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J Biol Chem, Vol. 274, Issue 40, 28420-28426, October 1, 1999
From INSERM, Unités 356 and 430, Université Pierre et
Marie Curie and the Hôpital Broussais (Assistance-Publique,
Hôpitaux de Paris), 75270 Paris, France
Extracellular lactic acid is a major fuel for the
mammalian medullary thick ascending limb (MTAL), whereas under anoxic
conditions, this nephron segment generates a large amount of lactic
acid, which needs to be excreted. We therefore evaluated, at both the functional and molecular levels, the possible presence of
monocarboxylate transporters in basolateral (BLMVs) and luminal (LMVs)
membrane vesicles isolated from rat MTALs. Imposing an inward
H+ gradient induced the transient uphill accumulation
of L-[14C]lactate in both types of vesicles.
However, whereas the pH gradient-stimulated uptake of
L-[14C]lactate in BLMVs was inhibited by
anion transport blockers such as The medullary thick ascending limb
(MTAL)1 is significantly
engaged in the active absorption of NaCl,
NH4+, and
HCO3 In most cells, transport of lactic acid occurs via monocarboxylate
transporters (MCTs) exhibiting the following properties: 1) cotransport
of H+ with a monocarboxylate (or its equivalent,
OH Although the mechanisms of lactate export and import have been
extensively investigated in a variety of tissues (for review, see Ref.
9), little information has been available concerning the mechanisms of
lactate transport by the distal nephron, and the molecular identity of
these transporters has not been established. To our knowledge, only one
study has been performed on the MTAL. Vinay et al. (10)
reported the presence of a
lactate/OH This study was initiated in an attempt to characterize, at both the
functional and molecular levels, the possible presence of
monocarboxylate transporters at the luminal and/or basolateral cell
surfaces of the MTAL. Isotopic flux studies performed on basolateral
(BLMVs) and luminal (LMVs) membrane vesicles isolated from rat MTALs
provide evidence for the presence of a CHC-sensitive lactate/H+ cotransporter restricted to BLMVs, whereas a
H+-independent organic anion exchanger has been detected in
LMVs. We also demonstrate, using immunofluorescence confocal microscopy and Western blot analysis of BLMVs and LMVs, that the recently cloned
MCT2 isoform (4) is specifically expressed on the basolateral domain of
the rat MTAL, whereas the MCT1 isoform could not be detected in this
nephron segment.
Preparation of MTAL Tubules
Male Harlan Sprague-Dawley rats (250-300 g) were anesthetized
with pentobarbital (50 mg/kg intraperitoneal), and the kidneys were
removed quickly, decapsulated, and sliced sagittally. The method used
for isolating MTAL tubules was the same as that previously described
(11, 12). Briefly, under stereomicroscopic control, the inner stripe of
the outer medulla was carefully excised. The resulting tissue was
subjected to collagenase treatment. In the final suspensions, no
significant activity of maltase, a marker of the proximal tubule
brush-border membrane, could be detected, whereas its specific activity
in homogenates from the immediately adjacent outer stripe of the outer
medulla was ~150 nmol/mg of protein/min, excluding significant
contamination of the starting material with tubule segments from the
outer stripe of the outer medulla.
Isolation of Plasma Membranes
Typically, the preparation began with 30-60 mg of protein from
MTAL tubules obtained from the kidneys of 20 rats. LMVs and BLMVs were
prepared from purified rat MTAL tubules as described previously in
detail (11, 12).
Apical membrane fractions were isolated from whole renal cortex using a
Ca2+ aggregation method (13). Differential and Percoll
gradient centrifugations were used for isolating basolateral membrane
fractions from whole renal cortex (14). Transport assays were performed after overnight storage of the vesicles at Isotopic Flux Measurements
L-[14C]Lactate and
36Cl Immunoblot Analysis
Fractionated basolateral and apical membranes were solubilized
and separated by SDS-7.5% polyacrylamide gel electrophoresis. Proteins
were transferred to nitrocellulose (Amersham Pharmacia Biotech). For
immunoblotting, nitrocellulose was first incubated in 10% nonfat dry
milk in phosphate-buffered saline (pH 7.4) for 1 h at room
temperature to block nonspecific binding of antibody, followed by an
overnight incubation at 4 °C with different diluted primary
antibodies. Membranes were then washed four times with phosphate-buffered saline containing 0.1% Tween 20 for 5 min each before incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA). Blots were washed as described above, and luminol-enhanced chemiluminescence (Amersham Pharmacia Biotech) was used to visualize bound antibodies on Polaroid film. Photographs of immunoblots were
numerized with NIH image software. For peptide inhibition experiments,
the anti-MCT1 and anti-MCT2 antibodies were preincubated with 100 µg/ml concentrations of the specific immunizing peptides prior to
immunoblotting basolateral membrane fractions.
Immunohistochemistry
Tissue Preparation--
Rat kidneys were fixed using 4%
paraformaldehyde in Dulbecco's modified Eagle's/Ham's F-12 medium.
Coronal kidney sections were incubated overnight at 4 °C in 4%
paraformaldehyde and then embedded in paraffin. Subsequently, 4-µm
sections of the paraffin block were deparaffinized, washed in graded
ethanol, rehydrated in Tris-buffered saline (pH 7.6) (TBS), and
subjected to microwave antigen retrieval.
Immunolabeling--
To reduce nonspecific binding, sections were
treated with background reducing buffer from Dako Corp. (Copenhagen,
Denmark). This was blotted away after 10 min, and the sections were
incubated with a 1:200 dilution of chicken anti-rat MCT2 peptide
polyclonal antibody (Chemicon International, Inc., Temecula, CA)
overnight in a humid chamber at room temperature. Sections were then
washed in TBS (3 × 5 min) and incubated with a 1:200 dilution of
biotinylated anti-chicken immunoglobulin (Vector Laboratories, Inc.,
Burlingame, CA), followed by Cy2-conjugated streptavidin (Amersham
Pharmacia Biotech) diluted 1:500 in TBS, each for 30 min at room
temperature with three TBS washes in between. Sections were mounted in
Glycergel solution (Dako Corp.). Slides were then observed using a
Leica TCS SP confocal laser microscope equipped with an ArKr laser
(excitation at 488 nm and detection at 502-601 nm). Control
experiments using anti-MCT2 antibody preadsorbed with a 100 µg/ml
concentration of the immunizing peptide revealed no labeling.
To ascertain the presence of MCT2 in medullary thick ascending limbs,
sections were dually labeled with anti-MCT2 antibody and an antibody to
NHE3, which specifically stains apical membranes of thick ascending
limbs within the inner stripe of the outer medulla (15). Microwaved
sections were first stained for MCT2 as described above. Sections were
then incubated with a 1:100 dilution of monoclonal anti-NHE3 antibody
(Chemicon International, Inc.), followed by Cy3-conjugated anti-mouse
immunoglobulin G (Amersham Pharmacia Biotech) diluted 1:200 in TBS,
each for 30 min with three TBS washes in between. Sections were then
washed in TBS (3 × 5 min). Sections were mounted and observed
using the confocal microscope. To avoid cross-talk (unwanted overlap
range) of emission spectra of Cy2 and Cy3, sequential scanning was
used. Cy2 was excited at 488 nm and detected at 502-601 nm, and then, on exactly the same field, Cy3 was excited at 568 nm and detected at
560-650 nm.
Materials
From Amersham Pharmacia Biotech, we obtained
L-[U-14C]lactic acid. H36Cl
obtained from NEN Life Science Products was titrated to
neutrality with tetramethylammonium (TMA) base to produce
TMA36Cl. Rabbit polyclonal antibody to NHE3 was a
gift of Dr. R. Alpern (University of Texas Southwestern Medical Center,
Dallas, TX). Mouse monoclonal antibody to NHE3 and chicken anti-rat
MCT2 peptide, anti-rat MCT1 peptide, and
anti-Na+/K+-ATPase polyclonal antibodies were
from Chemicon International Inc.
Statistical Methods
All data are represented as the means ± S.E., and error
bars in the figures also represent S.E. Comparison between groups was
generally carried out by ANOVA. For all analyses, statistical significance was accepted as p < 0.05.
Since outwardly directed OH To distinguish between these possibilities, we evaluated the effect of
CHC, a relatively specific inhibitor of H+-coupled
monocarboxylate transporters (16), on the initial rates of
L-[14C]lactate uptake under the same
conditions as in the Fig. 1 experiments. Fig.
2 shows that pH gradient-stimulated
L-[14C]lactate uptake in BLMVs was sensitive
to CHC inhibition with an IC50 of 63 ± 16 µM. In marked contrast, CHC, even at a concentration of 5 mM, had no effect on the pH gradient-stimulated
L-[14C]lactate uptake in LMVs. These results
are consistent with carrier-mediated lactate/H+ cotransport
(or lactate/OH
Polarized Expression of Different Monocarboxylate Transporters in
Rat Medullary Thick Limbs of Henle*
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyano-4-hydroxycinnamate,
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and
furosemide, it was unaffected by these agents in LMVs, indicating the
presence of a L-lactate/H+ cotransporter in
BLMVs, but not in LMVs. Under non-pH gradient conditions, however, the
uptake of L-[14C]lactate in LMVs was
transstimulated 100% by L-lactate, but by only 30% by
D-lactate. Furthermore, this L-lactate
self-exchange was markedly inhibited by
-cyano-4-hydroxycinnamate and DIDS and almost completely by 1 mM furosemide, findings consistent with the existence of a
stereospecific carrier-mediated lactate transport system in LMVs. Using
immunofluorescence confocal microscopy and immunoblotting, the
monocarboxylate transporter (MCT)-2 isoform was shown to be
specifically expressed on the basolateral domain of the rat MTAL,
whereas the MCT1 isoform could not be detected in this nephron segment.
This study thus demonstrates the presence of different monocarboxylate
transporters in rat MTALs; the basolateral H+/L-lactate cotransporter (MCT2) and the
luminal H+-independent organic anion exchanger are adapted
to play distinct roles in the transport of monocarboxylates in MTALs.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Extracellular lactate has been
shown to be a major fuel to support this large energy demand (for
review, see Ref. 1), indicating that lactate uptake is a crucial
process in the regulation of energy metabolism in this nephron segment.
Conversely, under anoxic conditions, the increase in lactic acid
production in the MTAL greatly exceeds the increase seen in other
nephron segments (2). There is thus a need for facilitating efflux of
glycolytically derived lactic acid from the MTAL.
/monocarboxylate exchange); 2) broad specificity for
short chain monocarboxylates, including lactate and pyruvate; 3)
inhibition by anion transport blockers such as
-cyano-4-hydroxycinnamate (CHC), DIDS, and phloretin; and 4)
transstimulation of L-lactate uptake by pyruvate,
L-lactate (but not by D-lactate), and other various substituted monocarboxylates. Seven different cDNAs have been identified that encode for MCTs in mammalians, designated MCT1 to
MCT7 (3-6). MCT1 and MCT2 show a broad tissue distribution and are
present in hamster (3, 4) and rat (7) kidneys. On the other hand, the
expression of rat MCT3 and MCT4 is restricted to the retinal pigment
epithelium (5) and muscle fibers (8), respectively. Three additional
MCT isoforms, MCT5, MCT6, and MCT7, have been identified in human
tissues (6).
(HCO3)
antiporter in preparations of basolateral membrane vesicles isolated
from the dog MTAL. That study, however, was derived from experiments in
membrane preparations that were more enriched with 
-glutamyltransferase, now recognized as a luminal marker of the MTAL
(11), than with Na+/K+-ATPase, a basolateral
marker. As a result, the allocation of a
lactate/OH(HCO3)
antiporter to the basolateral or luminal membrane domains and its
possible physiological role are not yet known.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
85 °C.
uptake in the membrane vesicles was
assayed at 20-25 °C by a rapid filtration technique. For each
experiment, the specific conditions are given in the figure legends.
The reaction was stopped with 1.4 ml of ice-cold stop solution
containing 20 mM Tris/Hepes (pH 7.40) and the desired
potassium gluconate concentration to maintain constant osmolarity. This
suspension was rapidly filtered on a 0.45-µm prewetted Millipore
cellulose filter (HAWP) and washed with an additional 16 ml of ice-cold
stop solution. In all experiments, vesicle uptake was corrected for
nonspecific isotopic binding to the filter. Radioactivity was
determined using a 
-scintillation counter.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gradients have been
reported to stimulate L-lactate uptake in partially
purified basolateral membrane vesicles isolated from the dog MTAL (10),
we investigated for the presence of this possible mode of lactate
transport in both BLMVs and LMVs. These experiments were conducted in
the presence of a valinomycin voltage clamp. As illustrated in Fig.
1, an inside alkaline pH gradient
(intravesicular pH (pHi) = 7.8, extravesicular pH
(pHo) = 5.5) stimulated the rate of L-[14C]lactate uptake compared with no pH
gradient (pHi = pHo = 7.8) and induced transient
accumulation of L-[14C]lactate to levels
~8- and 3-fold greater than equilibrium values in BLMVs and LMVs,
respectively. The pH gradient-stimulated uptake of lactate could be
explained by the transport of this anion via non-ionic diffusion of
lactic acid or via carrier-mediated H+/lactate
cotransport.
View larger version (19K):
[in a new window]
Fig. 1.
Time course of effect of H+
gradients on L-[14C]lactate uptake by BLMVs
and LMVs. Both types of vesicles were preincubated for 120 min
with pH 7.8 buffer containing 110 mM mannitol, 3 mM EGTA, 200 mM Tris/Hepes, and 100 mM potassium gluconate. Lactate (0.1 mM
L-[14C]lactate) uptake was then assayed by
1:11 dilution of vesicles into pH 5.5 buffer containing 100 mM mannitol, 3 mM EGTA, 100 mM
potassium gluconate, and 200 mM Tris/Mes. Uptake in the
absence of a pH gradient was determined by dilution of vesicles into
the reaction buffer identical to the preincubation buffer, except for
the presence of L-[14C]lactate. All vesicles
were pretreated for 120 min with valinomycin (10-15 µg/mg of
protein). Each data point represents the mean ± S.E. of six
experiments performed on two different BLMV and LMV preparations. When
S.E. is not shown, it was smaller than the symbols.
exchange) in BLMVs and with transport of
lactate via non-ionic diffusion of lactic acid in LMVs.
View larger version (21K):
[in a new window]
Fig. 2.
Inhibition of BLMV and LMV pH
gradient-stimulated uptake of lactate by CHC. BLMVs ( 
) and LMVs
(
) were preincubated for 120 min with pH 7.8 buffer containing
valinomycin (10-15 µg/mg of protein). Lactate (0.1 mM
L-[14C]lactate) uptake at 9 s was then
assayed by rapid 1:11 dilution of vesicles into pH 5.5 or 7.8 buffer
(see legend to Fig. 1 for composition of buffers). Values are
means ± S.E. of six determinations from different BLMV and LMV
preparations.
We next compared the effect of several less specific anion transport
blockers such as furosemide, DIDS, and phloretin (9) on the pH
gradient-stimulated transport of
L-[14C]lactate in BLMVs and LMVs. As shown in
Fig. 3, with the exception of phloretin,
DIDS and furosemide significantly inhibited the BLMV transporter. In
contrast, the pH gradient-stimulated transport of
L-[14C]lactate in LMVs was not significantly
inhibited by all of the tested agents. These findings confirm the view
that lactate/H+ cotransport is present only in BLMVs.
|
It is well established (3, 9, 17, 18) that MCTs have affinity for
several substituted monocarboxylates in addition to
L-lactate and H+ (or OH). To test
for monocarboxylates that might share the H+/lactate
cotransporter (or its equivalent, a OH/lactate
exchanger), we measured L-[14C]lactate uptake
in BLMVs in the presence of outwardly directed gradients of various
substituted and unsubstituted monocarboxylates (transstimulation).
These experiments were conducted at pH 7.8 under non-pH gradient
conditions using vesicles equilibrated with a highly buffered medium.
To further minimize possible alkalinization of the intravesicular
space, a valinomycin/FCCP pH clamp was used. As shown in Fig.
4, the uptake of
L-[14C]lactate in BLMVs was stimulated
183 ± 14% by prior incubation with unlabeled
L-lactate (lactate self-exchange) compared with the
gluconate non-gradient control, whereas D-lactate
stimulated L-[14C]lactate uptake by only
53 ± 8%. Because D-lactate is not available as a
sodium salt, these experiments were performed using either L-lactate as a lithium or sodium salt. The results were
identical; thus, the two sets of data were averaged. Prior loading of
BLMVs with other -substituted anions (pyruvate and
-hydroxybutyrate) and with -hydroxybutyrate also significantly
stimulated the uptake of L-[14C]lactate. In
contrast, we detected only marginal transstimulation with formate. The
other unsubstituted anions tested (acetate, propionate, and butyrate)
stimulated the uptake of L-[14C]lactate, but
the stimulation decreased progressively as the carbon chain length
increased from C2 to C4.
|
Because of the absence of lactate/H+ cotransport in LMVs,
we next considered whether an anion-exchange pathway for lactate may
represent an alternative transport system in these membranes. We tested
this hypothesis in two ways. In the first approach, we compared the
effects of outwardly directed L-and D-lactate gradients on the initial rates of
L-[14C]lactate uptake (transstimulation). As
shown in Fig. 5, the uptake of
L-[14C]lactate was stimulated nearly 100% by
prior incubation of LMVs with unlabeled L-lactate
(L-lactate self-exchange) compared with the gluconate
non-gradient control, but by only 30% with the D-isomer. Because these experiments were conducted under non-pH gradient conditions using a valinomycin/FCCP pH clamp and because L-
and D-lactate are stereoisomers with similar physical
properties, stimulation of L-[14C]lactate
uptake by an outward L-lactate gradient must be direct rather than secondary to alterations in pHi and/or
transmembrane potential. In the second approach, we examined the
possible effects of some anion transport blockers on
L-lactate self-exchange. As shown in Fig.
6,
L-[14C]lactate uptake was markedly inhibited
by DIDS and CHC and almost completely by furosemide. These findings are
therefore consistent with the presence of an anion-exchange pathway for
L-lactate in LMVs.
|
|
We next investigated whether additional mechanisms of lactate transport
were present in BLMVs and LMVs. Because the rat MTAL possesses both
luminal and basolateral Na+-independent
Cl/HCO3 exchangers (19),
we examined the effect of outwardly directed lactate gradients on
36Cl uptake under conditions in which the
vesicles were voltage- and pH-clamped. As indicated in Fig.
7, imposing an outward Cl
gradient stimulated by ~600% the rates of
36Cl influx in both BLMVs and LMVs compared
with their respective gluconate non-gradient controls, confirming the
presence Cl/HCO3
exchangers operating on the
Cl/36Cl exchange mode in both
preparations. In contrast, imposing an outward lactate gradient had no
significant effect on 36Cl uptake in BLMVs
and LMVs. Taken together, these results demonstrate that
L-lactate is not a substrate for the BLMV and LMV
Cl/HCO3 exchangers.
|
We also examined whether lactate could be cotransported with
Na+ across the basolateral and luminal membranes. As
illustrated in Fig. 8, for both
preparations, the uptake of L-[14C]lactate in
the presence of TMA+, used as a substitute for
Na+, slowly approached equilibrium. Imposing an inward
Na+ gradient caused no significant additional uptake of
L-[14C]lactate at the p < 0.05 level for incubation periods of 0.5, 1, and 3 min and failed to
induce an overshoot of this anion. Thus, these data argue against the
existence of direct coupling between the flux of Na+ and
the flux of L-[14C]lactate (i.e.
no Na+/lactate cotransport) in both BLMVs and LMVs.
|
Five independent preparations of BLMVs and LMVs were assessed by
immunoblotting for the presence of MCT1 and MCT2 polypeptides. As shown
in Fig. 9A, the
affinity-purified anti-MCT2 peptide antibody recognized a 37-kDa
protein that was enriched in basolateral membranes (B)
compared with luminal membranes (L). In contrast, MCT1 could not be detected neither in BLMV nor in LMV preparations isolated from
rat MTALs (Fig. 9A). In immunoblotting performed with apical and basolateral membrane-enriched preparations from rat renal cortex,
anti-MCT2 and anti-MCT1 antibodies recognized proteins with molecular
masses of 37 and 41 kDa, respectively, in basolateral, but not in
apical, membrane preparations. Labeling was blocked by the specific
immunizing peptides (data not shown). Blots were also probed with an
antibody to the Na+/K+-ATPase or with an
antibody against the Na+/H+ exchanger NHE3
(Fig. 9B), which serve as markers for basolateral and apical
membranes, respectively. Anti-NHE3 antibody reacted with a 85-kDa
protein that was enriched in luminal membranes compared with
basolateral membranes. Conversely, the
Na+/K+-ATPase -subunit identified as a
50-kDa protein was enriched in basolateral membranes and completely
absent from luminal membranes. These results confirm the view (11, 19)
that basolateral and luminal membranes isolated from rat MTALs are
largely separated from each other.
|
Fig. 10 shows the localization of MCT2
in the inner stripe of the outer medulla of the rat kidney using
indirect immunofluorescence on sections of paraformaldehyde-fixed
kidney embedded in paraffin. Anti-MCT2 antibody binds intensely to
thick ascending limbs and, to a lesser extent, to collecting ducts.
When anti-MCT2 peptide antibody was incubated with the specific peptide
antigen before application to a section, no binding of anti-MCT2
antibody was observed (data not shown).
|
To confirm the polarized expression of MCT2 in thick ascending limbs,
we performed dual labeling indirect immunofluorescence for MCT2 and the
apical NHE3 protein. In the inner stripe of the outer medulla, only the
apical membrane of the MTAL showed intense staining for NHE3. Anti-MCT2
antibody (Fig. 10A) labeled the same tubules more intensely
than did anti-NHE3 antibody (Fig. 10B). However, all
labeling of MCT2 did not spatially colocalize with that of NHE3 (Fig.
10C), indicating that MCT2 expression is restricted to the
basolateral domain.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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This study provides the first description, at both the functional and molecular levels, of distinct monocarboxylate transporters at the basolateral and luminal surfaces of rat MTALs. In this report, we found evidence compatible with the presence of a member of the MCT family in the basolateral membranes isolated from rat MTALs. First, an inside alkaline pH gradient induced uphill L-[14C]lactate accumulation (overshoot). Second, the pH gradient-stimulated transport of L-[14C]lactate was inhibited by CHC as well as by less specific anion transport blockers such as furosemide and DIDS. Third, L-[14C]lactate uptake was markedly transstimulated by prior equilibration of the vesicles with L-lactate, but only marginally with D-lactate. Fourth, the MCT2 isoform was shown by immunochemical methods to be expressed exclusively on the basolateral membranes of the MTAL, whereas MCT1 could not detected in this nephron segment.
In marked contrast, we found that L-lactate cannot be
transported across the luminal membrane in exchange for
OH (i.e. no lactate/OH exchange
or H+/lactate cotransport), but have detected the presence
of a carrier-mediated system for lactate transport in these membranes.
This conclusion is based on the following findings. First, the pH
gradient-stimulated transport of
L-[14C]lactate is completely resistant to
inhibition by anion transport blockers, suggesting that nonionic
diffusion of lactic acid is the mechanism underlying pH
gradient-stimulated lactate uptake. Second, under non-pH gradient
conditions, L-[14C]lactate uptake is markedly
transstimulated by prior equilibration of the vesicles with
L-lactate, but only marginally with D-lactate. Third, L-lactate self-exchange is sensitive to inhibition
by anion transport blockers such as DIDS, CHC, and furosemide.
Immunofluorescence confocal microscopy using antibodies to MCT1 and MCT2 revealed the presence of MCT2 at the basolateral surfaces of the MTAL, whereas MCT1 could not be detected in this nephron segment (Fig. 10). We also found that the antibody to MCT2 detected a band of 37 kDa on immunoblots of BLMVs isolated from rat MTALs, whereas this band was barely detectable on LMVs (Fig. 9). This apparent molecular mass of MCT2 on SDS-polyacrylamide gels is close to the value of 43 kDa reported by others on immunoblots from membranes of various hamster tissues (4). In agreement with our immunofluorescence microscopy experiments, Western blotting using the antibody to MCT1 showed no evidence for the presence of this isoform in both BLMVs and LMVs (Fig. 9). As a positive control, we found that the antibody to MCT1 detected a band of 41 kDa on immunoblots of basolateral membranes isolated from rat kidney cortex (Fig. 9). In apparent contrast with our data, previous immunofluorescent studies (4) have shown expression of MCT2 restricted to the basolateral surfaces of the inner medullary collecting ducts of the hamster kidney. Using immunofluorescence confocal microscopy, we have detected MCT2 not only at the basolateral surfaces of the MTAL (Fig. 10), but also at the basolateral domains of the inner medullary collecting ducts, cortical medullary thick ascending limbs, and distal convoluting tubules.2 These differences between the results of Garcia et al. (4) and those reported here for the localization of the MCT2 isoform in rat kidneys are not surprising. Indeed, as recently pointed out in detail by Jackson et al. (7), there are markedly different patterns of expression of MCT1 and MCT2 in hamster and rat tissues. For example, Garcia et al. (4) found MCT2 to be abundant in hamster heart, but not brain, whereas the opposite was found by Jackson et al. (7) in membrane fractions prepared from rat heart and brain.
The intense expression of the MCT2 polypeptide in the basolateral membranes of rat MTALs, as demonstrated here by immunochemical methods (Figs. 9 and 10), completely matches the robust functional expression of the H+/monocarboxylate cotransporter (Figs. 1-3). Conversely, the absence of MCT1 and MCT2 immunoreactivities in the luminal membranes of rat MTALs (Figs. 9 and 10) is consistent with our functional data demonstrating that carrier-mediated lactate transport in these membranes (Figs. 5 and 6) does not occur via operation of a H+/monocarboxylate cotransporter (Fig. 3).
Transstimulation experiments were used to investigate the substrate
specificity of the BLMV H+/lactate cotransporter (Fig. 4).
These studies were performed using vesicles equilibrated in a highly
buffered medium and in the presence of FCCP and valinomycin. It is
therefore highly unlikely that the organic acids tested caused
stimulation indirectly, i.e. either by alkalinizing the
intravesicular space via non-ionic diffusion and/or by generating an
interior positive. Consistent with this possibility is the observation
that L-lactate was more than three times as potent as the
D-isomer in stimulating
L-[14C]lactate uptake (Fig. 4). The BLMV
H+/lactate cotransporter (MCT2) is stereospecific and
exhibits a broad specificity for monocarboxylates, as expected for an
MCT isoform (9). L-Lactate and other -substituted anions
(pyruvate and -hydroxybutyrate) are the best substrates. The
possible importance of the 2-OH group in substrate recognition is
suggested by the observations that butyrate has no affinity for the
transporter and that -hydroxybutyrate is significantly less
effective than -hydroxybutyrate in stimulating
L-[14C]lactate uptake.
Since it is generally accepted that the proximal tubule depletes the tubular fluid in organic solutes and since MCT2 is restricted to the basolateral membrane of the MTAL (Figs. 9 and 10), it is likely that this isoform may play a major role in the uptake of lactate and other monocarboxylates. Consistent with this view, microperfusion studies indicated that lactate was effective to support Na+ transport only on the basolateral side of the cortical thick ascending limb (20), a nephron segment that exhibits a substrate preference comparable to that of the MTAL. The relatively high lactate concentration (10 mM) observed in rat medulla (21) probably maintains an inward lactate gradient sufficient to override the outward H+ gradient. The possibility exists, however, that MCT2 can mediate net lactate transport in either direction, depending on the physiological status and thus the actual driving forces. Under anoxic conditions, there is an increase in lactic acid production in all distal segments, and the maximum increase was observed in the MTAL (2), a situation that may be associated with marked intracellular acidification, which may damage the MTAL (2). Thus, a large thermodynamic driving force favoring net efflux of glycolytically derived lactic acid via MCT2 and via non-ionic diffusion is expected. The MTAL is particularly sensitive to anoxic damage because of the low medullary PO2 in the range of 10-20 mm Hg and the high rate of active NaCl transport (22). Thus, under anoxic conditions, stimulation of MCT2 activity may contribute to limit intracellular acidification by increasing efflux of glycolytically derived lactic acid. The resultant lactic acid accumulation in the medullary interstitium probably acidifies the interstitial fluid, which may stimulate H+ secretion in the adjacent collecting duct. Moreover, under normal conditions, lactic acid secretion in the tubular fluid across the luminal membrane via non-ionic diffusion is expected because there is no lactate in the lumen, and so, at least theoretically, there is a large gradient for lactate exit, and because pHi (23) is markedly lower than the pH of the tubular fluid (6.6 versus 7.4) (24).
The transport studies reported here on the luminal membrane vesicles
are preliminary and were designed to detect the presence in these
vesicles of an organic anion transporter, distinct from the
monocarboxylate transporters of the MCT family. Clearly, evaluation of
the physiological role of this new H+-independent organic
anion exchanger, demonstrated here, requires additional studies. With
the exception of HCO3, which is known
to be present at a non-negligible concentration (~20 mM)
in the tubular fluid of the rat MTAL (24), little information has been
available concerning the concentrations of other anions that might be
substrates for the newly described LMV anion exchanger. Regarding
HCO3, however, we have found, in
experiments not illustrated, that this anion is not a substrate for the
LMV organic anion transporter (i.e. no
lactate/HCO3 exchange). Recently, we
have also demonstrated that the BLMV H+/monocarboxylate
cotransporter has no affinity for HCO3
(25).
In conclusion, we have demonstrated the existence of distinct
monocarboxylate transporters in basolateral and luminal plasma membranes isolated from rat MTALs. The results of our
immunolocalization studies demonstrate that the basolateral transport
system is the H+/monocarboxylate cotransporter MCT2
isoform. Under normal conditions, MCT2 may be of special importance in
mediating the active uptake of L-lactate and of other
substituted monocarboxylates of physiological interest. Under hypoxic
conditions, MCT2 may limit the degree of intracellular acidification,
potentially damaging for the MTAL, by extruding glycolytically derived
lactic acid into the medullary interstitium. On the other hand, the
luminal anion exchanger identified in this report has not been
characterized molecularly, and its physiological function remains to be evaluated.
| |
FOOTNOTES |
|---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Established Investigator of INSERM. To whom correspondence should
be addressed: Inst. Biomédical des Cordeliers, 15 rue de l'Ecole
de Médecine, 75270 Paris Cedex 06, France. Tel.: 33/01-444137 10;
Fax: 33/01-44413717; E-mail: podevin@ccr.jussieu.fr.
2 D. Eladari, R. Chambrey, T. Irinopoulou, F. Leviel, F. Pezy, P. Bruneval, M. Paillard, and R.-A. Podevin, unpublished results.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
MTAL, medullary thick ascending limb;
MCT, monocarboxylate transporter;
CHC, -cyano-4-hydroxycinnamate;
DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
BLMV, basolateral
membrane vesicle;
LMV, luminal membrane vesicle;
TBS, Tris-buffered
saline (pH 7.6);
TMA, tetramethylammonium;
ANOVA, analysis of variance;
FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone;
Mes, 4-morpholineethanesulfonic acid;
NHE, Na+/H+ exchanger.
| |
REFERENCES |
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|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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