scholarly journals Characterization of the monocarboxylate transporter 1 expressed in Xenopus laevis oocytes by changes in cytosolic pH

1998 ◽  
Vol 333 (1) ◽  
pp. 167-174 ◽  
Author(s):  
Stefan BRÖER ◽  
Hans-Peter SCHNEIDER ◽  
Angelika BRÖER ◽  
Basim RAHMAN ◽  
Bernd HAMPRECHT ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Intracellular Ph ◽  
Expression System ◽  
Lactate Concentration ◽  
Monocarboxylate Transporter ◽  
Kinetic Properties ◽  
Xenopus Laevis Oocytes ◽  
Lactate Transport ◽  
Monocarboxylate Transporter 1

Several laboratories have investigated monocarboxylate transport in a variety of cell types. The characterization of the cloned transporter isoforms in a suitable expression system is nevertheless still lacking. H+/monocarboxylate co-transport was therefore investigated in monocarboxylate transporter 1 (MCT1)-expressing Xenopus laevis oocytes by using pH-sensitive microelectrodes and [14C]lactate. Superfusion with lactate resulted in intracellular acidification of MCT1-expressing oocytes, but not in non-injected control oocytes. The basic kinetic properties of lactate transport in MCT1-expressing oocytes were determined by analysing the rates of intracellular pH changes under different conditions. The results were in agreement with the known properties of the transporter, with respect to both the dependence on the lactate concentration and the external pH value. Besides lactate, MCT1 mediated the reversible transport of a wide variety of monocarboxylic acids including pyruvate, d,l-3-hydroxybutyrate, acetoacetate, α-oxoisohexanoate and α-oxoisovalerate, but not of dicarboxylic and tricarboxylic acids. The inhibitor α-cyano-4-hydroxycinnamate bound strongly to the transporter without being translocated, but could be displaced by the addition of lactate. In addition to changes in the intracellular pH, lactate transport also induced deviations from the resting membrane potential.

scholarly journals Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes

1999 ◽  
Vol 341 (3) ◽  
pp. 529-535 ◽  
Author(s):  
Stefan BRÖER ◽  
Angelika BRÖER ◽  
Hans-Peter SCHNEIDER ◽  
Carola STEGEN ◽  
Andrew P. HALESTRAP ◽  
...  
Keyword(s):  
Ketone Bodies ◽  
Anion Channel ◽  
Monocarboxylate Transporter ◽  
Xenopus Laevis Oocytes ◽  
Lactate Transport ◽  
High Affinity ◽  
Monocarboxylate Transport ◽  
Km Value ◽  
Decreasing Ph

Observations on lactate transport in brain cells and cardiac myocytes indicate the presence of a high-affinity monocarboxylate transporter. The rat monocarboxylate transporter isoform MCT2 was analysed by expression in Xenopus laevisoocytes and the results were compared with the known characteristics of lactate transport in heart and brain. Monocarboxylate transport via MCT2 was driven by the H+ gradient over the plasma membrane. Uptake of lactate strongly increased with decreasing pH, showing half-maximal stimulation at pH 7.2. A wide variety of monocarboxylates and ketone bodies, including lactate, pyruvate, β-hydroxybutyrate, acetoacetate, 2-oxoisovalerate and 2-oxoisohexanoate, were substrates of MCT2. All substrates had a high affinity for MCT2. For lactate a Km value of 0.74±0.07 mM was determined at pH 7.0. For the other substrates, Ki values between 100 μM and 1 mM were measured for inhibition of lactate transport, which is about one-tenth of the corresponding values for the ubiquitously expressed monocarboxylate transporter isoform MCT1. Monocarboxylate transport via MCT2 could be inhibited by α-cyano-4-hydroxycinnamate, anion-channel inhibitors and flavonoids. It is suggested that cells which express MCT2 preferentially use lactate and ketone bodies as energy sources.

An endogenous monocarboxylate transport inXenopus laevisoocytes

2000 ◽  
Vol 278 (5) ◽  
pp. R1190-R1195 ◽  
Author(s):  
M. Tosco ◽  
M. N. Orsenigo ◽  
G. Gastaldi ◽  
A. Faelli
Keyword(s):  
Xenopus Laevis ◽  
Monocarboxylate Transporter ◽  
Monocarboxylate Transporters ◽  
Xenopus Laevis Oocytes ◽  
Lactate Transport ◽  
Ph Gradients ◽  
Monocarboxylate Transport ◽  
Functional Features ◽  
Lactate Uptake ◽  
Uptake Studies

We investigated the existence of an endogenous system for lactate transport in Xenopus laevis oocytes.36Cl-uptake studies excluded the involvement of a DIDS-sensitive anion antiporter as a possible pathway for lactate movement.l-[14C]lactate uptake was unaffected by superimposed pH gradients, stimulated by the presence of Na+in the incubating solution, and severely reduced by the monocarboxylate transporter inhibitor p-chloromercuribenzenesulphonate (pCMBS). Transport exhibited a broad cation specificity and was cis inhibited by other monocarboxylates, mostly by pyruvate. These results suggest that lactate uptake is mediated mainly by a transporter and that the preferred anion is pyruvate. [14C]pyruvate uptake exhibited the same pattern of functional properties evidenced for l-lactate. Kinetic parameters were calculated for both monocarboxylates, and a higher affinity for pyruvate was revealed. Various inhibitors of monocarboxylate transporters reduced significantly pyruvate uptake. These studies demonstrate that Xenopus laevis oocytes possess a monocarboxylate transport system that shares some functional features with the members of the mammalian monocarboxylate cotransporters family, but, in the meanwhile, exhibits some particular properties, mainly concerning cation specificity.

scholarly journals Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes

1999 ◽  
Vol 341 (3) ◽  
pp. 529 ◽  
Author(s):  
Stefan BRÖER ◽  
Angelika BRÖER ◽  
Hans-Peter SCHNEIDER ◽  
Carola STEGEN ◽  
Andrew P. HALESTRAP ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Monocarboxylate Transporter ◽  
Xenopus Laevis Oocytes ◽  
High Affinity

Characterization of Transport Activity of SLC11 Transporters in Xenopus laevis Oocytes by Fluorophore Quenching

2021 ◽  
pp. 247255522110041
Author(s):  
Raffaella Cinquetti ◽  
Francesca Guia Imperiali ◽  
Salvatore Bozzaro ◽  
Daniele Zanella ◽  
Francesca Vacca ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Voltage Clamp ◽  
Expression System ◽  
Peptide Transporter ◽  
Xenopus Laevis Oocytes ◽  
Substrate Uptake ◽  
Transport Activity ◽  
Experimental Conditions ◽  
Metal Transporters ◽  
Electrode Voltage

Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. Xenopus laevis oocytes provide a powerful heterologous expression system for functional studies of these proteins. Typical experiments investigate transport using electrophysiology and radiolabeled uptake. A two-electrode voltage clamp is suitable only for electrogenic proteins, and uptake measurements require the existence of radiolabeled substrates and adequate laboratory facilities. Recently, Dictyostelium discoideum Nramp1 and NrampB were characterized using multidisciplinary approaches. NrampB showed no measurable electrogenic activity, and it was investigated in Xenopus oocytes by acquiring confocal images of the quenching of injected fluorophore calcein. This method is adequate to measure the variation in emitted fluorescence, and thus transporter activity indirectly, but requires long experimental procedures to collect statistically consistent data. Considering that optimal expression of heterologous proteins lasts for 48–72 h, a slow acquiring process requires the use of more than one batch of oocytes to complete the experiments. Here, a novel approach to measure substrate uptake is reported. Upon injection of a fluorophore, oocytes were incubated with the substrate and the transport activity measured, evaluating fluorescence quenching in a microplate reader. The technique permits the testing of tens of oocytes in different experimental conditions simultaneously, and thus the collection of significant statistical data for each batch, saving time and animals. The method was tested with different metal transporters (SLC11), DMT1, DdNramp1, and DdNrampB, and verified with the peptide transporter PepT1 (SLC15). Comparison with traditional methods (uptake, two-electrode voltage clamp) and with quenching images acquired by fluorescence microscopy confirmed its efficacy.

scholarly journals Comparison of Lactate Transport in Astroglial Cells and Monocarboxylate Transporter 1 (MCT 1) ExpressingXenopus laevisOocytes

1997 ◽  
Vol 272 (48) ◽  
pp. 30096-30102 ◽  
Author(s):  
Stefan Bröer ◽  
Basim Rahman ◽  
Gioranni Pellegri ◽  
Luc Pellerin ◽  
Jean-Luc Martin ◽  
...  
Keyword(s):  
Monocarboxylate Transporter ◽  
Lactate Transport ◽  
Astroglial Cells ◽  
Monocarboxylate Transporter 1

scholarly journals Functional Characterization of the Oxantel-Sensitive Acetylcholine Receptor from Trichuris muris

2021 ◽  
Vol 14 (7) ◽  
pp. 698
Author(s):  
Tina V. A. Hansen ◽  
Richard K. Grencis ◽  
Mohamed Issouf ◽  
Cédric Neveu ◽  
Claude L. Charvet
Keyword(s):  
Single Dose ◽  
Mouse Model ◽  
Xenopus Laevis ◽  
Acetylcholine Receptor ◽  
Drug Target ◽  
Functional Characterization ◽  
Xenopus Laevis Oocytes ◽  
Trichuris Muris ◽  
Electrode Voltage

The human whipworm, Trichuris trichiura, is estimated to infect 289.6 million people globally. Control of human trichuriasis is a particular challenge, as most anthelmintics have a limited single-dose efficacy, with the striking exception of the narrow-spectrum anthelmintic, oxantel. We recently identified a novel ACR-16-like subunit from the pig whipworm, T. suis which gave rise to a functional acetylcholine receptor (nAChR) preferentially activated by oxantel. However, there is no ion channel described in the mouse model parasite T. muris so far. Here, we have identified the ACR-16-like and ACR-19 subunits from T. muris, and performed the functional characterization of the receptors in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. We found that the ACR-16-like subunit from T. muris formed a homomeric receptor gated by acetylcholine whereas the ACR-19 failed to create a functional channel. The subsequent pharmacological analysis of the Tmu-ACR-16-like receptor revealed that acetylcholine and oxantel were equally potent. The Tmu-ACR-16-like was more responsive to the toxic agonist epibatidine, but insensitive to pyrantel, in contrast to the Tsu-ACR-16-like receptor. These findings confirm that the ACR-16-like nAChR from Trichuris spp. is a preferential drug target for oxantel, and highlights the pharmacological difference between Trichuris species.

scholarly journals miR-124 Suppresses Pancreatic Ductal Adenocarcinoma Growth by Regulating Monocarboxylate Transporter 1-Mediated Cancer Lactate Metabolism

2018 ◽  
Vol 50 (3) ◽  
pp. 924-935 ◽  
Author(s):  
De-Hai Wu ◽  
Hao Liang ◽  
Shou-Nan Lu ◽  
Hao Wang ◽  
Zhi-Lei Su ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Intracellular Ph ◽  
Molecular Mechanisms ◽  
Monocarboxylate Transporter ◽  
Human Pancreatic Cancer ◽  
Ductal Adenocarcinoma ◽  
Luciferase Reporter ◽  
Lactate Metabolism ◽  
Rt Pcr ◽  
Monocarboxylate Transporter 1

Background/Aims: Increasing evidence shows that reprogramming of energy metabolism is a hallmark of cancer. Considering the emergence of microRNAs as crucial modulators of cancer, this study aimed to better understand the molecular mechanisms of miR-124 in regulating glycolysis in human pancreatic cancer. Methods: RT-PCR was used to investigate the expression of monocarboxylate transporters (MCTs) in pancreatic ductal adenocarcinoma (PDAC) patient samples and the PANC-1 cell line. A public database and immunochemistry were used for comprehensive analysis of MCT1 expression. The targeting of MCT1 by miR-124 was predicted by software and validated for the MCT1 3’-UTR by dual-luciferase reporter analysis. Cell proliferation, apoptosis, migration, xenografting, and the intracellular pH and L-lactate levels were assessed. Hypoxia-inducible factor-α (HIF-1α) and lactate dehydrogenase A (LDH-A) expression levels were determined by RT-PCR and western blotting. Results: MCT1 expression was higher in PDAC tissue than in normal tissue. Inhibition of MCT1 affected lactate metabolism, resulting in a higher intracellular pH and less proliferation of PANC-1 cells. MCT1 was the target gene of miR-124. In in vitro experiments, miR-124 inhibited the glycolytic activity of PANC-1 cells by targeting MCT1, further decreasing the tumor phenotype by increasing the intracellular pH through LDH-A and HIF-1α. In in vivo experiments, overexpression of miR-124 and silencing of MCT1 significantly inhibited tumor growth. Conclusion: miR-124 inhibits the progression of PANC-1 by targeting MCT1 in the lactate metabolic pathway. Our findings provide novel evidence for further functional studies of miR-124, which might be useful for future therapeutic approaches to PDAC.

Functional characterization of leucine transport induced in Xenopus laevis oocytes injected with mRNA isolated from midguts of lepidopteran larvae (Philosamia cynthia).

1995 ◽  
Vol 198 (4) ◽  
pp. 961-966
Author(s):  
V F Sacchi ◽  
C Perego ◽  
S Magagnin
Keyword(s):  
Xenopus Laevis ◽  
Functional Characterization ◽  
Xenopus Laevis Oocytes ◽  
Fourfold Increase ◽  
Leucine Uptake ◽  
Lepidopteran Larvae ◽  
Nacl Concentration ◽  
Leucine Transport ◽  
The Difference

The injection of poly(A)+ mRNA prepared from Philosamia cynthia midgut caused time- and dose-dependent increases of leucine transport in Xenopus laevis oocytes, with an increase in leucine uptake 1.5-3 times that of oocytes injected with water. When the NaCl concentration was reduced from 100 to 5 mmol l-1, the difference between mRNA- and water-injected oocytes was greater and a fourfold increase of L-leucine uptake was measured. D-Leucine (10 mmol l-1) completely inhibited the induced uptake of 0.1 mmol l-1 L-leucine. The newly expressed component of L-leucine uptake increased at alkaline pH and was abolished by incubation for 15 min with 15 mmol l-1 phenylglyoxal. The mean Km values, calculated using Na+ activation curves of leucine uptake, were 23.3 +/- 6.1 mmol l-1 in water-injected oocytes and 0.4 +/- 0.2 mmol l-1 for the newly expressed component of leucine uptake in mRNA-injected oocytes. On the basis of these results, we conclude that the increase of L-leucine uptake in mRNA-injected oocytes was due to the expression of a new transport system, which differs from the endogenous ones and shares many features with that found previously in Philosamia cynthia midgut.

Rapid downregulation of the rat glutamine transporter SNAT3 by a caveolin-dependent trafficking mechanism in Xenopus laevis oocytes

2010 ◽  
Vol 299 (5) ◽  
pp. C1047-C1057 ◽  
Author(s):  
Sarojini Balkrishna ◽  
Angelika Bröer ◽  
Alice Kingsland ◽  
Stefan Bröer
Keyword(s):  
Xenopus Laevis ◽  
Dominant Negative ◽  
Monocarboxylate Transporter ◽  
Xenopus Laevis Oocytes ◽  
Independent Manner ◽  
Cultured Astrocytes ◽  
Glutamine Transporter ◽  
Liver And Kidney ◽  
The Brain ◽  
Glutamine Uptake

The glutamine transporter SNAT3 is involved in the uptake and release of glutamine in the brain, liver, and kidney. Substrate transport is accompanied by Na+ cotransport and H+ antiport. In this study, treatment of Xenopus laevis oocytes expressing rat SNAT3 with the phorbol ester PMA resulted in a rapid downregulation of glutamine uptake in less than 20 min. PMA treatment of oocytes coexpressing SNAT3 and the monocarboxylate transporter MCT1 reduced SNAT3 activity only, demonstrating the specificity of the regulatory mechanism. Single or combined mutations of seven putative phosphorylation sites in the SNAT3 sequence did not affect the regulation of SNAT3 by PMA. Expression of an EGFP-SNAT3 fusion protein in oocytes established that the downregulation was caused by the retrieval of the transporter from the plasma membrane. Coexpression of SNAT3 with dominant-negative mutants of dynamin or caveolin revealed that SNAT3 trafficking occurs in a dynamin-independent manner and is influenced by caveolin. Although system N activity was not affected by PMA in cultured astrocytes, a downregulation was observed in HepG2 cells.

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