Báo cáo khoa học: Role of the plasma membrane leaflets in drug uptake and multidrug resistance ppt

Role of the plasma membrane leaflets in drug uptake and multidrug resistance Hagar Katzir*, Daniella Yeheskely-Hayon*, Ronit Regev and Gera D. Eytan Department of Biology, The Technion – Israel Institute of Technology, Haifa, Israel Introduction P-glycoprotein [Pgp; multidrug resistance protein (MDR) 1] (ABCB1) [1] and the multidrug resistance- associated protein (MRP) 1 (ABCC1) [2] were recog- nized as serious impediments to cancer chemotherapy through their ability to eliminate drugs from cells. Both proteins are members of the ABC transporters superfamily [3]. Pgp efficiently exports amphipathic somewhat basic drugs, such as paclitaxel (taxol), anth- racyclines and Vinca alkaloids. The hydrophobic parts of these drugs allow their rapid insertion in the mem- brane. The hydrophilic residues prevent rapid flipping of the drug from the extracellular leaflet to the cyto- plasmic leaflet of the membrane, slowing down entry into the cell; indeed, for an anthracycline such as doxorubicin, this takes approximately 1 min, giving the Pgp pump ample opportunity to deal with the influx [4–6]. This rate of spontaneous flip-flop is rele- vant because estimates of the turnover number of Pgp Keywords MDR1; MRP1; multidrug resistance; P-glycoprotein; plasma membrane Correspondence G. D. Eytan, Department of Biology, The Technion – Israel Institute of Technology, Haifa, Israel Fax: +972 4 822 5153 Tel: +972 4 829 3406 E-mail: eytan@tx.technion.ac.il Website: http://biology.technion.ac.il *These authors contributed equally to this work (Received 5 November 2009, revised 13 December 2009, accepted 18 December 2009) doi:10.1111/j.1742-4658.2009.07555.x The present study aimed to investigate the role played by the leaflets of the plasma membrane in the uptake of drugs into cells and in their extrusion by P-glycoprotein and multidrug resistance-associated protein 1. Drug accumulation was monitored by fluorescence resonance energy transfer from trimethylammonium-diphenyl-hexatriene (TMA-DPH) located at the outer leaflet to a rhodamine analog. Uptake of dye into cells whose mito- chondria had been inactivated was displayed as two phases of TMA-DPH fluorescence quenching. The initial phase comprised a rapid drop in fluo- rescence that was neither affected by cooling the cells on ice, nor by activ- ity of mitochondria or ABC transporters. This phase reflects the association of dye with the outer leaflet of the plasma membrane. The sub- sequent phase of TMA-DPH fluorescence quenching occurred in drug- sensitive cell lines with a half-life in the range 20–40 s. The second phase of fluorescence quenching was abolished by incubation of the cells on ice and was transiently inhibited in cells with active mitochondria. Thus, the sec- ond phase of fluorescence quenching reflects the accumulation of dye in the cytoplasmic leaflet of the plasma membrane, presumably as a result of flip- flop of dye across the plasma membrane and slow diffusion from the inner leaflet into the cells. Whereas activity of P-glycoprotein prevented the sec- ond phase of fluorescence quenching, the activity of multidrug resistance- associated protein 1 had no effect on this phase. Thus, P-glycoprotein appears to pump rhodamines from the cytoplasmic leaflet either to the outer leaflet or to the outer medium. Abbreviations CCCP, carbonyl cyanide m-chlorophenylhydrazone; FRET, fluorescence resonance energy transfer; MDR, multidrug resistance; MRP1, multidrug resistance-associated protein; Pgp, P-glycoprotein; TMA-DPH, trimethylammonium-diphenyl-hexatriene; TMRM, tetramethylrhodamine methyl ester. 1234 FEBS Journal 277 (2010) 1234–1244 ª 2010 The Authors Journal compilation ª 2010 FEBS substrates are in the range 1–10 s )1 , which is fast com- pared to the flip-flop rates of drugs, such as doxorubi- cin [6,7]. Pgp has been proposed to function as a ‘hydropho- bic vacuum cleaner’, extracting its substrates directly from the lipid core of the membrane rather than from the aqueous phase [8]. This idea is supported by data showing that the apparent affinity of a drug for bind- ing to purified Pgp is highly correlated with its lipid– water partition coefficient [9]. Subsequently, this model has been refined and Pgp has been suggested to act as a plasma membrane flippase, moving drug molecules from the cytoplasmic leaflet to the extracellular leaflet [10]. Romsicki and Sharom [11] have shown that Pgp reconstituted into proteoliposomes transports lipid analogs from the cytoplasmic leaflet to the extracellu- lar leaflet. In contrast, it has been demonstrated that reconstituted Pgp and the bacterial multidrug trans- porter, LmrP, expel drugs from the cytoplasmic leaflet of the membrane to the aqueous medium rather than to the extracellular leaflet [12,13]. The question remains as to whether the release of drugs from the cytoplasmic leaflet of the plasma membrane into the cytoplasm is fast, resulting in a practical equilibrium between the drug concentrations in the cytoplasmic leaflet and the cytoplasm, or whether the release is slow, and drugs taken up into cells accumulate in the cytoplasmic leaflet prior to being released into the cytoplasm. In the latter case, Pgp functioning as a flippase will have the added advantage of capturing incoming drugs before they reach the cytoplasm and at a transient high local concentration. Moreover, in the latter case, Pgp is expected to handle incoming drugs more efficiently compared to drugs already present in the cell interior. By contrast, in the case where drug concentrations in the cytoplasm and the cytoplasmic leaflet are in equi- librium, Pgp is expected to treat incoming drugs and drugs already present within the cell in a similar manner. By contrast to Pgp, MRP1 functions as a glutathi- one–X conjugate pump. It not only transports a vari- ety of drugs conjugated to glutathione, sulfate or glucuronate, as well as anionic drugs and dyes, but also neutral ⁄ basic amphipathic drugs and even oxya- nions. Previously, it has been assumed that the oxya- nions arsenite and antimonite and the neutral ⁄ basic drugs are cotransported by MRP1 with glutathione [14]. However, recent data indicate that the mechanis- tic interaction between the transported neutral ⁄ basic drugs and the glutathione is more complicated [15]. The hydrophilic nature of some MRP1 substrates makes it unlikely that MRP1 functions as a flippase and extracts these substrates from the inner leaflet of the plasma membrane. Rather, MRP1 pumps these substrates directly from the cytoplasm. The experiments conducted in the present study were designed to dissect the cellular uptake of MDR-type drugs into its constituent steps: uptake into the extra- cellular leaflet, flip-flop across the lipid core of the membrane and movement to the cytoplasmic leaflet of the plasma membrane. First, an awareness of such data should help to resolve an outstanding question: is there a kinetic barrier between the cytoplasmic leaflet of the plasma membrane and the cytoplasm? Such a putative barrier would result in the cytoplasmic leaflet constituting a kinetic compartment separate from the extracellular leaflet and from the interior of the cell. In the case where the cytoplasmic leaflet does constitute a separate compartment, the accumulation of drug within this would be accomplished prior to saturation of the total cellular content of the drug. By contrast, in the case where there is no kinetic barrier, drug accu- mulation within the cytoplasmic leaflet would proceed in parallel with total drug accumulation within the cells. Second, measurement of drug accumulation in the cytoplasmic leaflet should help determine whether Pgp removes its substrates from the cytoplasmic leaflet, as predicted by the vacuum cleaner model, whereas MRP1 extracts its substrates from the cytoplasm. Tetramethylrhodamine methyl ester (TMRM) served as a highly fluorescent probe representing the MDR- type drugs [16]. TMRM accumulation in the plasma membrane leaflets was assayed in cells over-expressing either Pgp or MRP1 and their sensitive parental lines. TMRM accumulation was monitored as fluorescence resonance energy transfer (FRET) from trimethyl- ammonium-diphenyl-hexatriene (TMA-DPH) to the TMRM present in the membrane or very close to it. Because of its polar nature, TMA-DPH, unlike its ana- log diphenyl hexatriene, has a high specificity for the plasma membrane in intact cells [17,18]; TMA-DPH is located within the lipid bilayer close to the outer sur- face. The probe has been reported to be useful for measurements of plasma membrane fluidity and for studies on cellular exocytosis [19]. Kessel [20] found similar values for TMA-DPH accumulation in drug- resistant P388 cells and wild-type cells; no differences were observed in the fluorescence anisotropy and life- time of TMA-DPH between these cell lines, which would indicate that there are no MDR-related differ- ences in the binding of TMA-DPH to different cellular components. On the basis of the overlap between the fluorescence-emission spectrum of TMA-DPH and the excitation spectrum of TMRM, FRET can occur, pro- vided that the probe and the drug are sufficiently close. H. Katzir et al. Cytoplasmic leaflet in drug uptake and resistance FEBS Journal 277 (2010) 1234–1244 ª 2010 The Authors Journal compilation ª 2010 FEBS 1235 Thus, the degree of TMA-DPH fluorescence quenching by TMRM may provide information on the amount of TMRM associated with the plasma membrane, as described previously for synthetic and natural mem- brane vesicles [21,22]. Results FRET from TMA-DPH to TMRM in cells sensitive to anticancer drugs The association of the dye, TMRM, with the surface of cells was monitored as FRET from TMA-DPH located at the outer leaflet of the plasma membrane to this dye [23,24]. The background fluorescence of TMA-DPH in the aqueous medium appeared to be negligible. Immediately upon the addition of cells to a medium containing TMA-DPH, the fluorescence of the latter increased by at least a factor of 100 as a result of adsorption of dye on the outer leaflet of their plasma membrane [25]. Steady-state fluorescence was reached after < 5 min. The fluorescence of TMA- DPH was observed immediately at the periphery of the cells (data not shown). After prolonged incubation, additional fluorescence was observed within the cells. However, this fluorescence, presumably located in the mitochondria and endosomes [23], was faint compared to the fluorescence at the periphery of the cells. The quenching pattern of TMA-DPH fluorescence by TMRM was unaffected by the preincubation period of TMA-DPH with the cells (Fig. 1). Upon the addition of TMRM to cells preincubated with TMA-DPH, quenching of the fluorescence of the TMA-DPH occurred in two steps: an initial fast drop in fluores- cence followed by a slow further fluorescence quench- ing. The simultaneous addition of the two dyes to cells resulted in a slow quenching similar to the second step that was observed when TMRM was added after TMA-DPH. Presumably, when the two dyes are added together, the initial quenching of fluorescence occurred faster than the adsorption of TMA-DPH to the cells and fluorescence quenching as a result of the added TMRM prevented the rapid initial drop in fluorescence observed when TMRM was added to cells preincubat- ed with TMA-DPH. Thus, the measured FRET occurred from the TMA-DPH located at the surface of the plasma membrane and not from TMA-DPH located within the cells. The rapid initial quenching of the fluorescence was essentially complete within 1 s after the addition of TMRM. The extent of the initial quenching was linear with the outer concentration of TMRM up to a con- centration of 25 lm. Because the initial rapid drop in TMA-DPH was not modulated by low temperatures (Fig. 1), it reflects the absorption of TMRM to the outer leaflet of the plasma membrane. After the initial rapid quenching phase, a slower quenching phase was observed at ambient temperatures, although not on ice. Thus, the slower fluorescence quenching reflected TMRM crossing a lipid barrier located in the plasma membrane. The main intracellular accumulation site of rhodam- ines inside cells is the mitochondria. This accumulation could be eliminated by poisoning the mitochondria either with the uncoupler, carbonyl cyanide m-chloro- phenylhydrazone (CCCP), or the respiration inhibitor, sodium azide. Poisoning the mitochondria had no effect on the initial rapid phase of TMA-DPH fluores- cence quenching by TMRM. By contrast, poisoning the mitochondria accelerated the second phase of TMA-DPH fluorescence quenching by TMRM. The resulting curve could be fitted to a first-order reaction with half-lives in K562, GLC4 and 2008 cells of 36 ± 5, 19 ± 4 and 21 ± 6 s, respectively (Fig. 2). To determine whether the second phase of TMA- DPH fluorescence quenching by TMRM in the B A TMA-DPH fluorescence 3 min C D Fig. 1. TMA-DPH fluorescence quenching by TMRM. K562 cells were incubated in the presence of glucose and sodium azide (10 m M)at37°C and their fluorescence was monitored continu- ously using the excitation and emission wavelengths of TMA-DPH fluorescence. Trace A: 2 l M TMA-DPH was added at the time point marked by the thin arrow and 25 l M TMRM was added at the time point marked by the thick arrow. Trace B: 2 l M TMA-DPH was added at the time point marked by the thin arrow and, 5 s later, 25 l M TMRM was added. Trace C: 2 lM TMA-DPH and 25 lM TMRM were added together at the time point marked by the arrows. Trace D: Cells were incubated for 10 min at 37 °C with 2 l M TMA-DPH. Subsequently, the cells were cooled by incubation on ice for 5 min and, at the time point marked by the arrow, 25 l M TMRM was added. The extent of fluorescence drop presented in trace D was equivalent to 0.26 ± 0.05 of the fluorescence observed before the addition of the TMRM. Cytoplasmic leaflet in drug uptake and resistance H. Katzir et al. 1236 FEBS Journal 277 (2010) 1234–1244 ª 2010 The Authors Journal compilation ª 2010 FEBS presence of CCCP reflects the total cellular uptake of TMRM, the time course of the quenching was com- pared with the time course of TMRM uptake into the cells. The time course of TMRM uptake into cells con- sists of two stages: a first rapid stage that reflects bind- ing of TMRM to the outer leaflet of the plasma membrane and a subsequent uptake of TMRM into the cells [16]. The uptake of TMRM into K562 and GLC4 cells occurred with half-lives of 3.7 ± 0.4 and 1.3 ± 0.2 min, respectively (as calculated based on data presented in Fig. 3). Thus, the FRET was four- to six-fold faster compared to the total uptake of TMRM into the cells and the kinetics of the two movements are separate. A comparison of TMA-DPH fluorescence quenching by TMRM observed in normally respiring cells and in cells whose mitochondria had been poisoned reveals that the active uptake of TMRM into the mitochon- dria interferes with the second phase of TMA-DPH quenching (Fig. 2). Initially, there is significant inhibi- tion of the quenching in the respiring cells, which is subsequently relieved, presumably as a result of satura- tion of the mitochondria with TMRM. As shown in Table 1, poisoning of the mitochondria with either CCCP or sodium azide resulted in little change in their ATP content. These cells relied mainly on glycolysis for their ATP supply and only poisoning the mito- chondria and glucose deprivation lead to a reduction in cellular ATP content. Thus, the effect of the mito- chondrial poisons on the secondary fluorescence drop is not the result of an indirect effect mediated by ATP depletion. FRET from TMA-DPH to TMRM in multidrug resistant cells Over-expression of Pgp by K562 cells had no effect on the rapid initial drop in TMA-DPH fluorescence induced by TMRM. By contrast, it eliminated the sec- ond phase of drop in TMA-DPH fluorescence induced by TMRM (Fig. 4A). The activity of Pgp completely cancelled the slow phase of fluorescence drop, both in cells with active mitochondria and in cells whose mito- chondria had been poisoned. This effect of the over- expressed Pgp was partially reversed as a result of the K562 A B 5 min C TMA-DPH fluorescence GLC4 A 3 min B 2008 A 2 min B A B C Fig. 2. Effect of poisoning the mitochondria on TMA-DPH fluores- cence quenching by TMRM. (A) K562, (B) GLC4 or (C) 2008 cells were incubated at 37 °C either in the absence (trace A) or presence of either 1 l M CCCP (trace B) or 10 mM sodium azide (trace C). 2 l M TMA-DPH was added at the time points marked by the thin arrows and 25 l M TMRM was added at the time points marked by the thick arrows. TMA-DPH fluorescence was monitored continu- ously. The curves represent at least four separate experiments. The curves describing the second, slow, phase of TMA-DPH fluo- rescence quenching by TMRM in the presence of either CCCP or sodium azide were fitted to the first-order reaction y = a · exp(–k · t)+c, where t is the time period elapsed from the addition of the dye and k is the reaction constant; a and c represent the extent of the secondary fluorescence drop and the fluorescence remaining after both phases of fluorescence quenching, respectively. The k values obtained served to calculate the half-life of the fluorescence quenching. All fluorescence values are expressed as fractions of the TMA-DPH fluorescence exhibited by the cells just before the addition of the TMRM dye. The r 2 values obtained were > 0.95. H. Katzir et al. Cytoplasmic leaflet in drug uptake and resistance FEBS Journal 277 (2010) 1234–1244 ª 2010 The Authors Journal compilation ª 2010 FEBS 1237 modulation of Pgp activity by the chemosensitizers, cyclosporine A, verapamil and reserpine, or by deple- tion of cellular ATP. These treatments had no signifi- cant effect on either the initial fast phase of fluorescence in the Pgp-over-expressing cells or fluores- cence quenching in wild-type cells (Fig. 5). Inhibition of Pgp with cyclosporine A caused a parallel increase in the amount of TMRM taken up by the cells as well as the extent of the second phase of TMA-DPH fluo- rescence quenching by TMRM (Figs 6 and 7). By con- trast to over-expression of Pgp, over-expression of MRP1 had no apparent effect on fluorescence quench- ing of TMA-DPH by TMRM (Fig. 4B, C). As expected, MRP1 activity had no apparent effect on the rapid initial quenching of TMA-DPH fluorescence. Moreover, MRP1 over-expression did not affect the subsequent slow fluorescence quenching of TMA-DPH fluorescence, either in respiring cells or in cells whose mitochondria had been poisoned. Discussion Cellular uptake of the rhodamine, TMRM, was analy- sed using FRET from the dye TMA-DPH located at the surface of the cell plasma membrane to the incom- ing rhodamine dye. The quenching of TMA-DPH occurred in two distinct phases: an initial rapid phase followed by a slower phase with a measurable kinetics. Because the initial quenching phase was very rapid and was unaffected by low temperatures, it represents the adsorption of dye to the cell surface. The subse- quent phase was eliminated at low temperatures and thus involves transport across or into the lipid core of the plasma membrane. This temperature-dependent flu- orescence quenching phase exhibited the following characteristics. (a) In the presence of mitochondrial poisons, it occurred as a single first-order reaction. (b) Active uptake of the TMRM by respiring mitochon- dria transiently inhibited the temperature-dependent fluorescence quenching. This inhibition could be pre- vented by mitochondrial poisons such as the uncou- pler, CCCP, and the respiration inhibitor, sodium azide. Treatment of cells with these poisons did not deplete the ATP content of the cells. Thus, the fluores- cence quenching observed in the presence of these poisons, especially the hydrophilic azide ion, does not reflect a direct effect on the plasma membrane. (c) The temperature-dependent fluorescence quenching was prevented by the activity of over-expressed Pgp. Fluorescence quenching could be restored by modu- lation of Pgp activity, either by its specific inhibitors or by depletion of cellular ATP. The temperature-dependent fluorescence quenching reflects the transfer of TMRM from its location at the surface of the cells toward an inner location. Because the cationic rhodamine dye is amphipathic, it is practi- cally insoluble in the lipid core and is expected to be 3 3 GLC4 K562 2 2 1 1 0 0 102030 0.0 2.5 5.0 7.5 10.0 0 Time (min) Cell-associated TMRM (nmol 10 –6 cells) Fig. 3. TMRM uptake into K562 (left) and GLC4 (right) cells. K562 or GLC4 cells were incubated with 25 lM TMRM in the presence (circles) or absence (squares) of 1 l M CCCP. Samples were withdrawn at various time points and the amount of TMRM associated with the cells was determined by the quantitative procedure described in the Experimental procedures. The data describing the dye uptake into the cells whose mitochondria were poisoned with CCCP were fitted to a first-order reaction with r 2 > 0.95, as described in Fig. 2. Table 1. Effect of mitochondrial poisons on the cellular ATP con- tent of K562 cells. K562 cells or their Pgp over-expressing sub-line, K562 ⁄ ADR, were incubated for 30 min at 37 °C in the absence or presence of 10 m M glucose, 10 mM deoxyglucose, 1 lM CCCP or 1m M azide. Cell samples were withdrawn and their ATP content was determined. ATP content is expressed as a percentage of the ATP content of the control K562 cells and their Pgp over-express- ing cells (4.6 ± 0.6 and 5.3 ± 0.7 nmolÆ10 )6 cells, respectively). K562 wild-type Pgp over-expressing cells Control 100 100 Azide + glucose 91 ± 7 88 ± 6 Azide + deoxyglucose 11 ± 2 18 ± 5 CCCP + glucose 89 ± 8 91 ± 6 CCCP + deoxyglucose 16 ± 5 11 ± 3 Cytoplasmic leaflet in drug uptake and resistance H. Katzir et al. 1238 FEBS Journal 277 (2010) 1234–1244 ª 2010 The Authors Journal compilation ª 2010 FEBS localized at the surfaces of the plasma membrane. Theoretically, this fluorescence quenching could be the result of TMRM transfer from the cell surface further into the outer leaflet of the membrane or flip-flop across the membrane and residence in the inner leaflet of the membrane. The observation that active uptake of the TMRM by the mitochondria delays the temper- ature-dependent fluorescence quenching is inconsistent with the possibility that quenching occurs as a result of dye moving within the outer leaflet of the plasma membrane. The lipid core of the plasma membrane constitutes the main barrier to TMRM transport across the membrane and the mitochondria cannot affect the TMRM concentration bound at the outer leaflet. Thus, the temperature-dependent fluorescence quenching reflects the flip-flop of TMRM from the outer leaflet of the plasma membrane to the inner K562/ADR A 5 min B TMA-DPH fluorescence B TMA-DPH fluorescence GLC4/ADR A 2 min B TMA-DPH fluorescence 2008/MRP1 A 2 min B A B C Fig. 4. TMA-DPH fluorescence quenching by TMRM in Pgp or MRP1 over-expressing cells. Pgp over-expressing cells, (A) K562 ⁄ ADR, or MRP1 over-expressing cells, (B) GLC4 ⁄ MRP1 and (C) 2008 ⁄ MRP1, were incubated at 37 °C either in the absence (trace A) or presence (trace B) of 1 l M CCCP. 2 lM TMA-DPH was added and the cells were incubated for a further 10 min. 25 l M TMRM was added at the time points marked by the arrows. TMA- DPH fluorescence was monitored continuously. The curves repre- sent at least four separate experiments. The curves describing the second phase of TMA-DPH fluorescence quenching by TMRM in presence of CCCP were fitted as a first-order reaction with r 2 > 0.95, as described in Fig. 2. Sensitive K562 cells A B A B C D 3 min TMA-DPH fluorescence TMA-DPH fluorescence D E E Resistant K562/ADR cells B A B C D 3 min D E E Fig. 5. Effect of Pgp modulation on TMA-DPH fluorescence quenching by TMRM. (A) K562 cells or (B) their Pgp over-express- ing sub-line, K562 ⁄ ADR, were incubated at 37 °C in the presence of glucose and 1 m M azide and either in the absence (trace A) or presence of 10 l M cyclosporine (trace B), 100 lM verapamil (trace C) or 30 l M reserpine (trace D). Cells presented in trace E were depleted of ATP by incubation for 30 min at 37 °C in the presence of deoxyglucose instead of glucose and 1 m M azide. At the time points marked by the arrows, 2 l M TMA-DPH was added and, after a further 5 min of incubation, 25 l M TMRM was added. TMA-DPH fluorescence was monitored continuously. The curves represent at least four separate experiments. H. Katzir et al. Cytoplasmic leaflet in drug uptake and resistance FEBS Journal 277 (2010) 1234–1244 ª 2010 The Authors Journal compilation ª 2010 FEBS 1239 leaflet. The expected distance between TMRM located at the inner leaflet of the plasma membrane and TMA-DPH located at the outer leaflet is somewhat < 3 nm (i.e. a distance that could allow FRET between these dyes). The results obtained with FRET from TMA-DPH to TMRM located at the outer leaflet of the plasma membrane suggest that uptake of TMRM occurs in distinct steps: rapid binding to the outer surface of the cells, flip-flop across the plasma membrane, accu- mulation of dye in the cytoplasmic leaflet of the plasma membrane and release into the cell interior. The initial binding of dye to the cells, evident as the TMRM-mediated initial drop in TMA-DPH fluores- cence, appears to be instantaneous, even at low temperatures. Therefore, it is very rapid, possibly limited by the diffusion of dye toward the cell. Local- ization studies of multidrug-type drugs and modula- tors suggest that, upon association of the TMRM with the plasma membrane, it is located between the phosphate of the lipid headgroups and the upper segments of the lipid hydrocarbon chains [26]. The subsequent accumulation of dye in the cytoplas- mic leaflet of the plasma membrane comprises a fast process compared to the total uptake of dye into the cells, indicating that, in kinetic terms, the cytoplasmic leaflet comprises a compartment separate from the cytoplasm. The accumulation of dye in the cytoplas- mic leaflet is the outcome of a balance between the rate of flip-flop across the membrane from the outer leaflet to the cytoplasmic leaflet of the plasma mem- brane and the release from the cytoplasmic leaflet into the cell. Analysis using a kinetic model of drug uptake into cells similar to the previously reported models [27,28] suggests that a significant accumulation in the cytoplasmic leaflet of the plasma membrane takes place only when the release from the plasma mem- brane into the cytoplasm occurs at a rate similar to that of the flip-flop of dye across the plasma mem- brane. In the case where the release into the cytoplasm is fast compared to the flip-flop, the amount of dye accumulated in the cytoplasmic leaflet will be insignifi- cant. By contrast, in the case where diffusion into the cells is slower than the flip-flop across the plasma membrane, it will constitute the limiting step of dye uptake into the cells. The secondary drop in the TMRM-mediated TMA- DPH fluorescence observed in cells whose mitochon- dria were poisoned, reflects the flip-flop rate of dye from the outer leaflet of the plasma membrane to the cytoplasmic leaflet. The apparent half-life of the flip- flop was in the range 20–40 s in the various cell lines investigated in the present study. This half-life value was similar to the flip-flop value of doxorubicin observed in cell-free systems such as liposomes and iso- lated erythrocyte membranes [4,5]. The half-life of the flip-flop observed as the drop in TMA-DPH fluores- cence is a minimum value because the step subsequent 0.0 0.1 0.2 1.0 TMA-DPH fluorescence 5.0 K562 5 min Sensitive cells Fig. 6. Effect of various cyclosporine concentrations on TMA-DPH fluorescence quenching by TMRM in Pgp over-expressing cells, K562 ⁄ ADR, or K562 sensitive cells, were incubated in the presence of 1 l M CCCP, 2 lM TMA-DPH and various concentrations of cyclo- sporine A (l M concentrations are indicated) for 15 min and then 25 l M TMRM was added at the time points marked by the arrows. TMA-DPH fluorescence was monitored continuously. The curves represent at least four separate experiments. The curves describing the second phase of TMA-DPH fluorescence quenching by TMRM in presence of CCCP were fitted as a first-order reaction with r 2 > 0.95, as described in Fig. 2. 1.5 1.0 0.20 0.15 0.10 0.5 0.05 Cell associated TMRM (nmol 10 –6 cells) Extent of fluorescence drop (fraction of total fluorescence) 0.1 1.0 10 Sensitive cells 0.0 C y clos p orine [µM] Fig. 7. Effect of cyclosporine A on FRET from TMA-DPH to TMRM and TMRM uptake in Pgp over-expressing cells. K562 ⁄ ADR cells were treated as described in Fig. 6. The amount of TMRM that was associated with cells during 30 min of incubation (circles) was determined quantitavely as described in the Experimental proce- dures. The extent of the second phase of fluorescence quenching by TMRM (squares) was determined by fitting the relevant curves from Fig. 6 to equations describing a first-order reaction, as described in Fig. 2. Cytoplasmic leaflet in drug uptake and resistance H. Katzir et al. 1240 FEBS Journal 277 (2010) 1234–1244 ª 2010 The Authors Journal compilation ª 2010 FEBS to the flip-flop, namely the release of dye into the cyto- plasm, can appear to accelerate the rate at which dye accumulation in the cytoplasmic leaflet of the plasma membrane reaches steady-state. Fast release of dye will result in shorter apparent half-life of the flip-flop of dye across the membrane. Surprisingly, and of interest, the active uptake of dye into the mitochondria prevented the accumulation of dye in the cytoplasmic leaflet of the plasma mem- brane. Only after a prolonged period, TMRM was accumulated in the cytoplasmic leaflet of the plasma membrane, presumably as a result of saturation of the mitochondria, leading to diminished uptake of TMRM into the mitochondria. Because there are no reports of direct contact of mitochondria with the plasma mem- brane, we have to assume that the mitochondria do not pump the dye directly from the plasma membrane but, instead, from the cytoplasm adjacent to the mem- brane. On the basis of this observation, it can be deduced that the limiting step in the release of dye from the plasma membrane is not the actual release from the membrane but, instead, the movement away from the plasma membrane into the cell. The cytoplasm next to the plasma membrane is unstirred and dense with proteins. Moreover, TMRM and anti- cancer drugs, such as anthracyclines, are positively charged and therefore bind to acidic groups in proteins and membranes. Thus, their movement into the cell can be envisaged as a series of binding and releasing events rather than simple diffusion. However, it should be stressed that although, in kinetic terms, the com- partment of the plasma membrane includes the cyto- plasm layer adjacent to the plasma membrane, the drop in TMA-DPH fluorescence reflects almost exclu- sively the dye present in the cytoplasmic leaflet. This is a result of the partition of the dye into the plasma membrane in preference to remaining soluble in the aqueous cytoplasm. The data of the FRET from TMA-DPH to TMRM suggest that Pgp extracts its substrates directly from the cytoplasmic leaflet of the plasma membrane. This is consistent with the suggestion made by Higgins and Gottesman [10] that Pgp acts as a flippase transporting its substrates from the cytoplasmic leaflet of the lipid bilayer to the outer leaflet or to the external medium. The data reported in the present study, and obtained in living cells, confirm the finding obtained in reconsti- tuted proteoliposomes [11,12] and isolated membranes [29,30] indicating that Pgp and a bacterial multidrug ABC-transporter extract their substrates from the cyto- plasmic leaflet of the membrane. By contrast to Pgp, over-expression of MRP1 does not affect the presence of TMRM in the cytoplasmic leaflet, but appears to pump it directly from the cyto- plasm. Over-expression of MRP1 did not alter the pat- tern of the drop in TMA-DPH fluorescence observed in the sensitive parent cell lines. MRP1 transports, on the one hand, organic anions, such as glutathione con- jugates, and, on the other hand, basic hydrophobic drugs, such as daunorubicin and vincristine [14]. It has been suggested that MRP1 has two binding sites: one with high affinity for hydrophobic ligands and the other with high affinity for glutathione [31,32]. The results obtained in the present study suggest that both sites are not located within the plasma membrane, but at its surface. The difference in the transport mecha- nisms between Pgp and MRP1, as revealed with FRET from TMA-DPH to TMRM is not the result of higher resistance levels in the Pgp cells. Inhibition of Pgp with various concentrations of cyclosporin A allowed for corresponding levels of TMRM accumulation, although in no case was the pattern of TMA-DPH fluorescence drop similar to that observed in sensitive cells, as is the case in MRP1 over-expressing cells. The finding that the cytoplasmic leaflet of the plasma membrane constitutes a kinetic compartment separate from the cell interior emphasizes the relevance of Pgp as a flippase to multidrug resistance. Drugs taken up into cells stay in the cytoplasmic leaflet of the plasma membrane for a few seconds before reaching the cell interior. Thus, Pgp that extracts its substrates from the cytoplasmic leaflet of the plasma membrane has the opportunity to remove drugs from the cells before they reach the cell interior. Pgp is adapted to prevent drugs from entering cells rather than to remove drugs already present in the cells. By contrast, transporters such as MRP1 extract their substrates directly from the cytoplasm and are more adapted to remove drugs already present inside the cells than to prevent the access of drugs into the cells. This phe- nomenon is especially relevant to drug transcellular transport and multidrug resistance in cell monolayers such as the blood–brain barrier and the epithelia lining the intestine and the nephrons. It has been shown that the tight junctions pose a barrier to the movement of lipids between the outer leaflets of the apical and baso- lateral domains of the plasma membrane [33]. By con- trast, they do not interfere with the movement of lipids and presumably drugs between the cytoplasmic leaflets of these domains [33]. Transcellular movement across cell monolayers of certain drugs and dyes, such as TMRM, is expected to occur mainly by rapid incorpo- ration into the outer leaflet of the plasma membrane, flip-flop across the lipid core of the membrane, lateral movement in the cytoplasmic leaflet of the plasma membrane from one membrane domain to the other, H. Katzir et al. Cytoplasmic leaflet in drug uptake and resistance FEBS Journal 277 (2010) 1234–1244 ª 2010 The Authors Journal compilation ª 2010 FEBS 1241 flip-flop again across the lipid core of the membrane and, finally, release from the plasma membrane into the aqueous phase. Thus, drugs and dyes with a high partition coefficient (membrane ⁄ aqueous phase) are expected to cross cell monolayers via lateral movement in the cytoplasmic leaflet of the plasma membrane, with little access into the cells’ cytoplasm. Indeed, kinetic analysis of drug transport across kidney conflu- ent cell monolayers suggests that hydrophobic drugs cross the monolayer by lateral transport in the cyto- plasmic leaflet of the plasma membrane rather than via the cytoplasm [34]. Experimental procedures K562, a human leukemia cell line established from a patient with chronic myelogeneous leukemia in blast trans- formation [35], was purchased from ATCC (Rockville, MD, USA) and maintained in RPMI medium (Biological Industries, Beit-Haemmek, Israel). The K562 Pgp-over- expressing subline was obtained by sequential exposure of cells to increasing concentrations of doxorubicin and was maintained in the presence of 0.5 lm doxorubicin. 2008 parental cells and their MRP1 over-expressing subline [36] were kindly provided by P. Borst (Netherlands Cancer Institute, Amsterdam, The Netherlands) and grown in RPMI-1640 (Sigma-Aldrich, Rehovot, Israel) The CIR [37], GLC4 cells and MRP1-over-expressing GLC4 ⁄ ADR cells [38] were cultured in RPMI 1640 either in the absence or presence of 1 lm doxorubicin. All media were supplemented with 10% fetal bovine serum, 100 IUÆmL )1 penicillin and 100 lgÆmL )1 streptomycin (Invitrogen, Rehovot, Israel) and the cells were grown at 37 °C under 5% CO 2 ⁄ humidified air. TMRM, Silicone oil AR200 and mineral oil were purchased from Sigma-Aldrich. Cellular ATP content was measured by the luciferin-luciferase assay [39]. Measurement of FRET from TMA-DPH to TMRM Cells were labeled with TMA-DPH (2 lm) by incubation at 37 °C. The fluorescence of TMA-DPH was monitored con- tinuously with the temperature maintained at 37 °C. In a typical experiment, 2 · 10 6 cells were incubated with stir- ring in 2 mL of medium composed of NaCl (132 mm), KCl (3.5 mm), CaCl 2 (1 mm), MgCl 2 (0.5 mm), glucose (10 mm) and Hepes-Tris buffer (20 mm, pH 7.4). A concentration of 2 lm TMA-DPH was added, leading to a rapid rise in TMA-DPH fluorescence. After further incubation for 10– 15 min, TMRM (25 lm) was added. The TMA-DPH fluo- rescence was monitored continuously in a Varian Cary Eclipse fluorescence spectrophotometer (Varian Inc., Palo Alto, CA, USA) using an excitation wavelength of 366 nm and an emission wavelength of 426 nm. Quantitative determination of the amount of TMRM associated with cells For determination of the amount of TMRM associated with cells, cells were incubated with the dye in the medium described above. Samples containing 4 · 10 5 cells in 0.4 mL of medium were withdrawn and placed in an Eppendorf-style microfuge above a 0.2 mL cushion consist- ing of 95 parts Silicone oil AR 200 (d 20 = 1.049) and five parts mineral oil (d 20 = 0.89). After centrifugation for 4 min at 13 200 g at room temperature, the oil cushion was washed three times with water by suction. Subse- quently, all of the upper phase, including part of the oil cushion, was removed, leaving a fraction of the oil above the cell pellets. The cell pellets were dissolved by the addi- tion of 0.1 mL of guanidine HCl (5 m) buffered with Hepes-Tris (50 mm, pH 7.4), centrifugation for 5 min and incubation for at least 1 h at room temperature. The dissolved samples were mixed thoroughly with 0.5 mL of water and centrifuged for 5 min. Samples (0.4 mL) were withdrawn from the pellets dissolved in the aqueous phase. 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December 2009) doi:10.1111/j.1742-4658.2009.07555.x The present study aimed to investigate the role played by the leaflets of the plasma membrane in the uptake of drugs into cells and in their extrusion by P-glycoprotein and multidrug resistance- associated. accu- mulation of dye in the cytoplasmic leaflet of the plasma membrane and release into the cell interior. The initial binding of dye to the cells, evident as the TMRM-mediated initial drop in TMA-DPH. quenching could be the result of TMRM transfer from the cell surface further into the outer leaflet of the membrane or flip-flop across the membrane and residence in the inner leaflet of the membrane.