From the
Human erythrocytes parasitized with the malarial protozoan Plasmodium falciparum showed rates of L-lactate, D-lactate, and pyruvate uptake many fold greater than control
cells. Thus it was necessary to work at 0 °C to resolve true
initial rates of transport. Studies on the dependence of the rate of
transport on substrate concentration implied the presence in
parasitized cells of both a saturable mechanism blocked by
Once inside the red blood cell, the malarial parasite Plasmodium falciparum reproduces asexually, producing about 16
progeny every 48 h. Such rates of multiplication require intense
metabolic activity, in stark contrast to the meager metabolic
requirements of the uninfected erythrocyte. In particular, the energy
needs of the parasite, which is a homolactate fermenter, are
accompanied by a 20-100-fold increase in the rate of glucose
uptake and lactic acid extrusion by the infected
cells(1, 2, 3, 4, 5) . Since the
parasite is surrounded by three membranes, its own plasma membrane, the
parasitophorous vacuole membrane and the host cell plasma membrane, the
lactic acid must be transported across all three of them.
Alternatively, a parasitophorous duct might be present which would
allow direct access of the parasite to the extracellular
medium(6) , in which case only a transport mechanism across the
parasite plasma membrane would be required. Since glycolysis by the
parasite will produce lactic acid, this is most likely to be a
lactate/proton cotransporter as is now well established for the lactate
transporter of mammalian cells(7) .
If the parasitophorous
duct does not exist, a new pathway for lactic acid transport out of the
host cell would need to be induced in the host cell membrane since the
activity of the endogenous transporter is insufficient to account for
the increased fluxes of lactic acid observed in infected
cells(4, 7) . Studies from many laboratories have
demonstrated that the permeability properties of the host red blood
cell plasma membrane are substantially altered by the trophozoite stage
of infection. Thus, rates of entry of amino acids, glucose,
nucleosides, choline, anions, and cations into the infected red cell
are all increased, as is the permeability to other compounds, such as
mannitol, which are normally
impermeant(5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) .
It is probable that the enhanced permeability is caused by the export
of parasite-derived proteins to the host plasma membrane, but
parasite-induced changes in the properties of host cell plasma membrane
proteins such as Band 3 cannot be ruled
out(19, 20, 21, 22, 23, 24) .
It is also unclear whether there are a variety of new transport
pathways induced in the infected cell or whether a nonspecific channel
is opened which allows entry of any small molecule, but with a
preference for anions(18) . The enhanced entry of many different
compounds into infected cells is unsaturable and inhibited in a similar
manner by a range of inhibitors, many of which are chloride channel
blockers(4, 5, 13, 17, 18) .
However, in the case of nucleosides and choline, evidence has been
presented for the presence of two transport pathways in parasitized
cells, one saturable and one
not(12, 15, 16, 25) . A complication of
studying substrate transport into infected cells is that it is
difficult to discriminate between substrate uptake into the cytosol of
the red cell and uptake into the parasite itself. It is possible that
the saturable and unsaturable components of transport which have been
described represent these two processes.
In the present paper we
provide a detailed account of the characteristics of lactate and other
monocarboxylates transported into human red cells infected with P.
falciparum. We show the presence of both a saturable and an
unsaturable proton-linked transport mechanism in parasitized cells. Our
data are most easily interpreted if lactate and pyruvate have direct
access to the malarial parasite, perhaps via the proposed
parasitophorous duct or some close contact between the host cell and
parasite plasma membranes, with transport across the latter occurring
by both a proton-linked carrier (CHC
Recently donated A
Prior to measurement of transport, both
infected and uninfected cells were depleted of intracellular lactate by
two sequential incubations in bicarbonate-buffered saline for 30 min at
37 °C, as described previously(29) , before resuspending in
citrate buffer (78 mM sodium citrate, 15 mM HEPES, 1
mM EGTA, adjusted to pH 7.4 with Na
For measurement of pH
gradients and intracellular volumes the protocol was identical except
that
On-line formulae not verified for accuracy where L
In most cases data are presented as the means ± S.E.
of several separate experiments performed on separate cell cultures.
Statistical analysis of differences between data groups was performed
using either a paired or unpaired Student's t test as
appropriate.
On-line formulae not verified for accuracy Thus, the pH gradient determines the maximum uptake of lactate at
equilibrium. In our experiments we routinely measured the pH gradient
by replacing the 0.5 mM lactate by 0.5 mM DMO, which
should perturb the pH gradient to the same extent as lactate. The pH
gradient varied considerably from one experiment to the next but was
generally lower when parasitemia was high. However, the correlation was
not consistent, and variation in control cells was also considerable
from day to day. This inherent variation, and the use of saline media
in which little pH gradient is maintained, allowed us to study the
relationship between the intracellular concentration of lactate after
equilibrium had been reached with the pH gradient in both control and
parasitized cells. The data are presented in Fig. 4. It is clear
that in both cases the data fall on a similar line, strongly indicative
of a protogenic mechanism.
We have confirmed that the same pattern of inhibition
observed when L-lactate was used as substrate was also
observed when D-lactate was used. These data are also
presented in . We also investigated the effects of 40
µM phloridzin on the initial rate of transport of D-lactate transport into the infected erythrocytes and found
59.5 ± 4.0% inhibition. This concentration of inhibitor had no
effect on L-lactate transport into control erythrocytes.
The data we present in this paper confirm and greatly extend
the observations of Kanaani and Ginsburg(4) , who also
demonstrated a dramatic increase in L-lactate transport into
malaria-infected cells which was inhibited by CHC but not by PCMBS.
These authors were unable to demonstrate saturation of the transport
mechanism, but their time resolution for transport measurements was not
sufficient to allow determination of true initial rates. In contrast,
our measurements at low temperature did allow us to determine initial
rates of transport and demonstrated that there is a saturable component
of the transport mechanism. Our data ( Fig. 3and Fig. 4)
imply that this new lactate transport mechanism is protogenic like that
found in most other cells(7) . This is what might be predicted,
since the parasite's glycolytic pathway produces lactic acid
rather than lactate. Indeed, the V
In addition to the saturable component
there also appears to be a nonsaturable component, not blocked by CHC,
which becomes dominant at higher substrate concentrations. This may
represent either free diffusion of the undissociated acid or transport
via an anionic channel as has been suggested in other
cells(7, 35, 37) . Saturable and nonsaturable
components of the induced transport mechanism for choline and
nucleosides into parasitized erythrocytes have also been described by
some (12, 15, 16, 25) but not all
workers(13) . If an anionic pathway is responsible for the
nonsaturable component of lactate transport, it is unlikely to be that
described by Kirk et al.(18) since it is blocked by
neither CHC nor NPPB at concentrations that almost totally inhibit
Cl
A question that our data cannot resolve with certainty is the
location of the new transport pathway(s). For lactic acid to enter or
leave the parasite there are potentially three membranes that must be
crossed: the host cell plasma membrane, the parasitophorous vacuolar
membrane, and the parasite plasma membrane. Transport across all three
would have to be very rapid to keep up with the parasite's high
rates of glycolysis. However, an alternative possibility has been
suggested, which proposes that there is a parasitophorous duct that
allows the parasite direct access to the extracellular medium, without
the requirement for passage through the host cell cytosol(6) .
This proposal is still
controversial(40, 41, 42, 43, 44, 45, 46, 47) ,
but evidence in favor of this proposal has come from several sources.
First, confocal microscopy has shown the presence of such a
duct(6, 45, 46) , although this is not accepted
by all workers(41, 43) . Second, the ability of certain
iron chelators and phloridzin to inhibit parasite growth when added
directly to the extracellular medium, but not when incorporated into
the host cell cytosol by encapsulation, can best be explained by their
access to the parasite being direct rather than through the host
cell(48, 49, 50) . Third, antibodies added to
the outside of infected cells appear to have access to antigens located
on the parasitophorous vacuolar membrane (47). Fourth, 2-deoxyglucose
can enter the parasite even when the host cell glucose transporter and
the induced pore are both blocked by the presence of 50 µM cytochalasin B and 500 µM niflumic acid(51) .
Our own data are also most easily explained if there is direct access
of lactate to the parasite which it then enters via a CHC-inhibitable
proton-linked transporter (saturable) and CHC-insensitive diffusion of
the free acid (nonsaturable), both of which would be sensitive to the
transmembrane pH gradient. In the absence of the duct, lactate would
have to cross the host cell plasma membrane by means of the induced
permeability channel described above, followed by passage across the
parasitophorous vacuole membrane by means of the large nonspecific
channels present in this membrane(52) . Such a process would
probably lead to rapid equilibration of lactate with the host cell
cytosol before equilibration with the parasite cytosol, but we have
found no evidence for more than one compartment transporting lactate at
a rate sufficient to account for the observed rates of lactate efflux
during active metabolism. However, if the membranes of the host cell,
parasite, and parasitophorous vacuole are in close proximity this might
effectively allow direct access of lactate to and from the parasite
without equilibration with the host cytosol.
Whatever the pathway
that lactate takes to and from the parasite plasma membrane, it would
seem most likely that a protogenic carrier catalyzes the net efflux of
lactic acid out of the parasite as in the case in other highly
glycolytic cells(7, 39) . Indeed, in another blood-borne
protozoa, Trypanosoma brucei, there is strong evidence for a
specific proton-linked pyruvate transporter in the plasma membrane with
a K
The number of people at risk from malaria worldwide is more than 2
billion, with about 270 million new cases of the disease being reported
each year. Of these some 2 million a year will die, mainly from P.
falciparum, which is the most severe form of infection. These
numbers are set to increase as resistance to the most widely prescribed
drugs such as chloroquine becomes widespread(55, 56) .
The most important alternative anti-malarial strategy is the
development of vaccines. This has proved to be a more complex task than
first thought, although prospects remain hopeful(57) . However,
new approaches to chemotherapy are still required urgently(58) ,
and any biochemical feature of the parasite or the infected cell which
distinguishes it from a normal red cell is a potential target of drug
therapy. Whatever the nature and locus of the induced lactate transport
pathway are, the pathway could be a locus for future chemotherapeutic
intervention since its inhibition might cause intracellular lactic acid
accumulation and consequent impairment of parasite growth. Indeed,
Kanaani and Ginsburg (59) have shown that cyanocinnamate
derivatives do inhibit malarial parasite growth in culture, as do a
variety of other anion transport inhibitors such as phloretin, niflumic
acid, and phloridzin(60) , which we have shown also inhibit
lactate transport in parasitized cells. However, since these compounds
also inhibit the general increase in permeability of infected cells to
a range of substrates(18, 59, 61, 62) ,
they cannot necessarily be considered to act by causing lactic acid
accumulation.
The
competing substrate and 0.5 mM [
The
uptake of 0.5 mML- or D-[
The uptake of either 0.6 mML-[
-cyano-4-hydroxycinnamate (CHC) and a nonsaturable mechanism
insensitive to CHC. The former was dominant at physiological substrate
concentrations with K
values for pyruvate
and D-lactate of 2.3 and 5.2 mM, respectively, with
no stereoselectivity for L- over D-lactate. CHC was
significantly less effective as an inhibitor of lactate transport in
parasitized erythrocytes than in uninfected cells, whereas p-chloromercuribenzenesulfonate, a potent inhibitor in control
cells, gave little or no inhibition of lactate transport into
parasitized erythrocytes. Inhibition of transport into infected cells
was also observed with phloretin, furosemide, niflumic acid,
stilbenedisulfonate derivatives, and
5-nitro-2-(3-phenylpropylamino)benzoic acid at concentrations similar
to those that inhibit the lactate carrier of control erythrocytes.
These compounds were more effective inhibitors of the rapid transport
of chloride into infected cells than of lactate transport, whereas CHC
was more effective against lactate transport. This implies that
different pathways are involved in the parasite-induced transport
pathways for lactate and chloride. The transport of L-lactate
into infected erythrocytes was also inhibited by D-lactate,
pyruvate, 2-oxobutyrate, and 2-hydroxybutyrate. The intracellular
accumulation of L-lactate at equilibrium was dependent on the
transmembrane pH gradient, suggesting a protogenic transport mechanism.
Our data are consistent with lactate and pyruvate having direct access
to the malarial parasite, perhaps via the proposed parasitophorous duct
or some close contact between the host cell and parasite plasma
membranes, with transport across the latter by both a proton-linked
carrier (CHC-sensitive, saturable, and the major route) and free
diffusion of the undissociated acid (CHC-insensitive, unsaturable, and
a minor route).
(
)-sensitive,
saturable, and the major route) and free diffusion of the undissociated
acid (CHC-insensitive, unsaturable and a minor route).
Materials
L-[C]Lactate, D-[
C]lactate,
[1-
C]pyruvate,
H
O, and
Na
Cl were obtained from Amersham International, Amersham,
Bucks., U. K. [1-
C]Pyruvate was dissolved in
water and divided into 2.5-µCi aliquots that were freeze-dried and
stored at -20 °C. Stilbenedisulfonate derivatives were
obtained as described previously(26, 27) .
5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was obtained from
Research Biochemicals Inc., Natick, MA. Silicone oil MS550 and dinonyl
phthalate were obtained from BDH Chemicals, Poole, Dorset, U. K. Unless
stated otherwise, all other chemicals and biochemicals were obtained
from the sources given previously(28, 29) .
Acetazolamide was used as a 0.1 M stock solution in dimethyl
sulfoxide and stored at -20 °C.
4,4-Diisothiocyanostilbene-2,2`-disulfonate (DIDS) was made up freshly
prior to use as a 2.5 mM solution in H
O.
erythrocytes and human serum,
types A
, AB
, or AB
,
were obtained from the South West Regional Blood Transfusion Centre,
Southmead Hospital, Bristol, U. K. P. falciparum, strain ITO4,
used for all experiments, was a gift of Dr. Barry Elford, University of
Oxford, U. K.
Culture of Malaria-infected
Erythrocytes
P. falciparum, strain ITO4, was grown
under continuous culture conditions following the method of Trager and
Jensen(30) . Infected A erythrocytes were
cultured in RPMI 1640 medium containing 20 mM HEPES (Life
Technologies, Inc. Ltd., Paisley, U. K.), supplemented with 5.5 mM glucose (BDH), 2 mM glutamine (Sigma, Poole, U. K.),
hypoxanthine 12.5 mg/liter (Life Technologies, Inc.), gentamicin
sulfate (Life Technologies, Inc.) at 10 mg/liter and 10% human serum,
types A
, AB
, or AB
.
Infected cells were synchronized by either gelatin flotation or
sorbitol hemolysis and harvested, at the trophozoite stage, by gelatin
flotation(31, 32, 33) . Parasitemia was
estimated from methanol-fixed Giemsa-stained smears.
Radioactive Influx Measurements
We have
used the silicone oil centrifuge stop technique (7, 34) to measure the uptake of lactate and pyruvate
into control and parasitized erythrocytes. In all experiments the
substrate was labeled with C, and
H
O was also added to allow for correction of
total water (intracellular and extracellular water) in the pellet.
Parallel experiments were always performed to measure the intracellular
volume using [
C]sucrose and
H
O, and these values were used in conjunction
with the total cell water in the uptake experiment to convert total
C present in the pellet to intracellular
C
(7, 34). Measurements of intracellular pH were also made routinely
using 0.5 mM
[2-
C]dimethyloxazolidine-2,4-dione (DMO) (35) since lactate transport is usually proton-linked and is
driven by the pH gradient across the plasma membrane(7) .
Transport can thus be maximized by generating an alkaline intracellular
pH, and this was achieved in the present experiments through the use of
a citrate buffer (7, 36).
HPO4),
containing 100 µM acetazolamide. Cells were then incubated
with DIDS (5 µM) at 0 °C for 1 h to ensure complete
inhibition of lactate transport via the anion transporter, Band
3(36) . Uptake of
C-substrate was initiated by
rapidly mixing 100 µl of erythrocytes (5% hematocrit) with 10
µl of citrate buffer containing
C-radiolabeled
substrate (1 µCi/ml),
H
O (10 µCi/ml),
and unlabeled substrate at the required concentration. The resulting
110-µl sample was transferred to a 0.3-ml microcentrifuge tube
containing 100 µl of oil (specific gravity 1.035) (MS550 silicone
oil:dinonyl phthalate; 65:35 v/v) layered on top of 50 µl of 10%
(v/v) HClO
containing 25% (v/v) glycerol. The cells were
carefully placed just above the oil layer and uptake terminated at the
required time by centrifugation at 10,000
g for 30 s
in a Beckman Microfuge E. The cells sedimented through the oil layer
into the HClO
, and the tubes were subsequently frozen in
liquid N
. The tip containing the HClO
/glycerol
layer and cell pellet was cut off, placed in a scintillation vial, and
mixed with 800 µl of H
O until the pellet had thawed.
After the addition of 10 ml of scintillant (Packard Emulsifier Safe)
the vials were capped and vigorously vortex-mixed twice to ensure that
all radioactivity was released from the tube. A 20-µl sample of the
supernatant was treated in the same manner and radioactivity determined
by dual isotope scintillation counting.
C-substrate was replaced by 0.5 mM [2-
C]DMO or
[
C]sucrose (1 µCi/ml), respectively.
Intracellular
C was calculated as (
C-substrate-
H
O) space -
([
C]sucrose-
H
O) space as
described previously(37) .
Presentation of Results
The data for
infected erythrocytes were fitted by nonlinear least squares regression
analysis to first-order rate Equation 1 is the lactate uptake at time t, L
is the total uptake of lactate at
equilibrium, k is the first-order rate constant, and x is the dead time of centrifugation (the time taken to sediment the
cells through the oil layer into perchloric acid), which averaged 8 s.
The additional term p represents a component of transport into
infected cells which was linear with respect to time and may represent
uptake into a different intracellular compartment such as the host cell
cytosol or contaminating uninfected erythrocytes. The initial rapid
rate of substrate uptake behaved as if only a single intracellular
compartment (referred to simply as ``parasitized cells'') was
involved, and initial rates of transport were either calculated from k and L
. (i.e.k
L
) or from the uptake at 10 or 15 s
assuming uptake to be linear with respect to time over this period (see
below).
Time Courses of Lactate and Pyruvate Entry into
Control and Parasitized Erythrocytes
In Fig. 1we
show that when L-lactate uptake was measured at 25 °C
using the silicone oil centrifuge stop technique, the time course
obtained was similar to that measured previously for human erythrocytes
using centrifugation stop in the absence of silicone oil (36). However,
for parasitized cells the rate of uptake was greatly enhanced, to the
extent that resolution of initial rates was not possible. Similar
observations have been made by others(4, 18) . The
fastest practical time point for centrifugation stop was 10 s after the
addition of C-substrate. This represents a total uptake
time of about 18 s when the dead time is included. Other techniques
that might allow faster time resolution such as membrane filtration or
inhibitor stop (7, 34) are not appropriate for use with
parasitized cells. No suitable inhibitor was available for the former
technique, whereas for membrane filtration it is necessary to wash the
extracellular medium away from the cells on the filter, and this was
not possible without losing intracellular substrate(7) . Thus,
to resolve the initial rates of transport we decreased the temperature
for the uptake experiments to 0 °C. In Fig. 2data are shown
for the time courses of L-lactate, pyruvate, and D-lactate transport into control and parasitized cells at 0
°C. These data do allow initial rates of transport to be determined
and confirm the great enhancement of transport rates seen in the
parasitized cells when compared with control cells. The effect is most
noticeable for D-lactate, which is transported little if at
all by the normal red cell transporter(7, 38) , and
least for pyruvate, which is the best substrate for the native
transporter(7, 36, 38) . The lack of
stereoselectivity of the uptake into parasitized cells is confirmed in Fig. 2d, where parallel time courses of L- and D-lactate into parasitized cells were determined in the same
experiment. The rates of transport of the two isomers was
indistinguishable. These data also confirm that the dead time for
centrifuge stop is about 8 s and show that the initial rate of
transport can be considered linear for at least the first 15-30
s. In subsequent studies, depending on the substrate and its
concentration, uptake at 10, 15, or 30 s was used as an estimate of
initial rate. It seems likely that previous studies of lactate
transport into parasitized erythrocytes which utilized higher
temperatures (18) or less accurate time resolution (4) were not capable of resolving initial rates of transport and
probably underestimated the true transport rate. The Dependence of Lactate Transport on the pH
Gradient-The lactate transporter of uninfected erytrocytes,
and indeed most other mammalian cells, is a lactate proton
cotransporter (7) which suits the physiological setting in which
metabolism both produces and uses lactic acid(7) . Since the
enhanced lactate transport across the plasma membrane of the
parasitized erythrocyte is required to remove glycolytically derived
lactic acid from the cell, it would seem highly probable that the
induced transport mechanism(s) were also proton-linked. To test this
possibility we have compared the uptake of L-lactate into
parasitized cells in both citrate medium (pH gradient 0.75 unit of
alkaline inside) with those in a HEPES-buffered saline medium (pH
gradient 0.02 alkaline inside). The data are presented in Fig. 3.
Although the initial rates of transport were similar in both media
(0.58 ± 0.020 and 0.48 ± 0.029 nmol/µl of
intracellular space/min in citrate and saline media, respectively), the
uptake at equilibrium was enhanced severalfold in the citrate medium
compared with the saline medium. The uptake at equilibrium for a
proton-linked lactate transporter is given by Equation 2.
Figure 1:
Time
courses for the uptake of L-lactate into parasitized and
uninfected erythrocytes at 25 °C. The uptake of 0.5 mML-lactate into parasitized () and control (
)
erythrocytes was measured as described under ``Experimental
Procedures.'' Data are presented as means ± S.E. (error
bars) of three separate experiments and were fitted to a
first-order rate equation. The
pH values (mean ± S.E.) for
parasitized and control erythrocytes were 0.53 ± 0.1 and 0.97
± 0.14, respectively, and the parasitemia ranged between 39.4
and 78.2%.
Figure 2:
Time courses for the uptake of pyruvate
and L- and D-lactate into parasitized and uninfected
erythrocytes at 0 °C. The uptake of 0.5 mML-lactate (panels a and d), pyruvate (panel b), and D-lactate (panels c and d) into parasitized () and control (
) erythrocytes
was measured as described under ``Experimental Procedures.''
Data are presented as the means ± S.E. (error bars) of
three separate experiments and were fitted to a first-order rate
equation. The
pH values (mean ± S.E.) for parasitized and
control erythrocytes were 0.72 ± 0.05 and 1.01 ± 0.06,
respectively, and the parasitemia ranged between 40 and 90%. In panels a-c different preparations of parasitized cells
were used for each substrate, whereas in panel dL-lactate (
, dashed line) and D-lactate (
, solid line) uptake were measured
in paired experiments on the same preparations of parasitized
cells.
Figure 3:
Comparison of the time courses of L-lactate uptake into parasitized erythrocytes in citrate or
HEPES-buffered saline medium. The uptake of 0.5 mML-lactate into parasitized erythrocytes was measured at 0
°C in citrate medium () or HEPES-buffered saline medium
(
). Data are presented as means ± S.E. (error
bars) of three separate experiments and were fitted to a
first-order rate equation with a dead time set at 8 s as described
under ``Experimental Procedures.'' The initial rates
(± S.E. of the fit shown) were 0.58 ± 0.02 and 0.48
± 0.03 nmol/µl of intracellular space/min for cells
incubated in citrate and saline media, respectively. The corresponding
pH values (means ± S.E.) were 0.75 ± 0.05 and 0.02
± 0.01, respectively, and the parasitemia ranged between 70 and
80%.
Figure 4:
The
relationship between the equilibrium uptake of L-lactate and
the pH gradient for parasitized and uninfected erythrocytes. Data for
equilibrium uptake of L-lactate (after 5 min) into parasitized
erythrocytes at 0 °C (,
) or control erythrocytes at 25
°C (
,
) were taken from separate experiments performed
in citrate medium (
,
) or HEPES-buffered saline medium
(
,
). The line drawn is a linear regression of the data (r = 0.939, p = 1.27
10
).
The Concentration Dependence of D-Lactate
and Pyruvate Transport
The concentration dependence of the
initial rates of both pyruvate and D-lactate transport were
determined, and the data are presented in Fig. 5. For this
purpose the uptake of substrate at 10 s (pyruvate) or 15 s (D-lactate) was taken to represent initial rates of uptake,
and this was confirmed at both the highest and lowest substrate
concentration used. D-Lactate was used in preference to L-lactate in these experiments to avoid any possibility that
uptake into contaminating nonparasitized cells might interfere.
However, from the data of Fig. 2d it is clear that this
effect is likely to be small; in a single experiment in which the
concentration dependence of L- and D-lactate
transport was studied in parallel, the results were indistinguishable
(not shown). It should be noted that the uptake of substrate at these
short times was not great, and by necessity the resulting error on
replicate determinations was of the order of 20%. Furthermore, the
variable parasitemia in the parasitized erythrocyte preparation also
introduced some variation. For these reasons the results of several
experiments were meaned before determination of the kinetic parameters
of transport. For both D-lactate and pyruvate the initial rate
of transport was clearly not linear with respect to substrate
concentration at lower concentrations, but a linear relationship was
observed at higher concentrations. The data were best fitted to an
equation incorporating both a saturable component and a nonsaturable
component as described previously(37) . In the presence of 2
mM CHC, only the unsaturable component remained, suggesting
that this may represent either diffusion of the free undissociated acid
across the membrane or the presence of an anionic channel as observed
in both isolated heart and liver cells, especially at low
temperatures(7, 27, 35, 36) . The K values (± S.E. for the fit
shown) for pyruvate and D-lactate were 2.25 ± 0.45 and
5.20 ± 0.82 mM, respectively, whereas the corresponding V
value (at 0 °C) for pyruvate was 3.84
± 0.57 nmol/min/µl of intracellular space and was fixed at
the same value for D-lactate. These values compare with K
values for pyruvate, L-lactate, and D-lactate of 1.9, 9.1, and >50
mM in uninfected cells with a corresponding V
value (at 10 °C) of of 2.0 nmol/min/µl of intracellular
space. The V
of the lactate transporter of
uninfected cells at 0 °C can be calculated from the activation
energy for transport between 0 and 10 °C of 148
kJ
mol
to be 0.2 nmol/min/µl of
intracellular space. Thus, the maximal rate of carrier-mediated
transport is increased about 20-fold in parasitized cells.
Figure 5:
Concentration dependence of the initial
rate of pyruvate and D-lactate uptake into parasitized
erythrocytes. The uptake of [C]pyruvate (panel a) or D-lactate (panel b) was
measured after a 10- and 15-s incubation at 0 °C, respectively,
over which period uptake was linear with time (Fig. 2). Data are
presented as the means ± S.E. (error bars) of five
(pyruvate) or three (D-lactate) experiments in the absence (solid lines and closed symbols) or presence (dashed lines and open symbols) of 2 mM CHC.
The initial rates of transport were calculated assuming a dead time of
8 s. The data in the absence of CHC were fitted to the equation
describing the kinetics of a mechanism combining a saturable and
unsaturable component: v = V
[S]/(K + [S])
+ k
[S], where v is the initial
rate of transport at substrate concentration [S], V
is the maximal velocity of the carrier, K is the Michaelis constant, and k is the first-order rate
constant for the nonsaturable component. For pyruvate all parameters
were fitted by least squares regression analysis and yielded values for V
, K, and k (± S.E.) of
3.84 ± 0.57 nmol/min/ml of intracellular space, 2.25 ±
0.45 mM, and 0.45 ± 0.03 min
,
respectively. For D-lactate the V
was
fixed at the same value and the values for K and k determined as 5.20 ± 0.82 mM and 0.64 ±
0.02 min
, respectively. Data in the presence of 2
mM CHC were fitted by linear regression to give slopes of
0.616 ± 0.014 and 0.417 ± 0.023 min
for pyruvate and D-lactate,
respectively.
Substrate Specificity of the Induced Lactate
Transporter of Parasitized Erythrocytes
To investigate the
substrate specificity of the induced transport pathway we have studied
the ability of other monocarboxylates to inhibit lactate transport into
parasitized erythrocytes. We have previously used this technique
successfully in studies of the substrate specificity of the lactate
transporter of control erythrocytes and cardiac
myocytes(7, 29, 36) . The data are presented in , where results are also presented for control erythrocytes
incubated under identical conditions. The inhibition given by D-lactate and pyruvate (32.6 and 43.5%, respectively) are
close to the values of 30 and 50%, respectively, which would be
predicted for these substrates acting as competitive inhibitors if it
is assumed that the K and K
values are the same. This assumption is
valid for the lactate carrier of other cells(7) . The data of also demonstrate that 2-oxobutyrate and
2-hydroxyisobutyrate are substrates for the transporter of parasitized
cells, whereas only the former is a substrate of the carrier of control
cells as shown previously(7) .
Inhibitor Specificity of Lactate Transport into
Parasitized Erythrocytes
Work from this and other
laboratories has characterized a wide range inhibitors of the lactate
transporter of erythrocytes (see 7). These include the cyanocinnamates
such as CHC, stilbenedisulfonates such as DIDS, organomercurials such
as p-chloromercuribenzenesulfonate (PCMBS), and miscellaneous
compounds such as phloretin and niflumic acid. In the
effects of these inhibitors on the initial rate of L-lactate
uptake into parasitized erythrocytes at 0 °C are compared with
their inhibition of transport into control erythrocytes at 25 °C.
In the majority of cases the extent of inhibition was similar, but
there were some significant differences. Thus PCMBS, which is a very
potent inhibitor of lactate transport into control cells(38) ,
was almost without effect on transport into parasitized cells. CHC was
significantly less effective in the parasitized cells than control
cells, but it should be noted that the K of CHC for monocarboxylate transport is critically dependent
on both the transmembrane pH gradient and the
temperature(7, 39) . Although the inhibition (mean
± S.E.) by 2 mM CHC of the rate of transport of 0.5
mML-lactate and D-lactate was 86.1 ±
5.8% (10) and 79.8 ± 9.4% (n = 3),
respectively, that for 0.5 mM pyruvate was only 67.0 ±
4.1%(3) . At 5 mMD-lactate or pyruvate the
inhibition dropped to only 62.5 ± 5.4% (3) and 41.9
± 7.0%(3) , respectively (see also Fig. 5). These
results are consistent with CHC inhibiting only the carrier-mediated
monocarboxylate transport. Since the rate of the CHC-insensitive
transport increases linearily with substrate concentration (Fig. 5) this pathway becomes proportionately greater at higher
substrate concentrations. In contrast to CHC and PCMBS, furosemide, and
NPPB were substantially more effective at inhibiting transport into
parasitized cells than control cells. Nonetheless, it should be noted
that NPPB is still a very potent inhibitor of the monocarboxylate
carrier in control erythrocytes, the K
value,
determined using variable concentrations of NPPB at 0.5 mML-lactate as described previously(26) , being 8.5
± 0.9 µM (mean ± S.E. of five separate
experiments).
Comparison of the Relative Potency of Inhibitors
against Chloride and Lactate Transport into Parasitized
Erythrocytes
Kirk et al.(18) have
suggested that the increase in permeability of parasitized erythrocytes
to a wide range of compounds, including lactate, is the result of the
induction of an anion-selective channel in the erythrocyte plasma
membrane. The main evidence for this was that entry of a wide range of
compounds including lactate and chloride was inhibited with similar
potency by well established anion channel inhibitors. To test whether
the enhanced rate of entry of lactate and pyruvate could be accounted
for by such a mechanism we compared the inhibition by a range of
compounds of the initial rate of 0.6 mML-lactate and
Cl in paired experiments. The results are presented
in I. In these experiments Cl
(and
lactate) transport into uninfected cells mediated by Band 3 was totally
inhibited by the addition of 5 µM DIDS. The infected cells
showed greatly enhanced rates of Cl
entry in
agreement with Kirk et al.(18) . Indeed, from time
courses of Cl
uptake at 0 °C (data not shown) it
was found to be necessary to measure transport at 10 s to ensure that
true values for initial rates of transport were determined. The rate of
transport of 0.6 mM Cl
measured in this way
was 1.31 ± 0.03 nmol/µl/min (mean ± S.E. of five
observations) compared with a value of 0.84 ± 0.06 for L-lactate transport. The initial rate of Cl
transport was strongly inhibited by 2.5 µM NPPB, 25
µM niflumate, 200 µM DIDS, and 25 µM furosemide. Surprisingly, 2.5 mM pyruvate and 5
mML-lactate also inhibited the initial rate of
Cl
uptake by 27.4 ± 6.6 and 39.2 ±
8.7%, respectively (I). In contrast Cl
does not appear to inhibit the rate of lactate transport since
the initial rates observed in citrate medium (no Cl
)
and saline medium (150 mM Cl
) were very
similar (0.58 ± 0.020 and 0.48 ± 0.029 nmol/µl
intracellular space/min, respectively; Fig. 3). When the effects
of inhibitors were tested on the initial rates of lactate and
Cl
transport in paired experiments (I)
it was found that for 200 µM DIDS and 25 µM
furosemide, the inhibition of both processes was of a similar
magnitude. For 2.5 µM NPPB and 25 µM niflumate the inhibition of lactate transport was significantly
less than for Cl
, whereas 200 µM CHC
gave sigificantly greater inhibition of lactate transport than that of
Cl
. At 2 mM CHC inhibition of L-lactate and Cl
transport was 88.3 ±
5.0 and 96.6 ± 4.7%, respectively, whereas for 100 µM NPPB, which totally inhibited chloride transport, the inhibition
of D-lactate transport was 83.4 ± 4.3%. Thus, the
residual lactate transport that occurs in the presence of CHC or NPPB
is unlikely to be due to the induced anion channel proposed by Kirk et al.(18) . Taken together, these data imply that
lactate and Cl
do not enter the parasitized cell by
an identical mechanism, although they do not rule out some lactate
entering by the same mechanism as Cl
.
of the
transporter is similar to that determined for Ehrlich Lettre tumor
cells, which are also almost exclusively glycolytic for their energy
requirements(39) .
transport into infected cells. This conclusion is
strengthened by the demonstration that there are inhibitors such as CHC
which inhibit L-lactate transport into the parasitized cells
more than Cl
, whereas others, such as NPPB and
niflumate, are less potent toward lactate than Cl
.
for pyruvate of about 2
mM(53) , similar to the value reported here for the
malarial protozoa. The trypanosomal carrier is also inhibited by
cyanocinnamate derivatives and DIDS with a potency similar to that
observed P. falciparum(54) . However, the trypanosomal
carrier appears to be specific for pyruvate and other keto acids and is
not inhibited by either L- or D-lactate. This is
entirely consistent with the metabolism of T. brucei, which
produces and excretes pyruvic acid as an end product of glycolysis.
Substantial differences in the affinity of different isoforms of the
mammalian lactate carrier for pyruvate and L- and D-lactate are well documented(7) , and thus the
trypanosomal and malarial monocarboxylate carriers may be related.
Table: Inhibition of L-lactate uptake into
control and parasitized erythrocytes by other monocarboxylates
C]lactate were added simultaneously and
transport terminated by centrifugation after 15 s at 0 °C for
parasitized cells and 90 s at 25 °C for control cells. Data are
presented as the means ± S.E. of four separate experiments for
which mean parasitemias (percent trophozoites) varied from 66 to 69%.
Table: Inhibition of L-lactate uptake into
control and parasitized erythrocytes by various inhibitors
C]lactate was measured as described
in Table I. Inhibitors were added at the concentrations shown 1 min
before the addition of lactate. The pH gradient was shown to be
unaffected by any of the inhibitors used. All data are expressed as
mean values ± S.E. for n experiments for which mean
parasitemias (percent trophozoites) varied from 62 to 76%. The
statistical significance of differences between inhibition of control
and parasitized cells was calculated using a two-tailed Student's t test (*p < 0.01,**p < 0.001).
Table: Comparison of the inhibition by various
compounds of L-lactate and chloride transport into parasitized
erythrocytes
C]lactate (30 s) or
Cl
(10 s) into parasitized cells at 0
°C was measured in the presence and absence of inhibitor at the
concentration shown. Both lactate and Cl
uptakes were
performed on the same cell preparations (parasitemias ranged from 48 to
72%). Control rates of transport assuming linearity of uptake with time
and a deadtime of 8 s were 0.84 ± 0.06 and 1.31 ± 0.03
nmol/µl/min for lactate and chloride, respectively. The ratio of
the inhibition of lactate to Cl
transport was used to
assess significant differences in inhibitor potency using a paired
Student's t test (*p < 0.05,**p < 0.02,***p < 0.01).
-cyano-4-hydroxycinnamate; DIDS,
4,4`-diisothiocyanostilbene-2,2`-disulfonate; DBDS,
4,4`-dibenzamidostilbene-2,2`-disulfonate; DNDS,
4,4`-dinitrostilbene-2,2`-disulfonate; PCMBS, p-chloromercuribenzenesulfonate; NPPB,
5-nitro-2-(3-phenylpropylamino)benzoic acid; DMO,
dimethyloxazolidine-2,4-dione.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.