Inhibition of P-glycoprotein-mediated transport by a hydrophobic contaminant in commercial gluconate salts

1999 ◽  
Vol 276 (6) ◽  
pp. C1439-C1442 ◽  
Author(s):  
Carlos G. Vanoye ◽  
Guillermo A. Altenberg ◽  
Luis Reuss
Keyword(s):  
Membrane Transport ◽  
Ringer Solution ◽  
Chloroform Extract ◽  
Rhodamine 123 ◽  
Transport Mechanisms ◽  
Sodium Gluconate ◽  
Chloroform Extraction ◽  
P Glycoprotein ◽  
Dependent Transport

The substitution of gluconate for Cl− is commonly used to characterize Cl− transport or Cl−-dependent transport mechanisms. We evaluated the effects of substituting gluconate for Cl− on the transport of the P-glycoprotein substrate rhodamine 123 (R123). The replacement of Ringer solution containing Cl−(Cl−-Ringer) with gluconate-Ringer inhibited R123 efflux, whereas the replacement of Cl− by other anions (sulfate or cyclamate) had no effect. The inhibition of R123 efflux by gluconate-Ringer was absent after chloroform extraction of the sodium gluconate salt. The readdition of the sodium gluconate-chloroform extract to the extracted gluconate-Ringer or to cyclamate-Ringer inhibited R123 efflux, whereas its addition to Cl−-Ringer had no effect. These observations indicate that the inhibition of P-glycoprotein-mediated R123 transport by gluconate is due to one or more chloroform-soluble contaminants and that the inhibition is absent in the presence of Cl−. The results are consistent with the fact that P-glycoprotein substrates are hydrophobic. Care should be taken when replacing ions to evaluate membrane transport mechanisms because highly pure commercial preparations may still contain potent contaminants that affect transport.

Expression and functional activity of P-glycoprotein in cultured hepatocytes fromOncorhynchus mykiss

2001 ◽  
Vol 281 (4) ◽  
pp. R1119-R1126 ◽  
Author(s):  
Armin Sturm ◽  
Christina Ziemann ◽  
Karen I. Hirsch-Ernst ◽  
Helmut Segner
Keyword(s):  
Multidrug Resistance ◽  
Cyclosporin A ◽  
Transport Mechanism ◽  
Protein S ◽  
Rhodamine 123 ◽  
Cultured Hepatocytes ◽  
Hydrophobic Compounds ◽  
P Glycoprotein ◽  
Dependent Transport ◽  
Conserved Epitope

P-glycoproteins encoded by multidrug resistance 1 ( mdr1) genes are ATP-dependent transporters located in the plasma membrane that mediate the extrusion of hydrophobic compounds from the cell. Using cultured isolated rainbow trout hepatocytes, we characterized an mdr1-like transport mechanism of the teleost liver. Immunoblots with the monoclonal antibody C219, which recognizes a conserved epitope of P-glycoproteins, revealed the presence of immunoreactive protein(s) of 165 kDa in trout liver and cultured hepatocytes. In trout liver sections, the immunohistochemistry with C219 stained bile canalicular structures. Compounds known to interfere with mdr1-dependent transport (verapamil, vinblastine, doxorubicin, cyclosporin A, and vanadate) all increased the accumulation of rhodamine 123 by hepatocytes. Verapamil, vinblastine, and cyclosporin A decreased the efflux of rhodamine 123 from hepatocytes preloaded with rhodamine 123. By contrast, the substrate of the canalicular cation transporter tetraethylammonium and the inhibitor of the multidrug resistance-associated protein MK571 had no effect on rhodamine 123 transport. The results demonstrate the presence of an mdr1-like transport system in the teleost liver and suggest its function in biliary excretion.

scholarly journals Pulchinenosides from Pulsatilla Chinensis Increase P-Glycoprotein Activity and Induce P-Glycoprotein Expression

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Yali Liu ◽  
Ling Zhang ◽  
Shaofeng Wei ◽  
Jinyang Cai ◽  
Zhenzhong Zang ◽  
...  
Keyword(s):  
Membrane Vesicles ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Intestinal Epithelial ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Human Intestinal Epithelial Cells ◽  
Pulsatilla Chinensis ◽  
Dependent Transport ◽  
Cytotoxic Studies

Five pulchinenosides (pulchinenoside B3, pulchinenoside BD, pulchinenoside B7, pulchinenoside B10, and pulchinenoside B11) isolated from Pulsatilla chinensis (Bge) Regel saponins extract exhibited strong antitumor activities but poor gastrointestinal absorption properties. The enteric induction of P-glycoprotein (P-gp) is understood to restrict the oral bioavailability of some pharmaceutical compounds and lead to adverse drug reactions. Therefore, the present investigation was intended to delineate the impacts of pulchinenosides on cellular P-gp function and expression using Sf9 membrane vesicles and LS180 cells as a surrogate of human intestinal epithelial cells. Preliminary cytotoxic studies showed that 10 μM was an acceptable concentration for cytotoxicity and antiproliferation studies for all pulchinenosides using the alamarBlue assay. The cell cycle of LS180 cells detected by flow cytometry was not significantly influenced after 48 hours of coincubation with 10 μM of pulchinenosides. In the presence of pulchinenosides, the ATP-dependent transport of N-methyl-quinidine mediated by P-glycoprotein was stimulated significantly. The upregulation of P-glycoprotein and mRNA levels was found by Western blot and real-time PCR analysis in LS180 cells. Parallel changes indicate that all pulchinenosides are exposed to pulchinenosides-mediated transcriptional regulation. In conclusion, pulchinenosides could induce P-glycoprotein expression and directly increase its functional activity.

Examination of the Functional Activity of P-glycoprotein in the Rat Placental Barrier Using Rhodamine 123

2003 ◽  
Vol 305 (3) ◽  
pp. 1239-1250 ◽  
Author(s):  
Petr Pavek ◽  
Frantisek Staud ◽  
Zdenek Fendrich ◽  
Hana Sklenarova ◽  
Antonin Libra ◽  
...  
Keyword(s):  
Functional Activity ◽  
Rhodamine 123 ◽  
Placental Barrier ◽  
P Glycoprotein

scholarly journals Analysis of P-glycoprotein-mediated membrane transport in human peripheral blood lymphocytes using the UIC2 shift assay

Cytometry ◽  
2003 ◽  
Vol 53A (2) ◽  
pp. 67-78 ◽  
Author(s):  
Suk W. Park ◽  
Noureddine Lomri ◽  
Luiz A. Simeoni ◽  
John P. Fruehauf ◽  
Eugene Mechetner
Keyword(s):  
Membrane Transport ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Peripheral Blood Lymphocytes ◽  
Blood Lymphocytes ◽  
Human Peripheral Blood Lymphocytes ◽  
P Glycoprotein

The involvement of selected membrane transport mechanisms in the cellular uptake of 177Lu-labeled bombesin, somatostatin and gastrin analogues

2015 ◽  
Vol 42 (1) ◽  
pp. 1-7 ◽  
Author(s):  
M. Volková ◽  
J. Mandíková ◽  
A. Lázníčková ◽  
M. Lázníček ◽  
P. Bárta ◽  
...  
Keyword(s):  
Membrane Transport ◽  
Cellular Uptake ◽  
Transport Mechanisms

scholarly journals Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms

2015 ◽  
Vol 29 (6) ◽  
pp. 2583-2594 ◽  
Author(s):  
Kate L. Widdows ◽  
Nuttanont Panitchob ◽  
Ian P. Crocker ◽  
Colin P. Please ◽  
Mark A. Hanson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Computational Modeling ◽  
Membrane Transport ◽  
Transport Mechanisms ◽  
Amino Acid Exchange ◽  
Transport Studies ◽  
Acid Exchange

scholarly journals Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system.

1996 ◽  
Vol 7 (10) ◽  
pp. 1485-1498 ◽  
Author(s):  
M Ramachandra ◽  
S V Ambudkar ◽  
M M Gottesman ◽  
I Pastan ◽  
C A Hrycyna
Keyword(s):  
Drug Resistance ◽  
Atpase Activity ◽  
Cyclosporin A ◽  
Transient Expression ◽  
Expression System ◽  
Rhodamine 123 ◽  
Wild Type ◽  
P Glycoprotein ◽  
Transient Expression System

Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that confers multidrug resistance to otherwise sensitive cells. A mutation in Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines that are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutation leads to an altered drug resistance profile, we used a transient expression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of many aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human osteosarcoma (HOS) cells by infection with a recombinant vaccinia virus encoding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant and wild-type proteins, photolabeling with [3H]azidopine and [125I]iodoarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodamine 123, and accumulation of radiolabeled vinblastine and colchicine were evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant than for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosporin A inhibited the ATPase activity of the mutant more efficiently than that of the wild-type. This latter observation explains the enhanced potency of cyclosporin A as an inhibitor of the mutant Pgp. No differences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblastine in cells expressing the mutant protein, as compared with those expressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase activity and altered interaction with substrates and the inhibitor cyclosporin A.

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