA with IC(50) values of 6.0 and 0.12 micromol/l, respectively. Western blots and RT-PCR demonstrate that rat hepatocytes do express all three subunits (alpha, beta, gamma) of ENaC. Coexpression of hSGK with rENaC in Xenopus oocytes reveals that the kinase stimulates ENaC by a factor of 4. Moreover, hSGK decreases the affinity to amiloride (increase of IC(50) from 0.12 to 0.26 micromol/l) and increases the affinity to EIPA (decrease of IC(50) from 250 to 50 micromol/l). In conclusion, rat hepatocytes express ENaC, which is activated by the cell volume-sensitive kinase hSGK. ENaC may contribute to the Na(+) channels activated by osmotic cell shrinkage in hepatocytes, whereby the relatively low amiloride and high EIPA sensitivity of the channel could at least be partially due to modification by SGK, which decreases the amiloride and increases the EIPA sensitivity of ENaC.

Amiloride↗
PubMed2000

Acute regulation of the betaine/GABA transporter BGT-1 expressed in Xenopus oocytes by extracellular pH.

Besides uptake of Na(+) and Cl(-), mammalian cells counteract osmotic cell shrinkage also by Na(+)-coupled uptake of osmolytes, e. g., myo-inositol, taurine or betaine. The expression of the corresponding transporters is transcriptionally regulated by the ambient pH and osmolarity and is increased upon cell shrinkage, a process requiring hours. The present study has been performed to disclose rapid regulation by pH of osmolyte transport via BGT-1. Transport of GABA was investigated by using the two-electrode voltage-clamp technique with BGT-1 expressing Xenopus oocytes. GABA was used as a substrate, because of the low oocyte endogenous transport activity. Extracellular acidification to pH 5.5 reversibly decreased and extracellular alkalinization to pH 8.5 increased GABA-induced currents. Kinetic analysis revealed that extracellular alkalinization increases the affinity for Cl(-) as reflected by a decrease of the apparent K(m)-value for Cl(-) from >500 mM to 55.8 +/- 4.7 mM upon an increase of the pH from 7.0 to 8.5. The apparent K(m)- values for Na(+) and GABA remained unaltered in the pH range from 6.0 to 8.5. Instead, alkalinization increased the maximal current induced by saturating Na(+) and GABA concentrations. The results are compatible with a model of interference of H(+) ions with Cl(-) binding and a pH-dependent reduction of V(max) for Na(+) and GABA.

Animals↗
PubMed1999

Functional characterization of the Betaine/gamma-aminobutyric acid transporter BGT-1 expressed in Xenopus oocytes.

Betaine is an osmolyte accumulated in cells during osmotic cell shrinkage. The canine transporter mediating cellular accumulation of the osmolyte betaine and the neurotransmitter gamma-aminobutyric acid (BGT-1) was expressed in Xenopus oocytes and analyzed by two-electrode voltage clamp and tracer flux studies. Exposure of oocytes expressing BGT-1 to betaine or gamma-aminobutyric acid (GABA) depolarized the cell membrane in the current clamp mode and induced an inward current under voltage clamp conditions. At 1 mM substrate the induced currents decreased in the following order: betaine = GABA > diaminobutyric acid = beta-alanine > proline = quinidine > dimethylglycine > glycine > sarcosine. Both the Vmax and Km of GABA- and betaine-induced currents were voltage-dependent, and GABA- and betaine-induced currents and radioactive tracer uptake were strictly Na+-dependent but only partially dependent on the presence of Cl-. The apparent affinity of GABA decreased with decreasing Na+ concentrations. The Km of Na+ also depended on the GABA and Cl- concentration. A decrease of the Cl- concentration reduced the apparent affinity for Na+ and GABA, and a decrease of the Na+ concentration reduced the apparent affinity for Cl- and GABA. A comparison of 22Na+-, 36Cl--, and 14C-labeled GABA and 14C-labeled betaine fluxes and GABA- and betaine-induced currents yielded a coupling ratio of Na+/Cl-/organic substrate of 3:1:1 or 3:2:1. Based on the data, a transport model of ordered binding is proposed in which GABA binds first, Na+ second, and Cl- third. In conclusion, BGT-1 displays significant functional differences from the other members of the GABA transporter family.

Animals↗
PubMed1999

Effect of verapamil enantiomers and metabolites on cardiac K+ channels expressed in Xenopus oocytes.

The effect of verapamil and its enantiomers and metabolites on cardiac action potential repolarizing potassium channels was tested. For this purpose, the potassium channels Kv1.1, Kv1.5, Kir2.1, and HERG, and the IsK subunit of the IKs-channel complex were expressed in Xenopus oocytes and two-electrode voltage-clamp experiments were performed. Verapamil induced a concentration-dependent block of Kv1. 1-, Kv1.5-, IKs-, and HERG-induced currents with IC50 values of 14.0 +/- 2.7 microM (n = 4), 5.1 +/- 0.5 microM (n = 6), 161.0 +/- 26.3 microM (n = 4), and 3.8 +/- 0.2 microM (n = 5), respectively. The same potency of HERG channel inhibition was observed for the optical enantiomers (+)-verapamil (IC50 = 3.5 +/- 0.4 microM, n = 5) and (-)-verapamil (IC50 = 4.0 +/- 0.7 microM, n = 4), as well as the derivatives norverapamil (D591; IC50 = 3.8 +/- 0.3 microM, n = 4) and D703 (IC50 = 2.2 +/- 0.4 microM, n = 4). The verapamil metabolites D620 and D617 did not block HERG-induced currents at concentrations of up to 30 microM (n = 3). These results demonstrate that cardiac delayed rectifier potassium currents are sensitive targets to calcium channel blockers.

Animals↗
PubMed1999

The astroglial ASCT2 amino acid transporter as a mediator of glutamine efflux.

Glutamine release from astrocytes is an essential part of the glutamate-glutamine cycle in the brain. Uptake of glutamine into cultured rat astrocytes occurs by at least four different routes. In agreement with earlier studies, a significant contribution of amino acid transport systems ASC, A, L, and N was detected. It has not been determined whether these systems are also involved in glutamine efflux or whether specific efflux transporters exist. We show here that ASCT2, a variant of transport system ASC, is strongly expressed in rat astroglia-rich primary cultures but not in neuron-rich primary cultures. The amino acid sequence of rat astroglial ASCT2 is 83% identical to that of mouse ASCT2. In Xenopus laevis oocytes expressing rat ASCT2, we observed high-affinity uptake of [U-14C]glutamine (Km = 70 microM) that was Na(+)-dependent, concentrative, and unaffected by membrane depolarization. When oocytes were preloaded with [U-14C]glutamine, no glutamine efflux was detected in the absence of extracellular amino acids. Neither lowering intracellular pH nor raising the temperature elicited efflux. However, addition of 0.1 mM unlabeled alanine, serine, cysteine, threonine, glutamine, or leucine to the extracellular solution resulted in a rapid release of glutamine from the ASCT2-expressing oocytes. Amino acids that are not recognized as substrates by ASCT2 were ineffective in this role. Extracellular glutamate stimulated glutamine release weakly at pH 7.5 but was more effective on lowering pH to 5.5, consistent with the pH dependence of ASCT2 affinity for glutamate. Our findings suggest a significant role of ASCT2 in glutamine efflux from astrocytes by obligatory exchange with extracellular amino acids. However, the relative contribution of this pathway to glutamine release from cells in vivo or in vitro remains to be determined.

Amino Acid Sequence↗
PubMed1998

Angiotensin II stimulates vesicular H+-ATPase in rat proximal tubular cells.

Two mechanisms of H+ ion secretion in the proximal tubule that mediate bicarbonate reabsorption have been identified: the brush border Na/H exchanger and electrogenic H+ ion secretion. Angiotensin II (AII) has been shown to be a regulator of the luminal Na+/H+ exchanger and the basolateral Na+/HCO3- cotransporter. In the present study, we examined the effects of AII on H+-ATPase activity in isolated proximal tubule fragments. H+-ATPase activity was assessed by monitoring intracellular pH after Na+ removal from the bath. In addition, we investigated the effects on pH recovery of the proton pump inhibitor bafilomycin A1, removal of Cl-, and of colchicine. pH was continuously measured with the pH-sensitive fluorescent dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Recovery of cell pH was observed in the absence of external Na+ and was significantly accelerated by AII. The AII-stimulated pH recovery was completely abolished by bafilomycin A1, by removal of Cl-, by NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate; a potent Cl- channel blocker], and by colchicine. We conclude from these studies that AII stimulates proton extrusion via H+-ATPase by a Cl--dependent process involving brush border insertion of vesicles. This process may contribute to up-regulation of HCO3- reabsorption along the proximal tubule when tubules are exposed to AII.

Angiotensin II↗
PubMed1998

Chloride conductance and Pi transport are separate functions induced by the expression of NaPi-1 in Xenopus oocytes.

Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl- conductance (GCl), organic anion transport and Na+-dependent Pi-uptake. In the present study we investigated the relation between the NaPi-1 induced GCl and Pi-induced currents and transport. NaPi-1 expression induced Pi-transport, which was not different at 1-20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1-2 days after cRNA injection. In contrast, GCl was augmented at increased amounts of cRNA injection (1-20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. Pi-induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of Pi (>/= 1 mm Pi). The amplitudes of Ip correlated well with GCl. Ip was blocked by the Cl- channel blocker NPPB, partially Na+-dependent and completely abolished in Cl- free solution. In contrast, Pi-transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na+ and weakly affected by Cl- substitution. Endogenous Pi-uptake in water-injected oocytes amounted in all experiments to 30-50% of the Na+-dependent Pi-transport observed in NaPi-1 expressing oocytes. The properties of the endogenous Pi-uptake system (Km for Pi > 1 mM; partial Na+- and Cl--dependence; lack of NPPB block) were similar to the NaPi-1 induced Pi-uptake, but no Ip could be recorded at Pi-concentrations </=3 mM. In summary, the present data suggest that Ip does not reflect charge transfer related to Pi-uptake, but a Pi-mediated modulation of GCl.

Animals↗
PubMed1998

The Na+-phosphate cotransport system (NaPi-II) with a cleaved protein backbone: implications on function and membrane insertion.

1. Renal handling of inorganic phosphate (Pi) involves a Na+-Pi cotransport system which is well conserved between vertebrates. The members of this protein family, denoted NaPi-II, share a topology with, it is thought, eight transmembrane domains. The transporter is proposed to be proteolytically cleaved within a large hydrophilic loop in vivo. 2. The consequences of an interrupted backbone were tested by constructing cDNA clones encoding different N- (1-3 and 1-5) and C-terminal (4-8 and 6-8) complementary fragments of NaPi-II from winter flounder. When the cognate fragments were used in combination (1-3 plus 4-8; 1-5 plus 6-8) they comprised the full complement of the putative transporter domains. 3. None of the four individual fragments or the 1-5 plus 6-8 combination when expressed in Xenopus oocytes increased Pi flux. Coexpression of fragments 1-3 plus 4-8 stimulated transport activity identical to that for expressed wild-type NaPi-II with regard to pH dependency and Km for Na+ and Pi binding; however, the maximal transport rate (vmax) was lower. 4. Immunohistochemistry on cryosections confined the functionally active 1-3 plus 4-8 combination to the oocyte membrane. This was not the case for the 1-5 plus 6-8 combination or any of the individual fragments, all of which failed to induce fluorescence. 5. A second immunohistochemical approach using intact oocytes allowed determination of the extracellular regions of the protein. Epitopes within the loop between transmembrane domains 3 and 4 enhanced fluorescence. Neither N- nor C-terminal tags induced fluorescence.

Animals↗
PubMed1998

Effect of extracellular pH on the myo-inositol transporter SMIT expressed in Xenopus oocytes.

The myo-inositol transporter SMIT is expressed particularly at high extracellular osmolarity and serves to accumulate the osmolyte myo-inositol. Transport of myo-inositol is coupled to the cotransport of Na+ and is electrogenic. In Xenopus oocytes injected with mRNA encoding SMIT but not in water-injected oocytes, myo-inositol creates an inward current that is dependent on the ambient Na+ concentration. The present study has been performed to elucidate the pH dependence of myo-inositol-induced currents. Therefore, Xenopus oocytes were injected with mRNA encoding SMIT and two-electrode voltage-clamp studies were performed. The myo-inositol-induced currents in oocytes expressing SMIT were found to have a sigmoidal dependence on the ambient pH between pH 5.5 and 8.5 with an apparent Ki of 0.21+/-001 microM H+ and a Hill coefficient of 1.80+/-0.16. Kinetic analysis of the myo-inositol-induced currents at pH 8.0 and -90 mV holding potential revealed a Hill coefficient of 0.93+/-0.07 and an apparent Km for myo-inositol of 0.031+/-0.003 mM as well as a Hill coefficient of 1. 64+/-0.24 and an apparent Km of 38.8+/-4.1 mM for Na+. A decrease of the Na+ concentra-tion from 150 mM to 50 mM significantly altered the maximal observed current and increased the apparent Km for myo-inositol. Acidification to pH 6.5 significantly increased the apparent Km for myo-inositol and for Na+ to 0.057+/-0.005 mM and 73. 9+/-4.8 mM, respectively. The Hill coefficients for myo-inositol and Na+ were not affected and remained close to 1 for myo-inositol and 2 for Na+. In summary, acidification impedes SMIT-mediated myo-inositol transport at least partially by decreasing the affinity of the carrier for Na+. The impaired Na+ binding subsequently decreases binding and transport of myo-inositol.

Animals↗
PubMed1997

Electrophysiological characterization of the flounder type II Na+/Pi cotransporter (NaPi-5) expressed in Xenopus laevis oocytes.

The two electrode voltage clamp technique was used to investigate the steady-state and presteady-state kinetic properties of the type II Na+/Pi cotransporter NaPi-5, cloned from the kidney of winter flounder (Pseudopleuronectes americanus) and expressed in Xenopus laevis oocytes. Steady-state Pi-induced currents had a voltage-independent apparent K(m) for Pi of 0.03 mM and a Hill coefficient of 1.0 at neutral pH, when superfusing with 96 mM Na+. The apparent K(m) for Na+ at 1 mM Pi was strongly voltage dependent (increasing from 32 mM at -70 mV to 77 mM at -30 mV) and the Hill coefficient was between 1 and 2, indicating cooperative binding of more than one Na+ ion. The maximum steady-state current was pH dependent, diminishing by 50% or more for a change from pH 7.8 to pH 6.3. Voltage jumps elicited presteady-state relaxations in the presence of 96 mM Na+ which were suppressed at saturating Pi (1 mM). Relaxations were absent in non-injected oocytes. Charge was balanced for equal positive and negative steps, saturated at extremes of potential and reversed at the holding potential. Fitting the charge transfer to a Boltzmann relationship typically gave a midpoint voltage (V0.5) close to zero and an apparent valency of approximately 0.6. The maximum steady-state transport rate correlated linearly with the maximum Pi-suppressed charge movement, indicating that the relaxations were NaPi-5-specific. The apparent transporter turnover was estimated as 35 sec-1. The voltage dependence of the relaxations was Pi-independent, whereas changes in Na+ shifted V0.5 to -60 mV at 25 mM Na+. Protons suppressed relaxations but contributed to no detectable charge movement in zero external Na+. The voltage dependent presteady-state behavior of NaPi-5 could be described by a 3 state model in which the partial reactions involving reorientation of the unloaded carrier and binding of Na+ contribute to transmembrane charge movement.

Animals↗
PubMed1997

Aging and lymphocyte subsets in the spleen and peripheral blood of the Sprague-Dawley rat.

This study was undertaken to determine the effects of aging on lymphocyte subsets in the peripheral blood and spleens of Sprague-Dawley rats. Rats aged 3,13 and 26 months were used in the study. Analyses of dual labeled lymphocytes from the 26 month animals show decreases in the numbers of lymphocytes due to decreased cellularity (spleen) or reduced lymphocyte percentages within the total white blood cell population (peripheral blood). In the spleens and blood of the oldest rats, there were reduced numbers of Total T, T helper/amplifier (Th/a), virgin Th and natural killer (NK) cells. Other changes were observed in the spleen but not peripheral blood. The numbers of T cytotoxic/suppressor cells (Tc/s) B cells, "autoimmune" B cells and NK cells were reduced in the spleen but remained within normal limits in peripheral blood. The data show aging exerts different effects on the peripheral blood and splenic compartments of the immune system. These differences may have teleological significance in relation to immune responses to xenobiotics and neoplastic cells.

Aging↗
PubMed1996

Obligatory amino acid exchange via systems bo,+-like and y+L-like. A tertiary active transport mechanism for renal reabsorption of cystine and dibasic amino acids.

Mutations in the rBAT gene cause type I cystinuria, a common inherited aminoaciduria of cystine and dibasic amino acids due to their defective renal and intestinal reabsorption (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-426; Calonge, M. J., Volipini, V., Bisceglia, L., Rousaud, F., De Sanctis, L., Beccia, E., Zelante, L., Testar, X., Zorzano, A., Estivill, X., Gasparini, P., Nunes, V., and Palacín, M.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9667-9671). One important question that remains to be clarified is how the apparently non-concentrative system bo,+-like, associated with rBAT expression, participates in the active renal reabsorption of these amino acids. Several studies have demonstrated exchange of amino acids induced by rBAT in Xenopus oocytes. Here we offer evidence that system bo,+-like is an obligatory amino acid exchanger in oocytes and in the "renal proximal tubular" cell line OK. System bo, +-like showed a 1:1 stoichiometry of exchange, and the hetero-exchange dibasic (inward) with neutral (outward) amino acids were favored in oocytes. Obligatory exchange of amino acids via system bo,+-like fully explained the amino acid-induced current in rBAT-injected oocytes. Exchange via system bo,+-like is coupled enough to ensure a specific accumulation of substrates until the complete replacement of the internal oocyte substrates. Due to structural and functional analogies of the cell surface antigen 4F2hc to rBAT, we tested for amino acid exchange via system y+L-like. 4F2hc-injected oocytes accumulated substrates to a level higher than CAT1-injected oocytes (i.e. oocytes expressing system y+) and showed exchange of amino acids with the substrate specificity of system y+L and L-leucine-induced outward currents in the absence of extracellular sodium. In contrast to L-arginine, system y+L-like did not mediate measurable L-leucine efflux from the oocyte. We propose a role of systems bo,+-like and y+L-like in the renal reabsorption of cystine and dibasic amino acids that is based on their active tertiary transport mechanism and on the apical and basolateral localization of rBAT and 4F2hc, respectively, in the epithelial cells of the proximal tubule of the nephron.

Amino Acid Transport Systems, Basic↗
PubMed1996

Expression of a renal type I sodium/phosphate transporter (NaPi-1) induces a conductance in Xenopus oocytes permeable for organic and inorganic anions.

Two distinct molecular types (I and II) of renal proximal tubular brush border Na+/Pi cotransporters have been identified by expression cloning on the basis of their capacity to induce Na+-dependent Pi influx in tracer experiments. Whereas the type II transporters (e.g., NaPi-2 and NaPi-3) resemble well known characteristics of brush border Na+/Pi cotransport, little is known about the properties of the type I transporter (NaPi-1). In contrast to type II, type I transporters produced electrogenic transport only at high extracellular Pi concentrations (> or =3 mM). On the other hand, expression of NaPi-1 induced a Cl- conductance in Xenopus laevis oocytes, which was inhibited by Cl- channel blockers [5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) > niflumic acid >> 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid]. Further, the Cl- conductance was inhibited by the organic anions phenol red, benzylpenicillin (penicillin G), and probenecid. These organic anions induced outwardly directed currents in the absence of Cl-. In tracer studies, we observed uptake of benzylpenicillin with a Km of 0.22 mM; benzylpenicillin uptake was inhibited by NPPB and niflumic acid. These findings suggest that the type I Na+/Pi cotransporter functions also as a novel type of anion channel permeable not only for Cl- but also for organic anions. Such an apical anion channel could serve an important role in the transport of Cl- and the excretion of anionic xenobiotics.

Animals↗