From the
P-glycoprotein functions as an ATP-driven active efflux pump for
many cytotoxic drugs. We now show that hydrophobic peptides and
ionophores also interact with the multidrug transporter.
Multidrug-resistant cells are cross-resistant to several hydrophobic
peptides and ionophores, but not to some other membrane-active species.
Linear peptides, cyclic peptides, and ionophores stimulated the ATPase
activity of P-glycoprotein in plasma membrane vesicles by up to
2.5-fold. Drugs and chemosensitizers were able to block P-glycoprotein
ATPase stimulation by verapamil, however, peptides and ionophores (with
the exception of cyclosporine A) were unable to do so. Peptides and
ionophores also effectively inhibited ATP-dependent drug transport by
P-glycoprotein in plasma membrane vesicles. The median effect analysis
was used to extract quantitative parameters from the drug transport
inhibition data. Unlike drug substrates and cyclic peptides, linear
peptides did not inhibit photoaffinity labeling of P-glycoprotein by
[
The resistance of tumors to multiple chemotherapeutic drugs is a
serious barrier to the treatment of human cancer. A common form of
multidrug resistance is caused by the overexpression of a
170-180-kDa plasma membrane protein, the P-glycoprotein or
multidrug transporter. This protein is a member of the ABC (ATP-binding
cassette)
(1) or traffic ATPase
(2) superfamily, and is
proposed to function as an ATP-driven drug efflux pump. Recently, we
reconstituted P-glycoprotein into proteoliposomes, and demonstrated
that it is indeed an active transporter, pumping drugs up a
concentration gradient, powered by concomitant ATP hydrolysis
(3) . One intriguing aspect of P-glycoprotein biochemistry
concerns its ability to interact with, and transport, many structurally
distinct classes of compounds, which suggests the possibility of
multiple or overlapping drug-binding domains on the transporter. Drugs
transported by P-glycoprotein are, in general, lipophilic, and it has
been proposed that the drug-binding site(s) may reside within the
transmembrane domains. This hypothesis is consistent with reports that
these regions of P-glycoprotein are photolabeled by hydrophobic ligands
(4) , and site-directed mutations within the transmembrane
segments are able to modulate drug specificity
(5, 6, 7) .
Two P-glycoprotein isoforms are
expressed in normal tissues: overexpression of Class I and II
P-glycoproteins ( e.g. human MDR1, mouse
mdr1/3, hamster pgp1/2) leads to multidrug
resistance, whereas overexpression of Class III proteins (human
MDR3, mouse mdr2, hamster pgp3) does not.
Recent studies of mdr2 ``knockout'' mice suggest
that the mdr2 gene product plays an essential role in the
liver in the export of phospholipid from the apical surface of the
canalicular membrane into the bile
(8) . This proposal was
confirmed by Ruetz and Gros
(9) , who demonstrated that the
mdr2 protein acts as a phosphatidylcholine translocase (or
flippase).
The physiological substrates for Class I and II
P-glycoproteins have not yet been identified. However, the sequence
similarity of P-glycoprotein with other ABC transporters known to
export peptides, both in prokaryotes ( e.g. the Escherichia
coli hemolysin exporter HlyB (10) ; the
oligopeptide permease of Salmonella typhimurium (11) )
and eukaryotes ( e.g. the yeast ste6 a-factor exporter
(12) ; the Tap-1/2 peptide transporters in the
endoplasmic reticulum
(13) ), suggests that peptides may also
serve as P-glycoprotein substrates in vivo. Recently, there
have been reports which indicate that P-glycoprotein may transport
peptides, although the evidence for this has, to date, been indirect.
Sharma et al. showed that mdr1-expressing cells were
resistant to the hydrophobic tripeptide ALLN
Chemosensitizers,
compounds which reverse drug resistance, show promise when combined
with chemotherapeutic agents in cancer treatment. The identification of
new clinically effective chemosensitizers is an important goal in
developing strategies to overcome MDR. The cyclic peptide cyclosporine
A, a well-known chemosensitizer, was recently reported to be
vectorially exported by monolayers of MDR cells
(19) and brain
endothelial cells
(20) , indicating that it is also a substrate
for transport by P-glycoprotein. Peptides thus represent an important,
and hitherto largely unexamined, class of P-glycoprotein substrates and
chemosensitizers, which may offer the hope of substantially lower
toxicity.
In the present study, we show that many hydrophobic
peptides and ionophores both block drug transport by P-glycoprotein,
and stimulate its ATPase activity, in an in vitro membrane
vesicle system. In addition, we correlate these data with
cross-resistance of MDR cells to peptides, and investigate their
ability to inhibit azidopine photoaffinity labeling of P-glycoprotein.
The results establish that certain hydrophobic peptides and ionophores
are P-glycoprotein substrates, and also indicate that these classes of
compounds interact with the transporter at different sites from those
associated with other drugs and chemosensitizers. Export of hydrophobic
peptides may thus be an important endogenous function for
P-glycoprotein, which suggests that further exploration of this class
of compounds as potential chemosensitizers is warranted.
On-line formulae not verified for accuracy
The ATPase activity of CH
Previous reports in the literature have suggested that
P-glycoprotein may transport hydrophobic peptides and ionophores,
including ALLN
(14) and gramicidin D
(16) . The present
study establishes that various hydrophobic peptides and ionophores
(both linear and cyclic) are P-glycoprotein substrates. Criteria used
to classify a particular compound in this way include cross-resistance
in MDR cells, and the ability to both stimulate P-glycoprotein ATPase
activity, and inhibit colchicine transport by P-glycoprotein in a
CH
We have used the
median effect analysis to derive the quantitative parameters
D
The
equilibrium inhibition data for all the compounds examined fitted well
to the median effect equation, as indicated by the straight line plots
in Fig. 4. The resulting D
Any
investigation of the effect of various compounds on transport in a
vesicle system must consider the possibility of nonspecific
permeabilization. This is especially important for studies involving
P-glycoprotein, where many putative substrates and chemosensitizers are
amphiphilic and/or membrane-active. Three of the peptides in this study
(gramicidin S, melittin, and alamethicin) were not P-glycoprotein
substrates based on the criteria of cross-resistance and ATPase
stimulation, yet they inhibited drug transport. They were shown to
permeabilize the CH
The other parameter used to
identify a compound as a P-glycoprotein substrate is stimulation of
ATPase activity. Assuming that the ATPase activity originating from
other membrane ATPases is the same in CH
There appears to be
little correlation between the turnover rate of ATP hydrolysis
following stimulation by peptides and ionophores, and their affinity
for P-glycoprotein, as assessed by their ability to compete with
colchicine for transport in plasma membrane vesicles. Some compounds,
especially the linear tripeptides ALLN, ALLM and leupeptin, and the
cyclic ionophore nonactin, induced the highest levels of ATPase
activity of all the species tested, yet showed high
D
The
results of verapamil blocking and azidopine photoaffinity labeling
experiments allow us to come to some conclusions about the relationship
between the interaction sites for these drugs and those for the linear
and cyclic peptides. Although drugs such as vinblastine and
trifluoperazine are able to block ATPase activation by verapamil in a
concentration-dependent manner, all of the linear peptides tested were
unable to do so. This finding indicates that the P-glycoprotein
interaction site for the linear peptides does not overlap with, and is
not linked to, the site where verapamil binds. Linear peptides were
also incapable of blocking azidopine photolabeling of P-glycoprotein,
which suggests that the binding site on the transporter for linear
peptides is also distinct from that for azidopine.
Of the cyclic
structures tested, the only one capable of abrogating verapamil
stimulation was cyclosporine A, which was highly effective at
relatively low concentrations. In contrast, cyclosporine A,
valinomycin, and nonactin all inhibited photolabeling at concentrations
similar to those which blocked drug transport, which suggests that the
site of interaction of these cyclic peptides/ionophores within
P-glycoprotein either overlaps with the azidopine site, or is linked to
it in a negative allosteric fashion. Thus the interaction site on
P-glycoprotein for cyclic peptides/ionophores appears to overlap with,
or be linked to, the verapamil and azidopine sites to varying degrees.
It is also clear from this study that not all P-glycoprotein substrates
can be identified on the basis of their ability to block azidopine
photolabeling.
The peptide ionophore gramicidin D deserves a special
mention. Several studies have shown that it is undoubtedly a
P-glycoprotein substrate ( e.g. Refs. 16 and 43), and thus it
might be expected to inhibit drug transport and stimulate
P-glycoprotein ATPase activity. However, its extremely low aqueous
solubility is clearly a problem at the experimental level; we
demonstrated only partial inhibition of drug transport
(Fig. 3 A), and ATPase stimulation was not observed.
After completion of this work, Sarkadi et al. (17) reported that the ATPase activity of the human MDR1 protein
overexpressed in Sf9 insect cell membranes was stimulated by various
bioactive hydrophobic peptides. In general, for those peptides tested
in both our study and theirs, comparable results were obtained.
However, leupeptin was ineffective at P-glycoprotein ATPase stimulation
in their system, whereas we have clearly shown that it is a
P-glycoprotein substrate on the basis of both ATPase stimulation and
inhibition of drug transport. It is possible that the insect cell
membrane system contains surface proteases which bind or degrade
certain peptides. In addition, the cytotoxic pentapeptide dolastatin
10, a promising new anti-tumor agent, appears to be a P-glycoprotein
substrate
(44) . Various prenylated cysteine compounds, which
are quite hydrophobic, also stimulate P-glycoprotein ATPase activity in
the Sf9 system
(18) . In both the latter cases, some inhibition
of [
We suggest that the ability of a compound to
inhibit drug transport in the CH
The relatively low D
We thank Dr. Victor Ling, Ontario Cancer Institute,
for providing the MDR cell lines used in this study.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H]azidopine. Taken together, these results
indicate that certain hydrophobic peptides and ionophores are
P-glycoprotein substrates, however, they affect the transporter in a
different manner from drugs. Linear peptides interact with
P-glycoprotein at a site distinct from those for verapamil and
azidopine, whereas the interaction site for cyclic peptides and
ionophores appears to be linked to these sites to varying degrees.
Export of hydrophobic peptides may be an important physiological
function of P-glycoprotein.
(
)(14) . In addition, the ability of mdr1 to
complement ste6 mutations
(15) implies that
P-glycoprotein can export the a-factor mating peptide from
Saccharomyces cerevisiae, although its efficiency remains
uncertain. More recently, we have demonstrated that the channel-forming
linear hydrophobic peptide gramicidin D is a substrate for the
multidrug transporter, which interferes with the ability of the peptide
to form a functional dimeric cation channel in the membrane
(16) . In addition, prenylcysteine methyl esters and various
hydrophobic peptides have been reported to stimulate the ATPase
activity of P-glycoprotein in human mdr1-transfected Sf9 cell
membranes
(17) and MDR breast cancer cells
(18) , which
suggests that they interact with the transporter.
MDR Cell Lines and Plasma Membrane
Preparation
The drug-sensitive parent Chinese hamster ovary cell
line (AuxB1) and an MDR cell line selected for colchicine resistance
(CHC5)
(21) , were grown as described previously
(22, 23, 24) . Plasma membrane vesicles from
AuxB1 and CH
C5 were isolated by a method involving cell
disruption by nitrogen cavitation followed by centrifugation on a 35%
(w/w) sucrose cushion
(22) . Plasma membrane vesicles were
stored at
70 °C for no longer than 3 months before use.
Cross-resistance of MDR Cells
Cross-resistance of
CHC5 cells to peptides and ionophores, and
chemosensitization by verapamil, were determined by growth inhibition
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
dye reduction assay, as described previously
(16, 25) .
The fold resistance was calculated as the ratio of the IC
values for CH
C5 relative to the AuxB1 parent. The
chemosensitization index was calculated as the ratio of the IC
for CH
C5 in the presence of 20 µM
verapamil, to that in its absence
(16, 25) . A value of
the chemosensitization index of 1 indicates that resistance was not
reversed by verapamil, whereas a value >1 indicates that resistance
was reversed.
Protein Determination
The protein content of
CHC5 plasma membrane was determined by a microplate
adaptation of the Bradford assay
(26) , using bovine serum
albumin as a standard.
Measurement of ATPase Activity
The ATPase activity
of P-glycoprotein in CHC5 plasma membrane vesicles was
determined as described previously
(3, 24, 27) ,
by measuring the release of inorganic phosphate from ATP, using a
colorimetric method. Samples contained 1-2 µg of
CH
C5 plasma membrane with 2 mM ATP and 5
mM Mg
, which gave maximal ATPase activity.
The assay buffer did not contain either Na
or
K
, to avoid contributions to activity from the
Na
K
-ATPase; addition of 1 mM
ouabain to the assay did not affect the measured ATPase activity.
Membrane vesicles were preincubated with peptides and ionophores for 5
min before initiation of the assay by addition of ATP. Peptides,
ionophores, and drugs were added as stock solutions in
Me
SO, and controls contained the appropriate levels of
Me
SO (which never exceeded 1%, v/v).
Measurement of Drug Transport
Steady-state
[H]colchicine uptake into CH
C5 plasma
membrane vesicles was determined using a protocol previously developed
in our laboratory
(23) . Briefly, membrane vesicles (25-35
µg of protein in a 100-µl final volume of buffer) were mixed
with 1 µM [
H]colchicine (0.3
µCi/sample), 5 mM Mg
, and 1 mM
ATP plus a regenerating system (creatine phosphate/creatine kinase).
After various times at 23 °C, the vesicles were harvested by rapid
filtration on Whatman GF/F filters using a Hoeffer filtration manifold,
and immediately washed with 5 ml of ice-cold buffer. Filters were dried
and radioactivity was quantitated by liquid scintillation counting.
Peptides, ionophores, and drugs were added as stock solutions in
Me
SO, and controls contained the appropriate levels of
Me
SO (which never exceeded 1%, v/v). Drug binding to
filters, and nonspecific uptake into the vesicles, were determined in
the absence of membrane vesicles, and in the absence of ATP and a
regenerating system, respectively.
Median Effect Analysis
The median effect equation
(28, 29) describes the relationship between any
concentration of a compound, and its effect on the system being
studied. In this case, the inhibition of
[H]colchicine transport into CH
C5
plasma membrane vesicles was measured at various concentrations of the
test compound(s). The basic median effect equation is as follows,
Assay for Membrane Permeabilization
The effect of
peptides and ionophores on the integrity of lipid bilayers was
determined by a modification of the method of Ertel et al. (30) . Briefly, 2.5 mg of egg phosphatidylcholine (Sigma)
in ethanol was dried under a stream of Nand then pumped
in vacuo for 30 min. The lipid was then suspended in 1 ml of
70 mM calcein (Sigma), 50 mM NaCl. After
freeze-thawing 10 times in liquid N
, large unilamellar
vesicles (LUV) were prepared by high pressure extrusion through 100-nm
polycarbonate filters, as described
(31) . Untrapped dye was
removed by gel filtration chromatography on Sephadex G-50. The void
volume fractions containing LUV were pooled, and the lipid content
determined using a microscale Bartlett assay
(32) . Aliquots
(0.5-1.0 µg of lipid) were dispensed into individual wells of
a 96-well microplate, in a total volume of 100 µl of Hepes-buffered
saline (10 mM Hepes, 0.15 M NaCl, 5 mM EDTA,
pH 7.0), and increasing concentrations of the test compounds in
Hepes-buffered saline were added. The release of calcein from the LUV
was quantitated after 30 min at 23 °C, using fluorescence
measurements on a cytofluorimeter (Millipore Cytofluor 2300;
= 485 nm,
= 535
nm). [
H]Azidopine Photoaffinity
Labeling-Photoaffinity labeling of P-glycoprotein in
CH
C5 membrane vesicles with
[
H]azidopine (200 nM, 52 Ci/mmol;
Amersham) was carried out as described previously
(16, 25, 33, 34) , in the presence of
various concentrations of peptides and ionophores. Membrane vesicles
were then analyzed by SDS-polyacrylamide gel electrophoresis on a 7.5%
polyacrylamide gel, followed by fluorography.
MDR Cells Are Cross-resistant to Peptides and
Ionophores
The MDR Chinese hamster ovary cell lines
CHC5 and CH
B30 were previously selected for
resistance to colchicine, and overexpress predominantly the Class I
isoform of P-glycoprotein
(35) . We previously reported that
CH
C5 cells and mdr1-transfectants were highly
cross-resistant to the linear channel-forming peptide gramicidin D
(16) . We have now investigated cross-resistance of MDR cell
lines to a selected set of linear and cyclic hydrophobic peptides and
ionophores, and chemosensitization of this resistance by verapamil
(). CH
C5 displayed high levels of resistance to
the protease inhibitor ALLN, and moderate levels were noted for the
related peptide ALLM and the cyclic peptide ionophore valinomycin. A
low, but significant level of resistance was observed for nonactin, a
cyclic non-peptide ionophore. The CH
B30 cell line, which
was derived from CH
C5, has substantially higher levels of
drug resistance and P-glycoprotein expression, and displayed 5-fold
resistance to nonactin, indicating that cross-resistance to this
compound is genuine. No cross-resistance was observed to the
membrane-active linear peptides melittin and alamethicin, or to the
cyclic membrane-active peptide gramicidin S, and the cyclic peptide
chemosensitizer cyclosporine A. Verapamil was able to reverse
resistance to ALLM, ALLN, gramicidin D, valinomycin, and nonactin,
which suggests that they are all P-glycoprotein substrates, but had no
effect on resistance to melittin, alamethicin, gramicidin S, and
cyclosporine A ().
P-glycoprotein ATPase Activity Is Stimulated by Drugs and
Chemosensitizers
CHC5 plasma membrane vesicles
displayed a high intrinsic Mg
-ATPase activity (0.273
µmol/min/mg) compared to plasma membrane from the drug-sensitive
parent line AuxB1 (0.056 µmol/min/mg, see Ref. 24). In previous
studies, we confirmed that this additional ATPase activity in the
drug-resistant cell line arises from the presence of P-glycoprotein
(24) . The ATPase activity of CH
C5 membrane was
inhibited by vanadate (IC
of 1.5 µM; 80%
inhibition at 10 µM), which was previously observed to
inhibit both P-glycoprotein-mediated drug transport
(23) and
the ATPase activity of a partially purified P-glycoprotein preparation
(24) .
C5 plasma
membrane was stimulated over 2.2-fold by the chemosensitizer verapamil.
Trifluoperazine also produced a large activation, while the drug
substrates vinblastine and colchicine increased activity by 40%.
Maximal stimulation of P-glycoprotein ATPase was reached at 10
µM for verapamil, trifluoperazine, and vinblastine, and
100 µM for colchicine. The ATPase activity of plasma
membrane from the drug-sensitive AuxB1 parent cell line was not
stimulated significantly by any of the compounds tested. Thus,
P-glycoprotein ATPase in a native membrane environment is activated by
certain chemosensitizers and drugs. Similar results have been reported
for plasma membrane vesicles from other P-glycoprotein-expressing
cells. CH
C5 membrane vesicles therefore provide a simple
and convenient system for screening various compounds for their effect
on P-glycoprotein ATPase activity. We previously demonstrated that the
ATPase activity of P-glycoprotein in detergent solution is also
modulated by drugs
(24, 36) , although to a lesser
extent.
Peptides and Ionophores Stimulate P-glycoprotein ATPase
Activity
Linear and cyclic peptides and ionophores were examined
for their ability to stimulate P-glycoprotein ATPase in
CHC5 plasma membrane. The linear peptides ALLN, ALLM,
pepstatin A, and leupeptin (Fig. 1 A), and the cyclic
ionophores valinomycin and nonactin (Fig. 1 B) produced
maximal enhancement of ATPase activity of around 2-2.5-fold.
These six compounds all exhibited a pattern of activation which did not
decrease at high concentrations, unlike the biphasic pattern noted
previously by our group
(3, 24, 36) and others
(37, 38) for chemosensitizers and drugs. The
membrane-active linear peptides melittin, alamethicin, and gramicidin D
did not stimulate the ATPase (Fig. 1 A); instead they
caused varying degrees of inhibition as the concentration increased.
Activity was also stimulated (up to 1.4-fold) by cyclosporine A, which
displayed a biphasic pattern, whereas gramicidin S did not activate the
ATPase (Fig. 1 B).
Figure 1:
Stimulation of P-glycoprotein ATPase
activity in CHC5 plasma membrane by peptides and
ionophores. CH
C5 plasma membrane vesicles (1.5-2.0
µg of protein) were assayed for Mg
-ATPase
activity in the presence of various linear peptides ( A) ALLM
(
), ALLN (▾), leupeptin (
), pepstatin A (
),
gramicidin D (
), melittin (
), alamethicin (
); and
various cyclic peptides and ionophores ( B) gramicidin S
(
), valinomycin (
), cyclosporine A (
), nonactin
(▾). Data are presented as percent control ATPase activity (means
± S.E., n = 3), measured in the absence of
peptides.
The concentrations of each peptide
or ionophore that induced half-maximal stimulation of P-glycoprotein
ATPase activity (SCvalues) were interpolated from the
curves in Fig. 1, A and B, and are listed in
, together with the maximal fold-stimulation observed for
each. The SC
values are useful indicators of the relative
``affinity'' of each compound for interaction with
P-glycoprotein. Cyclosporine A showed the lowest SC
value
of all the compounds tested (around 10 nM), whereas the
SC
value for valinomycin was less than 1 µM,
comparable to that for vinblastine. Nonactin and pepstatin A gave
half-maximal ATPase stimulation in the low micromolar range, while
ALLN, ALLM, and leupeptin had SC
values between 40 and 100
µM. In general, only compounds to which CH
C5
cells were cross-resistant stimulated ATPase activity; peptides such as
melittin, alamethicin, and gramicidin S, to which MDR cells did not
display cross-resistance, did not induce ATPase stimulation.
Drugs Abolish Verapamil Stimulation of P-glycoprotein
ATPase, whereas Peptides and Ionophores Do Not
As demonstrated
above, verapamil stimulates the ATPase activity of CHC5
plasma membrane by over 2.2-fold. It was of interest to determine
whether peptides and ionophores block verapamil-induced ATPase
activation, since this would give some indication of whether these
classes of compounds compete with verapamil for a common binding site
on P-glycoprotein. As shown in Fig. 2, cyclosporine A and
vinblastine completely abrogated verapamil stimulation of
P-glycoprotein ATPase activity at low concentrations (5 and 50
µM, respectively), as did other drugs and
chemosensitizers, including daunomycin, trifluoperazine, quinine, and
quinidine (not shown). In contrast, linear peptides such as ALLM and
pepstatin A were unable to do so, even at a concentration of 200
µM. Valinomycin inhibited verapamil stimulation slightly
at 100 µM. ALLN, leupeptin, and nonactin were also unable
to block verapamil stimulation of ATPase activity (not shown). These
data support the hypothesis that linear peptides interact with
P-glycoprotein at a binding site distinct from that for verapamil. On
the other hand, cyclosporine A and other drugs and chemosensitizers
reside in a site which either overlaps with the verapamil-binding site,
or is allosterically linked to it, so that occupancy affects the
ability of verapamil to bind to the transporter, or signal the ATPase
catalytic site once bound.
Figure 2:
Effect of peptides and ionophores on
stimulation of P-glycoprotein ATPase activity by verapamil.
CHC5 plasma membrane vesicles (1.5-2.0 µg of
protein) were assayed for Mg
-ATPase activity in the
presence of 10 µM verapamil, and increasing concentrations
of ALLM (
), pepstatin A ( PEPA,
), valinomycin
( VAL, ▾), cyclosporine A ( CSA,
), and
vinblastine ( VBL,
). Data are presented as percent
control ATPase activity (means ± S.E., n = 3),
measured in the presence of verapamil alone. The basal level of ATPase
activity determined in the absence of verapamil is indicated by the
horizontal dashed line.
Peptides and Ionophores Inhibit Drug Transport by
P-glycoprotein
Peptides and ionophores were examined for their
ability to block active, ATP-dependent colchicine transport into
CHC5 plasma membrane vesicles, using methodology previously
established in our laboratory
(3, 23) . As shown in
Fig. 3
, a large number of peptides and ionophores inhibited
accumulation of drug in the vesicle lumen, in a concentration-dependent
manner. Cyclosporine A and vinblastine (Fig. 3 B) were
highly effective competitors of colchicine transport, with >90%
inhibition observed at 1 and 3 µM, respectively.
Valinomycin (Fig. 3 B) also blocked transport very
effectively in the low micromolar concentration range. Linear peptides
(Fig. 3 A) inhibited colchicine transport in the order of
effectiveness pepstatin A > leupeptin > ALLM, and the cyclic
ionophore nonactin was the least potent inhibitor. Gramicidin D could
not be tested above 50 µM due to poor solubility, but it
blocked drug accumulation by >35% at this concentration.
Figure 3:
Inhibition by peptides and ionophores of
P-glycoprotein-mediated drug transport. Equilibrium uptake of 1
µM [H]colchicine into
CH
C5 membrane vesicles was measured in the presence of 1
mM ATP and a regenerating system, together with increasing
concentrations of ( A) nonactin ( NON, ▾), ALLM
(
), leupeptin ( LEU,
), pepstatin A
( PEPA,
); and ( B) valinomycin ( VAL,
), vinblastine ( VBL,
), and cyclosporine A
( CSA,
). Data are presented as percent control drug
uptake relative to membrane vesicles in the absence of peptides, and
represent the mean ± S.E. ( n =
3).
Median Effect Analysis of Transport Inhibition
Data
The transport inhibition data presented in
Fig. 3
cannot be analyzed by enzyme kinetic models, since it
represents steady-state drug accumulation, rather than initial rates of
transport. As an alternative means for evaluation of the transport
inhibition data, we employed the median effect analysis, which was
developed by Chou (for reviews, see Refs. 28 and 29). This method is
derived from the law of mass action, and requires no assumptions about
mechanism (indeed, it is mechanism-independent), or estimates of
binding or kinetic constants. It permits the experimenter to quantitate
the relationship between the concentration of any compound, D,
and its effect on the system being studied (see ``Materials and
Methods''). In this case, the fractional inhibition of colchicine
accumulation in CHC5 plasma membrane vesicles (data shown
in Fig. 3) was used as the parameter
f
, and median effect plots of log
( f
/ f
)
versus log D were constructed for each of the test
compounds. The drug accumulation inhibition data gave a series of
straight line median effect plots (shown in Fig. 4), covering a
wide range of concentrations. The plot for cyclosporine A (the most
effective transport inhibitor) appears at the far left of the series,
and the plot for nonactin (the least effective transport inhibitor) is
shown at the far right. The parameter D
was determined for each plot from the zero intercept on the x axis, and represents the concentration of test compound required
to inhibit drug accumulation by 50% (see ). The
D
values thus provide a means of
quantitating the efficacy of each test compound as an inhibitor of drug
transport by P-glycoprotein. All of the peptides that stimulated ATPase
activity also inhibited colchicine transport, which indicates that they
are likely to be P-glycoprotein transport substrates. The linear
peptide pepstatin A and the cyclic peptide ionophore valinomycin are
comparable to, or more effective than verapamil as inhibitors of the
multidrug transporter.
Figure 4:
Median
effect analysis of transport inhibition data. Median effect plots of
log ( f/ f) versus log D for nonactin
(▾), ALLM (), leupeptin (
), pepstatin A (
),
valinomycin (
), cyclosporine A (
), and vinblastine
(
).
The slope of the median effect plot for each
test compound yields the parameter m, which is an indicator of
the sigmoidal nature of the plot, analogous to a Hill coefficient.
Compounds with m values approximating 1 (non-sigmoidal)
include ALLM, leupeptin, pepstatin A, nonactin, and vinblastine. Three
peptides (ALLN, valinomycin, and cyclosporine A) and verapamil showed
m values close to 3, which indicates a high level of
sigmoidicity in the median effect plot.
Membrane Permeabilization by Peptides
Three of the
peptides tested (melittin, alamethicin, and gramicidin S) were
nonspecific membrane-active agents, and displayed anomalous effects on
drug transport by P-glycoprotein. As shown in Fig. 5 A,
increasing concentrations of these peptides reduced colchicine
accumulation below the level of the control with no ATP. We have
previously demonstrated that colchicine uptake into CHC5
plasma membrane vesicles in the absence of ATP represents diffusional
equilibration of the drug across the bilayer into the vesicle lumen
(23) . Thus, it seems likely that these three peptides
nonspecifically permeabilize the membrane, and prevent sequestration of
drug in the vesicle lumen by diffusional equilibration. This premise
was confirmed by the use of an assay which monitored release of the
self-quenching fluorescent dye, calcein, from the lumen of large
unilamellar phospholipid vesicles. As shown in Fig. 5 B,
melittin and alamethicin released calcein from LUV over the
concentration ranges 0.017-0.17 and 0.5-5 µM,
respectively, and gramicidin S permeabilized the vesicles at around 3
µM (not shown). The permeabilization curve for the
nonionic detergent Triton X-100 is displayed for comparison; it
released calcein from the vesicle lumen at about 0.01% (v/v). It was
also necessary to address the issue of whether any of the other
compounds tested in this study inhibited transport as a result of
nonspecific permeabilization, rather than an effect on P-glycoprotein.
Further experiments showed that none of the other peptides, ionophores,
drugs, or chemosensitizers listed in were able to release
calcein from the vesicle lumen within the relevant concentrations
ranges; typical data resembled those for leupeptin
(Fig. 5 B). Taken together, these observations indicate
that melittin, alamethicin, and gramicidin S inhibited drug transport
by nonspecific permeabilization of the CH
C5 membrane
vesicles, whereas the other compounds tested did so as a result of an
effect on P-glycoprotein. These results are consistent with other data
indicating that MDR cells are not cross-resistant to these three
peptides, and that they do not stimulate P-glycoprotein ATPase activity
().
Figure 5:
Drug transport inhibition and membrane
permeabilization. A, inhibition of P-glycoprotein-mediated
drug transport. Equilibrium uptake of 1 µM
[H]colchicine into CH
C5 membrane
vesicles was measured in the presence of 1 mM ATP and a
regenerating system, together with increasing concentrations of
alamethicin ( ALA,
), gramicidin S ( GMS,
), and melittin ( MEL, ▾). Data points are given
as percent control drug uptake relative to membrane vesicles in the
absence of added peptides, and represent the mean ± S.E. ( n = 3). B, permeabilization of LUV by various
peptides. LUV of egg phosphatidylcholine preloaded with the fluorescent
dye calcein were incubated with increasing concentrations of various
peptides, including leupeptin ( LEU,
), alamethicin
( ALA,
), and melittin ( MEL, ▾). After 30
min at 23 °C, the release of calcein from the lumen of the vesicles
was monitored (
= 485 nm,
= 535 nm). Results for the nonionic detergent Triton X-100
( TRIT,
) are shown for
comparison.
Effect of Peptides and Ionophores on Azidopine
Photoaffinity Labeling of P-glycoprotein
The ability to inhibit
photoaffinity labeling of P-glycoprotein by the drug azidopine has
frequently been used as an indicator of whether a particular compound
is a P-glycoprotein ``substrate''
(33, 34) .
In particular, it is believed that compounds which compete with
azidopine for a common binding site on the multidrug transporter will
be able to block photolabeling. For example, the drug vinblastine
abolishes azidopine photolabeling of P-glycoprotein very effectively,
with half-maximal inhibition at 5 µM. In contrast, linear
peptides were completely unable to block azidopine photolabeling of
P-glycoprotein (see ), as shown for ALLN and pepstatin A in
Fig. 6
, A and B, even at concentrations
comparable to, or higher than, those inhibiting drug transport. This
observation suggests that the binding site on P-glycoprotein for linear
peptides is distinct from that for azidopine. Alamethicin displayed
inhibition of photolabeling, possibly as a result of nonspecific
membrane disruption, at concentrations substantially higher than those
inhibiting transport. We previously noted that certain amphiphiles,
including Triton X-100 and Nonidet P-40, were able to block azidopine
labeling of P-glycoprotein in CHC5 plasma membrane
(25) .
Figure 6:
Effect of peptides and ionophores on
photoaffinity labeling of P-glycoprotein by
[H]azidopine. CH
C5 membrane vesicles
(20 µg of protein) were incubated with
[
H]azidopine in the presence of increasing
concentrations of: A, ALLN; B, pepstatin A;
C, valinomycin; and D, nonactin. After 1 h at room
temperature, the samples were subsequently irradiated with UV light for
30 min. After separation by SDS-polyacrylamide gel electrophoresis, the
intensity of P-glycoprotein photolabeling was detected by fluorography;
the only visible band was P-glycoprotein, with a molecular mass of
170-180 kDa. Peptide/ionophore concentrations in µM
are indicated along the bottom of each gel. Arrows and numbers to the left of the gels indicate the position
of molecular mass markers in kDa.
Of the cyclic peptides and ionophores tested,
cyclosporine A, valinomycin, and nonactin inhibited photolabeling
(Fig. 6, C and D, and ), whereas
gramicidin S, which does not appear to be a P-glycoprotein substrate,
did not. Valinomycin and cyclosporine A abolished photolabeling at
half-maximal concentrations which were of the same order of magnitude
as those required for half-maximal inhibition of colchicine transport
(see ). It thus seems likely that the site of interaction
of these cyclic structures within P-glycoprotein either overlaps with
the azidopine site, or is negatively allosterically linked to it.
C5 plasma membrane vesicle system. This vesicle model
system is unique in that it permits concurrent quantitation of both the
latter parameters. We conclude that the CH
C5 plasma
membrane vesicle system will prove both useful and convenient for rapid
screening of putative P-glycoprotein substrates.
and m from the equilibrium
transport inhibition data gathered in this study. Several previous
reports in the literature have mistakenly applied Michaelis-Menten
kinetic analysis to this type of data, which is clearly incorrect,
since initial rates of transport were not measured. Experiments in our
laboratory
(
)
have shown that colchicine transport
by P-glycoprotein in the CH
C5 membrane vesicle system is
too fast (on the subsecond time scale) for rigorous measurement of true
initial rate kinetics using conventional rapid filtration methodology.
Specialized instrumentation for rapid kinetic measurements will be
necessary to determine true initial rates of drug transport.
values
quantitate the ability of each compound to block P-glycoprotein drug
transport, and are useful indicators of the relative affinity of the
interaction of each with the transporter. Values for m, which
is a parameter analogous to a Hill number, were also determined for
each species. All the compounds tested had m values close to
either 1 (not sigmoidal) or 3 (highly sigmoidal). The significance of
the value of m with respect to P-glycoprotein function is not
yet clear, although it must reflect the underlying molecular
interaction. The median effect analysis therefore appears to be a very
useful method for quantitatively analyzing equilibrium drug uptake
data, which have been reported for several different vesicle systems
containing P-glycoprotein
(3, 23, 40, 41, 42) .
C5 plasma membrane vesicles in the same
concentration range, and thus we concluded that they affected transport
in a nonspecific fashion, rather than by an interaction with
P-glycoprotein itself. These results indicate that caution is necessary
when dealing with membrane-active compounds in transport experiments
which depend on membrane integrity.
C5 and the parent
cell line, then approximately 80% of the measured activity in
CH
C5 membrane can be attributed to P-glycoprotein. Thus,
the actual stimulation of P-glycoprotein ATPase activity by verapamil
is likely around 2.8-fold. This is similar to the 3.5-fold stimulation
observed in plasma membrane from Sf9 insect cells overexpressing the
human mdr1 gene product
(37) , and lower than the
5-fold stimulation noted for plasma membrane from a Chinese hamster
ovary cell line selected for a high level of P-glycoprotein
overexpression
(39) . Different levels of ATPase stimulation by
other drugs were also reported in these two studies. It is possible
that these variations arise from differences in the properties of the
gene products themselves (human versus hamster), or
differences in post-translational modification of P-glycoprotein,
especially phosphorylation, which is known to modify drug resistance in
intact cells. We recently investigated the phosphorylation state of
P-glycoprotein in CH
C5 plasma membrane, and determined that
it is not fully phosphorylated
(36) .
values for inhibition of transport,
indicating that they interact with P-glycoprotein with relatively low
affinity. In contrast, some drug substrates, such as vinblastine,
stimulated smaller increases in P-glycoprotein ATPase activity, yet
were clearly very high affinity transport substrates on the basis of
their D
values. However, the
concentration of compound required for half-maximal ATPase stimulation
(SC
) correlated well with the D
values for transport inhibition for all the linear peptides
tested. In the case of the cyclic peptides and ionophores, ATPase
stimulation occurred at concentrations substantially lower
(6-70-fold) than those observed to inhibit drug transport.
H]azidopine photolabeling was observed,
although at concentrations much higher than those needed to stimulate
ATPase activity.
C5 membrane vesicle system
in vitro is a much more reliable indicator of whether it is a
P-glycoprotein substrate than the ability of the compound to either
stimulate P-glycoprotein ATPase activity, or block azidopine
photoaffinity labeling, both of which have been proposed as possible
one-step ``screens'' for putative substrates. In addition,
the median effect analysis allows quantitation of the inhibition
process.
values
measured for several of the hydrophobic peptides and ionophores tested
in this study, in many cases comparable to those for drugs and
chemosensitizers, indicates that this class of compounds interacts with
P-glycoprotein with high affinity. These results strengthen the
argument that export of hydrophobic peptides may be the true
physiological function of the multidrug transporter. Many of the
compounds tested in this study are rich in leucine and/or valine, and
it is possible that this is one structural characteristic of peptides
that is recognized by P-glycoprotein. We are currently examining the
interaction of a series of leucine-rich synthetic hydrophobic peptides
with the multidrug transporter, in an effort to determine how the chain
length, charge, and amino acid R-group affect their interaction with
the protein. Preliminary experiments using
I-labeled
peptides indicate that they are, in fact, transported by
P-glycoprotein. Further investigation of the interaction of
P-glycoprotein with hydrophobic peptides will provide information
necessary for the development and design of new peptide-based
chemosensitizers for use in clinical treatment.
Table:
Effect of peptides and ionophores on various
aspects of P-glycoprotein function
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.