(Received for publication, June 26, 1995; and in revised form, October 5, 1995)
From the
The aim of the present study was to demonstrate that the
modulation of P-glycoprotein (Pgp) ATPase activity by peptides, drugs,
and chemosensitizers takes place on a common drug pharmacophore. To
this end, a highly emetine-resistant Chinese hamster ovary cell line
was established, in which Pgp constituted 18% of plasma membrane
protein. Reconstituted proteoliposomes, the Pgp content of which was up
to 40%, displayed a basal activity of 2.6 ± 0.45 µmol of
P/min/mg of protein, suggesting the presence of an
endogenous Pgp substrate. This basal ATPase activity was stimulated (up
to 5.2 µmol of P
/min/mg of protein) by valinomycin and
various Pgp substrates, whereas, to our surprise, gramicidin D, an
established Pgp substrate, was inhibitory. Taking advantage of this
novel inhibition of Pgp ATPase activity by gramicidin D, a drug
competition assay was devised in which gramicidin D-inhibited Pgp
ATPase was coincubated with increasing concentrations of various
substrates that stimulate its ATPase activity. Gramicidin D inhibition
of Pgp ATPase was reversed by Pgp substrates, including various
cytotoxic agents and chemosensitizers. The inhibition of the basal
ATPase activity and the reversal of gramicidin D inhibition of Pgp
ATPase by its various substrates conformed to classical
Michaelis-Menten competition. This competition involved an endogenous
substrate, the inhibitory drug gramicidin D, and a stimulatory
substrate. We conclude that the various MDR type substrates and
chemosensitizers compete on a common drug binding site present in Pgp.
Inherent as well as acquired antitumor drug resistance continue
to pose major obstacles toward the successful chemotherapeutic
treatment of various human malignancies(1) . Resistance to a
broad spectrum of hydrophobic cytotoxic drugs including Vinca alkaloids, anthracyclines, epipodophyllotoxins, actinomycin D,
taxoids (e.g. taxol), actinomycin D, and dolastatin 10 has
been termed multidrug resistance (MDR(); for reviews, see (2, 3, 4, 5) ). Mammalian cells with
the MDR phenotype, express increased levels of a heavily glycosylated
170-kDa plasma membrane protein known as P-glycoprotein (Pgp). Pgp is a
tandemly duplicated 1280-amino acid polypeptide, each half of which
contains six transmembrane
-helices and one cytoplasmic nucleotide
triphosphate binding site(6, 7) . It has been shown
that MDR cells contain markedly decreased intracellular drug
concentrations(8, 9, 10) ; this was shown to
result from an ATP-dependent drug efflux (for reviews, see (2, 3, 4, 5) and 11). Pgp-containing
plasma membrane vesicles from tissue-cultured cells (12) or
from rat liver canalicular membrane vesicles (13) displayed an
ATP-dependent drug transport.
Recently, Sharom et al.(14) showed an ATP-dependent transport of colchicine into proteoliposomes reconstituted with hamster Pgp, whereas Eytan et al.(15) demonstrated an ATP-driven transport of the hydrophobic peptide ionophores gramicidin D and valinomycin into proteoliposomes reconstituted with rat and hamster Pgp. Pgp has been shown to bind the photoaffinity ATP analogue 8-azido-ATP(16, 17) , and amino acid substitutions of either or both the ATP-binding domains resulted in abolished function of drug transport. In this respect, several initial reports have demonstrated that Pgp has a low ATPase activity(18, 19) , whereas more recent studies have shown Pgp to contain a high drug-stimulatable ATPase activity(20, 21, 22, 23) . Recently, Smit et al.(24) , used targeted MDR1 gene disruption in transgenic mice and found these mice to be 100-fold more sensitive to ivermectin, a central nervous system neurotoxin, thus strongly suggesting that the physiological overexpression of Pgp in the blood brain barrier endothelium (25) is presumably responsible for cytotoxins extrusion. Taken together, these results strongly suggest that Pgp functions as an ATP-dependent efflux transporter of multiple hydrophobic cytotoxic drugs.
A major unresolved problem in
the multidrug resistance field is how a single integral membrane
transporter can transport various hydrophobic peptides, cytotoxic
drugs, and chemosensitizers bearing a wide array of molecular
structures(4) . Several genetic and biochemical approaches have
been taken in an attempt to resolve this major question; these included
an examination of the relationship between the structure and function
of Pgp using mutational analysis of this transporter, as well as its
photoaffinity labeling with substrate and substrate analogues (for a
recent review, see (26) ). The first point mutation described
in Pgp, involving a Gly Val substitution at position 185, was
detected in a highly colchicine-selected MDR KB-C1 cell line; this
single amino acid substitution was shown to change the specificity of
the transporter so that colchicine transport was improved, whereas
vinblastine and actinomycin D transport was
decreased(27, 28) . Similarly, mutations of Gly
Ala and Ala
Pro at positions 338 and 339, respectively, in
transmembrane domain 6 of Chinese hamster Pgp (29) , appeared
to decrease the resistance to several drugs, while maintaining normal
resistance to actinomycin D. Furthermore, a site-directed mutation
leading to conversion of Ser
Phe at residue 941 in the mouse mdr1 gene in transmembrane domain 11 (30, 31) altered drug specificity such that the
transport of vinblastine was intact, whereas that of colchicine and
doxorubicin was drastically decreased.
Several photoaffinity labels including Pgp substrates such as anticancer drugs, hydrophobic peptides, and chemosensitizers bind to two symmetrical sites on Pgp (i.e. transmembrane domains 6 and 12; see (4) ). Various hydrophobic peptides, drugs, and chemosensitizers were shown to competitively inhibit the photoaffinity labeling of Pgp with radiolabeled substrate analogues, or chemosensitizers' analogues, in isolated plasma membrane vesicles, or in whole MDR cells (reviewed in (26) ). Using Pgp-reconstituted proteoliposomes, we have recently shown an ATP-driven transport of valinomycin and gramicidin D(15) ; this active intravesicular accumulation of peptide ionophores was blocked by established Pgp substrates including the anthracyclines doxorubicin, daunorubicin, the Vinca alkaloid vinblastine as well as the tripeptide N-acetyl-leucyl-leucyl-norleucinal (ALLN; see (32) ).
Taken collectively, these different studies pointed out that a common drug pharmacophore may exist in Pgp, which binds the structurally and functionally distinct drugs and chemosensitizers that participate in the MDR phenomenon(2, 3, 4, 33, 34) . Indeed, using proteoliposomes reconstituted with Pgp ATPase, we show here that various hydrophobic peptides, cytotoxic drugs, and MDR chemosensitizers compete on a common drug pharmacophore present in Pgp. We therefore suggest that substrate competition using a drug-modulatable Pgp ATPase activity, is a functional assay useful for the screening of Pgp substrates and MDR chemosensitizers.
Emetine-resistant variants were derived from wild type AA8
cells using a stepwise selection protocol of increasing drug
concentrations. Drug selection was performed by first seeding 5
10
wild type cells/25-cm
tissue culture flask
(Nunc) in growth medium (5 ml) containing 0.15 µM emetine
(
3 times the 50% lethal dose (LD
) for parental AA8
cells). Following growth to midconfluence, emetine-selected cells were
detached by trypsinization, counted, and replated as above in the
presence of a 50-100% increment in the emetine concentration. The
gradual increase in emetine concentrations was terminated at 1
µM (
20-fold LD
).
Plasma membrane
vesicles were isolated from the microsomal fraction by discontinuous
density gradient centrifugation as described by Riordan and Ling (38) with the modification of Al-Shawi and Senior(21) .
The routine yields for microsomes and plasma membrane vesicles were,
respectively: 0.4-0.6 mg of protein/10 cells and
50-100 µg of protein/10
cells. The membrane
fractions were frozen in liquid nitrogen and stored at -75 °C
until analysis.
Proteoliposomes were formed by dilution
of the protein and lipid solution into 25 volumes of vigorously-stirred
reconstitution medium containing PMSF as the only protease inhibitor.
The proteoliposomes were recovered by centrifugation at 130,000 g for 45 min, suspended in reconstitution medium, and rapidly
frozen in liquid nitrogen. Samples were stored for weeks at -75
°C without any detectable loss of Pgp ATPase activity. Due to the
high lipid content of the samples, protein was determined according to
Esen(39) , using bovine serum albumin (Fraction V, Sigma) as a
standard.
Regression analysis and reiterative best-fit computer-simulated curves were generated using Sigmaplot software distributed by Jandel Scientific Software.
Figure 1:
Autoradiogram of Southern blot
performed with genomic DNA from parental AA8 cells and their various
emetine-resistant sublines. High molecular weight DNA (10 µg/lane)
extracted from wild type cells (lane 1) and their
emetine-resistant variants stably growing in 0.15, 0.3, 0.6, and 1
µM emetine (lanes 2-5, respectively) was
digested with EcoRI, fractionated on 0.8% agarose gels,
transferred to Zetabind filter membrane, and probed with a P-oligolabeled hamster MDR cDNA sequence
(0.66-kilobase EcoRI insert of pCHP-1). The blots were then
washed under stringent conditions and autoradiographed for 6-18
h.
Emt cells displayed a 45-fold
resistance to the selecting agent emetine, as well as a 15-95
cross-resistance to MDR-type drugs including doxorubicin, colchicine,
vinblastine, taxol, and the novel microtubule depolymerizing anticancer
peptide, dolastatin 10 (Table 1). The ionophore resistance
pattern in Emt
cells (Table 1) was consistent with
our previous findings of a high level resistance to the channel-forming
pentadecapeptide ionophore gramicidin D and a low level resistance to
the carrier-type ionophore valinomycin in GD
cells(36) . In contrast, Emt
cells
maintained wild type sensitivity to cytotoxic agents that are not
handled by Pgp including the hydrophilic antifolate methotrexate and
the hydrophobic peptide antibiotic viscosin. Taken together these data
suggest that upon stepwise selection to the antiamebic drug emetine,
mammalian cells acquire a classic MDR phenotype via a prominent MDR gene amplification and consequent Pgp overexpression. The
excessive overproduction of Pgp in Emt
plasma membrane
makes this cell line an excellent source for Pgp.
When examined with established Pgp
substrates, the ATPase activity of Pgp- reconstituted proteoliposomes
was linear for at least 1 h at 37 °C (Fig. 2). The basal (i.e. when no substrates were added) ATPase activities
obtained with proteoliposomes reconstituted with Emt microsomal and plasma membrane proteins were: 1.1 ± 0.25
and 2.6 ± 0.45 µmol of P
/min/mg of protein (mean
± S.E.), respectively. Thus, in accord with the 2.2-fold
increased Pgp content in proteoliposomes reconstituted with plasma
membrane proteins was their 2.3-fold elevated ATPase activity (Fig. 3). This basal ATPase activity was vanadate sensitive (see
below), and under the sodium-free assay conditions used, the ATPase
activity was ouabain-insensitive. Furthermore, this ATPase activity was
inhibited by the well established Pgp substrate gramicidin D and
stimulated by a variety of known Pgp substrates including hydrophobic
peptides, cytotoxic drugs, and chemosensitizers (see below). In
contrast, proteoliposomes reconstituted with AA8 cell membrane proteins
did not display any drug-modulatable ATPase activity. Thus, the basal
ATPase activity present in proteoliposomes reconstituted with
Emt
membrane proteins is attributable to Pgp.
Figure 2: Linearity of ATPase activity of Pgp-reconstituted proteoliposomes incubated with various stimulatory and inhibitory Pgp substrates. Proteoliposomes were incubated for variable times with 33 µM gramicidin D, 100 µM verapamil, 100 µM valinomycin, or with 5 µM dolastatin 10. After incubation at 37 °C for up to 1 h, the ATPase activity was measured colorimetrically by following the production of phosphate (see ``Experimental Procedures'').
Figure 3:
Modulation by valinomycin and gramicidin D
of the ATPase activity of proteoliposomes reconstituted with Pgp.
Proteoliposomes reconstituted with detergent-soluble proteins from
Emt microsomal (A) or plasma membrane fraction (B) were incubated for 1 h at 37 °C in an ATP-containing
medium (for details see ``Experimental Procedures'') in the
presence of increasing concentrations of the peptide ionophores
valinomycin and gramicidin D, after which the ATPase activity was
determined. Data shown in panel A were derived from 10
independent experiments, and the lines depicted were obtained by a
computer-aided best-fit analysis using , described under
``Experimental Procedures,'' for a two-substrate competition
system consisting of a putative endogenous substrate and an added MDR
drug.
A quantitative description of the inhibition of
basal Pgp ATPase by gramicidin D and stimulation by valinomycin is best
described in terms of competition between these substrates and a
putative endogenous substrate responsible for the basal ATPase activity
(see ``Discussion''). Both the endogenous and added
substrates are described in classical Michaelis-Menten terms of K and V
. A stimulatory
substrate such as valinomycin exhibits a high V
,
whereas an inhibitory substrate such as gramicidin D is characterized
by a relatively low V
. As shown in Fig. 3A, the experimental data could be best-fit to a
computer simulation of classical competition between the putative
endogenous substrate and either gramicidin D or valinomycin (see
``Experimental Procedures'' for and explanations
thereof).
Figure 4:
Modulation of the ATPase activity of
Pgp-reconstituted proteoliposomes by hydrophobic bioactive peptides,
cytotoxic agents, and chemosensitizers. Proteoliposomes reconstituted
with Pgp from Emt cells were incubated in the absence or
presence of varying concentrations of established Pgp substrates
including anticancer drugs and cytotoxic agents (A),
chemosensitizers of the MDR phenotype (B), as well as various
bioactive hydrophobic peptides (C). After determination of the
ATPase activity, the -fold modulation was calculated as the ratio
between the activity observed in the presence of the exogenously added
Pgp substrate and the basal activity observed in the absence of any
added drug. The minimal activity obtained after incubation on ice of
Pgp-reconstituted proteoliposomes was considered background and was
therefore routinely subtracted from all measurements. Proteoliposomes
reconstituted with n-octylglucoside-soluble microsomal and
plasma membrane proteins consistently showed a barely detectable ATPase
activity that was constant after incubation with high concentrations of
the various drugs.
As a control, the effect of drugs that do not participate in the MDR phenomenon, including methotrexate, metoprine, and the peptide ionophore alamethicin, was also examined. These cytotoxic agents had no effect on the ATPase activity even at high concentrations (Table 2). It should be emphasized that no drug-dependent stimulation of ATPase activity was detected in proteoliposomes reconstituted with microsomal proteins extracted from wild type AA8 cells.
We have examined the relationship between the
polarity of peptides and their ability to stimulate the Pgp-associated
ATPase activity. A series of bioactive linear tripeptides bearing
increasing polarity was chosen. At a 1 mM concentration, the
highly hydrophobic (i.e. due to formylated NH terminus and esterified carboxyl terminus) white blood cell
chemoattractants f-NLP-ME and f-MLP-ME potently stimulated Pgp ATPase
activity by 2.6- and 2.3-fold, respectively (Fig. 4C).
At a 1 mM concentration, the less hydrophobic calpain
inhibitors ALLN and ALLM (Fig. 4C), which are
established Pgp substrates(32) , as well as leupeptin, a polar
protease inhibitor increased the ATPase activity by 1.7-, 1.3-, and
1.2-fold, respectively (Fig. 4C). The less hydrophobic
tripeptides (Fig. 4C) and n-acetyl-Ala-Ala-Ala-p-nitroanilide failed to
increase the basal ATPase activity. These results suggest an inverse
relationship between the polarity of peptide substrates and their
ability to stimulate Pgp ATPase activity.
In summary, the effect of
Pgp substrates and chemosensitizers varied between partial inhibition
as displayed by gramicidin D and stimulation as shown with valinomycin,
dolastatin 10, verapamil, and progesterone, whereas some classical MDR
drugs such as colchicine, doxorubicin, and trimetrexate did not
modulate Pgp ATPase activity. As shown in Fig. 5, this complex
effects could be computer-simulated assuming competition between the
added substrates and a putative endogenous substrate responsible for
the basal Pgp ATPase activity. Substrates with a high V stimulate Pgp ATPase activity, those with a
low V
partially inhibit it, whereas substrates
with an intermediate V
appear silent as they do
not alter Pgp ATPase activity.
Figure 5:
Computer simulation of possible effects of
drugs on Pgp ATPase. Possible effects of Pgp substrates on its ATPase
activity were analyzed, assuming competition of the added substrate
with a putative endogenous substrate responsible for the basal ATPase
activity. The curves depicted were obtained using a two-term
version of , described under ``Experimental
Procedures.'' The K of both
substrates were assumed to be 1 mM and the V
of the endogenous substrate equal to twice the basal rate of
ATPase activity.
Figure 6: Competition between gramicidin D and various established Pgp substrates that stimulate its ATPase activity. Pgp-reconstituted proteoliposomes were incubated at 37 °C for 1 h in the following gramicidin D concentrations: 0 (closed circles), 4 (open circles), 16 (squares), 31 (triangles), 63 (inverted triangles), 125 (diamonds), and 250 µM (hexagons) in the simultaneous presence of increasing concentrations of verapamil (A), valinomycin (B), progesterone (C), dolastatin 10 (D), vinblastine (E), and emetine (F). The relative ATPase activity was then calculated as described in Fig. 4legend.
Figure 7: Lineweaver-Burk plots of ATPase activity in the presence of various concentrations of gramicidin D. The experimental data presented in Fig. 6describing the competition between gramicidin D and either verapamil or dolastatin 10 were redrawn as Lineweaver-Burk plots (panels B and D, respectively). The ATPase rates of Pgp observed in the presence of the various gramicidin D concentrations and in the absence of added stimulatory drug were subtracted from the corresponding rates observed in the presence of these drugs. The lines presented in panels B and D were obtained by linear regression analysis of the experimental data. Computer simulation of a competition on a common Pgp pharmacophore by three substrates including a putative endogenous substrate, an inhibitory substrate (i.e. gramicidin D), and a stimulatory substrate consisting either verapamil or dolastatin 10, are presented in panels A and C. The curves were generated using a three-term version of the equation described under ``Experimental Procedures'' after subtraction of the rates generated in the absence of stimulatory drugs. The gramicidin D concentrations were assumed to be similar to those in panels B and D.
In contrast, orthovanadate, a well established inhibitor of P-type ATPases and of Pgp (21) blocked 90% of the ATPase activity of Pgp-reconstituted proteoliposomes (Fig. 8); however, unlike the competitive inhibition of gramicidin D, the inhibition of Pgp ATPase by vanadate could not be reversed even with a 100-fold molar excess of the potent stimulator, valinomycin (Fig. 8).
Figure 8: The effect of vanadate on Pgp ATPase in the presence or absence of valinomycin. Pgp-reconstituted proteoliposomes were incubated in the presence of several concentrations of the P-type and Pgp ATPase inhibitor, orthovanadate, in the simultaneous presence of increasing concentrations of valinomycin. The relative ATPase activity was then determined as described in Fig. 4legend.
Several lines of evidence establish that gramicidin D is a substrate of the multidrug transporter, Pgp. (a) Single-dose exposure or stepwise selection with increasing gramicidin D concentrations result in a prominent MDR gene amplification and Pgp overexpression(36) . (b) Mammalian cells stably transfected with the human MDR1 cDNA display a typical MDR phenotype with a collateral resistance to gramicidin D(47) . (c) Various MDR cell lines obtained by selection to different established pleiotropic drugs exhibit a marked cross-resistance to gramicidin D (36, 48) ; this gramicidin D resistance was attributed to a consistent decrease in the number of gramicidin D channels in the plasma membrane (36, 49) . (d) We have recently described an ATP-driven transport of gramicidin D and valinomycin into proteoliposomes reconstituted with Pgp from rat liver or from highly MDR cells(15) . Surprisingly, however, instead of stimulating the Pgp ATPase activity like valinomycin and like a variety of other Pgp substrates, gramicidin D was inhibitory. Taking advantage of this ATPase inhibition by gramicidin D, a drug competition assay was devised in which well established Pgp substrates, which stimulate its ATPase activity, were examined for their ability to competitively overcome the gramicidin D inhibition of Pgp ATPase. We demonstrated that various established Pgp substrates including the cytotoxic agents valinomycin, vinblastine, dolastatin 10, and emetine, the chemotactic tripeptides f-NLP-ME and f-MLP-ME, the calpain tripeptide inhibitors ALLN and ALLM (data not shown), as well as the chemosensitizers verapamil and progesterone reverse the gramicidin D inhibition of Pgp ATPase. The basal ATPase activity and the reversal of gramicidin D inhibition of Pgp ATPase by its various substrates conformed to classical Michaelis-Menten competition. This competition involved an endogenous substrate, the inhibitory drug gramicidin D, and a stimulatory substrate. We therefore conclude that the various Pgp substrates including peptides, cytotoxic agents, and chemosensitizers compete for a common pharmacophore (i.e. drug binding site) present in Pgp.
The conclusion of a common drug pharmacophore present in Pgp agrees with several previous studies. 1) A wide spectrum of Pgp substrates including anticancer drugs and MDR chemosensitizers compete with various photoaffinity labels of Pgp(26) . 2) Various point mutations leading to single amino acid substitutions in mammalian Pgp markedly alter the original resistance pattern of MDR cells to multiple hydrophobic cytotoxic drugs that participate in the MDR phenomenon (27, 28, 29, 30, 31) . 3) Structure-activity relationship studies using a photoactivatable vinblastine analog as a probe together with a semi-synthetic series of structurally homologous reserpine and yohimbine analogues pointed out that the Pgp pharmacophore requires two planar aromatic domains and a basic nitrogen atom(50) .
A major question arises as to the
underlying basis for the surprising gramicidin D inhibition of Pgp
ATPase. The most likely explanation is that the bulky three-dimensional
helical structure of gramicidin D may be difficult
to handle by Pgp, thereby leading to a decreased translocation rate. In
this respect, this pentadecapeptide that spans a whole hemilayer (51, 52) is to date the largest Pgp substrate (M
1900) that was shown to be transported.
We
observe here a strong basal ATPase activity of 2.6 µmol of
P/min/mg of protein (K
= 7
s
) in Pgp-reconstituted proteoliposomes under
conditions where no exogenous substrate was added. We, as well as
others(17, 18, 19, 20, 21, 22, 23, 53) ,
have detected this basal ATPase activity in microsomes prepared from
Pgp-overexpressing MDR cells. Thus, it appears that the putative
endogenous substrate(s) is a lipid-soluble component present in lipid
bilayers. This prominent basal ATPase activity has been well documented
in plasma membrane vesicles from MDR cells (17, 53) and in functionally reconstituted
proteoliposomes(14, 20, 21, 22, 23) .
Thus, we here propose a mechanistic basis for this marked basal ATPase
activity involving an endogenous lipid-soluble substrate(s), which
continuously stimulates Pgp ATPase activity. In this respect, the
various lipids used for reconstitution studies are crude extracts
prepared from liver(23) , brain(21, 23) ,
soybean(15, 22, 23) , and Escherichia
coli(20) , all of which are rich in a variety of
hydrophobic compounds. Thus, lipid-soluble compounds copurifying during
lipid extraction will remain associated with the membrane of the
proteoliposomes. Some of these lipid-associated hydrophobic compounds
could be recognized as substrates by Pgp, thereby serving as endogenous
substrate(s) which continuously stimulate Pgp ATPase activity. The
latter property may physiologically be crucial, inasmuch as, in
vivo, the multidrug transporter is continuously exposed to
exogenous (i.e. from the diet) or endogenous Pgp substrates,
all of which are hydrophobic. Therefore, once extruded, they easily
diffuse back to the membrane, thereby continuously stimulating Pgp
ATPase.
The assay of competitive reversal of gramicidin D inhibition
of Pgp ATPase offers an excellent functional system for the rapid
screening of novel Pgp inhibitors, or alternatively, agents that are
recognized by Pgp. This functional assay is best suited for the
screening of potent MDR chemosensitizers as it combines
drug-modulatable ATPase activity and most importantly the ability to
obtain quantitative parameters including apparent K and V
for each drug and chemosensitizer.
Furthermore, their competitive inhibitory or stimulatory effects on Pgp
ATPase could be reliably and rapidly determined.
In the present
study, we used the eukaryotic protein synthesis inhibitor emetine,
which is an ipecac alkaloid currently used in the treatment of severe
invasive amebiasis(54, 55) . It was recently reported
that emetine-resistant Entamoeba histolytica variants
overexpress two mRNA species encoded by two P-glycoprotein genes
(EhPgp1 and EhPgp2; see (56) and (57) ). The open
reading frames for these EhPgps showed 40% positional identity with the
human mdr1 gene. Furthermore, a phylogenetic tree showed that Entamoeba P-glycoproteins are more related to mammalian Pgps
than to those from the parasitic protozoa Plasmodium and Leishmania. This prominent homology between EhPgps and
mammalian Pgp led us to examine whether the MDR phenotype emerges as a
major protective mechanism upon selection of mammalian cells with
emetine. Several lines of evidence support that emetine is an active
participant in the Pgp-dependent MDR phenotype. First, like various Pgp
substrates, emetine stimulated the ATPase activity of Pgp-reconstituted
proteoliposomes. Moreover, consistent with various MDR-type drugs,
emetine competitively reversed the gramicidin D inhibition of Pgp
ATPase in reconstituted proteoliposomes. Second, a dramatic MDR gene amplification was observed in Chinese hamster ovary cells
selected for stepwise resistance to emetine. Consistently,
emetine-resistant Entamoeba histolytica overexpressed EhPgps
mRNA. Third, Emt cells expressed extremely high levels of
Pgp and consequently displayed a typical MDR to multiple hydrophobic
cytotoxic agents. Fourth, Emt
cells failed to accumulate
rhodamine 123, a chromophoric substrate of Pgp; however, upon
coincubation with the established MDR chemosensitizer reserpine, a wild
type level of rhodamine 123 accumulation was resumed in Emt
cells (data not shown). We therefore conclude that acquisition of
a Pgp-dependent MDR phenotype in mammalian cells and in amebae is an
efficient means of functional protection against the cytotoxicity of
emetine.
We note here that, although valinomycin is a bona fide substrate of Pgp, the highly MDR Emt cell line, which
displays a strong MDR phenotype including a 100-fold resistance to
gramicidin D, exhibited only a modest cross-resistance to valinomycin.
We have previously documented various MDR cell lines and found them all
to be only marginally resistant to valinomycin but highly resistant to
gramicidin D(36) . Valinomycin, a cyclic decapeptide
mobile-carrier ionophore possesses a rapid diffusion rate across
membrane (10
to 10
s). In
contrast, the linear pentadecapeptide ionophore gramicidin D forms a
functional K
-channel only after two gramicidin D
monomers residing in opposite membrane leaflets undergo hydrogen
bonding and dimerization. Thus, the rate-limiting step in gramicidin D
channel formation is the slow (i.e. minutes; see (51) and (52) ) flip-flop from the outer hemilayer to
the inner membrane leaflet. Based on the turnover rate of Pgp ATPase at
maximal drug stimulation (K
= 15 ATP
s
; (23) and this paper) and an estimated
near stoichiometric substrate transport to ATP hydrolysis (see (58) ), the multidrug transporter can efficiently expel
gramicidin D monomers thus abolishing its cytotoxicity. In contrast,
valinomycin is 3 orders of magnitude more rapid in traversing the lipid
bilayer than the V
of Pgp ATPase (Fig. 3); valinomycin molecules extruded by Pgp rapidly diffuse
back into the plasma membrane and rapidly traverse it. Consequently,
Pgp overexpression confers upon MDR cells a strong resistance to
gramicidin D but only, if at all, a marginal protection from
valinomycin cytotoxicity. This study points out that peptide substrates
of Pgp, particularly ionophores, which are primarily confined to the
membrane, are an invaluable dissection tool for probing the
intramembranal mechanism of action of the mammalian multidrug
transporter.