©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reconstitution of Drug Transport by Purified P-glycoprotein (*)

Adam B. Shapiro (§) , Victor Ling (¶)

From the (1)Division of Molecular and Structural Biology, The Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 500 Sherbourne Street, Toronto, Ontario M4X 1K9, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

P-glycoprotein confers multidrug resistance upon cells in which it is highly expressed, reducing the effectiveness of numerous cytotoxic drugs, including many of those used for chemotherapy of cancer. Although P-glycoprotein is widely believed to function as an ATP-dependent drug efflux pump, the unusually broad substrate specificity of P-glycoprotein has engendered the proposal of other, less direct mechanisms. None of the hypothetical mechanisms has been definitively tested, however, in a purified system where other cellular components and processes are absent. We have used a fluorescent substrate of P-glycoprotein, Hoechst 33342, to measure transport activity in real-time of highly purified P-glycoprotein in a reconstituted liposome system in which the P-glycoprotein has a uniformly inside-out orientation. Using this system, we demonstrated MgATPdependent, chemosensitizer-inhibitable transport of Hoechst 33342. Transport was prevented by omission of Mg, by substitution of nonhydrolyzable adenylyl-,-imidodiphosphate for ATP, by inhibition of the ATPase activity of P-glycoprotein with vanadate and N-ethylmaleimide, and by the chemosensitizers verapamil and amiodarone. Measurements of intraliposomal pH during Hoechst 33342 transport detected no large pH changes in P-glycoprotein-containing liposomes. These results are inconsistent with a mechanism in which P-glycoprotein affects drug accumulation by directly altering intracellular pH. The Hoechst 33342 transport assay results are consistent with mechanisms in which P-glycoprotein alone is sufficient to transport drugs out of the membrane bilayer.


INTRODUCTION

P-glycoprotein is a 170-kDa mammalian plasma membrane ATPase belonging to a large superfamily of integral membrane transport proteins, the ATP Binding Cassette (ABC) superfamily or traffic ATPases (Higgins, 1992; Doige and Ames, 1993). P-glycoprotein is unusual, however, in having a very broad specificity for dissimilar lipophilic or amphiphilic substrates (Chin et al., 1993). There are three P-glycoprotein genes in rodents and two (classes I and III) in humans (Childs and Ling, 1994). Cells which express class I or class II, but not class III, P-glycoprotein in substantial amounts exhibit the multidrug resistance phenotype, in which they are resistant to a wide variety of structurally unrelated cytotoxic compounds (Gottesman and Pastan, 1993; Endicott and Ling, 1989). Multidrug resistance, including that mediated by P-glycoprotein, is a serious problem limiting the effectiveness of cancer chemotherapy. It is of clinical as well as biochemical interest, therefore, to develop an understanding of the mechanism by which P-glycoprotein confers multidrug resistance upon cells.

Reduced drug accumulation in P-glycoprotein-overexpressing cells is the result of an increased rate of drug efflux (Nielsen and Skovsgaard, 1992; Altenberg et al., 1994) and in some cases a decreased rate of drug influx (Stein et al., 1994; Homolya et al., 1993; Frezard and Garnier-Suillerot, 1991; Nielsen and Skovsgaard, 1992). Several mechanisms have been hypothesized to explain how P-glycoprotein performs this function. In the conventional model for a transporter, P-glycoprotein binds substrates in the cytoplasm and expels them directly into the extracellular medium, using ATP hydrolysis as an energy source. In the ``hydrophobic vacuum cleaner'' model (Gottesman and Pastan, 1993), P-glycoprotein interacts directly with substrates in the plasma membrane and pumps them out of the cell, accounting for both increased drug efflux and decreased drug influx rates (the substrates are expelled prior to entering the cytoplasm), as well as for the hydrophobic or amphiphilic nature of the known substrates. According to the ``flippase'' model (Higgins and Gottesman, 1992), P-glycoprotein encounters drugs in the inner leaflet of the plasma membrane and flips them to the outer leaflet from which they diffuse into the extracellular medium. In a completely different model, P-glycoprotein raises the intracellular pH (Roepe, 1992) and/or depolarizes the plasma membrane electrical potential (Roepe et al., 1993) of the cell by acting as a proton pump, a chloride channel, or by a less direct means, thereby reducing intracellular accumulation of weakly basic, cationic, lipophilic compounds or reducing pH-dependent binding of the compounds to their intracellular targets. This model avoids the necessity of P-glycoprotein having to recognize numerous, structurally diverse compounds.

For the most part, P-glycoprotein function has been studied with whole cell or plasma membrane systems in which numerous proteins and other cellular components and processes may complicate interpretation of the data. With studies of P-glycoprotein function, these confounding variables include differences between multidrug resistant and sensitive cells, such as differences in intracellular pH and plasma membrane electrical potential, membrane lipid composition, and energy metabolism. In addition, it has been proposed that drug transport by P-glycoprotein involves other cellular components such as glutathione S-transferase (West, 1990) and cytochrome P450 (reviewed in Gatmaitan and Arias, 1993). Distinguishing definitively between hypotheses would be greatly facilitated by the availability of a means of measuring drug transport by purified P-glycoprotein reconstituted into unilamellar lipid vesicles where the pH and membrane potential can be controlled or monitored, the lipid composition is the same for control and P-glycoprotein-containing liposomes, the only source of energy is ATP hydrolysis by P-glycoprotein, and systems for chemically modifying xenobiotic substances are not present. Sharom et al.(1993) reported transport of radiolabeled colchicine by partially purified and reconstituted P-glycoprotein. We described previously (Shapiro and Ling, 1994) a method for purifying class I hamster P-glycoprotein to a high degree and reconstituting it into a lipid milieu with retention of ATPase activity. In this paper, we describe fluorescence-based methods for continuous monitoring of transport of Hoechst 33342 by purified, reconstituted P-glycoprotein. This assay demonstrates for the first time that highly purified P-glycoprotein is capable of multidrug transport and has been used to distinguish between hypothetical mechanisms of P-glycoprotein function.


EXPERIMENTAL PROCEDURES

P-glycoprotein Purification

P-glycoprotein was purified as described (Shapiro and Ling, 1994) with a few modifications. The pre-elution wash of the immunoaffinity column with 10 ml of 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 2 mM CHAPS()(Calbiochem) was pooled with the subsequent two fractions. These latter fractions were doubled in volume to 10 ml each. The 30-ml pool was then concentrated to 600-800 µl. These modifications doubled the yield of P-glycoprotein from the immunoaffinity column without loss of purity.

Reconstitution

For most experiments, about 10 µg of purified P-glycoprotein in 50 µl of 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 2 mM CHAPS was mixed with 0.5 mg of sonicated, crude soybean L--phosphatidylcholine (Sigma), 17.5 mM octyl glucoside (Sigma), 50 mM Tricine-NaOH (pH 7.4), 125 mM NaCl, and 1 mM EDTA in 150 µl. The phosphatidylcholine pellets were suspended in water at 20 mg/ml with a Dounce homogenizer and sonicated for 15 min under nitrogen in a glass tube in a bath-type sonicator at room temperature. The mixture was incubated 45 min on ice, then applied to a 0.8 19-cm column of Sephadex G-50-80 equilibrated with 50 mM Tricine-NaOH (pH 7.4), 125 mM NaCl, and 1 mM EDTA. The column was eluted with the same buffer. Liposomes were collected from the void volume between about 3.4 and 4.1 ml of eluted volume. Control liposomes were prepared with the same composition but lacking P-glycoprotein.

The quantities of control and P-glycoprotein-containing liposomes were compared by their ultraviolet-visible absorbance spectra between 250 and 550 nm, a combination of absorbance and light scattering. The spectra for the two types of liposomes were parallel except for a small excess absorbance centered at about 280 nm in the P-glycoprotein-containing liposomes due to the absorbance of the protein.

For experiments in which pyranine (Molecular Probes) was included in the liposomes, except for the experiment shown in Fig. 1, 1 mM neutralized pyranine was included in the reconstitution mixture. In addition, 200 µl of 1 mM pyranine in buffer was preloaded onto the Sephadex columns prior to application of the reconstitution mixture to prevent loss of the pyranine from the reconstitution mixture by gel filtration. See the legend to Fig. 1for the details of the reconstitution performed for the experiments shown there.


Figure 1: Effect of octyl glucoside concentration in the reconstitution mixture on P-glycoprotein incorporation, ATPase activity, and internal volume of liposomes. Panel A, reconstitution mixtures of 200 µl containing 0.5 mg of sonicated, crude soybean L--phosphatidylcholine, 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5 mM CHAPS, 10 mM pyranine, about 2 µg of P-glycoprotein, and various concentrations of octyl glucoside were incubated for 45 min on ice, then applied to 0.8 19-cm columns of Sephadex G-50-80 equilibrated with 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM EDTA. Two-hundred µl of the same buffer containing 10 mM pyranine was drained into the top of each column just before application of the reconstitution mixtures. The liposomes were collected from the void volumes of the columns in about 800 µl. The amount of incorporated pyranine was measured by absorbance spectroscopy, correcting for light scattering by the liposomes. Portions of the liposomes were used for ATPase assays. Data are shown as percentages of the level of liposomes made with 14 mM octyl glucoside. Open and solidsymbols represent data from two separate experiments. Squares, pyranine incorporation. Circles, ATPase activity. Panel B, the remainder of the liposomes were subjected to SDS-polyacrylamide gel electrophoresis on an 8% gel as described in Shapiro and Ling (1994). The gel was stained with silver. Samples from the two experiments are shown separately, with octyl glucoside concentrations indicated.



Internal Volume of Liposomes

The internal volume of the liposome preparations was determined by incorporating pyranine or dithiothreitol-IAEDANS (IAEDANS (Molecular Probes) reacted with a 2-fold molar excess of dithiothreitol) into the liposome interior using the procedures described above, except that 10 mM pyranine or 19 mM dithiothreitol-IAEDANS was used instead of 1 mM pyranine. The pyranine and dithiothreitol-IAEDANS concentrations of the resulting liposome suspensions were determined by absorbance spectroscopy, using extinction coefficients of 21,000 M-cm at 454 nm for pyranine and 57,000 M-cm at 334 nm for IAEDANS, with correction for light scattering due to the liposomes. The internal volume of 10 mM pyranine or 19 mM dithiothreitol-IAEDANS required to yield the measured concentration in the measured volume of liposome suspension was calculated. The internal volume of liposomes prepared as described was 1.17 ± 0.09 µl (n = 5). About 92% of the included pyranine was unquenched by 10 mM of externally added DPX (Molecular Probes), a potent quencher of pyranine fluorescence, demonstrating that nearly all the pyranine was localized internally and that the liposomes were tightly sealed.

Measurement of Hoechst 33342 Transport

A 300-µl aliquot of control or P-glycoprotein-containing liposomes having the same amount of liposomes was placed in a quartz fluorescence cuvette with a 2-mm path length for excitation and a 10-mm path length for emission. Fluorescence intensity was measured with an Aminco-Bowman Series 2 Luminescence Spectrometer at 1-s intervals using 355 nm excitation and 457 nm emission. The excitation and emission bandwidths were 0.5 and 16 nm, respectively. The narrow excitation bandwidth was used to eliminate photobleaching caused by strong illumination. After 100 s of data acquisition, 3 µl of 2.5 mM Hoechst 33342 in water was added. Hoechst 33342 binding to the liposomes was essentially complete within 30 s, as judged by the fluorescence increase. About 90% of the Hoechst 33342 added became bound to the lipid. This was determined by pelleting the liposomes by ultracentrifugation and measuring the Hoechst 33342 concentration in the supernatant spectrophotometrically. After a further 900 s, 16 µl of 50 mM Tricine-NaOH (pH 7.4), 60 mM MgCl, 30 mM NaATP, and 40 mM dithiothreitol was added to initiate transport. The addition did not alter the external chloride concentration. Dithiothreitol was included because it slightly stimulates ATP hydrolysis by P-glycoprotein (Shapiro and Ling, 1994). Other additions were made as described in the figure legends. Mixing was performed manually, which required 30 s, during which data acquisition was halted. Transport was monitored for 2400 s.

For all transport measurements, fluorescence intensity versus time traces were normalized to an intensity value of 1 just prior to the addition of MgATP. To show the effect of P-glycoprotein, the normalized trace for control liposomes was often subtracted from the normalized trace for P-glycoprotein-containing liposomes. The signal noise in many measurements was very large due to the high photomultiplier gain needed because of the low intensity of the exciting light used to avoid photobleaching. To reduce this noise, time points were often averaged over a moving 15-s window. When comparing subtracted traces, the vertical positions of some traces were shifted for clarity. Occasional large, temporary, vertical deviations from the trace base line are instrument artifacts.

Measurements of Intraliposomal pH

The pH-sensitive fluorophore pyranine was used to measure the intraliposomal pH (Kano and Fendler, 1978; Young, 1992). Fluorescence excitation spectra of 10 µM pyranine in 50 mM Tricine-NaOH, 125 mM NaCl, and 1 mM EDTA buffers of various pH at 37 °C were measured to establish a pH calibration curve for pyranine. The emission wavelength was 515.2 nm. The excitation and emission bandwidths were both 4 nm. The ratio of fluorescence at the pH-sensitive excitation wavelength, 452.6 nm, to the fluorescence at the pH-insensitive excitation wavelength, 419.0 nm, was plotted against the pH of the buffer, measured at 37 °C after the spectra were collected. The resulting calibration curve (Fig. 8A) was linear over the pH range of 6.5-7.5.


Figure 8: Measurements of pH of liposome suspensions and interiors using pyranine fluorescence. Details of the experiment are given under ``Experimental Procedures.'' Panel A, calibration curve for the 452.6/419 nm fluorescence excitation ratio as a function of pH. Panels B and C, each panel shows the 515.2-nm pyranine fluorescence at the pH-insensitive excitation wavelength of 419.0 nm and the pH-sensitive excitation wavelength of 452.6 nm, as well as the ratio of 452.6 to 419 nm excitation, which is directly proportional to the pH. The traces are labeled for control (C) and P-glycoprotein-containing (P) liposomes. Times of addition of H0, Hoechst 33342 (H), and ATP are indicated. Panel B, measurements for liposomes to which water was added instead of Hoechst 33342. Panel C, measurements for liposomes with 10 mM external DPX to which Hoechst 33342 was added.



For measurements of intraliposomal pH during transport of Hoechst 33342, liposomes containing 1 mM pyranine were used. Pyranine fluorescence was monitored at 515.2 nm while the excitation wavelength switched between 452.6 and 419.0 nm. One ratio measurement was made every 5 s, and the corresponding pH values were obtained from the calibration curve.

Where DPX was used, it was added to 10 mM to the liposome suspension from a concentrated stock solution in 50 mM Tricine-NaOH (pH 7.4), 125 mM NaCl, and 1 mM EDTA.

P-glycoprotein ATPase Activity Measurements

The ATPase activity of reconstituted P-glycoprotein was measured as described in Shapiro and Ling(1994).


RESULTS

P-glycoprotein Reconstitution

Crude soybean L--phosphatidylcholine was chosen as the source of lipid for reconstituting P-glycoprotein for transport measurements. The advantages of this lipid are its low cost, ease of handling, and its formation of tightly sealed unilamellar vesicles. For 0.5 mg of lipid and 10 µg of P-glycoprotein in 200 µl of the reconstitution mixture, the concentration of the detergent octyl glucoside was varied in order to optimize the reconstitution for retention of ATPase activity, assuming that ATP hydrolysis is coupled to transport, and maximal intraliposomal volume, so that there will be somewhere for the transported substrate to go. The internal volume, measured by the amount of pyranine incorporated, was maximal when 17.5 mM octyl glucoside was used (Fig. 1A). (The reconstitution mixture also contained 0.5 mM CHAPS, which causes a small amount of reconstitution in the absence of octyl glucoside (Shapiro and Ling, 1994).) The ATPase activity of the resulting P-glycoprotein-containing liposomes, however, was maximized by minimizing the octyl glucoside concentration, but no pyranine was incorporated into the resulting liposomes. Nearly half the ATPase activity was lost when 17.5 mM octyl glucoside was used. This appears to reflect a reduction in the amount of P-glycoprotein incorporated into the liposomes (Fig. 1B). In contrast, higher detergent concentrations improved the incorporation of P-glycoprotein but resulted in even greater losses of ATPase activity, suggesting either that the protein was damaged by high octyl glucoside concentrations or that the catalytic sites were primarily inside the liposomes. For transport experiments, 17.5 mM octyl glucoside was used in the reconstitution mixture to maximize the internal volume of the liposomes while maintaining acceptable ATPase activity. ATP was added only externally.

Orientation of Reconstituted P-glycoprotein

We attempted to determine the orientation of P-glycoprotein in the liposome membranes using the CHAPS permeabilization method of Sharom et al. (1993), who found that a 2-fold increase in the ATPase activity of reconstituted, partially purified P-glycoprotein occurred in the presence of 2 mM CHAPS to permeabilize the liposomes to ATP. The interpretation of this result was that 50% of the P-glycoprotein was right-side-out, i.e. with its catalytic sites inside the liposomes. Our liposomes were also permeabilized by 2 mM CHAPS: 94% of the fluorescence of 10 mM entrapped pyranine was quenched by the addition of 2 mM CHAPS in the presence of 10 mM external DPX, an impermeant quencher of pyranine fluorescence (data not shown). In contrast to the results of Sharom et al.(1993), however, the ATPase activity of our reconstituted P-glycoprotein was not increased by the addition of 2 mM CHAPS. Instead, the ATPase activity was slightly decreased (Fig. 2A). This result is consistent with the P-glycoprotein having a uniformly inside-out orientation.


Figure 2: Orientation of reconstituted P-glycoprotein. Panel A, enhancement of ATPase activity of P-glycoprotein reconstituted with 17.5 mM octyl glucoside by CHAPS. The ATPase activity of the P-glycoprotein-containing liposomes was measured in the presence of 0-10 mM CHAPS. ATPase activity is expressed as nanomoles of inorganic phosphate released/hour/100 µl of liposome suspension. Panel B, inhibition of the ATPase activity of reconstituted P-glycoprotein by SDM and subsequent enhancement by CHAPS. P-glycoprotein-containing liposomes reconstituted with 17.5 mM octyl glucoside were incubated for 15 min at 37 °C with or without 100 µM SDM, then passed through 3-ml Sephadex G-50-80 centrifuge columns (Penefsky, 1977) to remove excess SDM. ATPase activity was then measured in the presence or absence of 8 mM CHAPS. Bar heights represent the means of duplicate samples, expressed as nanomoles of inorganic phosphate released/hour/150 µl of liposome suspension.



Higher CHAPS concentrations increased the ATPase activity, with a 6-fold increase occurring at 6-10 mM CHAPS (Fig. 2A). This result was surprising because concentrations of CHAPS in excess of 2 mM were previously found to inhibit the ATPase activity of P-glycoprotein in detergent solution (Shapiro and Ling, 1994). The specific ATPase activity of reconstituted P-glycoprotein in the presence of 8 mM CHAPS is much higher (at least 1.3 µmol/min-mg assuming a recovery of 5 µg of P-glycoprotein in the liposomes) than the specific activity of the purified P-glycoprotein prior to reconstitution (0.3 µmol/min-mg; Shapiro and Ling, 1994). CHAPS may stimulate the ATPase activity of reconstituted P-glycoprotein, much as chemosensitizers like verapamil do (Shapiro and Ling, 1994).

The ATPase activity of P-glycoprotein is inhibited by N-ethylmaleimide (Shapiro and Ling, 1994) and other reagents that react with cysteine residues (Sharom et al., 1992). Inhibition by N-ethylmaleimide can be prevented by inclusion of MgATP (Al-Shawi et al., 1994), which suggests that cysteine residues in or near the catalytic sites are the targets for the inhibitors. To confirm the orientation of reconstituted P-glycoprotein, we treated it with 100 µM 4-acetamido-4`-maleimidylstilbene-2,2`-disulfonic acid (SDM); Molecular Probes), a membrane-impermeant maleimide, to inhibit external catalytic sites. SDM did not cause leakage of the liposomes, according to the pyranine quenching assay mentioned above. After removal of the excess reagent, the liposomes were permeabilized with 8 mM CHAPS. If there were internal catalytic sites, the addition of CHAPS should have elicited catalytic activity from them. Instead, there was virtually no ATPase activity (Fig. 2B). The above results are completely consistent with the conclusion that the reconstituted P-glycoprotein was uniformly in the inside-out orientation. Furthermore, these results indicate that the increased ATPase activity in the presence of CHAPS is due to stimulation of the ATPase activity of P-glycoprotein rather than exposure of internal catalytic sites.

Hoechst 33342 Transport

The lipophilic nature of the substrates is a serious problem in designing an assay for transport by P-glycoprotein reconstituted into liposomes because of the high lipid-to-protein ratio, typically 50:1 by mass, as compared to about 1:1 for plasma membranes. The substrates concentrate in the lipid phase, creating a very high background of bound substrate. Thus the substrate transported into the liposomes is difficult to detect above the background of lipid-bound substrate. To overcome this problem we used fluorescence techniques to continuously monitor transport. By collecting many data points over time, we were able to detect small changes in fluorescence intensity due to transport against a high background fluorescence. In addition, continuous monitoring allows an entire time course to be collected from a single specimen.

Hoechst 33342 was previously shown to be excluded from multidrug-resistant cells expressing P-glycoprotein (Lalande et al., 1981; Chen et al., 1993) and is apparently a substrate for P-glycoprotein. Hoechst 33342 may have potential use in chemotherapy because it inhibits topoisomerase I (Chen et al., 1993). Hoechst 33342 competes with the photoaffinity reagent and P-glycoprotein substrate azidopine for binding to P-glycoprotein (Fig. 3), demonstrating that Hoechst 33342 interacts directly with P-glycoprotein. That the inhibition of photolabeling was not due simply to absorption of the ultraviolet light by Hoechst 33342 is demonstrated by the presence of a second photolabeled band at low molecular weight, the labeling intensity of which is unchanged by Hoechst 33342. Hoechst 33342 at 25 µM has no effect on the ATPase activity of P-glycoprotein-containing liposomes (data not shown). Other known P-glycoprotein substrates such as daunorubicin and colchicine also have little or no effect on ATPase activity (Shapiro and Ling, 1994), whereas chemosensitizers such as verapamil, amiodarone, and trifluoperazine, and some substrates, such as vinblastine, cause substantial enhancements of the ATPase activity.


Figure 3: Inhibition of [H]azidopine photolabeling of P-glycoprotein in plasma membranes by Hoechst 33342. Sucrose gradient-purified plasma membranes from CHB30 cells (Shapiro and Ling, 1994) were incubated for 1 h at room temperature in darkness with 0.2 µM [H]azidopine (Amersham, specific activity = 52 Ci/mmol) and 0-100 µM Hoechst 33342, exposed to ultraviolet light for 10 min on ice in a Stratalinker (Stratagene) (Georges et al., 1991), and electrophoresed on an 8% SDS-polyacrylamide gel. The gel was fixed in 50% methanol and 10% acetic acid, soaked in Amplify scintillant (Amersham), dried, and fluorographed for 7 days at -70 °C against Kodak X-OMAT XAR5 film.



Hoechst 33342 is widely used for its property of exhibiting vastly increased fluorescence upon binding to DNA. We found that Hoechst 33342 fluorescence was also greatly increased by binding to liposomes (Fig. 4). We made use of this property in designing an assay for transport by purified, reconstituted P-glycoprotein. Fig. 4shows the degree of fluorescence enhancement of Hoechst 33342 as a function of the concentration of degraded herring sperm DNA (Sigma) or crude soybean phosphatidylcholine liposomes. The effect saturated at about 0.5 mg/ml lipid and a fluorescence enhancement of about 750-fold, about half the fluorescence enhancement achieved with DNA. A molecule of Hoechst 33342 experiences essentially a 100% fluorescence decrease upon going from the lipid to the aqueous phase. Therefore, if P-glycoprotein transports Hoechst 33342 from the liposome membrane to the interior aqueous space of liposomes, the Hoechst 33342 fluorescence should decrease. The fractional decrease in fluorescence intensity will be equal to the fraction of lipid-bound Hoechst 33342 transported.


Figure 4: Fluorescence enhancement of Hoechst 33342 by DNA and lipid. Hoechst 33342 (1 µM) was added to 0-5 mg/ml solutions of neutralized, degraded herring sperm DNA or crude, sonicated soybean L--phosphatidylcholine in 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM EDTA. Fluorescence was monitored with a Perkin-Elmer LS-3 fluorescence spectrometer at ambient temperature with excitation and emission wavelengths of 363 and 440 nm, respectively. Signals due to the DNA or lipid alone at each concentration were subtracted.



As can be seen in Fig. 5, after addition of ATP the Hoechst 33342 fluorescence of P-glycoprotein-containing liposomes decreased with time with respect to the control liposomes as the Hoechst 33342 was removed from the membrane by P-glycoprotein. The large, variable, instantaneous fluorescence drop occurring at 1000 s was due to the addition of the ATP-containing solution. A 5% drop was caused by dilution. Additional loss of fluorescence may have been caused by binding of Hoechst 33342 to ATP, thereby reducing the amount of lipid-bound Hoechst 33342. Finally, a slight acidification due to addition and dilution of the concentrated MgATP stock solution contributed to the instantaneous decrease in Hoechst 33342 fluorescence. The typical size of this drop was about 15%, but there was variation between experiments between about 5 and 20%. The reason for this variation is unknown. There was no consistent difference between control and P-glycoprotein-containing liposomes in the size of the instantaneous fluorescence drop.


Figure 5: Hoechst 33342 transport into liposomes. Details of the experiment are given under ``Experimental Procedures.'' Times of addition of Hoechst 33342 (H) and ATP are indicated. Normalized traces for control (C) and P-glycoprotein-containing (P) liposomes are shown. Inset, difference between P-glycoprotein-containing and control liposomes from the point at which MgATP was added.



The extent of the slow, usually linear fluorescence decrease due to Hoechst 33342 transport by P-glycoprotein following addition of MgATP was typically about 5% in 40 min, varying between 3 and 7%. This variation was probably due to variations in the amount of P-glycoprotein reconstituted as well as in the activity of the P-glycoprotein from different preparations. In some cases, it was necessary to begin the reconstitution with 20 µg of P-glycoprotein rather than 10 µg to achieve the 5% fluorescence decrease. This signal represents a net rate of transport of about 9 pmol/min, or about 6 nmol/min/mg of P-glycoprotein, assuming a typical 50% recovery of P-glycoprotein in the reconstitution. The turnover number was therefore 1 molecule of Hoechst 33342 transported/min/molecule of P-glycoprotein. This slow rate represents the difference between the rate of Hoechst 33342 transport out of the membrane and the rate of rebinding of transported Hoechst 33342 to the membrane. The actual rate of transport is likely to be much faster than the measured rate because the rebinding rate is high; about 90% of 25 µM added Hoechst 33342 binds to the liposomes within 30 s (see ``Experimental Procedures'' and Fig. 5). The rate of ATP hydrolysis is about 50 times faster than the apparent rate of transport.

By incorporating DNA into the aqueous interior of the liposomes, an increasing fluorescence due to transport could be seen,()demonstrating that the Hoechst 33342 was transported into the liposome interior.

As discussed below (Fig. 8C), 25 µM Hoechst 33342 can cause liposome aggregation and fusion. Fusion causes transient leakage of the liposome membranes. There does not appear to be any difference in the rate of fusion between control and P-glycoprotein-containing liposomes. Liposome aggregation and fusion do not affect the transport measurement, which detects removal of Hoechst 33342 from the lipid phase, not concentration of the substrate in the aqueous phase.

Requirement for ATP Hydrolysis for Hoechst 33342 Transport

The purified transport system allows us to determine whether transport is coupled to the ATPase activity of P-glycoprotein. Our previous studies of the ATPase activity of purified, reconstituted P-glycoprotein, as well as studies of the ATPase activity of P-glycoprotein done by others (Shapiro and Ling, 1994 and references therein), demonstrated that the ATPase activity of P-glycoprotein requires divalent cations such as Mg, and is inhibited by low micromolar concentrations of sodium orthovanadate and N-ethylmaleimide. Fig. 6A shows that transport of Hoechst 33342 by P-glycoprotein was inhibited by omission of Mg and by 50 µM sodium orthovanadate (NaVO), a concentration which completely inhibits P-glycoprotein ATPase activity. Fig. 6B shows that Hoechst 33342 transport was inhibited by reaction of the reconstituted P-glycoprotein with 50 µMN-ethylmaleimide. Inhibition was partially prevented by inclusion of 3 mM MgATP during the reaction with N-ethylmaleimide. Al-Shawi and Senior(1993) showed that MgATP protects P-glycoprotein ATPase activity against inhibition by N-ethylmaleimide. Furthermore, transport of Hoechst 33342 by P-glycoprotein was not supported by the nonhydrolyzable ATP analog adenylyl-,-imidodiphosphate (AMP-PNP) (Fig. 6C). These results demonstrate that Hoechst 33342 transport by P-glycoprotein requires ATP hydrolysis.


Figure 6: Dependence of Hoechst 33342 transport into liposomes on ATPase activity of P-glycoprotein. Details of the experiment are given under ``Experimental Procedures.'' Panel A, traces for control experiment (MgATP, bottom), experiment in which Mg was omitted (top), and experiment in which 50 µM NaVO was included with MgATP (middle). The traces represent the differences between normalized traces for P-glycoprotein-containing and control liposomes from the point at which ATP or MgATP was added. Panel B, inhibition of Hoechst 33342 transport by N-ethylmaleimide. P-glycoprotein-containing and control liposomes were divided into three equal portions. The liposomes were treated with either 50 µMN-ethylmaleimide, or 3 mM NaATP, 6 mM MgCl, and 50 µMN-ethylmaleimide, or were untreated for 10 min at 37 °C. The liposomes were then passed through 3-ml Sephadex G-50-80 centrifuge columns (Penefsky, 1977) equilibrated with 50 mM Tricine-NaOH (pH 7.4), 125 mM NaCl, and 1 mM EDTA to remove unreacted N-ethylmaleimide, MgCl, and ATP. The liposomes were used for transport measurements as described under ``Experimental Procedures.'' Bottom trace, untreated liposomes. Middle trace, liposomes treated with N-ethylmaleimide, MgCl and ATP. Top trace, liposomes treated with only N-ethylmaleimide. The traces show the differences between P-glycoprotein-containing and control liposomes from the point at which MgATP was added. Panel C, normalized traces for P-glycoprotein-containing liposomes from the point at which either MgATP (lower trace) or MgAMP-PNP (upper trace) were added. The vertical positions of some of the traces were shifted for clarity.



Inhibition of Hoechst 33342 Transport by Chemosensitizers

Numerous compounds act as multidrug resistance modulators, or chemosensitizers, restoring drug sensitivity to multidrug-resistant cells. The purified transport system allows us to determine whether chemosensitization is due to a direct effect on P-glycoprotein. In our assay, neither verapamil nor amiodarone appeared to cause substantial inhibition of Hoechst 33342 transport at 25 µM Hoechst 33342 (data not shown). Inhibition by verapamil and amiodarone was apparent, however, when the Hoechst 33342 concentration was reduced to 2 µM (Fig. 7). It may be that the chemosensitizers do not compete effectively with Hoechst 33342 for binding to P-glycoprotein under the conditions of high lipid concentration in the transport assay.


Figure 7: Inhibition of Hoechst 33342 transport by verapamil and amiodarone. Details of the experiment are given under ``Experimental Procedures,'' except that only 2 µM Hoechst 33342 was used. The normalized traces for the P-glycoprotein-containing liposomes are shown from the point at which MgATP was added for untreated liposomes (bottom), liposomes treated with 33 µM amiodarone (middle), and liposomes treated with 33 µM verapamil (top). Verapamil was dissolved in water. Amiodarone was dissolved in ethanol, with the final ethanol concentration in the assay being 0.3%, which has no effect on transport. Verapamil and amiodarone were added at the same time as Hoechst 33342. The vertical positions of the traces were shifted for clarity.



Measurements of pH during Hoechst 33342 Transport

Because of the hypothesis that P-glycoprotein may transport drugs indirectly by raising intracellular pH (Roepe, 1992), we monitored the intraliposomal pH using the pH-sensitive, hydrophilic fluorophore pyranine entrapped in the liposomes. In the absence of Hoechst 33342, the pH inside the control liposomes was stable (Fig. 8B). After the addition of MgATP, which caused a slight acidification of the external pH, the internal pH of the control and P-glycoprotein-containing liposomes gradually decreased as the small proton gradient dissipated as protons leaked into the liposomes. Of possible significance was a small but reproducible pH rise inside P-glycoprotein-containing liposomes with no Hoechst 33342, which was not observed in control liposomes. The reason for this is unknown. It could not have been an energy-dependent process because it occurred prior to the addition of MgATP. There were no large differences in intraliposomal pH between control and P-glycoprotein-containing liposomes in the absence of Hoechst 33342 following ATP addition, which demonstrates that P-glycoprotein does not act as an ATP-dependent proton translocator.

During the course of intraliposomal pH measurements in the presence of Hoechst 33342, we discovered that, as a function of time, Hoechst 33342 caused the liposomes to aggregate and leak, probably due to liposome fusion. Liposome leakage allowed pyranine to escape from the liposomes so that the pH measurement did not represent only the internal pH. By adding the non-lipid permeant pyranine quencher DPX to the outside of the liposomes, we restricted the pH measurement at each time point to those liposomes which had not yet undergone fusion and leakage (Fig. 8C). At the 10 mM concentration of DPX used, pyranine fluorescence was completely quenched if the liposomes had released their pyranine or allowed entrance of DPX. In the absence of Hoechst 33342, leakage of the liposomes does not occur, and the results look like those of Fig. 8B (). The large decrease in pyranine fluorescence at both excitation wavelengths after addition of Hoechst 33342 shows that, over time, most of the liposomes eventually become leaky. The ratiometric method of determining the pH is insensitive to the fluorescence intensity, however. In the experiment shown in Fig. 8C, the internal pH of the P-glycoprotein-containing liposomes was slightly lower than that of the control liposomes (). This difference was not consistently observed, however; in another experiment (not shown), the P-glycoprotein-containing liposomes had a slightly higher internal pH than the control liposomes.

After addition of MgATP, there was no significant change in the difference in internal pH between control and P-glycoprotein-containing liposomes (Fig. 8C and ). If P-glycoprotein were acting as an ATP-dependent proton pump, but were only present in a fraction of the liposomes, the acidification might be missed. The pH measurement is sensitive to a change of about 0.01 pH units, although the measurement variation is somewhat greater, at most 0.1 pH units. Although we do not know what fraction of the liposomes contain one or more active P-glycoprotein molecules, if we conservatively estimate that only 20% of the liposomes contained proton-pumping P-glycoprotein, which created an internal acidification of one pH unit from 7.0 to 6.0, then the measured internal pH would appear to be 6.55 for the entire liposome population. No such large changes specific to P-glycoprotein-containing liposomes occurred after addition of MgATP in our experiments (). Furthermore, the protonophore carbonyl cyanide m-chlorophenylhydrazone had no effect on the internal pH (). It is therefore quite unlikely that P-glycoprotein transports Hoechst 33342 indirectly by creating a pH gradient.


DISCUSSION

By using purified, reconstituted P-glycoprotein, we have demonstrated for the first time that P-glycoprotein by itself is capable of direct drug transport. The reconstituted system behaves qualitatively like P-glycoprotein in vivo with respect to the requirement for ATP hydrolysis and inhibition by chemosensitizers. Our system makes it possible to test definitively hypotheses about P-glycoprotein function (such as the ``hydrophobic vacuum cleaner'' hypothesis (Gottesman and Pastan, 1993)). A fluorescence method was devised specifically to distinguish movement of the transported substrate between lipid and aqueous phases, which is not possible with assays that measure the uptake of radioactive substrates. The results of the Hoechst 33342 transport assay support the concept that transport occurs from the membrane because the assay measures loss of Hoechst 33342 from the lipid phase. An essential criterion for an active transporter is that it be able to pump its substrate against a concentration gradient. Multidrug-resistant cells typically maintain intracellular drug concentrations well below the extracellular concentrations. Sharom et al.(1993) reported accumulation of colchicine against a 5.6-fold concentration gradient by reconstituted, partially purified P-glycoprotein. Similarly, Ruetz and Gros(1994) reported accumulation of colchicine by P-glycoprotein-containing yeast secretory vesicles to 7-fold higher internal than external concentration. Schlemmer and Sirotnak(1994) demonstrated concentrative uptake of vinblastine by P-glycoprotein-containing inside-out plasma membrane vesicles. As stated above, the leakage of liposomes caused by Hoechst 33342-induced aggregation in our system makes it difficult to determine the amount by which P-glycoprotein concentrates Hoechst 33342 inside the liposomes. Another consideration is that the majority of the Hoechst 33342 is bound to the lipid membranes. The concentration of Hoechst 33342 in the external aqueous phase is therefore much lower than the 25 µM average concentration, whereas the concentration in the lipid phase, which has a very small volume compared to the external aqueous phase, is very much higher. This situation makes discussion of substrate gradients problematic under conditions where the concentration of lipids is high, as in proteoliposomes. In our assay, P-glycoprotein was actually transporting Hoechst 33342 down its concentration gradient, but this required energy because of the strong partitioning of the dye into the lipid phase.

The lack of enhanced acidification of the liposome interior by P-glycoprotein enables us to conclude that P-glycoprotein is not directly responsible for the increased intracellular pH of some multidrug-resistant cell lines, and that this alkalinization is not necessary to account for P-glycoprotein-mediated decreases in intracellular drug accumulation. Similarly, our measurements allow us to rule out alteration of the membrane potential as the means by which P-glycoprotein affects drug accumulation. In our experiments, there was no membrane potential set up across the liposome membranes initially, nor was there at any time a chloride gradient by which P-glycoprotein could create a membrane potential by acting as a chloride channel, as has been proposed (Valverde et al., 1992; Gill et al., 1992). The lack of acidification of the interior of P-glycoprotein-containing liposomes relative to the interior of control liposomes rules out the generation of a membrane potential by proton pumping by P-glycoprotein. Another demonstration that the mechanism of action of P-glycoprotein is not to alter the plasma membrane electrical potential is the observation by Ruetz and Gros(1994) that P-glycoprotein-containing yeast secretory vesicles could take up vinblastine against an unfavorable membrane potential.

The apparent rate of Hoechst 33342 transport by P-glycoprotein in proteoliposomes was very slow. ATP-dependent transporters usually exhibit tight coupling of ATP hydrolysis to substrate transport. P-glycoprotein, however, has a high basal rate of ATP hydrolysis (Shapiro and Ling, and references therein). In our transport assay, P-glycoprotein hydrolyzed about 50 ATP molecules/molecule of Hoechst 33342 transported. This poor coupling represents excess ATP hydrolysis due to both the basal rate of ATP hydrolysis and futile cycling of the Hoechst 33342 between lipid and aqueous phases. It will be a challenge for the future to devise a method to prevent rebinding of the substrate to the lipid in order to determine the true rate of Hoechst 33342 transport by P-glycoprotein.

Many questions remain regarding the function of P-glycoprotein. If P-glycoprotein encounters its substrates in the membrane, as our results and those of others suggest (Gottesman and Pastan, 1993), then in which leaflet of the membrane is the substrate encountered, and how is the energy released by ATP hydrolysis transduced into vectorial substrate movement? Why do some chemosensitizers increase the rate of ATP hydrolysis by P-glycoprotein while inhibiting drug transport? Why do some substrates have no effect on the basal ATPase activity of P-glycoprotein, and why does P-glycoprotein have ATPase activity in the absence of any substrate? How can P-glycoprotein recognize and actively transport numerous chemically distinct compounds? Does P-glycoprotein function as a monomer or an oligomer (see Poruchynsky and Ling, 1994)? What role does post-translational modification, such as phosphorylation and glycosylation, play in drug transport? By studying purified P-glycoprotein reconstituted into liposomes, we have begun to develop a system which is enabling us to solve many of these questions about P-glycoprotein.

  
Table: Summary of pH measurements inside liposomes

The two pH measurements for each cell in the table represent the initial pH measured just after the addition and the final pH measured for that phase of the experiment. Details of the experiment are given under ``Experimental Procedures.'' Measurements of pH were made with 10 mM external DPX when Hoechst 33342 was present, and without DPX in the absence of Hoechst 33342. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added to 2.5 µM.



FOOTNOTES

*
This work was supported in part by a grant from the National Cancer Institute of Canada and United States Public Health Service Grant CA 37130 from the National Institutes of Health (to V. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Medical Research Council of Canada postdoctoral fellowship.

To whom correspondence should be addressed. Tel.: 416-924-0671 (Ext. 4988); Fax: 416-323-3858.

The abbreviations used are: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate; AMP-PNP, adenylyl-,-imidodiphosphate; DPX, p-xylene-bispyridinium bromide; IAEDANS, 5-((((iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid; octyl glucoside, n-octyl--D-glucopyranoside; pyranine, 8-hydroxypyrene-1,3,6-trisulfonic acid; SDM, 4-acetamido-4`-maleimidylstilbene-2,2`-disulfonic acid.

A. B. Shapiro and V. Ling, unpublished results.


ACKNOWLEDGEMENTS

We are deeply grateful to Dr. Ian Tanock for allowing us extensive use of his spectrofluorometers and to Dr. David Hedley for the suggestion to try Hoechst 33342 in the transport assay. We are also grateful to Francis Tan for many valuable scientific discussions, to Monika Duthie and Farida Sarangi for technical support, and to Stephanie Sulpizi for large scale cell culture.


REFERENCES
  1. Al-Shawi, M. K., and Senior, A. E. (1993) J. Biol. Chem.268, 4197-4206 [Abstract/Free Full Text]
  2. Al-Shawi, M. K., Urbatsch, I. L., and Senior, A. E. (1994) J. Biol. Chem.269, 8986-8992 [Abstract/Free Full Text]
  3. Altenberg, G. A., Young, G., Horton, J. K., Glass, D., Belli, J. A., and Reuss, L. (1993) Proc. Natl. Acad. Sci. U. S. A.90, 9735-9738 [Abstract]
  4. Altenberg, G. A., Vanoye, C. G., Horton, J. K., and Reuss, L. (1994) Proc. Natl. Acad. Sci. U. S. A.91, 4654-4657 [Abstract]
  5. Chen, A. Y., Yu, C., Bodley, A., Peng, L. F., and Liu, L. F. (1993) Cancer Res.53, 1332-1337 [Abstract]
  6. Childs, S., and Ling, V. (1994) in Important Advances in Oncology, (DeVita, V. T., Hellman, S., and Rosenberg, S. A., eds) pp. 21-36, J. B. Lippincott Company, Philadelphia, PA
  7. Chin, K.-V., Pastan, I., and Gottesman, M. M. (1993) Adv. Cancer Res.60,157-180 [Medline] [Order article via Infotrieve]
  8. Doige, C. A., and Ames, G. F.-L. (1993) Annu. Rev. Microbiol.47, 291-319 [CrossRef][Medline] [Order article via Infotrieve]
  9. Doige, C. A., Yu, X., and Sharom, F. J. (1992) Biochim. Biophys. Acta1109, 149-160 [Medline] [Order article via Infotrieve]
  10. Endicott, J. A., and Ling, V. (1989) Annu. Rev. Biochem.58, 137-171 [CrossRef][Medline] [Order article via Infotrieve]
  11. Frézard, F., and Garnier-Suillerot, A. (1991) Eur. J. Biochem.196, 483-491 [Abstract]
  12. Gatmaitan, Z. C., and Arias, I. M. (1993) Adv. Pharmacol.24, 77-97 [Medline] [Order article via Infotrieve]
  13. Georges, E., Zhang, J.-T., and Ling, V. (1991) J. Cell. Physiol.148, 479-484 [Medline] [Order article via Infotrieve]
  14. Gill, D. R., Hyde, S., Higgins, C. F., Valverde, M. A., Mintenig, G. M., and Seplveda, F. V. (1992) Cell71, 23-32 [Medline] [Order article via Infotrieve]
  15. Gottesman, M. M., and Pastan, I. (1993) Annu. Rev. Biochem.62, 385-427 [CrossRef][Medline] [Order article via Infotrieve]
  16. Higgins, C. F. (1992) Annu. Rev. Cell Biol.8, 67-113 [CrossRef]
  17. Higgins, C. F., and Gottesman, M. M. (1992) Trends Biochem. Sci.17, 18-21 [CrossRef][Medline] [Order article via Infotrieve]
  18. Homolya, L., Holló, A., Germann, U. A., Pastan, I., Gottesman, M. M., and Sarkadi, B. (1993) J. Biol. Chem.268, 21493-21496 [Abstract/Free Full Text]
  19. Kano, K., and Fendler, J. H. (1978) Biochim. Biophys. Acta509, 289-299 [Medline] [Order article via Infotrieve]
  20. Lalande, M. E., Ling, V., and Miller, R. E. (1981) Proc. Natl. Acad. Sci. U. S. A.78, 363-367 [Abstract]
  21. Nielsen, D., and Skovsgaard, T. (1992) Biochim. Biophys. Acta1139, 169-183 [Medline] [Order article via Infotrieve]
  22. Penefsky, H. S. (1977) J. Biol. Chem.252, 2891-2899 [Abstract]
  23. Poruchynsky, M. S., and Ling, V. (1994) Biochemistry33, 4163-4174 [Medline] [Order article via Infotrieve]
  24. Ruetz, S., and Gros, P. (1994) J. Biol. Chem.269, 12277-12284 [Abstract/Free Full Text]
  25. Ruetz, S., Raymond, M., and Gros, P. (1993) Proc. Natl. Acad. Sci. U. S. A.90, 11588-11592 [Abstract]
  26. Roepe, P. D. (1992) Biochemistry31, 12555-12564 [Medline] [Order article via Infotrieve]
  27. Roepe, P. D., Wei, L. Y., Cruz, J., and Carlson, D. (1993) Biochemistry32, 11042-11056 [Medline] [Order article via Infotrieve]
  28. Schlemmer, S. R., and Sirotnak, F. M. (1994) J. Biol. Chem.269, 31059-31066 [Abstract/Free Full Text]
  29. Shapiro, A. B., and Ling, V. (1994) J. Biol. Chem.269, 3745-3754 [Abstract/Free Full Text]
  30. Sharom, F. J., Yu, X., and Doige, C. A. (1993) J. Biol. Chem.268, 24197-24202 [Abstract/Free Full Text]
  31. Simon, S., Roy, D., and Schindler, M. (1994) Proc. Natl. Acad. Sci. U. S. A.91, 1128-1132 [Abstract]
  32. Stein, W. D., Cardarelli, C., Pastan, I., and Gottesman, M. M. (1994) Mol. Pharmacol.45, 763-772 [Abstract]
  33. Valverde, M., Diaz, M., Seplveda, F. V., Gill, D. R., Hyde, S. C., and Higgins, C. F. (1992) Nature355, 830-833 [CrossRef][Medline] [Order article via Infotrieve]
  34. Young, X. K. (1992) Metabolite Transport across the Chloroplast Inner Envelope Membrane, Ph. D Thesis, Cornell University
  35. West, I. C. (1990) Trends Biochem. Sci.15, 42-46 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.