(Received for publication, May 18, 1995; and in revised form, July 13, 1995)
From the
Verapamil increases the net uptake and cytotoxicity of structurally diverse hydrophobic molecules in many multidrug-resistant mammalian cell lines. This compound has also been reported to reverse chloroquine resistance in the human malaria parasite Plasmodium falciparum (Martin, S. K., Oduola, A. M. J., and Milhous, W. K.(1987) Science 235, 899-901). Although the mechanism of this [Medline] reversal is unknown, it apparently involves an increase in the amount of chloroquine present in erythrocytes infected with the resistant parasites. Chloroquine is a diprotic weak base that accumulates in acidic organelles as a function of the pH gradient present between the organelle and the external medium. By changing the external medium pH, this property of chloroquine was used to alter the cytotoxicity phenotype of genetically chloroquine-sensitive and -resistant trophozoites. Verapamil was also found to be toxic for malaria trophozoites, although this toxicity was independent of external pH and consistently about 3-4-fold higher against resistant strains. When verapamil was tested for its effects on chloroquine cytotoxicity under conditions of phenotypic reversal, it was still found to exert only a measurable effect on the genetically resistant trophozoites. In short time incubations, verapamil was found to increase net chloroquine accumulation in erythrocytes infected with both chloroquine-sensitive and -resistant organisms, but only to affect the chloroquine susceptibility of the latter. Analysis of our data demonstrates that verapamil works independently of the overall pH gradient concentrating chloroquine into a trophozoite's lysosome. Instead, we propose that it inhibits the activity of a membrane ion channel indirectly responsible for determining chloroquine transit within the parasite's cytoplasm.
Human falciparum malaria (caused by an intracellular parasite of the genus Plasmodium) remains a serious disease in much of the tropical and subtropical world. There are at least 300 million new infections every year causing an estimated 2 million deaths mostly of young children. Due to its specificity, stability, and safety, chloroquine has been one of the most successful and widely used antimalarial drugs (reviewed in (2) ). The biological activity of chloroquine is directed against the intraerythrocytic stage of Plasmodium infection. During this stage, Plasmodium falciparum trophozoites obtain much of their amino acid requirements by degrading erythrocyte hemoglobin within an acidic food vacuole, a specialized organelle with some resemblance to mammalian lysosomes(3, 4, 5) . Heme is released during this process and rendered into a nontoxic, crystalline polymer called hemozoin or malaria pigment (6, 7) . Chloroquine is believed to exert its specific antimalarial effects by inhibiting this polymerization process(8, 9) . Accumulation of toxic amounts of heme substrate is probably then ultimately responsible for parasite cell death.
The evolution and geographical spread of P.
falciparum trophozoites resistant to chloroquine has greatly
reduced the clinical effectiveness of this
compound(2, 10, 11) . Identification of the
biochemical mechanism responsible for chloroquine resistance would
therefore assist in the development of alternative chemotherapeutic
strategies. The heme polymerizing activity contained in extracts of
both chloroquine-resistant and -sensitive trophozoites has similar
sensitivity to inhibition by chloroquine(2) , while a chemical
modification of chloroquine by resistant organisms has never been
demonstrated(12) . ()Therefore, the mechanism of
resistance must involve either the differential sequestration and/or
uptake and transport of chloroquine within the parasite.
Many
investigators have associated a reduced accumulation of chloroquine in
infected erythrocytes with the acquisition of chloroquine resistance,
suggesting that resistant trophozoites lower their lysosomal
chloroquine concentration below that necessary to disrupt hemoglobin
catabolism. Two mechanisms to account for this reduction in
accumulation of chloroquine have been proposed. The weakened proton
pump model postulates an increase in vacuolar pH leading to reduced
weak base-driven accumulation of chloroquine (a diprotic weak base)
into the lysosomes of resistant parasites(13, 14) .
Although plausible, the central predication of this model concerning
changes in vacuolar pH have not been detected, and it remains
controversial. Similarities to mammalian tumor cell multidrug
resistance (MDR), ()chiefly the reversal of chloroquine
resistance by drugs such as verapamil (a Ca
channel
blocker) and phenothiazines lead Martin et al.(1) to
propose that the two phenomena were similar(1) . However,
correlations between either mutation or overexpression of known malaria mdr genes and chloroquine resistance have not been detected in
genetic selection experiments.
The reversing effect of verapamil on chloroquine resistance has been confirmed in numerous studies; however, its biochemical target(s) and mechanism of action in malaria trophozoites are unknown. Bray et al.(15, 16) have shown that verapamil increases chloroquine accumulation in resistant parasites. To further understand the biochemical mechanism behind this observation, we have used the weak base phenomena to phenotypically alter the chloroquine susceptibility of both genetically chloroquine-sensitive and -resistant trophozoites. Verapamil exerted no effect on the toxicity of chloroquine for sensitive trophozoites under any experimental conditions examined, while it continued to increase the chloroquine susceptibility of resistant organisms even when rendered phenotypically sensitive by weak base manipulation. Verapamil was also found to induce a saturable increase of net chloroquine accumulation into both chloroquine-sensitive and -resistant organisms, while its mechanism of reversing chloroquine resistance is shown to be separable from any direct toxic action. We conclude that verapamil specifically increases chloroquine accumulation in genetically resistant P. falciparum trophozoites independently of weak base processes and phenotypic susceptibility due to external pH manipulations.
Martin et al.(1) reported that nontoxic verapamil concentrations
reverse chloroquine resistance and yet have no effect on chloroquine
susceptibility of sensitive strains. By using pH manipulations to reverse phenotype, we found that the verapamil
reversal effect is specific for chloroquine-resistant genotype, but
independent of actual chloroquine sensitivity. For RCQ parasites, 1
µM verapamil reduced chloroquine IC
regardless of whether their phenotypic susceptibility was
hyper-resistant (for example, 584 nM at pH
6.55)
or sensitive (for example, 29 nM at pH
7.8; 15
nM at pH
8.50) (Fig. 1). The
verapamil-induced increase in RCQ chloroquine susceptibility is
therefore independent of whether the parasites are phenotypically
hyper-resistant, resistant, or susceptible to chloroquine. On the other
hand, identical verapamil concentrations have no effect on genetically
chloroquine-sensitive parasites, even under conditions in which they
are phenotypically resistant. For example, at pH
6.35, the
SCQ strain has an IC
of 210 nM independent of the
presence of verapamil (Fig. 1). Furthermore, the decrease in RCQ
chloroquine IC
by verapamil is remarkably similar at all
pH
values, regardless of actual IC
. 1
µM verapamil reduced RCQ chloroquine IC
an
average of 73 ± 6% across all pH
tested. On the
other hand, verapamil had no effect on SCQ chloroquine IC
(average change of -4 ± 9%). These data demonstrate
that the reversal of chloroquine resistance by verapamil is specific
for parasite genotype and not phenotype.
Figure 1:
Effect of verapamil on the chloroquine
susceptibility of P. falciparum trophozoites as a function of
extracellular pH. Parasites were co-cultured at increasing chloroquine
concentrations in the presence of either 1 µM verapamil or
equivalent volumes of media. Data are expressed as the drug
concentration necessary for 50% growth inhibition (IC).
RCQ data are shown in open circles, SCQ in open
squares. 1 µM verapamil addition data are depicted as filled symbols. The lines represent the nonlinear
least squares fit to the data.
Figure 2: Susceptibility of P. falciparum to verapamil. Parasites were cultured in the presence of increasing concentrations of verapamil. Verapamil dose-response curve under normal growth conditions for RCQ (open circles) and SCQ (open squares). Data are expressed as percent growth inhibition in the presence of increasing verapamil concentration. A representative experiment is shown; data points are mean ± S.E., and the lines represent the best fit log dose-response curve.
These findings raise the following questions. 1) Is reversal of
chloroquine resistance an indirect consequence of differential
verapamil toxicity (as concentrations of verapamil used in these and
other published studies are primarily within toxic ranges for Dd2
parasites? (2) Can the chloroquine IC of sensitive
parasites be lowered in the presence of more verapamil? To address
these issues we measured chloroquine IC
in the presence of
increasing verapamil concentrations (Fig. 3). RCQ chloroquine
IC
decreases with increasing verapamil concentrations
until it plateaus at 10 nM in the presence of 5 µM verapamil. Although verapamil toxicity continues to increase,
suppressing parasite growth by 90% at 15 µM, no further
changes in chloroquine IC
are detected. The
chloroquine-sensitive D10 parasites, on the other hand, undergo no
changes in chloroquine IC
as a function of verapamil
concentration, even at highly toxic verapamil concentrations
(20-30 µM). Therefore, the chloroquine resistance
reversing property and direct toxic actions of verapamil are not
related. Furthermore, reversal of chloroquine resistance is not an
indirect outcome of further stressing a
``verapamil-weakened'' parasite, as toxic concentrations of
verapamil do not alter chloroquine sensitivity in the SCQ strain.
Figure 3:
Changes in chloroquine susceptibility as a
function of increasing external verapamil. Parasites were co-cultured
at increasing chloroquine concentrations in the presence of various
verapamil concentrations. Data are expressed as the chloroquine
concentration necessary for 50% growth inhibition (IC).
RCQ data are shown in closed circles, SCQ in closed
squares. The lines represent the nonlinear least squares
fit to the data. Each data point represents mean ± S.D. of at
least two different experiments.
Figure 4: Chloroquine accumulation in trophozoite-infected erythrocytes. Trophozoite-infected or uninfected erythrocytes were incubated with 4 nM radiolabeled chloroquine alone (A) or in the presence or absence of 1 µM verapamil (B). A shows chloroquine accumulation data for uninfected erythrocytes (open triangles), SCQ (open squares), and RCQ (open circles) as a function of time. The line represents the linear regression fit to the data. Each point represents mean ± S.D. of at least two experiments. B is a representative experiment of RCQ chloroquine accumulation in the presence (filled circles) or absence (open circles) of 1 µM verapamil. Control accumulation into an uninfected erythrocyte is shown as filled triangles. Each point represents mean ± S.E. of at least two samples. The line represents the linear regression fit to the data.
Figure 5: Chloroquine accumulation rates as a function of verapamil concentration. Chloroquine accumulation rates over a 10-min period for SCQ (filled squares) and RCQ (filled circles). The lines represent the nonlinear least squares fit to the data. Each data point represents mean ± S.D. of at least two different experiments.
Interestingly, chloroquine accumulation does not correlate perfectly
with chloroquine sensitivity. At pH 7.35, RCQ parasites are
4-5-fold less sensitive to the toxic effects of chloroquine but
accumulate only about 2-3-fold less drug. Manipulation of the
weak base effect by alteration of pH
results in chloroquine
accumulation variations. Even under conditions in which resistant
parasites accumulate more chloroquine than sensitive trophozoites (Fig. 6), chloroquine sensitivity is still markedly higher in
SCQ trophozoites (chloroquine IC
of 35 ± 5 at
pH
6.7 versus RCQ IC
of 90 ±
10 at pH
of 7.6).
Figure 6:
Chloroquine accumulation in
trophozoite-infected erythrocytes as a function of time and external
conditions. Infected and uninfected erythrocytes were incubated with 4
nM radiolabeled chloroquine. Chloroquine accumulation in RCQ
parasites (open circles) was measured in alkalinized media
(pH 7.6) and accumulation into SCQ parasites (open squares) in acidified media (pH
of
6.7). Each point represents mean ± S.E. of at least two samples
of a representative experiment. The line represents the linear
regression fit to the data.
Figure 7: Kinetics of chloroquine accumulation in trophozoite-infected erythrocytes. Chloroquine accumulation data for SCQ trophozoites (open squares) and RCQ trophozoites (open circles) as a function of time. The lines represent the linear regression fits to the data. Each data point represents mean ± S.D. of at least two data points.
Chloroquine-sensitive and -resistant P. falciparum trophozoites develop apparently identically within human erythrocytes and produce similar amounts of hemozoin pigment. As chloroquine resistance does not appear to involve a change either in the process of hemoglobin proteolysis, heme polymerization, or drug metabolism, resistance can only be achieved by lowering the effective concentration of chloroquine within the trophozoite's food vacuole(2) . Chloroquine and related antimalarial quinolines are hydrophobic weak bases known to concentrate within low pH vacuoles(24) . For example, Ginsburg and Geary (25) estimate that at nanomolar external concentrations (pH 7.2) diprotic chloroquine can reach millimolar levels within a trophozoite lysosome (estimated pH in the range 4.8-5.2). Three different mechanisms by which chloroquine-resistant trophozoites could reduce the amount of chloroquine that accumulates in this organelle can be envisaged. 1) Reducing the magnitude of the weak base effect by raising lysosomal pH, 2) rapid efflux of chloroquine from the parasite cytoplasm or lysosome, 3) trapping a charged form of chloroquine in the cytoplasm such that the rate of entry into the lysosome is slowed.
We and others have shown that the toxicity of chloroquine for both
chloroquine-sensitive and -resistant trophozoites is dependent on
pH, as predicted by the weak base theory of drug uptake.
Assuming that the vacuolar pH of chloroquine-sensitive trophozoites is
about 4.8, then changing the pH gradient from 3 (pH
= 7.8) to 2 (pH
= 6.8) results in a
4-fold increase in chloroquine IC
. Under physiological
conditions (pH
= 7.35), chloroquine-resistant
trophozoites typically display a 5-10-fold decrease in their
chloroquine sensitivity. To account for this by a direct change in the
pH gradient from external medium to lysosome would clearly require a
vacuolar pH in excess of 6.0. However, attempts to directly measure
even a small difference in lysosomal pH between chloroquine-sensitive
and -resistant trophozoites have so far been unsuccessful(21) .
The results of Goldberg et al.(3, 4) also
indicate that the rate of lysosomal hemoglobin degradation would be
markedly slower if vacuolar pH values were above 6.0, again
inconsistent with the equal rates of hemozoin formation observed in
chloroquine-sensitive and -resistant trophozoites.
Evidence for the
existence of a rapid efflux mechanism for the selective expulsion of
chloroquine from erythrocytes infected with chloroquine-resistant but
not chloroquine-sensitive trophozoites has been presented, although the
finding is controversial (summarized in (2) and (11) ). Using the D10 and Dd2 strains of chloroquine-sensitive
and chloroquine-resistant P. falciparum, we have only been
able to detect a small and inconsistent difference in the rate of
chloroquine efflux between the two strains. For example, after loading
trophozoites for 45-60 min at pH 7.35 with 4 nM chloroquine, average drug efflux after 10 min was 32 ± 14%
from SCQ trophozoites (n = 6) and 43 ± 3% from
RCQ trophozoites (n = 4). (
)This agrees with
the results of Bray and colleagues (13, 14, 15, 16) that chloroquine
resistance correlates with net chloroquine uptake rather than rapid
drug efflux from infected erythrocytes. Furthermore, our observation
that 2 µM verapamil increases net chloroquine uptake about
2-fold in both SCQ and RCQ trophozoites also argues against the
importance of a verapamil-inhibitable chloroquine efflux mechanism
specific for chloroquine-resistant trophozoites.
By manipulating
pH, we have found that the chloroquine cytotoxicity
phenotype of genetically chloroquine-sensitive and -resistant
trophozoites can be reversed. Verapamil lowered the chloroquine
IC
of RCQ trophozoites about 75% irrespective of whether
the trophozoites were forced to be phenotypically sensitive (pH
> 7.8) or hyper-resistant (pH
> 6.8). In marked
contrast, the chloroquine susceptibility of SCQ trophozoites was
completely refractory to coincubation with verapamil, even when they
were made phenotypically resistant by lowering pH
below
6.5. Therefore, the verapamil-inhibitable mechanism of chloroquine
resistance is specific for genetically chloroquine-resistant parasites
and does not simply result from a change in the pH gradient from
external medium to lysosome.
In RCQ trophozoites exposed to
verapamil, the decrease observed in chloroquine IC only
depends on verapamil concentration and not on the rate of chloroquine
accumulation. A likely explanation for this observation is that a given
amount of verapamil inhibits a constant fraction of the activity of
some undefined protein. If verapamil acted to reduce vacuolar pH
(thereby increasing the rate of chloroquine accumulation by directly
raising the magnitude of the pH gradient between external medium and
lysosome), it would be expected to proportionately exert a more
pronounced effect on chloroquine cytotoxicity when the pH gradient was
small. However, this is clearly not consistent with our data. It is
much more likely that the protein target for verapamil modulates
chloroquine accumulation and intracellular partitioning independent of
vacuolar pH, and that the reversing agent blocks the activity of the
same fraction of this putative protein irrespective of the pH gradient
present. Consistent with this proposal, in the presence of 1
µM verapamil, the chloroquine IC
of RCQ
trophozoites responds to changes in pH
in a similar way to
SCQ parasites cultured without the drug (Fig. 1). We conclude
that the overall weak base pH gradient driving chloroquine
concentration from the external medium into the lysosome of a
trophozoite is not the site for verapamil reversal of chloroquine
resistance.
Given the greater sensitivity of chloroquine-resistant trophozoites to the direct toxic effects of verapamil, it has been proposed that the synergism observed between chloroquine and verapamil is due to a nonspecific ``weakening'' of the resistant parasite by the later compound, indirectly making them more sensitive to chloroquine. However, ultrastructural studies have shown that the early morphological changes characteristic of chloroquine toxicity in a sensitive strain of P. falciparum are also apparent in resistant trophozoites upon simultaneous exposure to verapamil and chloroquine, arguing against two different mechanisms for cell killing (26) . Our data confirm this by demonstrating that verapamil-weakened SCQ trophozoite remain completely refractory to any changes in their susceptibility to chloroquine. The direct toxic effects of verapamil therefore appear to be unrelated to its reversal properties. However, it would be interesting to test whether other chloroquine resistance reversal agents, such as desipramine and cymetidine, also demonstrate differential toxicity between strains.
The site and molecular identity of the verapamil-sensitive target in
chloroquine-resistant trophozoites is unknown. One way in which a
trophozoite could exploit the weak base effect to become
chloroquine-resistant is to reduce the pH of its cytoplasm, analogous
to an experimental reduction in external pH. This would increase the
fraction of protonated chloroquine molecules present and thereby reduce
the rate of further passage of the unprotonated form into the lysosome.
For example, a reduction of cytoplasmic pH by 0.1 unit would decrease
uncharged chloroquine by 22%; a further 0.1 change would result in 37%
less uncharged chloroquine available for entry into the vacuole.
Several published reports have documented cytoplasmic pH
(pH) alterations in mammalian tumor MDR cell lines. For
example, the steady state efflux of the weak base doxorubicin has been
shown to strongly correlate with pH
(27) . Possible
mechanisms of pH
alteration include modifications in
Cl
, H
, or
HCO
transport and other indirect changes
in membrane potential and/or cell volume. Overexpression of murine MDR1
in CHO fibroblasts results in an inhibition of Cl
and
HCO
-dependent pH
homeostasis
concomitant with the drug resistance phenotype (28, 29) . A Cl
ion conductance
associated with the MDR protein has been
observed(30, 31) , while others have shown that a
monoclonal antibody raised against MDR1 inhibits chloride conductance
in pancreatic zymogen granules, but recognizes a different protein of
65 kDa(32) . The transmembrane movement of Cl
ions is an important component in the regulation of
pH
, volume, and membrane potential in mammalian cells, and
it has recently been proposed that tumor multidrug resistance mediated
by P-glycoprotein occurs secondary to perturbations in these
parameters(33) . Verapamil could thus act by
``correcting'' an anomalous Cl
conductance.
Indeed, evidence that verapamil reverses the altered
Cl
-dependent pH
behavior of
MDR-transfected mammalian cell lines has been presented(34) .
Members of the ATP-binding cassette (ABC) family of transmembrane
transporters such as the multidrug resistance (MDR) and cystic fibrosis
transmembrane regulator (CFTR) proteins have been associated with
increased Cl
channel activity. This channel regulator
function is separate from any putative transport
activities(35) . We therefore hypothesize that the mutation
conferring chloroquine resistance in P. falciparum up-regulates a Cl
channel regulator protein,
which alters ion conductances that indirectly control drug transit
within the parasite's cytoplasm by setting the cytoplasmic pH. It
is this chloride channel regulator which is inhibited by verapamil
during reversal of resistance. Interestingly, we have recently found
that changes in the ionic composition of the external medium, as well
as the presence of specific blockers of several known pH and volume
regulators, alters chloroquine uptake into erythrocytes infected with
chloroquine-resistant trophozoites. (
)Resolution of the role
of ion channels in drug transport is critical both to the further
understanding of chloroquine resistance and to the ultimate goal of
pharmacological intervention intended to overcome this problem.