Alternatively spliced isoform of apical
Na+-K+-Cl
cotransporter gene
encodes a furosemide-sensitive
Na+-Cl
cotransporter
Consuelo
Plata1,
Patricia
Meade1,
Amy
Hall2,
Rick C.
Welch3,
Norma
Vázquez1,
Steven C.
Hebert2, and
Gerardo
Gamba1
1 Molecular Physiology Unit, Instituto Nacional de Ciencias
Médicas y Nutrición Salvador Zubirán and Instituto de
Investigaciones Biomédicas, Universidad Nacional Autónoma
de México, Mexico City CP 14000, Mexico; 2 Department
of Cellular and Molecular Physiology, Yale University Medical
School, New Haven, Connecticut 06520; and 3 Division of
Nephrology, Department of Medicine, Vanderbilt University Medical
Center, Nashville, Tennessee 37232
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ABSTRACT |
In the absence of
vasopressin, medullary thick ascending limb cells express a
K+-independent, furosemide-sensitive
Na+-Cl
cotransporter that is inhibited by
hypertonicity. The murine renal specific
Na+-K+-2 Cl
cotransporter gene
(SLC12A1) gives rise to six alternatively spliced isoforms.
Three feature a long COOH-terminal domain that encodes the
butmetanide-sensitive Na+-K+-2 Cl
cotransporter (BSC1-9/NKCC2), and three with a short COOH-terminal domain, known as mBSC1-A4, B4, or F4 (19). Here we have
determined the functional characteristics of mBSC1-A4, as expressed in
Xenopus laevis oocytes. When incubated at normal
oocyte osmolarity (~200 mosmol/kgH2O),
mBSC1-4-injected oocytes do not express significant Na+ uptake over H2O-injected controls, and
immunohistochemical analysis shows that the majority of mBSC1-4
protein is in the oocyte cytoplasm and not at the plasma membrane. In
contrast, when mBSC1-4 oocytes are exposed to hypotonicity (~100
mosmol/kgH2O), a significant increase in Na+
uptake but not in 86Rb+ uptake is observed. The
increased Na+ uptake is Cl
dependent,
furosemide sensitive, and cAMP sensitive but K+
independent. Sodium uptake increases with decreasing osmolarity between
120 and 70 mosmol/kgH2O (r = 0.95, P < 0.01). Immunohistochemical analysis shows that in
hypotonic conditions mBSC1-A4 protein is expressed in the plasma
membrane. These studies indicate that the mBSC1-A4 isoform of the
SLC12A1 gene encodes a hypotonically activated, cAMP- and
furosemide-sensitive Na+-Cl
cotransporter.
Thus it is possible that alternative splicing of the BSC1 gene could
provide the molecular mechanism enabling the
Na+-Cl
-to-Na+-K+-2Cl
switching in thick ascending limb cells.
bumetanide; protein kinase A; adenosine 3',5'-cyclic monophosphate; thick ascending limb of Henle
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INTRODUCTION |
INCREASING NET NACL REABSORPTION in
the thick ascending limb of Henle (TAL) by hormones such as
vasopressin, which generate cAMP via their respective
Gs-coupled receptors, is a fundamental mechanism for
regulating salt transport in this nephron segment (13, 15,
16). The effects of these hormones are crucial to the normal
functioning of the TAL in reabsorbing 10-15% of filtered NaCl,
providing for normal diluting and concentrating power, and regulating
divalent mineral excretion.
Vasopressin increases transepithelial reabsorption in the TAL by
activating both Na+, K+, and Cl
uptake and K+ recycling through stimulation of the
Na+-K+-2Cl
cotransporter and
apical K+ conductance (14). This coupling of
NaCl with K+ during ion reabsorption has very important
implications for the cellular physiology of the TAL, because
K+-recycling is largely responsible for the generation of
the positive luminal potential difference that drives paracellular
cation transport. Vasopressin appears to directly activate the apical
Na+-K+-2Cl
cotransporter in mouse
TAL by a mechanism that is not completely understood but that includes
switching of the K+ dependence of Na+
cotransport in the apical membrane. In the absence of vasopressin, furosemide-sensitive Na+-Cl
cotransport was
observed in the mouse TAL, whereas in the presence of hormone,
furosemide-sensitive Na+ transport became K+
dependent (23). Thus vasopressin can switch cotransport in mouse TAL from a completely K+-independent, but
nevertheless loop diuretic-sensitive, Na+-Cl
mode, to a K+-dependent
Na+-K+-2Cl
mode
(23). Evidence for switching between
K+-independent and K+-dependent
Na+-Cl
cotransporters in TAL had been
previously observed by Eveloff and Calamia (5) in rabbit
mTAL cells. They showed that extracellular osmolarity alters the
K+ dependency of Na+-Cl
cotransport. In normal mammalian osmolarity (~300
mosmol/kgH2O), the apical Na+ pathway is mainly
Na+-Cl
transport, whereas when the
extracellular osmolarity is increased, the NaCl pathway exhibited the
classic characteristics of
Na+-K+-2Cl
cotransport. Both
transport systems were sensitive to the loop diuretic furosemide
(5). Taken together, these findings indicate that at the
luminal side of the TAL the predominant Na+ cotransport
system appears to be determined by hormonal stimuli and cell volume.
Supporting the functional evidence that two different loop
diuretic-sensitive cotransport systems are present in TAL, two binding
sites with distinct affinities for the tracer loop diuretics
[3H]bumetanide or [3H]piretanide have
been identified in crude plasma membrane preparations from mouse
(11) and dog (8, 9) kidney, respectively.
Furthermore, photolabeling mouse kidney membranes with the
photosensitive bumetanide analog
[3H]4-benzoyl-5-sulfamoyl-3(3-thenyloxy)benzoic
acid revealed that the high- and low-[3H]bumetanide
bindings sites exhibited incorporation of the label in two regions: one
of ~150 kDa and another of ~75 kDa, respectively, suggesting that
either the activation and inhibition of two distinct proteins or the
dimerization of one polypeptide could account for the switching between
Na+-Cl
and
Na+-K+-Cl
cotransport mode.
The recent identification of several isoforms of the mouse renal
specific bumetanide-sensitive
Na+-K+-2Cl
cotransporter gene
(SLC12A1) provides new insights into the mechanisms of salt
transport regulation in TAL (19). A total of six
alternatively spliced isoforms are encoded by SLC12A1 gene.
These isoforms are produced after the combination of two independent
splicing events (19). One is the utilization of an
alternative polyadenylation site that predicts two type 1 bumetanide-sensitive
Na+-K+-2Cl
cotransporter
(BSC1) proteins identical at the entire NH2-terminal and transmembrane domains, as well as in the first 74 amino acid residues of the COOH-terminal domain but different in the sequence and
length of the remaining COOH terminus. The longer isoform (1,095 amino
acids) contains 383 residues that are not present in the shorter
isoform. In contrast, the shorter, truncated isoform (770 amino acids)
exhibits a COOH terminus with 55 residues that are not present in the
longer isoform. Interestingly, the long and short COOH-terminal domains
contain different putative protein kinase A (PKA) and protein kinase C
(PKC) phosphorylation sites. We have designated the longer and shorter
transcripts as mBSC1-9 and mBSC1-4, respectively
(19). The other splicing event is due to the presence of
three mutually exclusive variants of coding exon 4, denoted A, B, and F
(17). The combination of both splicing mechanisms results
in the production of three mBSC1-9 (mBSC1-A9/B9/F9) isoforms that
encode the bumetanide-sensitive
Na+-K+-2Cl
cotransporter
(21) and three mBSC1-4 (mBSC1-A4/B4/F4) proteins, with unknown function. Interestingly, however, interaction between mBSC1-9 and mBSC1-4 isoforms appears to be critical for
vasopressin activation of the
Na+-K+-2Cl
cotransporter because
mBSC1-4 exerts a dominant negative effect on the cotransporter
function that can be prevented by PKA activation (21). In
the present paper we demonstrate that the mBSC1-A4 isoform encodes a
K+-independent furosemide-sensitive
Na+-Cl
cotransporter that is activated by
hypotonicity and inhibited by PKA activation. Our results provide a
molecular mechanism that can account for switching between
Na+-Cl
and
Na+-K+-2Cl
cotransporters in the
mammalian TAL.
 |
METHODS |
Xenopus laevis oocyte preparation and injection.
In the present study we used the X. laevis oocytes
heterologous expression system. Oocytes were harvested from
anesthetized (0.17% tricaine) frogs and incubated for 1 h under
vigorous shaking at room temperature in a Ca2+-free ND-96
(in mM: 96 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES/Tris, pH 7.4)
containing 2 mg/ml of collagenase B (Boehringer, Mannheim, Germany).
Oocytes were washed three times in standard ND-96 (in mM: 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES/Tris, pH
7.4), manually defolliculated, and incubated overnight at 17°C in
incubation medium (ND-96 supplemented with 2.5 mM
Na+-pyruvate and 5 mg/100 ml of gentamicin). Stage V-VI
oocytes (4) to be used for controls were either
noninjected or injected with a 1-mM Tris solution (50 nl), and
experimental oocytes were injected with either mBSC1-A4 or mBSC1-F9
cRNA (25 ng/oocyte in 50 nl). After injection, oocytes were incubated
at 17°C in incubation medium for 3-5 days. During this period,
incubation medium was changed daily.
mBSC1 cDNAs isoforms.
The mBSC1 isoform cDNAs were in the pSPORT1 (Life Technologies)
plasmid, and their generation has been described in detail (19). For preparation of cRNA templates, each isoform cDNA
was linearized at the 3' end by using Not I or
Xba I restriction enzymes, and cRNA was then transcribed in
vitro by using the T7 RNA polymerase in the presence of Cap analog
(mMESSAGE, Ambion, Austin, TX). Transcription product integrity was
confirmed on agarose gels, and concentration was determined by
absorbance reading at 260 nm (DU 640, Beckman, Fullerton, CA). cRNA was
stored in aliquots at
80°C.
Functional characterization of mBSC1 isoforms.
Functional characteristics of the mBSC1 isoforms were assessed by
measuring tracer 22Na+ or
86Rb+ uptake in groups of 20-25 oocytes
under various osmolarities. 22Na+ uptake was
measured with the following protocol: a 30-min incubation period in a
hypotonic and Cl
-free medium (in mM: 25 Na+-gluconate, 4.0 Ca2+-gluconate, 1.0 Mg2+-gluconate, 5 HEPES/Tris, pH 7.4), with 1 mM ouabain
and 100 µM amiloride, followed by a 60-min uptake period in a
hypotonic uptake medium (in mM: 25 NaCl, 10 KCl, 1.8 CaCl2,
1 MgCl-2, 5 HEPES/Tris, pH 7.4) containing 2.5 µCi/ml of
22Na+ (NEN, Boston, MA) and the same drugs used
during the incubation period. Various degrees of tonicity were studied
by adding sucrose to the incubation and uptake media to obtain
solutions with osmolarities between 70 to 150 mosmol/kgH2O.
The hypotonic conditions inhibit the endogenous oocyte
Na+-K+-2Cl
cotransporter
(6). Ouabain was added to prevent Na+ exit via
Na+-K+-ATPase, and amiloride prevents
Na+ uptake via Na+ channels or
Na+/H+ antiporters. To perform uptakes in
isotonicity, we used an incubation medium containing 96 mM Na-gluconate
and regular ND-96 as the uptake medium. To determine the
K+- and Cl
- dependent fraction of
22Na+ uptake, paired groups of oocytes were
incubated in uptake media without Cl
(substituted by
gluconate) or without K+ (substituted by
N-methyl-D-glucamine) in the presence of the K+-Cl
cotransporter inhibitor
[(dihydroindenyl) oxy]alkanoic acid (DIOA; 100 µM)
(7).
86Rb+ uptake was also assessed under various
degrees of hypotonicity with the following protocol: 30-min incubation
period in a hypotonic K+- and Cl
-free
solution (in mM: 40 Na+-gluconate, 4.6 Ca2+-gluconate, 1.0 Mg2+-gluconate, 10 Ba-sulfate, 5 Hepes/Tris, pH 7.4) with 1 mM ouabain, followed by a
60-min uptake period using a hypotonic uptake medium (in mM: 40 NaCl,
2.0 KCl, 1.8 CaCl2, 1.0 MgCl2, 10 BaCl2, 5.0 HEPES, pH 7.4) with 1 mM ouabain, and 2.0 µCi/ml of 86Rb, specific activity 0.57 µCi/nmol (NEN).
22Na+ and 86Rb+ uptakes
were performed at 30°C and were linear over the first 60 min. At the
end of the uptake period, oocytes were washed five times in ice-cold
uptake solution without isotope to remove extracellular fluid tracer,
after which oocytes were dissolved in 10% sodium dodecyl sulfate, and
tracer activity was determined by
scintillation counting.
Immunohistochemical staining of oocytes.
Unfixed X. laevis oocytes injected with mBSC1-A4 cRNA were
embedded in OCT (Tissue Tek, Miles, Elkhart, IN) and slowly frozen at
54°C. Ten-micrometer sections were cut by using a Leica 3050 cryostat at
13°C. Sections were fixed for 3 min in
20°C
acetone. The sections were washed three times (5 min) with PBS-T
(0.05% Tween 20 in PBS, pH = 7.4) then blocked with 1%
BSA-PBS-4% normal goat serum for 30 min at room temperature. Slides
were incubated overnight at 4°C with 1:100 dilution of
affinity-purified rabbit anti-mouse mBSC1-4 antibody
(19) diluted in 1% BSA-PBS-4% normal goat serum. This
antibody is directed against the 55 unique amino acid residues present
in mBSC1-4 and not in mBSC1-9 isoforms. We have shown
previously that this antibody recognized only mBSC1-4 protein
(19). Sections were washed three times (5 min) with PBS-T,
then incubated for 1 h with anti-rabbit Alexa 594 conjugate antibody (Molecular Probes, Eugene, OR) diluted 1:5,000 in 1% BSA-PBS-4% normal goat serum. Sections were washed as above and mounted with Aquapolymount (Polysciences, Warrington, PA). Slides were
examined with a Nikon Eclipse 800 research microscope.
Animals and materials.
Adult female X. laevis frogs were purchased from Carolina
Biological Supply (Burlington, NC) and from Nasco (Fort Atkinson, MI).
Frogs were maintained at the animal facility under constant control of
room temperature and humidity at 16°C and 65%, respectively. Frogs
were fed with brittle dry frog food from Nasco, and water was changed
twice a week. Dibutyryl cAMP (DBcAMP), collagenase B, and all
restriction enzymes were from Boehringer. H89 was from Calbiochem. The
cRNA transcription kit mMESSAGE was from Ambion. Tracer sodium
(22Na+) and rubidium
(86Rb+) were purchased from DuPont-NEN.
Ouabain, amiloride, bumetanide, IBMX, and general chemicals were from
Sigma (St. Louis, MO).
Statistical analysis.
Statistical significance was defined as a two-tailed P < 0.05, and the results are presented as means ± SE. The
significance of the differences between groups were tested by the
one-way ANOVA with multiple comparison by using the Bonferroni
correction or by the Kruskal-Wallis ANOVA on ranks with the Dunn's
method for the multiple-comparison procedure, as needed.
 |
RESULTS |
Expression of the mBSC1-A4 isoform in oocytes.
We have previously shown that mBSC1-4 exhibits no functional
activity assessed by either 22Na+ or
86Rb+ uptake for osmolarities between 210 and
150 mosmol/kgH2O (21). A similar observation
is shown in the first four bars in Fig. 1. Na+ uptake in water or
mBSC1-A4 cRNA-injected oocytes was similar under isotonic (200 mosmol/kgH2O; 2,061 ± 176 vs. 2,391 ± 115 pmol · oocyte
1 · h
1,
respectively) or in slight hypotonic (150 mosmol/kgH2O;
236 ± 63 vs. 284 ± 48 pmol · oocyte
1 · h
1,
respectively) conditions. The significant decrease in
22Na+ uptake observed in solutions with
osmolarities between 210 and 150 mosmol/kgH2O in both water
and mBSC1-A4-injected oocytes was due to inhibition of the endogenous
Na+-K+-2Cl
cotransporter. The
last two bars in Fig. 1 show that exposing the mBSC1-A4 cRNA-injected
oocytes to a further reduction in osmolarity to 100 mosmol/kgH2O resulted in an increased
22Na+ uptake in mBCS1-4-injected oocytes
that was ~48-fold higher than in water-injected oocytes (8,685 ± 737 vs. 181 ± 35 pmol · oocyte
1 · h
1,
respectively, P < 0.0001). Figure
2 shows the effects of decreasing osmolarity from 150 and 70 mosm/kgH2O on
22Na+ uptakes in mBSC1-A4 cRNA-injected
oocytes. Thus mBSC1-A4 functional expression in oocytes is activated by
hypotonicity in a dose-dependent fashion below osmolarities of 120 mosmol/kgH2O.

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Fig. 1.
Hypotonic-induced functional expression of mBSC1-A4 in
Xenopus laevis oocytes. 22Na+ uptake
was measured in oocytes injected with water (open bars) or with 25 ng
of mBSC1-A4 cRNA (filled bars) under 3 different extracellular
osmolarities as indicated. NaCl concentrations in extracellular medium
were 96 mM in 200 mosmol/kgH2O, 62 mM in 150 mosmol/kgH2O, and 40 mM for the 100 mosmol/kgH2O solution. Each bar represents mean ± SE
of 22 oocytes. *Significantly different from uptake in the same group
incubated at 200 mosmol/kgH2O (P < 0.001).
Significantly different from all groups (P < 0.00001).
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Fig. 2.
Reductions in osmolarity below 120 mosmol/kgH2O increase 22Na+ uptake
in mBSC1-A4-expressing oocytes in an inverse linear fashion. Each point
represents mean ± SE of 20 oocytes.
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Hypotonicity alters localization of mBSC1-A4 protein from the
cytosol to the plasma membrane.
Figure 3 shows the immunolocalization of
mBSC1-A4 protein in representative oocytes incubated in either 150 (left) or 100 mosmol/kgH2O (right)
media. The incubation protocol was identical to that used for
22Na+ uptake. In oocytes incubated in the 150 mosmol/kgH2O medium, the majority of staining is localized
to the oocyte cytoplasm just beneath the plasma membrane. In contrast,
in oocytes exposed to the 100 mosmol/kgH2O medium, most of
the staining is localized in the plasma membrane. Thus in X. laevis oocytes hypotonicity alters the localization of mBSC1-A4
protein from the cytosol to the plasma membrane. This expression of
mBSC1-A4 protein at the plasma membrane with 100 mosmol/kgH2O hypotonicity is consistent with the change of
mBSC1-A4 from a nonfunctional to a functional transporter as shown in
Fig. 1.

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Fig. 3.
Hypotonicity alters the localization of mBSC1-A4 in oocytes.
mBSC1-A4 protein was localized in oocytes by using the rabbit
anti-mouse polyclonal antibody directed against the unique 55-amino
acid COOH-terminal domain of mBSC1-A4 described previously
(19). Immunostaining was detected after incubation of
oocytes in either 150 (left) or 100 mosmol/kgH2O
(right) media by using the same protocol described for
22Na+ uptakes. In left panel,
most of the staining is localized in the cytosol, whereas in
right panel most staining is localized to the plasma
membrane.
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mBSC1-A4 encodes the loop diuretic-sensitive
Na+-Cl
cotransporter.
Figure 4, A and B,
show 22Na+ uptakes in mBSC1-A4 cRNA-injected
oocytes in the absence or presence of 100 µM concentrations of furosemide (A) or bumetanide (B). The addition of
either of the loop diuretics resulted in a 75-79% inhibition of
the increased 22Na+ uptake (vs. water-injected
controls) in mBSC1-A4-expressing oocytes. Figure
5 compares the bumetanide
concentration-dependent inhibition of 22Na+
uptake induced by mBSC1-A4 or mBSC1-F9 (21). Both mBSC-1
isoforms exhibited similar IC50 values of
~10
6 M. Thus 22Na+ uptake
mediated by mBSC1-A4 is loop diuretic sensitive, and the difference in
the COOH termini between mBSC1-A4 and mBSC1-F9 does not appear to alter
this bumetanide sensitivity. Because thiazide diuretics inhibit the
related Na+-Cl
cotransporter TSC1 or NCC, we
examined the effect of 100-µM trichloromethiazide on
22Na+ uptake in mBSC1-A4 expressing oocytes.
This thiazide had no effect on mBSC1-A4 function (data not shown).

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Fig. 4.
Effect of loop diuretics on 22Na+ uptake in
mBSC1-A4-injected oocytes exposed to 100 mosmol/kgH2O
hypotonicity. A: combined results of 3 experiments in which
22Na+ uptake was assessed in oocytes injected
with water (open bar), mBSC1-A4-injected oocytes (filled bar), and
mBSC1-A4 oocytes in the presence of 100 µM furosemide (hatched bar).
Each bar represents mean ± SE of 95 oocytes, obtained from 3 different experiments. B: combined results of 9 experiments
in which 22Na+ uptake was assessed in oocytes
injected with water (open bar), mBSC1-A4-injected oocytes (filled bar),
and mBSC1-A4 oocytes in the presence of 100 µM bumetanide (hatched
bar). Each bar represents mean ± SE of 360 oocytes, obtained from
9 different experiments. *P < 0.001 vs.
mBSC1-A4-injected oocytes.
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Fig. 5.
Bumetanide concentration-dependent inhibition of mBSC1-A4
( , solid line) and mBSC1-F9 ( , dashed
line). Groups of 20 oocytes microinjected with mBSC1-A4 or mBSC1-F9
were exposed to increasing concentrations of bumetanide
(10 9 to 10 4 M) in the preincubation and
uptake media. Data were normalized as the percentage of maximal uptake
obtained in the absence of bumetanide. Uptake was assessed at
osmolarities of 100 mosmol/kgH2O for mBSC1-A4 and at 150 mosmol/kgH2O for mBSC1-F9 (21).
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Because K+-independent, bumetanide-sensitive
Na+-Cl
cotransport has been described in
mouse TAL (23), we assessed the ion dependency of
22Na+ uptake in mBSC1-A4 cRNA-injected oocytes.
Figure 6 shows that mBSC1-A4-expressing
oocytes exhibited an increased 22Na+ uptake
over water-injected oocytes (3,670 ± 592 vs. 52 ± 10 pmol · oocyte
1 · h
1,
respectively) that was not dependent on extracellular K+
(2,876 ± 438 pmol · oocyte
1 · h
1,
P = not significant) but that was significantly reduced
when Cl
was omitted from the extracellular medium
(1,297 ± 289 pmol · oocyte
1 · h
1,
P < 0.0005) or when 100 µM bumetanide was added to
the uptake medium (998 ± 293 pmol · oocyte
1 · h
1,
P < 0.0002). The uptakes shown in Fig. 6 were
performed in the presence of DIOA to inhibit any endogenous
K+-Cl
cotransport (20).
Simultaneous experiments in oocytes showed that DIOA inhibits the
endogenous K+-Cl
cotransporter but has no
effect on Na+-K+-2Cl
cotransporter activity (data not shown). Thus mBSC1-4 mediates 22Na+ uptake that is Cl
dependent
and bumetanide sensitive but K+ independent. To further
demonstrate the K+-independent nature of Na+
transport by mBSC1-A4 under hypotonic conditions, we assessed in the
same experiment bumetanide-sensitive 22Na+ and
86Rb+ uptakes in 100 mosmol/kgH2O
media. As shown in Fig. 7A,
100 mosmol/kgH2O hypotonicity resulted in a significant
increase in 22Na+ uptake in mBSC1-A4
cRNA-injected oocytes, compared with water-injected controls. The
increased 22Na+ uptake was abolished by
addition of 100 µM bumetanide to the uptake medium. In contrast,
hypotonicity had no effect on 86Rb+ uptake by
mBSC1-A4-expressing oocytes (Fig. 7B). The small reduction in 86Rb+ uptake in control and
mBSC1-A4-expressing oocytes is due to the endogenous
K+-Cl
cotransporter (20). Thus
with 100 mosmol/kgH2O hypotonic conditions, mBSC1-A4
mediates 22Na+ but not
86Rb+ uptake. This observation is consistent
with the lack of effect of K+ omission on
22Na+ uptake as shown in Fig. 6.

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Fig. 6.
Ion dependency and bumetanide sensitivity of
22Na+ uptake in X. laevis oocytes
injected with water or mBSC1-A4 cRNA, as stated. Tracer uptake was
performed in the presence of Na+, K+, and
Cl in the water injected group (H2O) and in
the mBSC1-A4 injected oocytes (filled bar), in the absence of
extracellular K+ (open bar), in the absence of
extracellular Cl (crosshatched bar) or in the presence of
all 3 ions and 100 µM bumetanide (hatched bar). Each bar represents
mean ± SE of 21 oocytes. *P < 0.001 vs. uptake
in the presence of all 3 ions in the mBSC1-4 injected oocytes.
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Fig. 7.
22Na+ and
86Rb+ uptakes in X. laevis oocytes
injected with either water or mBSC1-A4 cRNA, in the absence (open bars)
or presence (filled bars) of 100 µM bumetanide. Uptakes were
performed under 100 mosmol/kgH2O conditions. Each bar
represents mean ± SE of 20 oocytes. *P < 0.05 vs. H2O control. P < 0.05 vs. absence of
bumetanide.
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22Na+ uptake mediated by mBSC1-A4 is
reduced by PKA activation.
Because the switch from the Na+-Cl
to
Na+-K+-2Cl
transport mode in
mouse mTAL has been shown to be dependent on the present of vasopressin
(23), we assessed the effect of the cell-permeable cAMP
analog DBcAMP and the phosphodiesterase inhibitor IBMX on the
functional expression of mBSC1-A4. Figure
8 shows the result of a single experiment
assessing the separate and combined effects of cAMP and IBMX on
22Na+ uptake in mBSC1-A4-expressing oocytes
under 100 mosmol/kgH2O hypotonic conditions. The addition
of 1 mM DBcAMP to the uptake medium resulted in a slight, but not
significant, fall in 22Na+ uptake (6,611 ± 700 vs. 5,293 ± 663 pmol · oocyte
1 · h
1, with
and without DBcAMP, respectively). In contrast, the addition of 1 mM
IBMX resulted in a significant 50% reduction of
22Na+ uptake in mBSC1-A4-injected oocytes
(3,585 ± 487 pmol · oocyte
1 · h
1,
P < 0.01, vs. mBSC1-A4 control). Moreover, the
combination of DBcAMP+IBMX exhibited a synergistic effect that resulted
in a further reduction in 22Na+ uptake to a
value that was 70% lower than uptake in mBSC1-A4 control oocytes
(2,080 ± 514 pmol · oocyte
1 · h
1,
P < 0.01). This value was also significantly different
from the uptake in mBSC1-A4-expressing oocytes exposed to DBcAMP alone. We confirmed the inhibitory effect of 1 mM DBcAMP+1 mM IBMX on mBSC1-A4
function in two additional experiments by using oocytes from different
frogs (8,425 ± 912 vs. 1,230 ± 285 pmol · oocyte
1 · h
1 without
and with cAMP+IBMX, respectively; P < 0.00001). In
addition, Fig. 9 shows the effect of
inhibition of endogenous PKA activity with 20 µM H89 on
22Na+ uptake in mBSC1-A4-expressing oocytes
under 100 mosmol/kgH2O hypotonic conditions. The inhibition
of PKA resulted in a significant increase in mBSC1-A4 function
(2,989 ± 184 vs. 4,762 ± 424 pmol · oocyte
1 · h
1 in the
absence and presence of H89, respectively; P < 0.0001). Moreover, 22Na+ uptake in mBSC1-A4
cRNA-injected oocytes in the presence of H89 was abolished by 100 µM
bumetanide. Thus functional expression of mBSC1-A4 under hypotonic
conditions is regulated by PKA activity.

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Fig. 8.
Effects of dibutyryl-cAMP and/or IBMX on mBSC1-A4-induced
increase in 22Na+ uptake in X. laevis oocytes. Water control oocytes (hatched bar), mBSC1-A4
oocytes in the absence (open bars) or presence (filled bars) of 100 µM bumetanide. Addition of 1 mM dibutyryl-cAMP and/or 1 mM IBMX is
depicted. Each bar represents mean ± SE of 20 oocytes.
*P < 0.01 vs. mBSC1-A4 control. P < 0.01 vs. mBSC1-A4+cAMP alone.
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Fig. 9.
Effect of H89-induced protein kinase A inhibition on
mBSC1-A4 functional expression in hypotonic conditions. Open bar:
uptake in water-injected controls; filled bars, mBSC1-A4 cRNA-injected
oocytes as indicated. Each bar represents mean ± SE of 20 oocytes. *P < 0.01 vs. uptake in mBSC1-A4 in the
absence of H89.
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DISCUSSION |
The present work describes the functional properties of mBSC1-A4,
the COOH-terminal truncated, alternatively spliced isoform of the
murine SLC12A1 gene expressed in TAL cells. Using the
X. laevis oocyte heterologous expression system, we show
that the mBSC1-A4 isoform is a functional transporter that encodes a
K+-independent, loop diuretic-sensitive
Na+-Cl
cotransporter (Figs. 1, 2, 4-7).
Activation of the mBSC1-A4 cotransporter in oocytes requires exposure
to reductions in osmolarity below 120 mosmol/kgH2O (Figs. 1
and 2) and this is accompanied by a shift in expression of
cotransporter protein from cytosol to the plasma membrane (Fig. 3). In
addition, we demonstrate that the ion transport function of mBSC1-A4 in
hypotonic media is further regulated by cAMP/IBMX (Figs. 8 and 9).
Several lines of evidence have indicated the existence of a
K+-independent, furosemide-sensitive
Na+-Cl
cotransporter in mouse
(23) and rabbit (5) TAL. Sun et al. (23), using isolated perfused mouse medullary TAL tubules,
demonstrated that ouabain-induced cell swelling was absolutely
dependent on salt entry into cells through the loop diuretic-sensitive,
apical Na+-K+-Cl
cotransport
mechanism. In the absence of vasopressin (i.e., cAMP), ouabain-induced
swelling of the tubular cells was abolished by loop diuretic and by
removal of luminal Na+ or Cl
but not by
omission of luminal K+. When vasopressin was added to the
preparation, removal of luminal K+ resulted in prevention
of cell swelling, indicating the existence of a
Na+-K+-2Cl
cotransport system in
the apical membrane. Thus vasopressin shifted the mode of apical
cotransport from Na+-Cl
to
Na+-K+-2Cl
. In rabbit medullary
TAL cells, Eveloff and co-workers (1, 5) had also shown
evidence for the coexistence of furosemide-sensitive Na+-Cl
and
Na+-K+-2Cl
cotransport pathways
and their regulation by osmolality (1, 5). These data
taken together indicate that, in the TAL, when extracellular osmolarity
is low (or cell swelling occurs by other means) and in the absence of
vasopressin (or cAMP), the transepithelial salt reabsorption is mainly
due to an apical Na+-Cl
cotransporter,
whereas when extracellular osmolarity is increased and/or in the
presence of vasopressin, the major salt transport pathway is the
Na+-K+-2Cl
cotransporter. Both
mechanisms observed in TAL cells are sensitive to loop diuretics, a
finding consistent with our observations in mBSC1-A4- and
mBSC1-F9-injected oocytes (Fig. 5).
We previously identified six spliced isoforms of the mouse
Na+-K+-2Cl
cotransporter gene
(19). Our initial functional expression of these isoforms
revealed that the three long COOH-terminal domain mBSC1-9 proteins
(A, B, and F) encode the loop diuretic-sensitive Na+-K+-2Cl
cotransporter. The
shorter COOH-terminal domain isoforms (mBSC1-4A/B/F), however,
showed no expression under our standard experimental conditions, with
osmolarities between 150 and 210 mosmol/kgH2O (21). Because increasing the extracellular osmolarity
inhibits the K+-independent
Na+-Cl
cotransporter in rabbit TAL cells
(5), we reasoned that hypotonicity and/or increase in cell
volume could be an important condition to activate the cotransporter.
We now show that osmolarities below 120 mosmol/kgH2O are
required for functional expression of mBSC1-A4 in oocytes (Fig. 2). The
primary cytosolic localization of mBSC1-A4 protein at an osmolarity of
150 mosmol/kgH2O shown in Fig. 3, left, provides
an explanation for the lack of function of mBSC1-A4 at higher
osmolarities, above 120 mosmol/kgH2O (Fig. 2). Incubation of mBSC1-A4-injected oocytes in extracellular medium with an
osmolarities <120 mosmol/kgH2O resulted in dramatic
increases in Na+ uptake (Figs. 1 and 2) and was associated
with immunohistochemical localization of mBSC1-4 at the plasma
membrane (Fig. 3, right). A similar hypotonicity-induced
translocation-activation mechanism has been recently suggested by Watts
and Good (24) for the Na+/H+
antiporter, NHE3, in TAL cells. In addition, other members of the
electroneutral, cation-dependent Cl
cotransporter family
are also regulated by hypotonicity (or cell volume), some being
inhibited [by, e.g., endogenous
Na+-K+-Cl
cotransporter in
Xenopus oocytes, Fig. 1, (6, 22); the rat thiazide-sensitive Na+-Cl
cotransporter
(18)] and others being activated
[K+-Cl
(KCC) cotransporters (10,
20)].
However, the mechanism by which hypotonicity increases the localization
of mBSC1-4 at the plasma membrane and thus its activity as a
Na+-Cl
cotransporter is not clear from the
present study. Potential mechanisms include enhanced posttranslational
processing of the protein via activation of intracellular proteins such
as heat shock proteins or other chaperones or alterations in local
vesicular trafficking to the plasma membrane. In addition, it is also
not clear whether the fundamental mechanism to explain the
hypotonicity-induced activation of mBSC1-A4 is the increased cell
volume per se or other mechanisms such as activation or deactivation of
intracellular messengers or dilution of potential intracellular
inhibitors (i.e., cAMP or intracellular Cl
). These are
complex issues, and further studies will be necessary to clarify the mechanisms.
The activation of mBSC1-4 in the present study was observed when
we reduced the extracellular osmolarity below 130 mosmol/kgH2O. The normal osmolarity for oocytes is ~210
mosmol/kgH2O. Thus this reduction represents a decrease of
~40%. Because oocytes are relatively impermeable to water, this
percentage of reduction is required to develop hypotonicity-induced
cell swelling. Such a low osmolarity (130 mosmol/kgH2O)
will be rarely present in mammalian renal medulla. However, similar and
even higher percentages of reduction in renal medulla osmolarity
develop during brisk diuresis induced by water loading. In these
circumstances, interstitial NaCl and urea concentration decline rapidly
and renal medulla osmolarity can be reduced from 1,200 to 600 mosmol/kgH2O. In contrast, due to the high content of
osmolytes such as sorbitol, inositol, or betaine within the medullary
cells, when medullary interstitial osmolarity is reduced, cells take up
water and swell (3). For this circumstance, we suggest
that mBSC1-4 may be activated. According to our previous observations in mouse TAL (23),
Na+-K+-2Cl
cotransport should be
sensitive to PKA activation induced by Gs-coupled,
receptor-dependent generation of cAMP (e.g., by vasopressin). The major
observations are that activation of PKA-dependent processes 1) enhance the rate of net salt reabsorption, and hence
Na+-K+-2Cl
cotransporter
activity, by the TAL (15); and 2) change the mode of Na+-Cl
cotransport from
K+ independent to K+ dependent
(23). Because both mBSC1-9 and mBSC1-4 isoforms
coexist in mouse medullary TAL cells (19), regulation of
both cotransporter isoforms by PKA probably contributes to the
vasopressin-stimulated state of salt transport in the mTAL. As we show
in Fig. 8, addition of 1 mM cAMP+1 mM IBMX to the uptake medium
resulted in an ~70% reduction in 22Na+
uptake mediated by mBSC1-A4. In addition, inhibition of endogenous PKA
activity by H89 in oocytes resulted in an increase in mBSC1-4 function (Fig. 9). Moreover, we have previously shown that mBSC1-A4 has
a dominant negative effect on
Na+-K+-2Cl
cotransport mediated
by mBSC1-F9 and that activation of PKA abrogates this dominant negative
effect (21). The latter gives rise to an increase in
Na+-K+-2Cl
cotransport activity.
Thus PKA activation inhibits ion transport by mBSC1-A4 and abrogates
the dominant negative effect of mBSC1-A4 on mBSC1-F9. These effects
would inhibit K+-independent
Na+-Cl
cotransport and activate
Na+-K+-2Cl
cotransport. The
specific mechanism of the dominant negative-like effect of mBSC1-A4 on
mBSC1-F9 and its modulation by vasopressin are presently under investigation.
On the basis of these findings, we suggest a functional model for the
molecular physiology of salt reabsorption in the mouse TAL and its
regulation by vasopressin. Two distinct functional and molecular models
operate depending on the prevalent stimuli in the TAL. Figure
10A shows the functional
model that operates during water conservation, a situation in which the
osmolarity of the renal medulla is high and vasopressin is present. In
this model, both Na+-K+-2Cl
and
apical K+ conductance will increase due to PKA activation
(12). Coordinated function of both pathways ensures
K+ recycling and generation of a lumen positive voltage.
Because the apical membrane of the TAL is impermeable to water, the
intense reabsorption of salt dilutes tubular fluid and concentrates the medullary interstitium. In this model, the Na+
entry pathway in apical membranes is the
Na+-K+-2Cl
cotransporter encoded
by the mBSC1-9 isoforms. In contrast, Fig. 10B shows
the functional model that operates during maximal water diuresis, a
situation in which the washout of medullary tonicity provides a
relatively hypotonic environment and vasopressin secretion rate is low.
It has been shown that when the interstitial solute concentration in
the renal medulla decreases rapidly, the cells take up water due to the
high content of osmotically active substances such as glycine,
inositol, etc. (2, 3). Thus under these circumstances, TAL
cells swell and the Na+ pathway in the apical membrane
operates as a Na+-Cl
rather than as a
Na+-K+-2Cl
cotransporter. In this
circumstance in the mouse medullary TAL, the NaCl absorption rate is
reduced to ~50% of that present in the antidiuretic state and in the
presence of vasopressin (14, 23).

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Fig. 10.
Proposed model for thick ascending limb (TAL) function.
A: operation of water conservation. B: operation
during maximal water diuresis. See text for discussion.
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ACKNOWLEDGEMENTS |
We are grateful to Octavio Villanueva and Jesús López
for help with frog care and to members of the Molecular Physiology Unit
for suggestions and stimulating discussion.
 |
FOOTNOTES |
This work was supported by research Grants 97629m from the Mexican
Council of Science and Technology (CONACYT) and 75197-553601 from
the Howard Hughes Medical Institute to (G. Gamba) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-36803
(to S. C. Hebert and G. Gamba). C. Plata and P. Meade were
supported by scholarship grants from CONACYT and from the Dirección General del Personal Académico of the National
University of Mexico. G. Gamba is an International Scholar of the
Howard Hughes Medical Institute.
Part of this work was presented at the 32nd Meeting of the American
Society of Nephrology, held in 1999 in Miami, FL., and published as an
abstract (J Am Soc Nephrol 10: 1288, 1999).
Address for reprint requests and other correspondence: G. Gamba, Molecular Physiology Unit, Instituto Nacional de la
Nutrición Salvador Zubirán, Instituto de Investigaciones
Biomédicas, UNAM, Vasco de Quiroga No. 15, Tlalpan 14000, México City (E-mail: gamba{at}mailer.main.conacyt.mx).
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.
Received 8 June 2000; accepted in final form 17 November 2000.
 |
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