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Intracellular pH does not affect drug extrusion by P-glycoprotein

1996, Journal of Photochemistry and Photobiology B: Biology

The intracellular pH (pHi) of cells exhibiting multidrug resistance (MDR) related to the expression of the P-glycoprotein (Pgp) is often more alkaline than that of the parental cells, as also observed for the KB-V I/KB-3-1 system in this paper. The possible role of an elevated pHi in Pgp-related MDR has been investigated by shifting back the pHi of the MDR ÷ cells to a more acidic value using the mobile proton ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP). The influence of CCCP-evoked A pHi on relative daunorubicin (DNR) accumulation was similar in the ease of several Pgp positive and negative cell lines, in view of flow cytometric and radioactive drug accumulation studies and measuring DNR levels in the medium in a flow-through system. Our data argue against a significant effect of pHi on Pgp pumping efficiency. However, an indirect connection between pHi regulation and the MDR phenotype is suggested by the fact that acidification of the external medium in the presence of verpamil could be observed exclusively in MDR + cells.

d~malaf ~OI.OOY B:BIOLOGY ELSEVIER Journal of Photochemistry and Photobiology B: Biology 34 (1996) 177-182 Intracellular pH does not affect drug extrusion by P-glycoprotein Katalin Goda a, L~iszl6 Balkay b, Ter6z M&i~in b, Lajos Tr6n b, Adorj~in Aszal6s c, G~ibor Szab6 Jr. a,, a Department of Biophysics, b PET Center, University Medical School of Debrecen. 4012 Debrecen, Hungary c Division of Research and Testing. CDER HFD-471, Food and Drug Administration, 200 C St. SW, Washington, DC 20204, USA Received 17 August ! 995; accepted 13 November 1995 Abstract The intracellular pH (pHi) of cells exhibiting multidrug resistance (MDR) related to the expression of the P-glycoprotein (Pgp) is often more alkaline than that of the parental cells, as also observed for the KB-V I/KB-3-1 system in this paper. The possible role of an elevated pHi in Pgp-related MDR has been investigated by shifting back the pHi of the MDR ÷ cells to a more acidic value using the mobile proton ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP). The influence of CCCP-evoked ApHi on relative daunorubicin (DNR) accumulation was similar in the ease of several Pgp positive and negative cell lines, in view of flow cytometric and radioactive drug accumulation studies and measuring DNR levels in the medium in a flow-through system. Our data argue against a significant effect of pHi on Pgp pumping efficiency. However, an indirect connection between pHi regulation and the MDR phenotype is suggested by the fact that acidification of the external medium in the presence of verpamil could be observed exclusively in MDR + cells. Keywords: Multidrug resistance; Intraceilular pH; P-glycoprotein 1. Introduction Intracellular levels of supravital fluorescent drugs are controlled by various cellular parameters, including active drug export mechanism, e.g. the membrane pumps related to the multidrug resistance (MDR) phenomenon. One of these proteins, P-glycoprotein (Pgp; PI70), is considered as one of the main mechanisms acquired by cancer cells that prevent effective chemotherapy (for reviews see Refs. [ 1,2] ). This 170 kDa transport ATPase extrudes its substrates against a concentration gradient [ 3]. Based on sequence homologies and functional analogies, this pump glycoprotein belongs to a group of transport proteins that transfer hydrophobic molecules, peptides and various drugs across the cell membrane [4--6]. The drug-resistant phenotype can be functionally reversed by a wide range of chemicals, including calcium channel blockers [7], calmodulin inhibitors [2]2 lysosomotropic agents [8], some ionophores [9], etc., all of which greatly increase drug accumulation in P170-expressing cells. Competition for the substrate binding site (s) [ 2,7 ] and aUosteric coupling [ 10] have been suggested to account for the mechanism of this reversion. *Corresponding author. I 0 ! 1- 1344/96/$15.00 © ! 996 Elsevier Science S.A. All rights reserved SSD! 1011 - 1344 ( 95 ) 0 7 2 8 2 - 9 P170 substrates (as well as reversing agents) are usually amphiphilic compounds [ 11]. In view of their pH-dependent lipophilicity [ 12], the intracellular pH (pHi) may have a decisive role in the exit process mediated by PI70. pHi may influence drug partitioning between the cell and its environment and/or between the various subcellular compartments [ 13,14]. A large body of data suggest that the pHi of multidrug-resistant cell lines appears to be higher than that of their non-resistant counterparts [15-20]. The elevated pHi of resistant cells has been interpreted in terms of ( 1 ) a transport mechanism specific to protonated substrates [18], (2) a direct contribution of an alkaline pHi to the efficiency of the pump-m~dia:ed drug extrusion [ 21 ], (3) Pgp-generated changes in pHi and qt [20] that could be responsible for decreased drug accumulation and (4) Pgp-unrelated mechanisms [ 19,21 ]. To examine the above alternatives, we measured the drug accumulation of various resistant and sensitive cell Jines as a function of pHi, using daunorubicin (DNR), a fluorescent, DNA-binding, anticancer drug [22], and P170 substrate. Simon et al. [ 19] and Altenberg et al. [21 ] also examined the effect of pHi alterations on drug accumulation, supporting the last interpretation above. In view of the complexity of parameters possibly influencing the outcome of such studies 178 K. Goda et al. / Journal of Photochemistry and Photobiology B: Biology 34 (1996~ 177-182 (e.g. dependence of fluorescence quantum yield on drug compartmentalization [23], alteration of the HCO3-/CIexchange implicated in relation to the MDR phenotype [ 24 ] by the treatments used), we applied various methods complementing each other to address the above possibilities. In our experiments, pHi was acidified by the mobile proton ionophore carbonylcyanide m-chlorophenyihydrazone (CCCP) [25] applied at low extracellular potassium concentration ([Ko + ]), when an inside negative membrane potential drives the protons, carried by CCCP, into the cell. The flow cytometric drug accumulation measurements are sensitive to population heterogeneities and 3H-DNR uptake data are not biased by the possible changes in DNR fluorescence quantum yield. The flow-through system was also applied for getting information about the immediate effects of ionophore treatment; since it is not influenced by possible spectral effects, it is expected to be more sensitive than the flow cytometric detection system. In addition, the changes in extracellular pH (pHo) were also monitored in response to incubation of the resistant and sensitive cells with various Pgp substrates. 2. Materials and methods 2.1. Chemicals and reagents Cells were trypsinized 2 days prior to the experiments and maintained without vinblastine. In some experiments the A2780/2780 Av (human ovarian carcinoma [27] ) cell pair was also used. The cells were checked for mycoplasma by the mycroplasma T.C. rapid detection system with a 3Hlabelled DNA probe from Gen-Probe Inc. and were found to be negative. 2.3. Drug uptake measurements by flow cytometry The "140 mM K +'' buffer contained 140 mM KCI, 10 mM NaCI, 10 mM Na-Hepes and 5 mM glucose (pH 7.4 at room temperature). In the "2 mM K +'' buffer most of the KC! was isotonically replaced by NaCI (or by choline chloride, with identical results). Cells were resuspended in the appropriate buffer (at 4 × 105 cells mi-~) containing DNR (3.55/~M) and incubated for 40 min at 37 °C. The ionophore CCCP was added to the cells at a concentration of 25 or 50 /zM, simultaneously with DNR. The viability of cells after incubations was checked by propidium iodide staining [ 28 ]. Drug uptake and pHi were determined 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 (FSC) signal to exclude the dead cells and cell debris from the analysis. The argon ion laser was tuned to 488 nm and used at a powe~~of 500 mW. Emission was detected through a 540 nm broad band interference filter (IF) and a 620 nm Iongpass filter (for DNR). BCECF-AM (tetraacetoxymethylester of the dye 2',7bis(2-carboxyethyl)-5(and 6).-carboxyfluorescein) was obtained from Molecular Probes Inc. (Eugene, USA). Daunorubicin (DNR), rhodamine 123 (R123), CCCP (carbonylcyanide m-chlorophenylhydrazone), nigericin, valinomycin (Vai), verapamil (Ver), vinblastine (Vbl) and inorganic chemicals were provided by Sigma-Aldrich (Budapest, Hungary). Hepes (N-[2-hydroxyethyl]piperazine-N'-[ethanesulphonic acid]), PSC833 and [G-3H]daunorubicin/HC! (specific activity 1.6 Ci mmol-~) were purchased from SERVA (Heidelberg, Germany), Sandoz ( Basle, Switzerland) and Dupont de Nemours ( 's-Hertogenbosch, Netherlands) respectively. Cyclosporin A was FDA standard (A. Aszal6s). The above buffer solutions were used supplemented with 5% foetal calf serum. The samples contained 0.3 × l0 s cells ml- ~.The cells were incubated in the presence of I pM DNR (containing 2 nM 3H-DNR) and CCCP or reversing agents (at concentrations indicated in Fig. 3) at 37 °C. After incubation the cells were washed twice with ice-cold buffer solution. The cell pellets were transferred to Opti-phase Ill (LKB, Bromma, Sweden) scintillation liquid and then the accumulated 3H-DNR was measured by scintillation counting. 2.Z Cell lines 2.5. Flow-through experiments The drug-sensitive human epidermoid carcinoma cell line KB-3-1 and its vinblastine-selected multidrug-resistant variant KB-V 1 ( isolated from KB-3-1 by stepwise selection with increasing vinblastine concentrations [26]) were used in most of the measurements. These cell lines were grown as monolayer cultures at 37 °C in an incubator containing ';% CO2 and 95% air and maintained by regular passage in Dulbecco's minimal essential medium (supplemented with 10% heat-inactivated foetal calf serum, 2 mM L-glutamine, 100 units m l - ~penicillin and 100/zg ml- ! streptomycin). KBV 1 cells were cultured in the presence of 180 nM vinblastine. The flow-through system is described in detail by Lankelma et ai. [29]. Briefly, a monolayer of approximately 1 × 107 cells attached to a glass chamber (surface area 50 cm2, height 0.1 mm) was perfused with the appropriate buffer containing I or 2/.tM DNR, at a constant flow rate of 200/tl rain- ~at 37 °C, until a steady state level was reached. Pulse injections (15 ~!) of reversing agents (verpamil) or ionophores (CCCP) were introduced into the flowing perfusion medium using a high performance liquid chromatography (HPLC) injection valve. Changes in the 480-560 nm fluorescence were measured on-line in the perfusion medium at 2.4. ~H-DNR accumulation experiments 179 K. Goda et al. / Journal of Photochemistry and Photobiology B: Biology 34 (1996) 177-182 Table 1 Changes in pHi (A pHi) after ionophore treatment of KB-3- ! ( M D R - ) and KB-V! (MDR + ) cells, pHi was measured by flow cytometry (see Section 2) at 2 or 140 mM [Ko + ] and pHo= 7.4. Means + SEM were calculated from three independent experiments. CCCP and Vai were used at 50 and 9 /tM concentration respectively [Ko + ] (mM) Cell line CCCP CCCP+Val 2 KB-3-1 KB-V- ! - 0.40 ___0.08 - 0.42 4- 0.06 - 0.68 + 0.06 - 0.81 + 0.02 140 KB-3- l KB-V- 1 - 0.09 + 0.06 0.03 4- 0.03 0.02 4- 0.02 0.05 + 0.01 the outlet of the flow-through system, by a fluorescence detector (type 821-FP, Jasco, Haschioji City, Japan). 2.6. pH~ and pH,, measurements For measurement of pHi, cells (1 × 107 m l - ' ) in phosphate-buffered saline (PBS; 150 mM NaCl, 3.3 mM KCI, 8.6 mM Na2HPO4 × 12H20, 1.69 mM KH2PO4, pH 7.4) were loaded with 10/.~M (MDR- cells) or 15 pM (MDR ÷ cells) BCECF for 1 h at 37 °C. Calibration of pHi vs. fluorescence was carried out using nigericin to set pHi to known values [30]. pHi was determined within 5-10 min following ionophore treatment, by measuring the ratio of red and green fluorescence intensities [ 31 ] (see Section 2.3). For the measurement of changes in pHo upon pumping, the cells (used at 1 x 106 m l - ' ) were incubated with various MDR substrates in a "low K +'' buffer (see below), but containing 0.8 mM Hepes, at 37 °C for 1 h. The pH of this solution was stable without addition of cells. After incubation the pH of supernatants was measured by a conventional pH electrode. was stable in time as measured after 30 min incubation. Acidification by CCCP occurred in a pHo-dependent manner, as we could detect a larger pHi decrease at pHo = 6.9 (data not shown). The addition of CCCP is expected to reduce pHi, the protons being driven in by a negative inside membrane potenti~', (q/). This interpretation is also supported by the fact that valinomycin (Val), which is expected to hyperpolarize the membrane (as follows from the Goldman-Hodgkin-Katz equation [33]), promoted acidification caused by CCCP. Accordingly, in the "140 mM K +" buffer ( ~ = 0) the ionophores had only a minor effect on pHi (see Table 1). The significant pHi differences demonstrated between MDR ÷ and MDR- cells are in agreement with Refs. [ 1520] and in contrast with Refs. [ 34,35 ]. An increased pHi is expected to decrease DNR accumulation independently from Pgp, since the pH gradient is a strong driving force for (cellular) DNR uptake (towards the more acidic compartment; see trapping of weak bases into liposomes by proton gradients following the Henderson-Hasselbach rule [ 12,13 ] ). Were protons to have a more specific role related to the Pgp pumping mechanism, the pHi would differentially affect MDR ÷ and MDR- cells. 3.2. Effect of CCCP treatment on DNR accumulation 3.2.1. Flow cytometric and 3H-DNR uptake studies As Fig. 1 shows, KB-VI cells, without ionophore treatment, accumulated approximately five times less DNR than the KB-3-1 cells. The DNR fluorescence of either KB-V1 (MDR) ÷ or KB-3-1 (MDR-) cells was usually decreased to a minor extent (to 96.5%+19.7%; p < 0 . 1 ) by CCCP (added simultaneously with DNR). Addition of CCCP to MDR ÷ cells preloaded with DNR did not affect mean fluo350 i 3. Results and discussion 300 3.1. lntraceUular acidification by CCCP 250 For flow cytometric pHi measurements we used the BCECF double-ratio method of Balkay et al. [31 ], which excludes the possible artefacts related to the different BCECF accumulation of MDR ÷ and MDR- cells [32] since the measured fluorescence emission ratios (proportional to pHi; see Section 2.6) are independent of the actual intracellular dye concentration and cell volume. However, we rely only on the changes in and relative values of pHi in our conclusions. The pHi of untreated KB-3-1 (sensitive) cells was lower than that of the KB-VI (resistant) line (by 0.23 + 0.02 (mean +standard error of measurement ((SEM) pH unit; the difference was significant atp < 0.01, calculated from five independent experiments). As Table 1 shows, the addition of CCCP acidified both the resistant and parental cells by 0.30.5 pH unit in the "2 mM K ÷'' buffer. This pHi decrease .~ I i I i * IB I , i I I i I I I i i I i I i T II I 1 i ' [ I| I| II :: ll -- 150 . ".. _ '"i" _ 50 ll .. ,... - I ' ;11 f.*.• - ..i 0 I0 o 10 i 10 2 10 3 Fluorescence intensity Fig. 1. F!uorescence intensity distribution histograms of KB-V i and KB-31 cells incubated with 3.55 p M DNR for 30 rain. KB-V 1 cells were treated with 50 pM Ver (full curve), 50/tM CCCP (broken curve) or were analysed without these additions ( dotted curve ); lon~ dashes represent untreated KB3-1 cells. 180 K. Goda et al. /Journal of Photochemistry and Photobiology B: Biology 34 (1996) 177-182 140 m M K + 2 mM K* A 1400 g~ 1200 g~ 1000 O om ,tJ _= H O t .= 4' 800 ! 600 400 Z A r- 200 m j 0 2SIJMC I . . . . . S0pMC 10pMPSC S0pMVer 2 rnM K + 140 mM K + 1400 i....... 1200 m .2 1000 M = U 800 m 400 n . . . . . . . . . . . . . . i 600 W 200 m o 2$ltMC S0~MC 10pMPSC S0pMVer Fig. 2.3H-DNR accumulation of 2780 Al' ( MDR +, A) and A2780 ( MDR -, B ) cell lines after 40 min incubation with !/xM DNR (containing 2 nM ~HDNR) in the presence of CCCP (C) or reversing agents (Ver, verapamil; PSC, PSC833). Means of triplicate samples with SEM are shown (in the case of one representative experiment out of three). rescence levels (data not shown). No population heterogeneity emerged in either of these experiments as a consequence of ionophore treatment (in addition to the revertant cells present in some cultures). Similar results were obtained in the case of A2780 (MDR-) and 2780 AD (MDR +) cells (data not shown). The flow cytometric data agree with the results of 3H-DNR uptake measurements, as shown in Fig. 2. CCCP had no effect on drug accumulation (measured after 40 min incubation with the drug) in either MDR + or MDR- cells. The CCCPprompted acidification was stable in time as measured up to 30 min (data not shown). In spite of the relatively high ionophore concentrations applied, the cells were alive in terms of both membrane permeability (routinely checked in flow cytometric experiments by propidium iodide staining) and intracellularATP concentrations (greater than 2 mM, as measured by ion exchange HPLC; data not shown). The increase in DNR uptake evoked by verapamil (Ver) or the cyclosporin A analogue PSC833 demonstrates the sensitivity of our experimental systems (see Figs. 1 and 2A). CCCP treatment enhances the proton permeability of intracellular membrane systems (besides decreasing pHi at low [ Ko ÷ ] ), equilibrating the proton gradients in mitochondria and lysosomes. The increment of lysosomal pH was immediate (and stable for at least 30 min) after the addition of 50 /.LM CCCP (at both low and high [ Ko ÷ ] ) and it was even higher than that caused by 50 mM methylamine (our unpublished data obtained by the FITC-dextran method [36] ). However, a significant contribution of the lysosomal compartment to cellular drug accumulation can be ruled out, since alkalinization of the lysosomes by CCCP at 140 mM [ Ko ÷ ] (when the pHi was unchanged; see Table 1) did not affect 3H-DNR accumulation (see Fig. 2). 3.2.2. Flow-through measurements In contrast with the above data, a significant DNR influx was detected into KB-V 1 and KB-3-1 cells immediately after CCCP injection in the flow-through system, as shown in Fig. 3 (traces A and B), perhaps owing to a higher sensitivity of this method. The superior sensitivity of this system over flow cytometric detection is explained by the fact that most of the DNR fluorescence is quenched when it binds to DNA [ 23 ]. Furthermore, the effect of pHi decrease seems to dominate over the effect of increased lysosomal pHi on this time scale, as demonstrated by Demant et al. [ 37 ]. Their kinetic analysis of the possible roles of transmembrane pH gradients in intracellular antracycline accumulation showed that the change in pHi from 7.4 to 6.9 was followed by a moderate (35%) increase in steady state accumulation of DNR. When the pHi decrease is accompanied by the equilibration of lysosomal pH (as in our 3H-DNR accumulation and flow cytometric experiments), the overall CCCP effect on drug accumulation is expected to be even smaller. MDR ÷ and MDR- cells exhibited similar responses to CCCP. No shift in medium A CCCP . . . . g B . O e~ . . C ccc~ . . . . . . . V.r Sn~n O.| pllg time Fig. 3. Effect of 50/~M CCCP on steady state accumulation of DNR in KB3-1 (MDR-) and KB-VI (MDR + ) cells, measured in the flow-through system at 2 mM [Ko + ]. The agents listed below were injected into the perfusion medium after steady state between intracelhlar and medium DNR was attained. A decrease in the medium fluorescence of DNR is a consequence of the net celhlar DNR uptake, while an increase shows net efflux of DNR. Trace A shows the effect of 50/xM CCCP on the steady state accumulation of 2/~M DNR by KB-3-! cells. Traces B and C show the effects of 5 0 / , M CCCP and 25/~M Ver (repeated five times, within 2.5 rain) on the accumulation of I/~M DNR by KB-Vi cells. K. Goda et al. / Journal of Photochemistry and Photobiology B: Biology 34 (1996) 177-182 Table 2 ApHo after I h incubationofKB-3-1 (MDR-) and KB-VI (MDR+ ) cells with various MDR substrates (or reversing agents) The means of three independent experiments + SEM are shown in the case of Ver or CsA treatment; the other results were reproduced once; R123, Vbl and CsA designate rhodamin i 23, vinblastineand cyclosporinA respectively MDR substrate Concentration A pHo A pHo KB-V1 KB-3-1 RI23 13/~M 26/~M 0.02 0.04 0.09 0. ! 1 DNR 17.7 p,M 35.4 p,M 0.00 0.03 0.04 0.04 Vbl 5.45/~M i 0.9/~M 0.03 - 0.07 - 0.05 - 0.06 16.4 p,M - 0.07 - 0.08 Ver CsA 50/~M 5 ttg ml- t 0.28 + 0.03 0.02 + 0.00 - 0.01 -I-0.02 0.04 + 0.02 181 dam, Netherlands), Ursula A. Germann and Michael M. Gottesman (Bethesda, USA) for important suggestions and critical comments. The authors also acknowledge Paul Noordhuis for measuring the intracellular ATP levels. The muitidrug-resistant cell lines were kindly donated by Dr. M.M. Gottesman and Jan Lankelma. This publication has been sponsored by the US-Hungarian Science and Technology Joint Fund in cooperation between the Department of Biophysics, University Medical School of Debrecen and the Division of Research and Testing, Food and Drug Administration under Project JFNO 127. This work was also supported by OTKA fund T017592 and ETT grant T-01 449/ 93. Certain parts of the work were done with the financial support of the Hungarian-Dutch Association. References DNR was detected upon CCCP addition at 140 mM [K,, ÷ ] (data not shown) where pHi was unchanged (see Table 1 ). Thus a decreased pHi appears to facilitate drug accumulation as expected [ 12. ! 3 ], but in a Pgp-independent manner. If the Pgp-driven drug export were coupled to proton transport [ 18 ] or the activity of Pgp were dependent on pHi, it would have been reflected in a major difference between the drug accumulation of MDR ÷ and M D R - cells in response to the acidification of pHi. However, this was not seen, in agreement with the data of Altenberg et ai. [ 21 ], who could not detect an alteration in the unidirectional efflux of R123 in response to pHi changes. Furthermore, if proton transport accompanied drug extrusion by Pgp, it could have been reflected in the changing of pHo upon pumping [ 18]. However, the change in pH,, after 1 h incubation with DNR (35 /xM), R123 ( 2 6 / x M ) , vinblastine ( 16.4/.tM) or CsA ( 5 / z g ml - ~) did not exceed that of the untreated control in the case of either MDR + ( KB-V 1 and 2780 AD) or M D R - ( KB-3-1 and A2780) cells. The data for the KB cell lines are shown in Table 2. Although Ver ( 1 0 - 5 0 /.~M) induced a significant external acidification ( A p H o = 0 . 2 8 +0o03) only in the case of MDR + cells, this is probably due to the elevated lactic acid production specific to Ver-treated MDR + cells [38]. Accordingly, verapamil treatment decreases the pHi o f M D R ÷ cells exclusively [ 18 ]. Thus Pgp activity per se does not entail considerable proton transport nor is it affected by pHi. However, the facts that MDR ÷ cell lines are often distinguished from their parental cells by a higher pHi, as well as differential acidification in response to Ver (see above), imply that the MDR ÷ phenotype frequently involves an altered pHi regulation. Acknowledgements The authors thank Drs. S~indor Damjanovich (Debrecen, Hungary), Jan Lankelma and Hans V. Westerhoff (Amster- [I]J.A. Endicott and V. Ling, The biochemistry of P-glycoproteinmediated muitidrug resistance, Ann. Rev. Biochem., 58 (1989) 137171. [2] M.M. Gottesman and E ~-~astan,Biochemistryof multidmgresistance mediatedby the multidrugtransporter, Ann. Rev. Biochem., 62 (1993) 385-427. [3] K. Dane, Active outward transport of daunomycin in Erlich ascites tumor cells, Biochim. Biophys. Acta, 455 (1973) 152-162. [4] C.F. Higgins, ABC transporters: from microorganismsto man, Ann. Rev. Cell Biol., 8 (1992) 67-113. [5] C.A. Doige and G. Ferro-Luzzi Ames, ATP-dependent transport systems in bacteria and humans: relevance to cystic fibrosis and multidmgresistance,Ann. Rev. Microbiol.. 47 (1993) 291-319. [6] R.C. Sharma, S. Inoue, J. Roitelman,R.T. Schimke and R.D. Simoni, Peptide transport by the muitidmg resistance pump, J. Biol. Chem., 267 ( 1991) 5731-51734. [7] MM. Cornwell, I. Pastan and M.M. Gottesman, Certain calcium channel blockers bind specificallyto multidrug resistant human KB carcinoma membrane vesicles and inhibit drug binding to Pglycoprotein,J. Biol. Chem. 262 (1987) 2166-2170. [ 8] J.M. Zamoraand W.T. Beck, Chloroquineenhancementof ~ticancer drug cytotoxicity in multiple drug resistant human leukaemic cells, Biochem. Pharmacol., 35 (1986) 4303-4310. [9] J.L. Weaver, G. Szab6 Jr., M.M. Gottesman, S. Goldenberg and A. Aszalos, The effect of ion channel blockers, immunosuppressive agents, and other drugs on the activity of the multi-drugtransporter, Int. J. Cancer, 54 (1993) !--6. [ 10] D.R. Ferry, M.A. Russeland M.H. Cullen, P-glycoproteinpossessesa 1,4-dihydropyridine-selectivedrug acceptor site whichis allosterically coupled to a vinca-alkaloid-selectivebinding site, Biochem. Biophys. Res. Commun., 188 (1992) 440--445. [ 11] W.T. Beck, Tha cell biology of multiple drug resistance, Biochem. Pharmacoi., 36 (1987) 2879-2887. [ 12] T. Skovsgaard, Transport and binding of daunorubicin, adriamycin, and rubidazone in Ehrlich ascites tumor cells, Biochem. Pharmacol., 26 (1977) 215-222. [ 13] T.D. Madden,P.R. Harrigan,L.C.L. Tai, M.B. Bally,L.D. Mayer,T.E. Redelmeir, H.C. Loughrey, C.P.S. Tilcock, L.W. Reinish and 'LR. Cullis, The accumulationof drugs within large unilamellarvesicles exhibitinga proton gradient: a survey, Chem. Phys. Lipids, 53 (1990) 37-46. [ 14] L.D. Mayer, M.B. Bally and P.R. Cullis, Uptake of adriamycininto large unilamellar vesicles in response to a pH gradient, Biochem. Biophys. Acta, 857 (1986) 123-126. 182 K. Goda et al. / Journal of Photochemistry and Photobiology B: Biology 34 (1996) 177-182 [ 15] R.C. Lyon, J.S. Cohen, P.J. Faustino, F. Megnin and C.E. Myers, Glucose metabolism in drug-sensitive and drag-resistant human breast cancer cells monitored by magnetic resonance spectroscopy, Cancer Res., 48 (1988) 870-877, [16] H.G. Keizer and H. Joenje, Increased cytosolic pH in multidmgresistant human lung tumor cells: effect of verapamil, J. Natl. Cancer Inst.. 81 (1989) 706-709. [ 17] D. Boscoboinik, R.S. Gupta and R.M. Epand, Investigation of the relationship between altered intracellular pH and muitidmg resistance in mammalian cells, Br. J. Cancer, 61 (1990) 568-572. [18] F. Thiebaut, S.J. Currier, J. Whitaker, R.P. Haugland, M.M. Gottesman, I. Pastan and M.C. Wellingham, Activity of the multidrug transporter results in alkalinization of the cytosol: measurement of cytosolic pH by microinjection of a pH-sensitive dye, J. Histochem. Cytochem., 38 (1990) 685-690. [ 19] S. Simon, D. Roy and M. Schindler, Intraceilular pH and the control of multidrug resistance, Proc. Natl. Acad. Sci. USA, 91 (1994) 1128II32. [20] P.D. Roepe, L.Y. Wei, J. Cruz and D. Carlson, Lower electrical membrane potential and altered pHi homeostasis in multidrug-resistant (MDR) cells: further characterization of a series of MDR cell lines expressing different levels of P-glycoprotein, Bwche,ii~try, 32 (1993) 11 042-I i 056. [211 G.A. Altenberg, G. Young, J.K. Horton, D. Glass, J.A. Belli and L. Reus% Changes in intra- or extracellular pH do not mediate Pglycoprotein-dependent multidrug resistance, Proc. Natl. Acad. Sci. USA, 90 (1993) 9735-9738. [22] I. Calendi, A. di Ma,-co, B. Regiani, B. Scarpinato and E. Valentini, On physico-chemical intemc~ons between daunomycin and nucleic acids, Biochem. Biophys. Acta, 103 (!965) 25-49. [23] J. Lankelma, H.S. Miilder, F. van Mourik, ~,'.F Sang, R. Kraayenhof and R. van Grondeile, Cellular daunomycin fluorescence in multidrug resistant 2780 ^D cells and its relation to cellular drug localization, Biochim. Biophy. Acta, 1093 ( 1991 ) 147-152. [24] J.G. Luz, L.Y. Wei, S. Basu and P.D. Roepe, Transfection ofmu MDR I inhibits Na + -independentCI -/HCO~- exchange in chinese hamster ovary cells, Biochemistry, 33 (1994) 7239-7249. [25] J. Kasianowicz, R. Benz and S. Mclaughlin, The kinetic mechanism by which CCCP (catbonyl cyanide m-chlorophenylhydrazone) transports protons across membranes, J. Membr. Biol.. 82 ( ! 984) ! 79190. [26] D.-W. Shen, C. Cardarelli, J. Hwang, M.M. Cornwell, N. Riche,rt, S. Ishii, I. Pastan and M.M. Gottesman, Multiple drug-resistant human KB carcinoma cells independently selected for high level resistance to colhicine, adriamycine or vinblastine show changes in expression of specific proteins, J. Biol. Chem., 261 (1986) 7762-7770. [27] K.G. Louie, T.C. Hamilon, M.A. Winkler, B.C. Behrens, T. Tsumo, R.W. Klecker, W.M. Mchoy, K.R. Grotzinger, C.E. Meyers, R.C. Young and R.F. Ozols, Adriamycin accumulation and metabolism in adriamycin-sensitive and resistant human ovarian cancer cell lines, Biochem. Pharmacol., 35 (1986) 467--472. [28] A. Krishan, Rapid flow cytophotometric analysis of mammalian cell cycle by propidium iodide staining, J. Cell Biol., 66 (1975) 188-192. [29] J. Lankelma, E. Laurensse and H.M. Pinedo, A flow-through tissue culture system with fast dynamic response, Anal. Biochem., 127 (1982) 340-345. [30] S. Grinstein, S. Cohen and A. Rothstein, Cytoplasmic pH regulation in thymic lymphocytes by an amiloride-sensitive Na/H antiport, J. Gen. Physiol., 83 (1984) 341-370. [ 31 ] L. Balkay, T. Mfiririn,M. Emri and L. Tr6n, A novel method to measure intracellular pH. Effect of neutron irradiation on pHi of transformed cells, J. Photochem. Photobiol B: Biol,, 16 (1992) 367-375. [32] L. Homolya, Zs. Holl6, U.A. Germann, I. Pastan, M.M. Gottesman and B. Sarkadi, Fluorescent cellular indicators are extruded by the multidrug resistance protein, J. Biol. Chem., 268 (!993) 21 49321 496. [ 33 ] A.L. Hodgkin, The ionic basis of electrical activity in nerve and muscle, Biol. Rev., 26 (1951) 339-409. [ 34] E.C. Spoelstra, H.V. Westerhoff, H. Dekker and J. Lankelma, Kinetics of daunorubicine transport by P-glycoprotein of intact cancer cells, Eur. J. Biochem., 207 (1992) 567-579. [35] F. Frezard and A. Garnier-Suillerot, Comparison of the membrane transport of antracycline derivatives in drug-resistant and drugsensitive K562 cells, Eur. J. Biochem., 196 ( 1991 ) 483-491. [36] S. Ohkuma and B. Poole, Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents, Proc. Natl. Acad. Sci., 75 (1978) 3327-3331. [37] E.J.F. Demant, M. Sehested and P.B. Jensen, A model for computer simulation of P-glycoprotein and transinembrane dpH-mediated antracycline transport in multidrug-resistant tumor cells, Biochim. Biophys. Acta, 1055 (1990) 117-125. [39] H.J. Broxterman, H.M. Pinedo, C.M. Kuiper, G.J. Schuurhuis and J. Lankelma, Giycolysis in P-glycoptotein-overexpressinghuman tumor cell lines. Effect of resistance-modifying agents, FEBS Lett., 247 (1989) 405--410.