Alternatively spliced isoform of apical Na+-K+-Clminus cotransporter gene encodes a furosemide-sensitive Na+-Clminus 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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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
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INTRODUCTION
METHODS
RESULTS
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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 beta  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.


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ABSTRACT
INTRODUCTION
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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). dagger 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.

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.

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 (open circle , 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).

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. dagger P < 0.05 vs. absence of bumetanide.

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. dagger 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alvo, M, Calamia J, and Eveloff J. Lack of potassium effect on Na-Cl cotransport in the medullary thick ascending limb. Am J Physiol Renal Fluid Electrolyte Physiol 249: F34-F39, 1985[ISI][Medline].

2.   Beck, F, Dörge A, Rick R, and Thurau K. Osmoregulation of renal papillary cells. Pflügers Arch 405: S28-S32, 1985[ISI][Medline].

3.   Beck, F-X, Guder WG, and Schmolke M. Cellular osmoregulation in kidney medulla. In: Cell Volume Regulation, edited by Lang F.. Basel: Karger, 1998, p. 169-184.

4.   Dumont, JN. Oogenesis in Xenopus laevis (Daudin). Stages of oocyte development in laboratory maintained animals. J Morphol 136: 153-180, 1970.

5.   Eveloff, J, and Calamia J. Effect of osmolarity on cation fluxes in medullary thick ascending limb cells. Am J Physiol Renal Fluid Electrolyte Physiol 250: F176-F180, 1986[ISI][Medline].

6.   Gamba, G, Miyanoshita A, Lombardi M, Lytton J, Lee WS, Hediger MA, and Hebert SC. Molecular cloning, primary structure and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney. J Biol Chem 296: 17713-17722, 1994.

7.   Garay, RP, Nazaret C, Hannaert PA, and Gragoe EJ, Jr. Demonstration of a [K+,Cl-]-cotransport system in human red cells by its sensitivity to [(dihydroindenyl)oxy]alkanoic acids: regulation of cell swelling and distinction from the bumetanide-sensitive [Na+, K+, Cl-]-cotransport system. Mol Pharmacol 33: 696-701, 1988[Abstract].

8.   Giesen-Crouse, E, Fandeleur P, Schmidt M, Schwartz J, and Imbs JL. Loop diuretics bind to distinct receptors in renal medulla and cortex. J Hypertens, 3: Suppl 3: S211-S213, 1985[ISI].

9.   Giesen-Crouse, EM, Welsch C, Imbs JL, Schmidt M, and Schwartz J. Characterization of a high affinity piretanide receptor on kidney membranes. Eur J Pharmacol 114: 23-31, 1985[ISI][Medline].

10.   Gillen, CM, Brill S, Payne JA, and Forbush B, III. Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat and human. A new member of the cation-chloride cotransporter family. J Biol Chem 271: 16237-16244, 1996[Abstract/Free Full Text].

11.   Haas, M, Dunham PB, and Forbush B, III. [3H]bumetanide binding to mouse kidney membranes: identification of corresponding membrane proteins. Am J Physiol Cell Physiol 260: C791-C804, 1991[Abstract/Free Full Text].

12.   Hebert, SC. Roles of Na-K-2Cl and Na-Cl cotransporters and ROMK potassium channels in urinary concentrating mechanism. Am J Physiol Renal Physiol 275: F325-F327, 1998[Abstract/Free Full Text].

13.   Hebert, SC, and Andreoli TE. Control of NaCl transport in the thick ascending limb. Am J Physiol Renal Fluid Electrolyte Physiol 246: F745-F756, 1984[Abstract/Free Full Text].

14.   Hebert, SC, and Andreoli TE. Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle. II. Determinants of the ADH-mediated increases in transepithelial voltage and in net Cl- absorption. J Membr Biol 80: 221-233, 1984[ISI][Medline].

15.   Hebert, SC, Culpepper RM, and Andreoli TE. NaCl transport in mouse medullary thick ascending limbs. II. ADH enhancement of transcellular NaCl cotransport; origin of transepithelial voltage. Am J Physiol Renal Fluid Electrolyte Physiol 241: F432-F442, 1981[Abstract/Free Full Text].

16.   Hebert, SC, Culpepper RM, and Andreoli TE. NaCl transport in mouse medullary thick ascending limbs. III. Modulation of ADH effect by peritubular osmolality. Am J Physiol Renal Fluid Electrolyte Physiol 241: F443-F451, 1981[Abstract/Free Full Text].

17.   Igarashi, P, Vanden Heuvel GB, Payne JA, and Forbush B, III. Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter. Am J Physiol Renal Fluid Electrolyte Physiol 269: F406-F418, 1995.

18.   Monroy, A, Plata C, Hebert SC, and Gamba G. Characterization of the thiazide-sensitive Na+-Cl- cotransporter: a new model for ions and diuretics interaction. Am J Physiol Renal Physiol 279: F161-F169, 2000[Abstract/Free Full Text].

19.   Mount, DB, Baekgard A, Hall AE, Plata C, Xu J, Beier DR, Gamba G, and Hebert SC. Isoforms of the Na-K-2Cl transporter in murine TAL. I. Molecular characterization and intrarenal localization. Am J Physiol Renal Physiol 276: F347-F358, 1999[Abstract/Free Full Text].

20.   Mount, DB, Mercado A, Song L, Xu J, Geroge AL, Jr, Delpire E, and Gamba G. Cloning and characterization of KCC3 and KCC4, new members of the cation-chloride cotransporter gene family. J Biol Chem 274: 16355-16362, 1999[Abstract/Free Full Text].

21.   Plata, C, Mount DB, Rubio V, Hebert SC, and Gamba G. Isoforms of the Na-K-2Cl cotransporter in murine TAL. II. Functional characterization and activation by cAMP. Am J Physiol Renal Physiol 276: F359-F366, 1999[Abstract/Free Full Text].

22.   Shetlar, RE, Scholermann B, Morrison AI, and Kinne RKH Characterization of the Na+K+2Cl- cotransport system in oocytes from Xenopus laevis. Biochem Biophys Acta 1023: 184-190, 1990[ISI][Medline].

23.   Sun, A, Grossman EB, Lombardi M, and Hebert SC. Vasopressin alters the mechanism of apical Cl- entry from Na+:Cl- to Na+:K+:2Cl- cotransport in mouse medullary thick ascending limb. J Membr Biol 120: 83-94, 1991[ISI][Medline].

24.   Watts, BA, III, and Good GW. Hyposmolality stimulates apical membrane Na(+)/H(+) exchange and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in renal thick ascending limb. J Clin Invest 104: 1593-1602, 1999[Abstract/Free Full Text].


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