Selection for MDR1/P-glycoprotein enhances swelling-activated K+ and Cl- currents in NIH/3T3 cells

1994 ◽  
Vol 267 (2) ◽  
pp. C650-C658 ◽  
Author(s):  
D. B. Luckie ◽  
M. E. Krouse ◽  
K. L. Harper ◽  
T. C. Law ◽  
J. J. Wine
Keyword(s):  
Cell Lines ◽  
Response Curve ◽  
Drug Transport ◽  
Regulatory Volume Decrease ◽  
Absolute Difference ◽  
P Glycoprotein ◽  
Cl Channels ◽  
Selection For ◽  
Nih 3T3 ◽  
Cl Conductance

The relationship between multidrug resistance (MDR) P-glycoprotein expression and swelling-activated Cl- and K+ conductance was investigated in mouse NIH/3T3 fibroblasts and their colchicine-selected counterparts (COL1000, high P-glycoprotein). Whole cell patch-clamp and isotopic flux experiments confirmed that swelling-activated Cl- currents were induced by 20-30% bath dilution only in the MDR-expressing cell line. However, at bath dilutions > 30%, both cell lines developed Cl- currents that reached similar large magnitudes at higher dilution levels. Thus the apparent absolute difference in cell lines at lower dilutions is due to a shift in the response curve relating hypotonicity to Cl- conductance. At all dilutions and in both cell lines, the swelling-activated Cl- currents were outwardly rectifying, active at negative cell voltages, and inactivated at positive voltages. Verapamil (100 microM) and 1,9-dideoxyforskolin (100 microM), which inhibit P-glycoprotein drug transport, did not significantly inhibit the swelling-activated Cl- conductance efflux in the COL1000 cells also showed a leftward shift in the response curve to hypotonicity. These results indicate that response curve to hypotonicity. These results indicate that colchicine-selection for increased P-glycoprotein expression did not lead to the expression of swelling-activated Cl- channels, but instead enhanced a step in the pathway from bath dilution to regulatory volume decrease that is common to both K+ and Cl- channels.

Recruitment of purinergically stimulated Cl- channels from granule membrane to plasma membrane

1996 ◽  
Vol 271 (2) ◽  
pp. C612-C619 ◽  
Author(s):  
D. Merlin ◽  
X. Guo ◽  
K. Martin ◽  
C. Laboisse ◽  
D. Landis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Lines ◽  
Inhibitory Effects ◽  
Fungal Metabolite ◽  
Cl Secretion ◽  
Fusion Inhibitors ◽  
Granule Membrane ◽  
Cl Channels ◽  
Ht 29 ◽  
Cl Conductance

HT29-Cl.16E and HT29-Cl.19A are two different subcloned cell lines derived from the human adenocarcinoma cell line HT-29. They are similar in their ability to grow and differentiate to polarized epithelial cells but differ in that HT29-Cl.16E is goblet cell-like with many mucin granules, whereas HT29-Cl.19A lacks mucin granules. Extracellular ATP stimulates Cl- secretion in both cell lines through luminal purinergic P20 receptors and, in HT29-Cl.16E, also mucin secretion release. To evaluate whether fusion of mucin granules is associated with an increase in Cl- conductance of the plasma membrane, the effects of two fusion inhibitors on luminal Cl- conductance were measured. Blockage of actin depolymerization with phalloidin (1 microM) inhibited purinergically stimulated but not adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated luminal Cl- efflux by 50% in HT29-Cl.16E. The same treatment was without effect in HT29-Cl.19A. The fungal metabolite wortmannin, which is an inhibitor of regulated exocytosis in leukocytes, at 100 nM inhibited Cl- secretion by 70% in HT29-Cl.16E. This inhibition was not a direct effect on purinergically stimulated Cl- channels because wortmannin concentrations of up to 1 microM did not affect the secretory response in HT29-Cl.19A. The wortmannin inhibition of Cl- secretion is associated with an inhibition of granule fusion as judged by electron microscopy. The differential inhibition of Cl- secretion in the related HT-29 clones that differ with respect to the presence of mucin granules indicates that 1) the granule fusion inhibitors, phalloidin and wortmannin, have no direct inhibitory effects on purinergically and cAMP-activated Cl- channels, 2) a major portion of purinergically but not cAMP-activated Cl- channels is associated with granule fusion in HT29-Cl.16E, and 3) the signaling pathways for Cl- secretion and granule fusion are not completely identical.

Doxorubicin selection for MDR1/P-glycoprotein reduces swelling-activated K+ and Cl- currents in MES-SA cells

1996 ◽  
Vol 270 (4) ◽  
pp. C1029-C1036 ◽  
Author(s):  
D. B. Luckie ◽  
M. E. Krouse ◽  
T. C. Law ◽  
B. I. Sikic ◽  
J. J. Wine
Keyword(s):  
Cell Line ◽  
Anion Channel ◽  
Uterine Sarcoma ◽  
Protein Levels ◽  
P Glycoprotein ◽  
Whole Cell Patch Clamp ◽  
Cl Channels ◽  
Line Selection ◽  
Selection For ◽  
K Currents

To test the hypothesis that P-glycoprotein enhances swelling currents through regulation of volume-sensitive Cl- channels [recently termed VSOAC (volume-sensitive osmolyte and anion channel)], a human uterine sarcoma cell line (MES-SA) and its doxorubicin-selected counterpart (Dx5) were studied. P-glycoprotein mRNA and protein levels were detected only in Dx5 cells. However, whole cell patch-clamp experiments showed that swollen Dx5 cells (n = 5) produced smaller VSOAC currents than MES-SA cells (n = 4; 106 +/- 26 pA/pF vs. 232 +/- 76 pA/pF at 90 mV). In radioisotopic efflux experiments, both swelling-activated 125I (Cl-) currents (n = 15) and 86Rb (K+) currents (n = 8) were found to be two-to fourfold smaller in the Dx5 (high P-glycoprotein) cells. Inhibitors of P-glycoprotein showed no specificity for the doxorubicin-selected cells (Dx5). Dideoxyforskolin (100 microM) blocked swelling-activated 125I efflux equally in both cell lines, whereas 100 microM verapamil had no effect. Thus, in this cell line, selection for P-glycoprotein expression is associated with reduced swelling currents. These findings suggest that P-glycoprotein expression does not directly facilitate VSOAC.

scholarly journals Swelling-activated chloride channels in multidrug-sensitive and -resistant cells.

1994 ◽  
Vol 104 (6) ◽  
pp. 1129-1161 ◽  
Author(s):  
G R Ehring ◽  
Y V Osipchuk ◽  
M D Cahalan
Keyword(s):  
Cell Lines ◽  
Chloride Channels ◽  
Imaging Techniques ◽  
Chemotherapeutic Agents ◽  
Cell Swelling ◽  
Pipette Solution ◽  
P Glycoprotein ◽  
Cell Recording ◽  
Nih 3T3 ◽  
Resistant Cells

Resistance to chemotherapeutic agents in neoplastic cells is often mediated by expression of P-glycoprotein, which functions as a drug-efflux pump for a broad range of substrates. We have used a combination of patch clamp and video-imaging techniques to examine the expression and drug-efflux function of P-glycoprotein and to determine the possible correlation with swelling-activated chloride channels in drug-sensitive and -resistant cell lines. Two pairs of cell lines were used in these experiments: (a) control NIH-3T3 cells and a corresponding MDR1-transfectant; and (b) control 8226 myeloma cells and a derivative cell line selected for resistance to chemotherapeutic agents. Control cells lacked detectable P-glycoprotein expression based on Western blotting, immunofluorescence staining with a specific monoclonal antibody, and a functional assay of rhodamine-123 (R123) efflux. Resistant cells expressed P-glycoprotein at high levels and rapidly exported R123. During whole-cell recording using either hyperosmotic pipette solution or hypoosmotic Ringer solution, cell swelling was accompanied by Cl- channel opening in all four cell lines. The rates of induction, biophysical properties and magnitudes of Cl conductance (gCl) were indistinguishable between control and corresponding multidrug-resistant cells: gCl reached 0.96 +/- 0.31 (n = 14) and 0.83 +/- 0.31 nS/pF (mean +/- SD; n = 31) in NIH-3T3 and NIH-3T3/MDR cells, respectively; and 0.31 +/- 0.20 (n = 9) and 0.37 +/- 0.22 nS/pF (n = 7) in 8226 and 8226/Dox40 cells, respectively. gCl exhibited moderate outward rectification in symmetrical Cl- solutions, with a rectification ratio of 1.4 at +/- 50 mV. Cl- channels slowly closed during strong depolarization beyond +60 mV. Using video-imaging techniques with SPQ as a fluorescent probe, we monitored Cl(-)-channel opening in intact drug-sensitive and -resistant cells. gCl, measured either with whole-cell recording or SPQ imaging, was blocked by DIDS (voltage-dependent Kd < 50 microM at +40 mV), NPPB (Kd approximately 30 microM), and tamoxifen (complete and irreversible block approximately 10 microM). None of these blockers inhibited R123 efflux. NPPB accelerated R123 efflux, an effect that was mimicked by CCP, a mitochondrial uncoupler. In contrast, verapamil selectively blocked R123 efflux (Kd = 0.3 to 0.5 microM); 10 microM left gCl unaltered. Induction of gCl was not affected by vincristine or doxorubicin in the pipette solution. Moreover, the rate of R123 efflux did not change during cell swelling. We conclude that P-glycoprotein and swelling-activated chloride channels function independently and are separable by expression and by pharmacological sensitivities.

scholarly journals Differences in the expression of endogenous efflux transporters in MDR1-transfected versus wildtype cell lines affect P-glycoprotein mediated drug transport

2010 ◽  
Vol 160 (6) ◽  
pp. 1453-1463 ◽  
Author(s):  
Konstantin Kuteykin-Teplyakov ◽  
Carlos Luna-Tortós ◽  
Kamila Ambroziak ◽  
Wolfgang Löscher
Keyword(s):  
Cell Lines ◽  
Drug Transport ◽  
Efflux Transporters ◽  
P Glycoprotein ◽  
Wildtype Cell

Volume regulation in NIH/3T3 cells not expressing P-glycoprotein. II. Chloride and amino acid fluxes

1997 ◽  
Vol 272 (6) ◽  
pp. C1804-C1809 ◽  
Author(s):  
J. Moran ◽  
D. Miranda ◽  
C. Pena-Segura ◽  
H. Pasantes-Morales
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Regulatory Volume Decrease ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
3T3 Cells ◽  
P Glycoprotein ◽  
Arachidonic Acids ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The osmolyte function of amino acids and Cl in native NIH/3T3 cells not expressing the P-glycoprotein was examined by investigating the free amino acid concentration and the swelling-activated efflux of [3H]taurine, as representative of amino acids, and of 125I, as a tracer for Cl. Taurine and 125I efflux was activated by 20 and 30% hyposmotic solutions. At 50% hyposmotic solutions, the osmolyte pool was essentially depleted. The Cl channel blockers 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid inhibited the release of the two osmolytes by 80-95%. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (400 microM) decreased the efflux of taurine 80% without affecting that of 125I. Linolenic and arachidonic acids (5-20 microM) showed a concentration-dependent inhibitory effect on taurine and 125I fluxes. Omission of Ca decreased osmolyte fluxes by 16%. Verapamil inhibited the osmolyte release only at 500 microM. Nimodipine at 25 and 50 microM decreased the release of [3H]taurine and 125I by approximately 60 and 80%, respectively, but this effect was independent of the presence of extracellular Ca. These results indicate that amino acids and Cl function as osmolytes during regulatory volume decrease in native NIH/ 3T3 cells.

Volume regulation in NIH/3T3 cells not expressing P-glycoprotein. I. Regulatory volume decrease

1997 ◽  
Vol 272 (6) ◽  
pp. C1798-C1803 ◽  
Author(s):  
H. Pasantes-Morales ◽  
R. Sanchez Olea ◽  
D. Miranda ◽  
J. Moran
Keyword(s):  
Amino Acids ◽  
Regulatory Volume Decrease ◽  
K Channel ◽  
Gamma Aminobutyric Acid ◽  
3T3 Cells ◽  
P Glycoprotein ◽  
Volume Recovery ◽  
Nih 3T3 ◽  
Volume Decrease ◽  
Nih 3T3 Cells

Exposure of NIH/3T3 fibroblasts not expressing P-glycoprotein to 50, 30, 20, and 10% hyposmotic solutions led to cell volume increases of 70, 32, 21, and 12%, respectively. After swelling, NIH/3T3 cells exhibited regulatory volume decrease (RVD), attaining complete volume recovery after 30 min except in 50% hyposmotic solution, in which volume recovery was 76%. RVD was accelerated by gramicidin and inhibited by the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid and by the K channel, blocker quinidine. RVD was reduced 15% by removal of extracellular Ca. The pathway opened by hypotonicity was highly permeable to K and Rb and only partly permeable to other cations. Most anions were able to permeate, with a permeability ranking of nitrate > benzoate = iodide > thiocyanate > chloride > > gluconate. The pathway was permeable to neutral amino acids, with a permeability ranking of glycine > alanine > glutamate > taurine > gamma-aminobutyric acid > glutamine. The pathway was not permeable to basic amino acids. These results show that, despite the absence of P-glycoprotein, NIH/3T3 cells exhibit RVD with properties similar to those expressed in most cell types.

MDR1/P-glycoprotein function. II. Effect of hypotonicity and inhibitors on Cl- efflux and volume regulation

1996 ◽  
Vol 270 (5) ◽  
pp. C1453-C1460 ◽  
Author(s):  
J. L. Weaver ◽  
A. Aszalos ◽  
L. McKinney
Keyword(s):  
Cell Lines ◽  
Volume Regulation ◽  
Regulatory Volume Decrease ◽  
Channel Blockers ◽  
Intact Cells ◽  
Hypotonic Stress ◽  
Cl Channel ◽  
P Glycoprotein ◽  
Significant Difference ◽  
Mdr1 Expression

Resistance to anti-tumor drugs can be mediated by overexpression of the multidrug resistance 1 (MDR1) protein (P-glycoprotein). In three MDR1-transfected cell lines (Gill et al. Cell 71: 23-32, 1992; Altenberg et al. Cancer Res. 54: 618-622, 1994), a hypotonic stress-induced Cl- current has been demonstrated that can be inhibited by MDR1 substrates and Cl- channel blockers. We tested the hypothesis that MDR1 expression confers additional Cl- conductance by measuring regulatory volume decrease (RVD) in four pairs of isogenic cell lines and 36Cl efflux in two cell lines with and without hypotonic stress. The kinetics of RVD and response to Cl- channel blockers were indistinguishable in MDR and parental cells. Additionally, no significant difference was seen between 36Cl efflux rate constants under hypotonic conditions between NIH/3T3 and L1210 parental and MDR cells. We conclude that, in intact cells, the expression of MDR1 does not alter the rate of volume regulation or the rate 36Cl efflux under hypotonic conditions between parental and MDR cells.

scholarly journals Retraction Note: Flavonoids modulate multidrug resistance through wnt signaling in P-glycoprotein overexpressing cell lines

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
S. Mohana ◽  
M. Ganesan ◽  
N. Rajendra Prasad ◽  
D. Ananthakrishnan ◽  
D. Velmurugan
Keyword(s):  
Multidrug Resistance ◽  
Wnt Signaling ◽  
Cell Lines ◽  
P Glycoprotein ◽  
Overexpressing Cell

An amendment to this paper has been published and can be accessed via the original article.

Effects of ouabain on NIH/3T3 cells transformed with retrovirai oncogenes and on human tumor cell lines

1987 ◽  
Vol 40 (5) ◽  
pp. 653-658 ◽  
Author(s):  
Pierosandro Tagliaferri ◽  
Kazuyoshi Yanagihara ◽  
Fortunate Ciardiello ◽  
Neil Talbot ◽  
Ursula Flatow ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Human Tumor ◽  
Tumor Cell Lines ◽  
3T3 Cells ◽  
Human Tumor Cell ◽  
Human Tumor Cell Lines ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

scholarly journals Inhibition of P-Glycoprotein–Mediated Drug Transport

Circulation ◽  
1999 ◽  
Vol 99 (4) ◽  
pp. 552-557 ◽  
Author(s):  
Martin F. Fromm ◽  
Richard B. Kim ◽  
C. Michael Stein ◽  
Grant R. Wilkinson ◽  
Dan M. Roden
Keyword(s):  
Drug Transport ◽  
P Glycoprotein
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