Related Experiment Videos
Doxorubicin selection for MDR1/P-glycoprotein reduces swelling-activated K+ and Cl- currents in MES-SA cells
D B Luckie1, M E Krouse, T C Law
1Cystic Fibrosis Research Laboratory, Stanford University, California 94305, USA.
Abstract:
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.
Insights
P-glycoprotein expression in cancer cells did not enhance swelling-activated ion currents. Instead, high P-glycoprotein levels were linked to reduced volume-sensitive osmolyte and anion channel (VSOAC) activity.
Area of Science:
- Cell Physiology
- Ion Channel Regulation
- Cancer Cell Biology
Background:
- Volume-sensitive outwardly rectifying anion channels (VSOAC) are crucial for cell volume regulation.
- P-glycoprotein (P-gp) is a drug efflux pump often overexpressed in multidrug-resistant cancer cells.
- The relationship between P-gp expression and VSOAC activity remains unclear.
Purpose of the Study:
- To investigate whether P-glycoprotein enhances swelling currents via VSOAC regulation.
- To compare VSOAC activity in a human uterine sarcoma cell line and its P-gp overexpressing counterpart.
Main Methods:
- Utilized whole-cell patch-clamp electrophysiology to measure VSOAC currents in MES-SA and Dx5 cells.
- Employed radioisotopic efflux assays to quantify swelling-activated iodide (Cl-) and rubidium (K+) currents.
- Assessed the effect of P-gp inhibitors on ion currents in both cell lines.
Main Results:
- P-glycoprotein mRNA and protein were detected exclusively in doxorubicin-selected Dx5 cells.
- Swollen Dx5 cells exhibited significantly smaller VSOAC currents compared to MES-SA cells.
- Swelling-activated 125I and 86Rb efflux were reduced in high P-gp expressing Dx5 cells.
Conclusions:
- P-glycoprotein expression in this cell line is associated with diminished swelling-activated currents.
- These findings suggest P-glycoprotein does not directly facilitate VSOAC.
- Further research is needed to elucidate the mechanisms underlying reduced VSOAC activity in P-gp expressing cells.