Expression of Human Polyspecific Renal Organic Cation Transport Activity in Xenopus laevis Oocytes

1997 ◽  
Vol 86 (6) ◽  
pp. 753-755 ◽  
Author(s):  
Joanne K. Chun ◽  
Micheline Piquette-Miller ◽  
Lei Zhang ◽  
Kathleen M. Giacomini
Keyword(s):  
Xenopus Laevis ◽  
Organic Cation ◽  
Cation Transport ◽  
Organic Cation Transport ◽  
Xenopus Laevis Oocytes ◽  
Transport Activity

scholarly journals Expression of renal organic cation transporter in Xenopus laevis oocytes

1992 ◽  
Vol 283 (2) ◽  
pp. 409-411 ◽  
Author(s):  
R Hori ◽  
M Hirai ◽  
T Katsura ◽  
M Takano ◽  
M Yasuhara ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Transport System ◽  
Organic Cation ◽  
Expression System ◽  
Fold Increase ◽  
Organic Cation Transporter ◽  
Sucrose Density Gradient ◽  
Cation Transport ◽  
Organic Cation Transport ◽  
Xenopus Laevis Oocytes

The expression of the organic cation transport system of rat renal proximal tubules has been studied in Xenopus laevis oocytes injected with poly(A)+ RNA from the rat renal cortex. The effectiveness of the technique was confirmed by examining expression of the Na+/D-glucose co-transporter. Compared with water-injected and non-injected oocytes, the injection of total poly(A)+ RNA resulted in about a 3-fold increase in tetraethylammonium (TEA) uptake activity. TEA uptake by poly(A)(+)-RNA-injected oocytes was time-dependent and was inhibited by cimetidine and HgCl2, but not by p-aminohippurate. After size-fractionation on a sucrose density gradient, a 1.4-2.4 kb poly(A)+ RNA fragment was identified that expressed the organic cation transport system in oocytes. These results demonstrate that the renal organic cation transporter was expressed in oocytes and that this expression system can provide an effective assay procedure for cloning of the organic cation transporter.

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.

Functional Impairment of Renal Organic Cation Transport in Experimental Diabetes

2002 ◽  
Vol 90 (4) ◽  
pp. 181-186 ◽  
Author(s):  
Brett Grover ◽  
Christopher Auberger ◽  
Rangaprasad Sarangarajan ◽  
William Cacini
Keyword(s):  
Functional Impairment ◽  
Organic Cation ◽  
Experimental Diabetes ◽  
Cation Transport ◽  
Organic Cation Transport

Peritubular organic cation transport in isolated rabbit proximal tubules

1994 ◽  
Vol 266 (3) ◽  
pp. F450-F458 ◽  
Author(s):  
C. E. Groves ◽  
K. K. Evans ◽  
W. H. Dantzler ◽  
S. H. Wright
Keyword(s):  
Organic Cation ◽  
High Capacity ◽  
Organic Cation Transporter ◽  
Cation Transport ◽  
Proximal Tubules ◽  
Organic Cation Transport ◽  
Inhibitory Potency ◽  
Renal Proximal Tubules ◽  
Michaelis Constants ◽  
Maximal Capacity

The physiological characteristics of peritubular organic cation transport were examined by measuring the transport of the organic cation tetraethylammonium (TEA) in rabbit renal proximal tubule suspensions and isolated nonperfused rabbit renal proximal tubules. Peritubular organic cation transport in both single S2 segments and suspensions of isolated renal proximal tubules was found to be a high-capacity, high-affinity, carrier-mediated process. For tubule suspensions, the maximal capacity of the carrier for TEA (Jmax) and the concentration of TEA at 1/2 Jmax (Kt) (1.49 +/- 0.21 nmol.min-1.mg dry wt-1 and 131 +/- 16 microM, respectively), did not differ significantly from those measured in single S2 segments (Jmax, 1.16 +/- 0.075 nmol.min-1.mg dry wt-1; Kt, 108 +/- 10 microM). In addition, the pattern of inhibition of peritubular TEA transport by long-chain n-tetraalkylammonium compounds (n = 1-5) was both qualitatively and quantitatively similar in single S2 segments and tubule suspensions, exhibiting an increase in inhibitory potency with increasing alkyl chain length. For example, in tubule suspensions, apparent Michaelis constants for inhibition of TEA uptake ranged from 1.3 mM for tetramethylammonium (TMA) to 0.8 microM for tetrapentylammonium (TPeA). To determine whether these compounds were substrates for the peritubular organic cation transporter, their effect on the efflux of [14C]TEA from tubule suspensions was examined. A concentration of 0.5 mM of the short-chain tetraalkyls TMA or TEA increased the efflux of [14C]TEA (i.e., trans-stimulated) from tubules in suspension. The longer-chain tetraalkyls tetrapropylammonium, tetrabutylammonium, and TPeA all decreased the efflux of [14C]TEA from tubules in suspension; TPeA completely blocked efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of organic cation transport

2004 ◽  
Vol 449 (5) ◽  
pp. 423-441 ◽  
Author(s):  
Giuliano Ciarimboli ◽  
Eberhard Schlatter
Keyword(s):  
Organic Cation ◽  
Cation Transport ◽  
Organic Cation Transport

POSTNATAL DEVELOPMENT OF ORGANIC CATION TRANSPORT IN THE RAT LIVER

1998 ◽  
Vol 37 (2) ◽  
pp. 131-136 ◽  
Author(s):  
F. MARTEL ◽  
M.J. MARTINS ◽  
C. CALHAU ◽  
C. HIPÓLITO-REIS ◽  
I. AZEVEDO
Keyword(s):  
Postnatal Development ◽  
Rat Liver ◽  
Organic Cation ◽  
Cation Transport ◽  
Organic Cation Transport
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