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Amino acid transport in multidrug-resistant Chinese hamster ovary cells
S E Daly1, R W Inch, E R Tustanoff
1Department of Biochemistry, University of Western Ontario, London, Canada.
Abstract:
In the process of assessing the effect of anthracycline drugs on cellular membrane function in cultured multidrug resistant (MDR) and its parental cells, experiments were undertaken to investigate the kinetics of neutral amino acid membrane transport (the sodium dependent A and ASC systems). P-glycoprotein, a high molecular weight energy requiring integral membrane protein responsible for actively pumping drugs out of cells, has been shown to be overexpressed in MDR cells. It was our hypothesis that its presence might affect other membrane energy requiring systems such as amino acid transport. On establishing the concentrations of P-glycoprotein by western blotting in the two cell lines to be studied, the kinetics of membrane transport of the neutral amino acids alpha-aminoisobutyric acid (AIB) and serine (SER) were investigated using the CHRC5 multidrug resistant and AUX B1 parental Chinese hamster ovary (CHO) cells. In CHRC5 cells, the amount and rate (Vmax) of accumulated amino acids, was significantly depressed when compared to AUX B1 cells, however, there was no difference in the rates of amino acid efflux between these two cell lines. Using 1,6-diphenyl 1,3,5-hexatriene (DPH) polarization to evaluate the state of membrane fluidity in the two cell lines studied, it was seen that CHRC5 cells showed a slightly lower degree of polarization than that observed in AUX B1 cells. These results suggest, that the P-glycoprotein does not alter amino acid transport directly but may modify the activity or numbers of functional transport carriers.
Insights
Multidrug resistant (MDR) cells overexpressing P-glycoprotein show depressed amino acid transport, suggesting P-glycoprotein may alter amino acid carrier activity or numbers, not directly affecting transport kinetics.
Area of Science:
- Cell Biology
- Biochemistry
- Pharmacology
Background:
- Multidrug resistant (MDR) cells often overexpress P-glycoprotein, an integral membrane protein.
- P-glycoprotein actively pumps drugs out of cells, requiring significant energy.
- The impact of P-glycoprotein on other energy-dependent membrane transport systems remains unclear.
Purpose of the Study:
- To investigate the effect of P-glycoprotein on neutral amino acid membrane transport kinetics.
- To determine if P-glycoprotein overexpression in MDR cells influences amino acid uptake and efflux.
- To explore the relationship between P-glycoprotein and cellular membrane fluidity.
Main Methods:
- Western blotting to quantify P-glycoprotein levels in multidrug resistant (MDR) and parental Chinese hamster ovary (CHO) cells (CHRC5 and AUX B1).
- Kinetic analysis of neutral amino acid transport (alpha-aminoisobutyric acid and serine) using radiolabeled substrates.
- 1,6-diphenyl-1,3,5-hexatriene (DPH) polarization assay to assess membrane fluidity.
Main Results:
- MDR CHRC5 cells exhibited significantly lower Vmax for amino acid accumulation compared to AUX B1 cells.
- No significant difference in amino acid efflux rates was observed between the two cell lines.
- MDR CHRC5 cells displayed slightly lower membrane fluidity (higher DPH polarization) than AUX B1 cells.
Conclusions:
- P-glycoprotein overexpression in MDR cells does not appear to directly alter amino acid transport kinetics.
- The observed decrease in amino acid transport in MDR cells may be due to P-glycoprotein modifying the activity or number of functional amino acid transporters.
- Altered membrane fluidity in MDR cells could potentially influence transporter function.