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Reversal of Multidrug Resistance by Valinomycin is Overcome by CCCP
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219, 306–310 (1996)
0228
Reversal of Multidrug Resistance by Valinomycin is Overcome by CCCP Katalin Goda, Zoltán Krasznai, Rezso˝ Gáspár, Jan Lankelma,* Hans V. Westerhoff,† Sándor Damjanovich, and Gábor Szabó, Jr.1 Department of Biophysics, University Medical School of Debrecen, Debrecen, Hungary; and *Department of Medical Oncology, Free University Hospital, and †Department of Microbial Physiology, Faculty of Biology, Free University, Amsterdam, The Netherlands Received January 5, 1996 Reversal of P-glycoprotein-mediated multidrug resistance by valinomycin is overcome by the proton ionophore, CCCP. This effect, a complete suppression of the 5- to 10-fold valinomycin-induced reversal (“rereversal”), exhibits a sharp extracellular potassium concentration ([K+o]) dependence. It is observed at [K+o] > 2–4 mM and not at [K+o] ø 2 mM, in the case of the fluorescent substrates rhodamine 123 and daunorubicin. The fact that “re-reversal” is detected only for the combination of CCCP with valinomycin raises the possibility that a direct interaction between these ionophores may explain the phenomenon. We show spectroscopic evidence of such an interaction, with a [K+o]-dependence similar to that of the “re-reversal.” These data suggest that the reversal of P-glycoprotein activity by valinomycin can be compromised by anionic compounds such as CCCP due to complex formation. More generally, molecular interactions involving P-glycoprotein substrates or reversing agents may significantly affect drug accumulation in multidrug resistant cells. © 1996 Academic Press, Inc.
Tumours can develop resistance during chemotherapy or can be intrinsically resistant to various chemically unrelated anticancer agents (multidrug resistance, MDR). A clinically significant class of drug resistance correlates with the presence of a 170 kDa glycoprotein (Pgp, P170) in the cell membrane (for review see refs. 7, 8, 10). This glycoprotein belongs, based on sequence homologies and functional analogies, to a group of transport proteins that actively transfer hydrophobic molecules, peptides and various drugs across the cell membrane (12). The drug resistance phenotype can be functionally reversed by a wide range of chemicals including calcium channel blockers (6,13) and some ionophores (see, e.g. 5,17,22) that appear to compete (6,10) with the substrates examined or to modulate active pumping allosterically (9,20). Examining the possible interplay between drug accumulation, membrane potential and intracellular pH by measuring the effect of ionophores (CCCP for protons, Val for K+) on Pgp activity, we observed that reversal of the MDR phenotype by Val was completely overcome by CCCP at [K+o] > 2–4 mM. Here we show that this effect is most probably the consequence of a direct interaction between Val and CCCP, withdrawing Val from its P170 reversing role. MATERIALS AND METHODS Chemicals and reagents. R123, DNR, Val, CCCP, verapamil, vinblastine and inorganic chemicals were purchased from Sigma-Aldrich (Budapest, Hungary). HEPES (N-[2-Hydroxyethyl]piperazine-N9-[ethanesulfonic acid]) was provided by SERVA (Heidelberg, Germany). Cyclosporin A was a gift from A. Aszalós (Food and Drug Administration, Washington, D.C.). Cell lines. The drug-sensitive human epidermoid carcinoma cell line KB-3-1 and its vinblastine-selected multidrugresistant variant KB-V1 (isolated from KB-3-1 by stepwise selection with increasing vinblastine concentrations, see 18) were used in the measurements. The cells were grown as monolayer cultures and maintained by regular passage in Dulbecco’s minimal essential medium (supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin). KB-V1 cells were maintained in the presence of 180 nM vinblastine. Cells 1 To whom correspondence should be addressed at Department of Biophysics, University Medical School of Debrecen, H-4012, P.O. Box 39, Debrecen, Hungary. Fax: +36 52-412-623. e-mail: [email protected]. Abbreviations: R123, rhodamin 123; DNR, daunorubicin; Val, valinomycin; MDR, multidrug resistance; MDR+ multidrug resistant; Pgp, P-glycoprotein; CCCP, carbonylcyanide m-chlorphenylhydrazone.
306 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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were typsinized 2 days before the experiments and maintained until use without vinblastine. Pgp expression of MDR+ cells was verified by labelling with the mouse monoclonal antibody MRK-16 (kindly provided by Dr. T. Tsuruo, see 11). Drug-uptake measurements. Buffers contained 2-140 mM KCl (KCl was isotonically replaced by choline-chloride or, in some experiments by NaCl with similar results), 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES (pH 4 7.4) and 5 mM glucose. Cells were resuspended in the relevant buffer (at 4 × 105 cells/ml) containing R123 (1.3 mM) or DNR (3.55 mM) and incubated for 40 min at 37°C. The ionophores CCCP (49 mM) and Val (9 mM) were added simultaneously with the dyes and the mean fluorescence intensities were evaluated by flow-cytometry. The viability of cells after incubation was checked by propidium-iodide staining (14). Flow cytometry. Drug-uptake was determined by flow cytometry using a Becton Dickinson FACS Star Plus flow cytometer equipped with a Spectra Physics 164-08 argon-ion laser. The fluorescence signal was gated on the forward angle light scatter signal to exclude dead cells and cell debris from analysis. The argon-ion laser was tuned to 488 nm and used at a power of 500 mW. Emission was detected through a 540 nm broadband interference filter and a 620 nm long-pass filter (for R123 and DNR, respectively). Absorption measurements. The samples were incubated at 37°C for 40 min and the absorption spectra were obtained by a Shimadzu UV-visible recording spectrophotometer. Spectra of the samples containing Val were obtained with the ionophore present in both reference and sample cuvettes. Precipitation of CCCP during 40 min incubation of the ionophores (25 mM) at 37°C was measured comparing the 380 nm absorbance of the supernatants before Val addition and after sedimentation for 2 min at 13000g.
RESULTS AND DISCUSSION KB-V1 cells accumulated about 5 times less DNR and 25 times less R123 than KB-3-1 cells, in agreement with their high level Pgp expression (the mean fluorescence intensity of MRK16 labelled cells was approx. 80 times higher than that of the control cells). Table 1, shows the effect of the ionophores Val and CCCP, applied alone and in combination, on R123 and DNR accumulation by KB-V1 cells. Both at 2 mM and 140 mM [K+o], Val strongly increased the intracellular drug concentrations due to the reversal of drug efflux by Pgp (17,22). When CCCP was added together with Val, the accumulation of both R123 and DNR was greatly reduced at 140 mM [K+o] as compared to the Val-treated cells, while it was unchanged at 2 mM [K+o]. We refer to this phenomenon as “re-reversal”. When MDR+ cells were pre-loaded with DNR in the presence of Val at 140 mM [K+o] for 40 min, the subsequent addition of CCCP induced a gradual DNR release, to the level of Val-untreated cells (data not shown). The addition of 70 mM KCl to cells pre-loaded with R123 in the presence of CCCP and Val at 2 mM [K+o], also induced R123 efflux to the level of ionophore-untreated cells (data not shown). Surprisingly, the “re-reversal” was sharply [K+o]-dependent. It was observed at [K+o] > 2–4 mM and not at [K+o] ø 2, as shown for R123 and DNR, in parallel samples of the same experiment (Figure 1A). The effect exhibited an all-or-none feature in the sense that the number of cells
TABLE 1 The Effect of Ionophores on DNR and R123 Accumulation of KB-V1 (MDR+) Cells as Measured by Flow-cytometry [K+]0 2 mM K+ 140 mM K+
Accumulated dye
CCCP
Val
DNR
0.77 ± 0.16
3.85 ± 0.76
4.40 ± 0.84
R123 DNR R123
0.77 ± 0.11 0.81 ± 0.10 0.62 ± 0.02
9.54 ± 1.07 4.81 ± 0.35 6.14 ± 2.03
12.99 ± 2.39 0.87 ± 0.19 0.59 ± 0.01
CCCP + Val
The cells were incubated with the dyes ([DNR] 4 3.55 mM; [R123] 4 1.3 mM) and ionophores ([Val] 4 9 mM; [CCCP] 4 49 mM), at 2 mM or 140 mM [K+0], at 37°C for 30 min. Dye uptake is shown in relative units, as the ratio of the mean fluorescence of the ionosphore-treated and that of the untreated cells.
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FIG. 1. The [K+o]-dependence of R123 accumulation of KB-V1 cells after incubation with R123 and ionophores, as measured by flow cytometry (A) in comparison with the formation of Val-K+-CCCP aggregates (B). (A) R123 uptake is shown in relative units, as the ratio of the mean fluorescence of the ionophore-treated and that of untreated cells, from one representative measurement (out of three independent experiments). Grey bars, Val-treated samples; cross-hatched bars, Val + CCCP-treated samples, for either subpopulation when the fluorescence distribution was heterogeneous (at 3 mM K+, with z30% of the cells showing complete “re-reversal”). Samples contained 1.3 mM R123, 9 mM Val and 49 mM CCCP. (B) CCCP precipitation in the presence of Val, as a function of [K+] (cross-hatched bars, pH 4 6.9; grey bars, pH 4 7.4). Both ionophores were used at 25 mM.
involved in “re-reversal” and not the extent of drug accumulation changed when it was partial (around the transition [K+o]). CCCP alone decreased R123 and DNR accumulation only mildly (by about 20-40 %) in the case of both KB-V1 (see Table 1), and KB-3-1 cells (data not shown) and independently from [K+o]. The above results argued against the possibility that the “re-reversal” effect was due to the enhancement of the activity of Pgp by CCCP. When an increased dye accumulation was achieved by higher concentrations of DNR or R123 (in the absence of Val) similar to what was brought about by Val treatment, CCCP decreased dye accumulation only by 10–30 % (data not shown). Thus, CCCP effect independent of Val did not appear to affect drug uptake sufficiently to account for the “re-reversal”. The possible involvement of ionophore induced changes of the intracellular pH and membrane potential in “re-reversal” was also examined in detail and excluded (data not shown). The facts that “re-reversal” was peculiar to the combination of Val and CCCP (as opposed to the combination of CCCP with verapamil or with cyclosporin A, data not shown) and it was detected only at [CCCP] ù [Val] (data not shown), raised the possibility that a direct interaction between these ionophores might be responsible for the effect. In the case of the CCCP-analogue FCCP, [K+]-dependent formation of Val-K+-FCCP complexes 308
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at extremely high [K ] has been reported (15). Val is known to interact with other anionic protonophores as well (1,23). For CCCP, only indirect evidence has been available so far suggesting a tendency of its complex formation with Val (23). In spite of these reports, these ionophores are often applied together in various membrane biological studies (2-4,19,23,24). We have detected perturbations of the absorption spectrum of CCCP at different [K+] in the presence of Val, similarly to those reported for FCCP (15). CCCP in aqueous solutions had an absorption maximum at 380 nm, which shifted to the right (by z40 nm) and the height of the peak decreases (by 50–70%, depending on the concentration ratio of the ionophores) in the presence of Val, at [K+] > 2 and pH 4 7.4, as shown in Figure 2. Moreover, we could directly observe precipitation of CCCP with Val as a pellet formed at 13000g only at [K+] > 2–2.5 mM (Figure 1B), concomitant with the shift of the CCCP absorption spectrum (Figure 2). CCCP did not appear to form complexes with the classical MDR-reversing agent verapamil or cyclosporin A (in terms of the above criteria, in cell-free solution). Likewise, reversal by these agents was not antagonised by CCCP. Consequently, CCCP (probably its negatively charged form see Figure 1B) can form a complex with Val withdrawing VAl from its P170-reversing role. The sharp [K+o]-dependence of the “re-reversal” phenomenon could be due to a [K+o]-dependent formation of Val-K+-CCCP complexes that would no longer hamper R123 or DNR export by Pgp, as opposed to Val alone. Thus, direct molecular interaction involving a ligand recognized by Pgp could explain a conspicuous (5-10-fold) change in drug accumulation. This observation implies that besides competitive inhibition and allosteric effects, molecular interactions involving Pgp ligands could also significantly affect drug accumulation in MDR+ cells. This aspect is reinforced by the expanding
FIG. 2. Effect of Val on the absorption spectrum of CCCP in aqueous solutions. The absorption spectra of 25 mM CCCP alone (1) and after 30 min incubation with 25 mM Val (2) were recorded in the presence of (A) 164.5 mM NaCl, (B) 160.5 mM NaCl + 4 mM KCl, or (C) 154.5 mM NaCl + 10 mM KCl (in addition to 5 mM HEPES (pH 4 7.4), 2.0 mM CaCl2 and 1.0 mM MgCl2, as in the drug accumulation experiments). 309
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variety of novel reversing agents, including cyclic peptides that resemble Val in their structure (16,17). Since CCCP and Val are often applied together in membrane transport research (2– 4,19,23,24), our observation also emphasizes the possibility that data obtained in analogous experimental situations may be seriously affected by the formation of Val-K+-CCCP complexes. ACKNOWLEDGMENT This work was supported by the U.S.–Hungarian Science and Technology Joint Fund in cooperation with Dept. Biophysics, Univ. Med. School of Debrecen and Division of Research and Testing, Food and Drug Administration under Project JFNO 127. This work was also sponsored by OTKA fundings T14655 and 17592 and ETT Grant T-01 449/93 and by the Hungarian–Dutch Association. The KB cell lines were kindly donated by Dr. M. M. Gottesman.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Arents, J. C., Hellingwerf, K. J., Van Dam, K., and Westerhoff, H. V. (1981) J. Membrane. Biol. 60, 95–104. Bennekou, P. (1988) J. Membrane. Biol. 102, 225–234. Bennekou, P. (1988) J. Membrane. Biol. 106, 41–46. Bennekou, P., and Christophersen, P. J. (1986) J. Membrane. Biol. 93, 221–227. Borrel, M. N., Pereira, E., Fiallo, M., and Garnier-Suillerot, (1994) Eur. J. Biochem. 223, 125–133. Cornwell, M. M., Pastan, I., and Gottesman, M. M. (1987) J. Biol. Chem. 262, 2166–2170. Doige, C. A., and Ferro-Luzzi Ames, G. (1993) Annu. Rev. Microbiol. 47, 291–319. Endicott, J. A., and Ling, V. (1989) Annu. Rev. Biochem. 58, 137–171. Ferry, D. R., Russel, M. A., and Cullen, M. H. (1992) Biochem. Biophys. Res. Commun. 188, 440–445 1992. Gottesman, M. M., and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385–427. Hamada, H., and Tsuruo, T. (1986) Proc. Natl. Acad. Sci. 83, 7785–7789. Higgins, C. F. (1992) Annu. Rev. Cell. Biol. 8, 67–113. Kessel, D., and Wilberding, C. (1984) Biochem. Pharmacol. 33, 1157–1160. Krishan, A. (1975) J. Cell. Biol. 66, 188–192. O’Brien, T. A., Nieva-Gomez, D., and Gennis, R. B. (1978) J. Biol. Chem. 253, 1749–1751. Sharma, R. C., Inoue, S., Roitelman, J., Schimke, R. T., and Simoni, R. D. (1991) J. Biol. Chem. 267, 5731–5734. Sharom, F. J., DiDiodatos, G., Yu, X., and Ashbourne, K. J. D. (1995) J. Biol. Chem., 270, 10334–10341. Shen, D-W., Cardarelli, C., Hwang, J., Cornwell, M. M., Richert, N., Ishii, S., Pastan, I., and Gottesman, M. M. (1986) J. Biol. Chem. 261, 7762–7770. Smith, H. G., and Fager, R. S. (1991) Biophys. J. 59, 427–432. Spoelstra, E. C., Westerhoff, H. V., Pinedo, H. M., Dekker, H., and Lankelma, J. (1994) Eur. J. Biochem. 221, 363–373. Tsuruo, T., Iida, H., Nojri, M., Tsukagoshi, S., and Sakurai, Y. (1983) Cancer Res. 43, 2905–2910. Weaver, J. L., Szabó, G. Jr, Gottesman, M. M., Goldenberg, S., and Aszalos, A. (1993) Int. J. Cancer. 54, 1–6. Yamaguchi, A., and Anraku, Y. (1977) Biochem. Biophys. Acta 501, 136–149. Yoda, A., and Yoda, S. (1990) J. Membrane. Biol. 117, 153–161.
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Reversal of Multidrug Resistance by Valinomycin is Overcome by CCCP Katalin Goda, Zoltán Krasznai, Rezso˝ Gáspár, Jan Lankelma,* Hans V. Westerhoff,† Sándor Damjanovich, and Gábor Szabó, Jr.1 Department of Biophysics, University Medical School of Debrecen, Debrecen, Hungary; and *Department of Medical Oncology, Free University Hospital, and †Department of Microbial Physiology, Faculty of Biology, Free University, Amsterdam, The Netherlands Received January 5, 1996 Reversal of P-glycoprotein-mediated multidrug resistance by valinomycin is overcome by the proton ionophore, CCCP. This effect, a complete suppression of the 5- to 10-fold valinomycin-induced reversal (“rereversal”), exhibits a sharp extracellular potassium concentration ([K+o]) dependence. It is observed at [K+o] > 2–4 mM and not at [K+o] ø 2 mM, in the case of the fluorescent substrates rhodamine 123 and daunorubicin. The fact that “re-reversal” is detected only for the combination of CCCP with valinomycin raises the possibility that a direct interaction between these ionophores may explain the phenomenon. We show spectroscopic evidence of such an interaction, with a [K+o]-dependence similar to that of the “re-reversal.” These data suggest that the reversal of P-glycoprotein activity by valinomycin can be compromised by anionic compounds such as CCCP due to complex formation. More generally, molecular interactions involving P-glycoprotein substrates or reversing agents may significantly affect drug accumulation in multidrug resistant cells. © 1996 Academic Press, Inc.
Tumours can develop resistance during chemotherapy or can be intrinsically resistant to various chemically unrelated anticancer agents (multidrug resistance, MDR). A clinically significant class of drug resistance correlates with the presence of a 170 kDa glycoprotein (Pgp, P170) in the cell membrane (for review see refs. 7, 8, 10). This glycoprotein belongs, based on sequence homologies and functional analogies, to a group of transport proteins that actively transfer hydrophobic molecules, peptides and various drugs across the cell membrane (12). The drug resistance phenotype can be functionally reversed by a wide range of chemicals including calcium channel blockers (6,13) and some ionophores (see, e.g. 5,17,22) that appear to compete (6,10) with the substrates examined or to modulate active pumping allosterically (9,20). Examining the possible interplay between drug accumulation, membrane potential and intracellular pH by measuring the effect of ionophores (CCCP for protons, Val for K+) on Pgp activity, we observed that reversal of the MDR phenotype by Val was completely overcome by CCCP at [K+o] > 2–4 mM. Here we show that this effect is most probably the consequence of a direct interaction between Val and CCCP, withdrawing Val from its P170 reversing role. MATERIALS AND METHODS Chemicals and reagents. R123, DNR, Val, CCCP, verapamil, vinblastine and inorganic chemicals were purchased from Sigma-Aldrich (Budapest, Hungary). HEPES (N-[2-Hydroxyethyl]piperazine-N9-[ethanesulfonic acid]) was provided by SERVA (Heidelberg, Germany). Cyclosporin A was a gift from A. Aszalós (Food and Drug Administration, Washington, D.C.). Cell lines. The drug-sensitive human epidermoid carcinoma cell line KB-3-1 and its vinblastine-selected multidrugresistant variant KB-V1 (isolated from KB-3-1 by stepwise selection with increasing vinblastine concentrations, see 18) were used in the measurements. The cells were grown as monolayer cultures and maintained by regular passage in Dulbecco’s minimal essential medium (supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin). KB-V1 cells were maintained in the presence of 180 nM vinblastine. Cells 1 To whom correspondence should be addressed at Department of Biophysics, University Medical School of Debrecen, H-4012, P.O. Box 39, Debrecen, Hungary. Fax: +36 52-412-623. e-mail: [email protected]. Abbreviations: R123, rhodamin 123; DNR, daunorubicin; Val, valinomycin; MDR, multidrug resistance; MDR+ multidrug resistant; Pgp, P-glycoprotein; CCCP, carbonylcyanide m-chlorphenylhydrazone.
306 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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were typsinized 2 days before the experiments and maintained until use without vinblastine. Pgp expression of MDR+ cells was verified by labelling with the mouse monoclonal antibody MRK-16 (kindly provided by Dr. T. Tsuruo, see 11). Drug-uptake measurements. Buffers contained 2-140 mM KCl (KCl was isotonically replaced by choline-chloride or, in some experiments by NaCl with similar results), 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES (pH 4 7.4) and 5 mM glucose. Cells were resuspended in the relevant buffer (at 4 × 105 cells/ml) containing R123 (1.3 mM) or DNR (3.55 mM) and incubated for 40 min at 37°C. The ionophores CCCP (49 mM) and Val (9 mM) were added simultaneously with the dyes and the mean fluorescence intensities were evaluated by flow-cytometry. The viability of cells after incubation was checked by propidium-iodide staining (14). Flow cytometry. Drug-uptake was determined by flow cytometry using a Becton Dickinson FACS Star Plus flow cytometer equipped with a Spectra Physics 164-08 argon-ion laser. The fluorescence signal was gated on the forward angle light scatter signal to exclude dead cells and cell debris from analysis. The argon-ion laser was tuned to 488 nm and used at a power of 500 mW. Emission was detected through a 540 nm broadband interference filter and a 620 nm long-pass filter (for R123 and DNR, respectively). Absorption measurements. The samples were incubated at 37°C for 40 min and the absorption spectra were obtained by a Shimadzu UV-visible recording spectrophotometer. Spectra of the samples containing Val were obtained with the ionophore present in both reference and sample cuvettes. Precipitation of CCCP during 40 min incubation of the ionophores (25 mM) at 37°C was measured comparing the 380 nm absorbance of the supernatants before Val addition and after sedimentation for 2 min at 13000g.
RESULTS AND DISCUSSION KB-V1 cells accumulated about 5 times less DNR and 25 times less R123 than KB-3-1 cells, in agreement with their high level Pgp expression (the mean fluorescence intensity of MRK16 labelled cells was approx. 80 times higher than that of the control cells). Table 1, shows the effect of the ionophores Val and CCCP, applied alone and in combination, on R123 and DNR accumulation by KB-V1 cells. Both at 2 mM and 140 mM [K+o], Val strongly increased the intracellular drug concentrations due to the reversal of drug efflux by Pgp (17,22). When CCCP was added together with Val, the accumulation of both R123 and DNR was greatly reduced at 140 mM [K+o] as compared to the Val-treated cells, while it was unchanged at 2 mM [K+o]. We refer to this phenomenon as “re-reversal”. When MDR+ cells were pre-loaded with DNR in the presence of Val at 140 mM [K+o] for 40 min, the subsequent addition of CCCP induced a gradual DNR release, to the level of Val-untreated cells (data not shown). The addition of 70 mM KCl to cells pre-loaded with R123 in the presence of CCCP and Val at 2 mM [K+o], also induced R123 efflux to the level of ionophore-untreated cells (data not shown). Surprisingly, the “re-reversal” was sharply [K+o]-dependent. It was observed at [K+o] > 2–4 mM and not at [K+o] ø 2, as shown for R123 and DNR, in parallel samples of the same experiment (Figure 1A). The effect exhibited an all-or-none feature in the sense that the number of cells
TABLE 1 The Effect of Ionophores on DNR and R123 Accumulation of KB-V1 (MDR+) Cells as Measured by Flow-cytometry [K+]0 2 mM K+ 140 mM K+
Accumulated dye
CCCP
Val
DNR
0.77 ± 0.16
3.85 ± 0.76
4.40 ± 0.84
R123 DNR R123
0.77 ± 0.11 0.81 ± 0.10 0.62 ± 0.02
9.54 ± 1.07 4.81 ± 0.35 6.14 ± 2.03
12.99 ± 2.39 0.87 ± 0.19 0.59 ± 0.01
CCCP + Val
The cells were incubated with the dyes ([DNR] 4 3.55 mM; [R123] 4 1.3 mM) and ionophores ([Val] 4 9 mM; [CCCP] 4 49 mM), at 2 mM or 140 mM [K+0], at 37°C for 30 min. Dye uptake is shown in relative units, as the ratio of the mean fluorescence of the ionosphore-treated and that of the untreated cells.
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FIG. 1. The [K+o]-dependence of R123 accumulation of KB-V1 cells after incubation with R123 and ionophores, as measured by flow cytometry (A) in comparison with the formation of Val-K+-CCCP aggregates (B). (A) R123 uptake is shown in relative units, as the ratio of the mean fluorescence of the ionophore-treated and that of untreated cells, from one representative measurement (out of three independent experiments). Grey bars, Val-treated samples; cross-hatched bars, Val + CCCP-treated samples, for either subpopulation when the fluorescence distribution was heterogeneous (at 3 mM K+, with z30% of the cells showing complete “re-reversal”). Samples contained 1.3 mM R123, 9 mM Val and 49 mM CCCP. (B) CCCP precipitation in the presence of Val, as a function of [K+] (cross-hatched bars, pH 4 6.9; grey bars, pH 4 7.4). Both ionophores were used at 25 mM.
involved in “re-reversal” and not the extent of drug accumulation changed when it was partial (around the transition [K+o]). CCCP alone decreased R123 and DNR accumulation only mildly (by about 20-40 %) in the case of both KB-V1 (see Table 1), and KB-3-1 cells (data not shown) and independently from [K+o]. The above results argued against the possibility that the “re-reversal” effect was due to the enhancement of the activity of Pgp by CCCP. When an increased dye accumulation was achieved by higher concentrations of DNR or R123 (in the absence of Val) similar to what was brought about by Val treatment, CCCP decreased dye accumulation only by 10–30 % (data not shown). Thus, CCCP effect independent of Val did not appear to affect drug uptake sufficiently to account for the “re-reversal”. The possible involvement of ionophore induced changes of the intracellular pH and membrane potential in “re-reversal” was also examined in detail and excluded (data not shown). The facts that “re-reversal” was peculiar to the combination of Val and CCCP (as opposed to the combination of CCCP with verapamil or with cyclosporin A, data not shown) and it was detected only at [CCCP] ù [Val] (data not shown), raised the possibility that a direct interaction between these ionophores might be responsible for the effect. In the case of the CCCP-analogue FCCP, [K+]-dependent formation of Val-K+-FCCP complexes 308
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at extremely high [K ] has been reported (15). Val is known to interact with other anionic protonophores as well (1,23). For CCCP, only indirect evidence has been available so far suggesting a tendency of its complex formation with Val (23). In spite of these reports, these ionophores are often applied together in various membrane biological studies (2-4,19,23,24). We have detected perturbations of the absorption spectrum of CCCP at different [K+] in the presence of Val, similarly to those reported for FCCP (15). CCCP in aqueous solutions had an absorption maximum at 380 nm, which shifted to the right (by z40 nm) and the height of the peak decreases (by 50–70%, depending on the concentration ratio of the ionophores) in the presence of Val, at [K+] > 2 and pH 4 7.4, as shown in Figure 2. Moreover, we could directly observe precipitation of CCCP with Val as a pellet formed at 13000g only at [K+] > 2–2.5 mM (Figure 1B), concomitant with the shift of the CCCP absorption spectrum (Figure 2). CCCP did not appear to form complexes with the classical MDR-reversing agent verapamil or cyclosporin A (in terms of the above criteria, in cell-free solution). Likewise, reversal by these agents was not antagonised by CCCP. Consequently, CCCP (probably its negatively charged form see Figure 1B) can form a complex with Val withdrawing VAl from its P170-reversing role. The sharp [K+o]-dependence of the “re-reversal” phenomenon could be due to a [K+o]-dependent formation of Val-K+-CCCP complexes that would no longer hamper R123 or DNR export by Pgp, as opposed to Val alone. Thus, direct molecular interaction involving a ligand recognized by Pgp could explain a conspicuous (5-10-fold) change in drug accumulation. This observation implies that besides competitive inhibition and allosteric effects, molecular interactions involving Pgp ligands could also significantly affect drug accumulation in MDR+ cells. This aspect is reinforced by the expanding
FIG. 2. Effect of Val on the absorption spectrum of CCCP in aqueous solutions. The absorption spectra of 25 mM CCCP alone (1) and after 30 min incubation with 25 mM Val (2) were recorded in the presence of (A) 164.5 mM NaCl, (B) 160.5 mM NaCl + 4 mM KCl, or (C) 154.5 mM NaCl + 10 mM KCl (in addition to 5 mM HEPES (pH 4 7.4), 2.0 mM CaCl2 and 1.0 mM MgCl2, as in the drug accumulation experiments). 309
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variety of novel reversing agents, including cyclic peptides that resemble Val in their structure (16,17). Since CCCP and Val are often applied together in membrane transport research (2– 4,19,23,24), our observation also emphasizes the possibility that data obtained in analogous experimental situations may be seriously affected by the formation of Val-K+-CCCP complexes. ACKNOWLEDGMENT This work was supported by the U.S.–Hungarian Science and Technology Joint Fund in cooperation with Dept. Biophysics, Univ. Med. School of Debrecen and Division of Research and Testing, Food and Drug Administration under Project JFNO 127. This work was also sponsored by OTKA fundings T14655 and 17592 and ETT Grant T-01 449/93 and by the Hungarian–Dutch Association. The KB cell lines were kindly donated by Dr. M. M. Gottesman.
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