Laboratoire de Physiologie et Endocrinologie Cellulaire Rénale, Faculté de Médecine Broussais-Hôtel Dieu, Institut National de la Santé et de la Recherche Médicale, Unité 356, 75270 Paris, France
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ABSTRACT |
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We studied the
pathways for HCO3 transport in
basolateral membrane vesicles (BLMV) purified from rat medullary thick
ascending limbs (MTAL). An inward HCO
3 gradient in the presence of an inside-positive potential stimulated the
rate of 22Na uptake minimally and
did not induce a 22Na overshoot,
arguing against the presence of electrogenic
Na+-HCO
3
cotransport in these membranes. An inside-acid pH gradient stimulated
to the same degree uptake of
86Rb+
(a K+ analog) with or without
HCO
3. Conversely, applying an outward
K+ gradient caused a modest
intracellular pH (pHi) decrease
of ~0.38 pH units/min, as monitored by quenching of
carboxyfluorescein; its rate was unaffected by
HCO
3, indicating the absence of
appreciable
K+-HCO
3
cotransport. On the other hand, imposing an inward
Cl
gradient in the presence
of HCO
3 caused a marked pHi decrease of ~1.68 pH
units/min; its rate was inhibited by a stilbene derivative. Finally, we
could not demonstrate the presence of a
HCO
3/lactate exchanger in BLMV. In
conclusion, the presence of significant
Na+-,
K+-, or lactate-linked
HCO
3 transport could not be
demonstrated. These and other data suggest that basolateral Cl
/HCO
3
exchange could be the major pathway for HCO
3 transport in the MTAL.
medullary thick ascending limb; bicarbonate; lactate; membrane vesicles
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INTRODUCTION |
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THE CELLS OF THE MEDULLARY thick ascending limb (MTAL)
are significantly engaged in absorption of filtered
HCO3. In the rat and mouse MTALs, the
first step of this HCO
3 absorption is
believed to be mediated virtually completely by apical membrane
Na+/H+
exchangers (NHEs) (reviewed in Ref. 11), presumably via operation of
the NHE3 (1, 3) and/or the NHE2 (9, 29) isoforms.
In contrast, little is known about the specific pathways implicated in
exit of HCO3 across the basolateral membrane of these cells. Indeed, conflicting results regarding the
presence or absence of basolateral
Na+-HCO
3
cotransport have been obtained in whole tubule experiments by measuring
intracellular pH (pHi). Kikeri et al. (19) proposed that an electrogenic
Na+-nHCO
3
cotransporter mediates base exit in suspensions of mouse MTAL (s-MTAL).
However, a subsequent study (20) failed to obtain similar results in
rat s-MTAL. In the latter study, it was concluded that those cells
possess an electroneutral
Na+-independent
K+-HCO
3
cotransporter that is inhibited by DIDS. There is also conflicting
evidence for a basolateral
Cl
/HCO
3
exchanger in whole tubule experiments. Kikeri et al. (19) and Leviel et
al. (20) failed to detect Cl
/base exchange in rat and
mouse MTAL suspensions by measuring pHi. More recently, basolateral
Na+-independent and
Cl
-dependent
HCO
3 transport that was inhibited by
DIDS has been reported in isolated, perfused mouse MTAL by monitoring
pHi in response to changes in
peritubular fluid composition (28). However, in view of the multiple
pathways for HCO
3 and
H+ transport in the MTAL, it is
difficult to establish, with certainty, direct coupling between fluxes
of ions on a given plasma membrane by measuring
pHi.
We recently demonstrated the presence in basolateral membrane vesicles
(BLMV) purified from rat MTAL of a
Na+-independent,
stilbene-sensitive
Cl/HCO
3
exchanger activated by an internal pH-sensitive modifier site (10). It
was the purpose of these studies to reexamine directly, using these
plasma membrane vesicles, the other possible basolateral membrane
HCO
3 transport pathways. We were
unable to detect substantial coupling of either
Na+,
K+, or lactate flux to
HCO
3 gradients, indicating that
Na+-HCO
3
and
K+-HCO
3
cotransporters and a lactate/HCO
3 antiporter postulated to reside in the basolateral membrane of the MTAL
are not important for net
HCO
3 absorption. These
results indicate that the main physiological basolateral HCO
3 extrusion pathway in rat MTAL may
be a Na+-independent
Cl
/HCO
3 exchanger.
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METHODS |
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Preparation of MTAL tubules. Male
Sprague-Dawley rats (250-300 g) were anesthetized with
pentobarbital (50 mg/kg intraperitoneal), and the kidneys were removed
quickly, decapsulated, and sliced sagittally. Slices were transferred
in fresh iced (0-4°C) Hanks' modified medium containing (in
mM) 115 NaCl, 0.4 MgSO4, 0.5 MgCl2, 0.4 KH2PO4,
0.3 Na2HPO4,
25 NaHCO3, 10 HEPES, 4 KCl, 1.2 CaCl2, 5 glucose, and 5 L-leucine, as well as 1 mg/ml
bovine serum albumin, pH 7.40 (bubbled with 95%
O2-5%
CO2). Under stereomicroscopic control, the inner stripe of the outer medulla, recognized by its
reddish color, was carefully separated from each slice by removing
completely the outer part of the outer medulla as well as the inner
medulla. The resulting tissue was subjected to collagenase treatment as
previously described (3, 5). In the final suspensions, most of the
tubules (>95%) proved to be MTAL in origin, based on
immunofluorescent staining with Tamm-Horsfall protein (3), a specific
marker for the thick ascending limb. Moreover, we were unable to detect
significant activity of maltase, a marker of the proximal tubule
brush-border membrane, in homogenates from s-MTAL, whereas its specific
activity in homogenates from the immediately adjacent outer stripe of
the outer medulla averaged 160 ± 7 nmol · mg
protein1 · min
1
(n = 5 in each group), 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 15-20 mg protein of MTAL
tubules obtained from the kidneys of 10 rats. Luminal membrane vesicles
(LMV) and BLMV were prepared from purified rat MTAL tubules as recently
described in detail (3, 5). We have recently demonstrated that the BLMV
and LMV preparations have a right-side-out orientation (5). Compared with homogenate, the basolateral marker activity of
Na+-K+-ATPase
was enriched ~9-fold in the BLMV and only 0.5-fold in the LMV. In
contrast, the apical marker activity of -glutamyltransferase was
enriched >10-fold in the LMV and ~1.5-fold in BLMV. Utilizing 22Na+
uptake determinations, we have also recently evaluated the purity of
the membrane vesicle preparations in functional terms. In these studies, we detected bumetanide-sensitive
Na+-K+-2Cl
cotransport activity only in LMV, and not in BLMV (24).
Basolateral membranes were isolated from the superficial rat renal cortex by differential and Percoll gradient centrifugations, as described previously (8). Compared with the starting homogenate, the purification of these BLMVs was 10- to 15-fold, on the basis of enrichment in Na+-K+-ATPase.
Transport assays were performed after overnight storage of the vesicles
at 85°C.
Isotopic flux measurements.
22Na+,
86Rb+,
and [14C]lactate
uptakes into the membrane vesicles were assayed at ambient temperature (20-25°C) by a rapid filtration technique. Media were gassed
for at least 120 min with either humidified 100%
N2 or 5%
CO2-95% N2 as specified in the legends to
Figs. 1-9. For each experiment, pH and total
CO2 of incubation solutions were
measured. For highly buffered media that were used in the radioactive
experiments, it was found that total
CO2 and pH were stable for at
least 30 min after stopping equilibration with the appropriate gas.
Total CO2 values measured with a
CO2 analyzer (Ciba-Corning,
Halstead, UK) were inferior to 0.1 and 0.5 mM in nominally
HCO3-free solutions at pH 6.0 and 7.8, respectively. The vesicles were preequilibrated at room temperature for
2 h to load with desired constituents. For each experiment, the
specific conditions are given in the legends to Figs. 1-9. In
general, a 10-µl aliquot of plasma membrane vesicles (10-40 µg
protein) was added to 100-300 µl of appropriate reaction medium
containing either
22Na+,
86Rb+,
or [14C]lactate (~1
µCi/ml). Incubation periods of 9 s were timed with a metronome and
used to estimate initial rates. The reaction was stopped with 1.5-ml
ice-cold solution containing 20 mM Tris-HEPES, pH 7.4, and the desired
potassium gluconate concentration to maintain constant osmolarity. This
suspension was rapidly filtered on the center of a 0.45-µm prewetted
Millipore cellulose filter (HAWP) and washed with an additional 16 ml
ice-cold stop solution. In all experiments, vesicle uptake was
corrected for nonspecific isotopic binding to the filter. The filters
were dissolved in 3 ml of scintillant (Filter-count,
Packard), and radioactivity was determined using a beta scintillation counter.
Carboxyfluorescein fluorescence
measurements. The fluorescent dye carboxyfluorescein
(CF) was chosen for pHi
measurements because of its negligible leakage rate and because of the
availability of an anti-CF antibody (kindly provided by Dr. M. L. Zeidel), which permits complete quenching of extravesicular CF. This
method is also advantageous because, in contrast to the widely used
acridine orange method, the rate of change in fluorescence was constant over a wide range of membrane protein concentrations and unaffected by
the presence of amiloride and DIDS. For these experiments, BLMV and LMV
were prepared exactly as described previously (3, 5), except for the
presence of 0.5 mM CF in the homogenization buffer. Both types of
vesicles were washed by centrifugation at 400,000 g for 60 min at 0°C in a large
excess of an appropriate preincubation buffer (indicated in text) to
remove extravesicular CF. The pellets thus obtained were resuspended
(~1 mg/ml) in the preincubation buffer by passage through a 27-gauge
needle and equilibrated for 2 h at room temperature until used. The
vesicles and the media were bubbled with either 5%
CO2-95%
N2 or 100%
N2 prior to the fluorescence
measurements and gassed at the surface of the cuvette during the
fluorescence measurements. Fluorescence of the labeled vesicle
suspensions was measured at 20-25°C in a Jobin-Yvon
spectrofluorometer. All experiments were performed in the presence of
an excess anti-CF antibody to eliminate completely extravesicular
fluorescence. Excitation and emission wavelengths were 490 and 525 nm,
respectively, and 10-nm slits were used. Usually,
pHi measurements were performed by
adding 20 µl (corresponding to ~20 µg membrane protein) of either
BLMV or LMV to a cuvette containing 2 ml of the indicated stirred
medium. Calibration of pHi vs.
fluorescence was carried out for each experiment by adding 20 µl of
labeled vesicles in a cuvette containing 2 ml of the indicated stirred
medium. For experiments using
K+-loaded vesicles, calibration
was based on the nigericin-high-K+
technique of Thomas et al. (30). In brief, vesicles were subjected, in
the presence of nigericin (2 µM), to stepwise decrements in extravesicular pH (pHo) (over a
pHo range of 7.35-6.4) with
concentrated gluconic acid, resulting in quenching of entrapped CF.
Disruption of vesicles with 0.4% Triton X-100 resulted in almost
complete quenching of CF. Assuming that the ionophore sets
H+i/H+o = K+i/K+o
(where "o" indicates extravesicular and "i" indicates
intravesicular), we estimated an apparent
pHi value from the linear
correlation between relative fluorescence and pH. A typical experiment
is shown in Fig. 1. Increasing threefold
the vesicle membrane protein concentration did not affect the
calibration of the dye.
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In experiments designed to evaluate
Cl/HCO
3
exchange activity, a comparable linear correlation between relative fluorescence and pH was obtained using the protonophore FCCP (4 µM),
which sets H+i = H+o (data not shown).
dpHi/dt
represents the initial rate of change in
pHi calculated in the first 9 s
after addition of the vesicles. The intravesicular buffer capacity was
determined in vesicles preequilibrated in the presence and absence of
CO2/HCO
3
using the technique of rapid sodium acetate addition (21) where 25 mM
tetramethylammonium (TMA) acetate replaced TMA gluconate.
The acetic acid in the TMA acetate solution rapidly enters the
vesicles, leading to immediate vesicle acidification with no
significant pHi recovery for at
least 20 s.
Statistics. All data are represented as means ± SE. Comparisons among groups were carried out by ANOVA. Statistical significance was defined as P < 0.05.
Materials. From Amersham, we obtained L-[U-14C]lactic acid, carrier-free 22NaCl, and 86RbCl. DIDS was obtained from Research Organics (Cleveland, OH). All other reagents were from Sigma Chemical (St. Louis, MO).
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RESULTS |
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Na+-dependent
HCO3 transport in BLMVs purified
from s-MTAL and cortical tubules.
If an electrogenic
Na+-nHCO
3
cotransporter is present in MTAL BLMVs, then imposing an inwardly
directed HCO
3 gradient in the presence
of an inside-positive membrane potential should lead to a marked
stimulation of
22Na+
uptake. As can be seen in Fig. 2A,
however,
22Na+
uptake at 9 s was stimulated only minimally (36%) by an inwardly directed HCO
3 gradient
(pHi
6.0/pHo 7.8, HCO
3) and an inside-positive potential
compared with the same conditions but in the nominal absence of
HCO
3 (64 ± 3.1 vs. 47 ± 2.3 pmol · mg
protein
1 · 9 s
1,
P = 0.02). In the presence of inwardly
directed HCO
3 gradients,
22Na+
uptake was significantly inhibited by amiloride, an inhibitor of the
Na+/H+
exchangers, and at a lesser degree by DIDS, an established anion transport inhibitor (control, 64 ± 3.1; vs. 32 ± 4 and 48 ± 6 pmol/mg protein for amiloride and DIDS, respectively). These data suggest that, in addition to an
Na+/H+
exchanger, these membranes might also contain a
HCO
3-dependent and DIDS-sensitive
component of Na+ uptake. Because,
however, inward gradients of HCO
3 stimulated the rate of 22Na uptake
only marginally in these membranes, we performed control experiments
using BLMVs isolated from rat renal cortex, which contain a robust
Na+-nHCO
3
cotransporter (13). As shown in Fig.
2B,
imposing a HCO
3 and a pH gradient, under the conditions of Fig. 2A, in
these membrane vesicles, stimulated 22Na+
uptake 326% compared with the same experimental conditions but in the
nominal absence of HCO
3, a stimulation nearly 10 times greater than that we detected in the BLMV prepared simultaneously. Another major difference between the two types of BLMVs
was the differential sensitivity of
22Na+
uptake by basolateral membranes purified from the renal cortex to
amiloride and DIDS. The HCO
3
gradient-driven 22Na+
influx was inhibited 67% by DIDS (434 ± 58 vs. 143 ± 12 pmol/mg protein, P < 0.001), whereas amiloride had no significant effect. It should be noted
that, in the above-described experiments, the transmembrane
Na+ gradient was
unfavorable for operation of the
Na+/H+
exchangers due to preincubation of the membrane vesicles with 1 mM
disodium DIDS.
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Effects of
H+ and
HCO3 gradients on
86Rb+
uptake and intravesicular acidification.
To determine whether a K+-coupled
HCO
3 transporter was present in
basolateral membrane from the rat MTAL, we first tested whether a
transmembrane HCO
3 gradient would be
capable of driving
86Rb+
uptake, used as an analog of K+.
As shown in Fig. 5, an outward
H+ gradient
(pHi = 6.0, pHo = 7.8) in
CO2/HCO
3-buffered solutions stimulated
86Rb+
uptake by 56 ± 19 and 72 ± 12%, compared with uptake values
observed under non-pH gradient conditions at pH 6.0 and 7.8, respectively. However, imposing the same inside-acid pH gradient in the
absence of added
CO2/HCO
3
stimulated
86Rb+
uptake to the same degree (67 ± 16 and 54 ± 11% vs. non-pH
gradient conditions at pH 6.0 and 7.8, respectively), indicating that
the H+-activated
86Rb influx was on a
HCO
3-independent pathway. Figure 5
also shows that this H+-activated
86Rb influx was markedly abolished
(~70%) by Ba2+ (a blocker of
K+ channels), whether measured in
the presence of HCO
3 or in its
absence, suggesting that this movement of
86Rb+
occurred mainly via K+ channels.
These results, therefore, argue against the existence of direct
coupling between the flux of Rb+
and the flux of HCO
3 (i.e., no
K+-HCO
3
cotransport) in these membranes. Since this hypothetical basolateral
transporter has been described using s-MTAL (6, 20), and since this
approach cannot distinguish between transport events occurring at the
basolateral or at the apical surface of this epithelium, we next
searched for its possible existence in the luminal membrane. As
illustrated in Fig. 5, right, however,
imposing an inside-acid pH gradient in the presence or absence of
CO2/HCO
3
modestly stimulated
86Rb+
uptake compared with that measured under non-pH gradient conditions at
pH 6.0. Moreover, the stimulatory effects of outward
H+ gradients on
86Rb+
uptake become barely detectable compared with those measured in the
absence of a pH gradient at pH 7.8 instead of at pH 6.0. For example,
in HCO
3-buffered solutions,
86Rb+
influx in the presence of a pH gradient tended to be greater than under
non-pH gradient conditions at pH 7.8 (36 ± 3.5 vs. 28 ± 1.8),
but this difference was not significant at the
P < 0.05 level. This may be
attributed to an effect of pHi per
se, insofar as it has been demonstrated that intracellular
acidification inhibits the apical
K+ conductance of the thick
ascending limb (7, 18, 34).
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Lactate/HCO3
exchange in BLMV.
Since outwardly directed HCO
3
gradients have been reported to stimulate lactate uptake into BLMVs
purified from the dog MTAL (32), we investigated for the presence of this possible mode of HCO
3 transport
in the rat BLMV. Figure 8 shows the effects
of pH and/or HCO
3 gradients on
time-dependent
[14C]lactate uptake.
In these experiments, the membrane potential was clamped at 0 mV by
using valinomycin and equimolar internal and external
K+ concentrations. Imposing an
inside-alkaline pH gradient (pHi 7.8, pHo 5.5) caused the transient
accumulation of lactate to a level approximately eightfold higher than
equilibrium. However, the presence of outwardly directed
HCO
3 gradients of 55 mM:2.62 mM with
the same pH gradient caused no further stimulation of lactate uptake,
arguing against the existence of direct coupling between the flux of
lactate and the flux of HCO
3 (i.e., no
lactate/HCO
3 antiport) in the
basolateral membrane of the rat MTAL.
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DISCUSSION |
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The specific mechanism(s) implicated in the final step of
HCO3 absorption by the MTAL remains
largely unknown. In the present study, we used BLMVs purified from rat
MTAL to characterize directly the transport pathway(s) by which
HCO
3 exits across the basolateral
membrane of this nephron segment.
In this study, we found no evidence compatible with electrogenic
Na+-HCO3
cotransport across basolateral membrane in the MTAL:
1) an inwardly directed
HCO
3 gradient in the presence of an
inside-positive membrane potential increased only marginally the
initial rate of
22Na+
uptake (17 pmol · mg
1 · 9 s
1), nearly 20 times less
of the increment (330 pmol · mg
1 · 9 s
1) we detected using
cortical BLMVs as a positive control assay (Fig. 2);
2) an inward
HCO
3 gradient in the presence of an
inside-positive membrane potential failed to induce a
22Na+
overshoot in BLMV at markedly different
pHi values (Fig. 3); 3) studies using the pH-sensitive
dye CF revealed that imposing outwardly directed
Na+ gradients induced only
marginal intravesicular acidification in BLMV that was unaffected by
DIDS (Fig. 4). These findings, therefore, argue against the presence of
basolateral membrane electrogenic
Na+-nHCO
3
cotransport in the rat MTAL. This conclusion is consistent with recent
immunohistochemical (27) and in situ hybridization (25) studies which
have shown that the electrogenic
Na+-HCO
3
cotransporter from rat kidney, termed rNBC, localizes to the straight
portion of the rat proximal tubule, whereas rNBC mRNA expression and
staining by polyclonal antibodies to this transporter were not detected
in any other tubule segment. It must be noted, however, that Kikeri et
al. (19) provided evidence for a
Na+-nHCO
3
cotransporter in mouse s-MTAL by measuring
pHi; whether this is due to
species difference remains to be determined.
In this study, we found that a
K+-HCO3
cotransporter postulated to reside in the rat MTAL (6, 20), presumably at the basolateral membrane (20), cannot be detected by two complementary approaches. In studies using the pH-sensitive dye CF, we
found that imposing outwardly directed
K+ gradients induced only marginal
intravesicular acidification in both BLMV and LMV and at identical
rates, regardless of whether measured in the absence of
HCO
3 or measured in its presence (Fig.
6). These negative results cannot be accounted for by a higher buffer
capacity in the presence of
CO2/HCO
3, which was identical in the presence and absence of
HCO
3. Thus these fluorescence dye
measurements of pHi demonstrate
that K+ gradient-induced
acidification has no requirement for
CO2/HCO
3. Conversely, we have found that imposing an inside-acid pH gradient in
BLMV and in LMV induced identical stimulation of the rates of
86Rb+
uptake in the presence and absence of
CO2/HCO
3 (Fig. 5), further arguing against
K+-HCO
3
cotransport across both types of plasma membranes in the MTAL.
Our results showing directly an absence of coupling between the flux of
K+ and the flux of
HCO3 are in apparent contradiction with indirect studies evaluating
pHi changes in response to
application of K+ gradients in rat
s-MTAL (6, 20). In studies by Leviel and colleagues (20), dilution of
CO2-loaded tubules into
CO2/HCO
3-free buffer resulted in a near-immediate
pHi increase from ~7.2 to ~7.8, due to CO2 exit, followed
by pHi recovery. This
pHi recovery was significantly
slowed by DIDS, as well as by reducing
[K+]i
or increasing
[K+]o,
results consistent with the presence of a
K+-HCO
3
cotransporter in this nephron segment. However, the thick ascending
limb possesses multiple K+- and
Cl
-conductive pathways
(14-17, 23, 26, 33). In this regard, we have recently demonstrated
that generation of an inside-positive membrane potential markedly
stimulated uptake of
36Cl
in BLMV and LMV, indicating significant
Cl
-conductive pathways in
these plasma membranes (10). Accordingly, outward
K+ gradients would be expected to
cause a large hyperpolarization, which would activate a conductive
pathway for Cl
transport
leading to a decrease in
[Cl
]i.
The resulting increase in the size of the inward
Cl
gradient would then
acidify the cells via operation of
Cl
/HCO
3
exchangers, which have been recently shown to be expressed, under
isotonic conditions and in the absence of arginine vasopressin, in the
mouse (28) and rat MTAL (10). Importantly, we have recently shown (10)
that the BLMV
Cl
/HCO
3
exchanger is markedly activated at alkaline pHi values, i.e., under the
experimental conditions (pHi
7.3-7.8) that have been used to detect
K+-HCO
3
cotransport in rat s-MTAL (6, 20).
Further studies by Blanchard and colleagues (6), using rat s-MTAL, have
shown that increasing
[K+]i
from 60 to 120 mM markedly stimulated the initial rate of intracellular acidification under conditions in which
K+-conductive pathways
were assumed to be completely blocked by 0.1 mM verapamil
and 10 mM Ba2+. These findings
were taken as evidence of an important modifier role for internal
K+ as an allosteric activator of a
putative
K+-HCO3
cotransporter. The validity of this conclusion, however, is uncertain
since the adequacy of the voltage clamp was not verified. Furthermore,
the possibility exists that the degree of blockade of
K+-conductive pathways by
Ba2+ could be
[K+]i
dependent. Evidence to support this contention was provided in studies
using K+ channels from rabbit
muscle incorporated into planar bilayers (31) and MTAL of mouse (16),
which showed that the effectiveness of
Ba2+ block can be mitigated by
increasing K+ concentrations. If
at high
[K+]i
Ba2+ did indeed become less
effective at inhibiting
K+-conductive pathways, then it is
possible that most of the
[K+]i-induced
acidification may have been due to negative shifts in the membrane potential.
In our studies using
86Rb+,
we have obtained evidence compatible with some mode of coupling between
K+ and
H+ fluxes in the BLMV (Fig. 5).
The marked inhibition (80-90%) of BLMV
86Rb+
uptake by Ba2+ under non-pH
gradient conditions, at pH 6.0 and 7.8, suggested that most of these
movements of
86Rb+
were mediated by K+ channels.
Nevertheless, we found that ~30% of the pH gradient-activated uptake
of 86Rb persisted in the presence
of Ba2+. This could be due to the
fact that Ba2+ inhibition is
voltage dependent, insofar as Greger and coworkers (14) demonstrated
that the Ba2+ inhibition of
K+ channels was reduced by
hyperpolarization in the thick ascending limb of Henle's loop.
Because, however, these membranes exhibited low permeabilities to
H+ (24), an alternative
possibility is that the pH-stimulated Ba2+-insensitive
86Rb+
uptake could reflect carrier-mediated basolateral
K+/H+
exchange of very low magnitude. Graber and Pastoriza-Munoz (12) have
already reported the presence of a
Ba2+-insensitive
K+/H+
exchanger in the OK opossum kidney cell, presumably at the basolateral membrane. Two studies report the presence of a
K+/H+
exchanger in the rat MTAL, which has been located, albeit indirectly, on the apical membrane (2, 4), which disagrees with our findings.
Indeed, the experiments reported in Fig. 5 demonstrate conclusively
that an inside-acid pH gradient caused only marginal increments in the
rates of 86Rb uptake into LMV
(8-14
pmol · mg1 · 9 s
1), about six to nine
times less of the corresponding increments (72-82
pmol · mg
1 · 9 s
1) we detected using
BLMV. It is possible that the marginal
H+i-activated 86Rb+
uptake observed here in LMV represents contamination by basolateral membrane. The difference between our results and those of
Attmane-Elakeb et al. (4) can be readily explained by the fact that
their luminal fractions were heavily contaminated with basolateral
membranes, as judged by a threefold to approximately fivefold
enrichment in
Na+-K+-ATPase.
Of note, failure to detect appreciable
H+-activated
86Rb+
uptake in LMV cannot be attributed to a general defect in the transport
capacities of these membrane vesicles. Indeed, we recently demonstrated
robust bumetanide-sensitive
Na+-K+-2Cl
cotransport activity in these fractions, and not in the BLMV (24), as
well as uphill
36Cl
transport via operation of a
Na+-independent, DIDS-sensitive
Cl
/HCO
3
exchanger (10).
The present studies confirm the observations of Vinay et al. (32) that
outwardly directed OH
gradients can induce uphill lactate uptake into BLMVs purified from the
MTAL. The experiments reported in Figs. 8 and 9, however, demonstrate
that the combination of a pH gradient and an outward HCO
3 gradient did not provide
additional stimulation of lactate uptake at markedly different
pHi values, strongly suggesting that there is not direct coupling between the flux of lactate and the
flux of HCO
3 (i.e., no
lactate/HCO
3 exchange). Vinay et al.
(32) reported HCO
3 gradient
stimulation of lactate uptake, in the absence of a pH gradient, and
concluded that it was due to
lactate/HCO
3 exchange, because the
stimulated uptake persisted unchanged in the presence of a pH clamp
with K+i = K+o and nigericin. In this study,
however, the adequacy of the pH clamp was not demonstrated. In the
experiments of Vinay et al. (32), imposing an outward
HCO
3 gradient in media not gassed with
CO2 may have led to a
near-instantaneous pHi increase
due to CO2 exit, raising the
possibility that most of the HCO
3
gradient-induced lactate uptake may have been via nonionic diffusion of
lactic acid and/or by lactate-H+
cotransport or lactate/OH
exchange. Since we have found that the pH gradient-stimulated uptake of
L-lactate was sensitive to
inhibition by furosemide, carrier-mediated
H+-lactate cotransport is the
likely mechanism underlying pH gradient-activated lactate uptake in the
BLMV. This view is supported by immunocytochemical studies showing that
the proton-linked monocarboxylate transporter isoform MCT2 is expressed
in the basolateral membrane of the rat MTAL (D. Eladari and R. Chambrey, unpublished observations).
Finally, the observations that inward
HCO3 gradients did not affect
significantly
86Rb+
uptake (Fig. 5) and that K+
gradient-induced acidification was not significantly affected by the
presence of HCO
3 (Fig. 6) suggest that basolateral HCO
3 conductance could not
play an important role in net transcellular
HCO
3 absorption.
In conclusion, we found no evidence for the presence in BLMVs purified
from rat MTAL of appreciable Na+-,
K+-, or lactate-linked
HCO3 transport. It appears from this
and other recent studies (10, 28) that basolateral Cl
/HCO
3
exchange may be the major transport pathway by which this nephron
segment absorbs HCO
3.
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ACKNOWLEDGEMENTS |
---|
We thank F. Pezy for excellent technical assistance.
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FOOTNOTES |
---|
Portions of this work were presented at the Annual Meeting of the American Society of Nephrology, Philadelphia, PA, and have been published in abstract form (J. Am. Soc. Nephrol. 9: 8, 1998).
R.-A. Podevin is an Established Investigator of the Institut National de la Santé et de la Recherche Médicale.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R.-A. Podevin, Institut Biomédical des Cordeliers, 15 rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France (E-mail: podevin{at}ccr.jussieu.fr).
Received 9 July 1998; accepted in final form 18 February 1999.
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