From the
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
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.
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.
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.
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.
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 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.
By incorporating DNA into the
aqueous interior of the liposomes, an increasing fluorescence due to
transport could be seen,
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.
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.
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.
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
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, 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.
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.
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 Na
ATP, 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.
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.
P-glycoprotein ATPase Activity Measurements
The
ATPase activity of reconstituted P-glycoprotein was measured as
described in Shapiro and Ling(1994).
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).
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.
Figure 3:
Inhibition of
[H]azidopine photolabeling of P-glycoprotein in
plasma membranes by Hoechst 33342. Sucrose gradient-purified plasma
membranes from CH
B30 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.
(
)demonstrating that the
Hoechst 33342 was transported into the liposome interior.
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
(Na
VO
), 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
Na
VO
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 Na
ATP, 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.
Table: Summary of pH
measurements inside liposomes
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.
,
-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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.