Molecular and Cellular PharmacologyStructure–activity relationship studies of propafenone analogs based on P-glycoprotein ATPase activity measurements
Section snippets
Cell culture
The T-lymphoblast cell line CCRF-CEM and the doxorubicin-resistant subline CCRF ADR5000 were a gift from V. Gekeler (Byk Gulden, Konstanz, Germany). The cell lines have been characterized previously. For ADR5000 cells, Pgp expression has been shown to be the cause of their multidrug-resistant phenotype 10, 11, 12, 13. Cells were maintained under standard cell culture conditions. The resistant subline was cultured in the presence of 5 μg/mL doxorubicin. One week prior to the experiments, the
Effect of propafenones on P-glycoprotein ATPase activity
In the presence of ouabain, EGTA, and Na-azide, which are potent inhibitors of the membrane-bound Na/K-, Ca2+-, and mitochondrial ATPases, respectively, the Pgp-containing vesicles exhibited an ATPase activity of 39.3 ± 5 nmol/min/mg protein, which was approximately twofold higher than the activity in wt membranes. This difference is termed basal Pgp ATPase activity according to Garrigos et al. [20] and represents the sum of unstimulated Pgp ATPase and residual ATPase activities of unknown
Discussion
Measurement of Pgp ATPase activity as a function of modulator concentration is a means to obtain quantitative data describing interaction between these substances and Pgp [20]. In such assays, there is no need to simultaneously use a transported toxin such as daunorubicin or vinblastine. Potential Pgp-independent interferences between toxin and modulator, such as fluorescence quench phenomena or membrane displacement, are thus eliminated.
In the present study, propafenone analogs were shown to
Acknowledgements
This work was supported by grants from the Austrian Science Fund (P11760MOB) and from the Austrian National Bank (6899). We thank V. Gekeler (Byk Gulden, Konstanz, Germany) for providing the cell lines used in this study. We are indebted to A. Ellinger (Institute of Histology II, University of Vienna, Austria) for preparing electron micrographs of the plasma membrane preparations.
References (24)
P-glycoprotein—a mediator of multidrug resistance in tumour cells
Eur J Cancer
(1996)- et al.
mdr1/P-glycoprotein gene segments analyzed from various human leukemic cell lines exhibiting different multidrug resistance profiles
Biochem Biophys Res Commun
(1990) - et al.
Functional studies of P-glycoprotein in inside-out plasma membrane vesicles derived from murine erythroleukemia cells overexpressing MDR 3
J Biol Chem
(1994) A rapid and sensitive method for the quantification of microgram quantities of protein
Anal Biochem
(1976)- et al.
Separation of right-side-out-oriented subfractions from purified thymocyte plasma membranes by affinity chromatography on concanavalin A-sepharose
Anal Biochem
(1981) - et al.
A method for the determination of inorganic phosphate in the presence of labile organic phosphate and high concentrations of proteinApplication to lens ATPases
Anal Biochem
(1988) - et al.
inhibition of purine nucleobase transport in human erythrocytes and cell lines by papaverine. Investigations of structure-activity relationship
Biochem Pharmacol
(1994) - et al.
Characterization of the adenosine triphosphatase activity of Chinese hamster P-glycoprotein
J Biol Chem
(1993) - et al.
Structure–activity relationships of P-glycoprotein interacting drugsKinetic characterization of their effects on ATPase activity
Biochim Biophys Acta
(1997) - et al.
The role of passive transbilayer drug movement in multidrug resistance and its modulation
J Biol Chem
(1996)