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Am J Physiol Renal Physiol 290: F608-F616, 2006. First published September 6, 2005; doi:10.1152/ajprenal.00248.2005
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EDITORIAL FOCUS

cAMP increases surface expression of NKCC2 in rat thick ascending limbs: role of VAMP

Pablo A. Ortiz

Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan

Submitted 13 June 2005 ; accepted in final form 5 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
NaCl absorption by the thick ascending limb of Henle’s loop (TAL) is mediated by the apical Na-K-2Cl cotransporter NKCC2. cAMP increases NaCl absorption in the TAL by stimulating NKCC2. In oocytes, cAMP increases NKCC2 activity by regulating its trafficking. However, the mechanism by which cAMP stimulates NKCC2 in TALs is not clear. We hypothesized that cAMP increases surface expression of NKCC2 and NaCl absorption in TALs and that vesicle-associated membrane protein (VAMP) is involved in this mechanism. We used surface biotinylation of rat medullary TALs (mTAL) to examine surface and total NKCC2 levels. When mTAL suspensions were treated with dibutyryl cAMP (db-cAMP) or forskolin plus IBMX for 20 min, surface NKCC2 expression increased by 126 ± 23 and 92 ± 17% above basal, respectively (P < 0.03). No changes in total NKCC2 expression were observed, suggesting that cAMP increased translocation of NKCC2. We studied the role of VAMP in NKCC2 translocation and found that incubating mTALs with tetanus toxin (30 nM), which inhibits vesicle trafficking by inactivating VAMP-2 and -3, completely blocked the stimulatory effect of db-cAMP on surface NKCC2 expression (tetanus toxin = 100% vs. tetanus toxin + db-cAMP = 102 ± 21% of control; not significant). We studied VAMP-2 and -3 expression and localization in isolated perfused TALs by confocal microscopy and found that both of them were located in the subapical space of the TAL. Finally, in isolated perfused mTALs, db-cAMP increased net Cl absorption by 95.0 ± 34.8% (P < 0.03), and pretreatment of TALs with tetanus toxin blocked the stimulation of Cl absorption (from 110.9 ± 15.9 to 109.7 ± 15.6 pmol·min–1·mm–1; not significant). We concluded that cAMP increases NKCC2 surface expression by a mechanism involving VAMP and that NKCC2 trafficking to the apical membrane is involved in the stimulation of TAL NaCl absorption by cAMP.

Na-K-2Cl cotransporter; trafficking; vesicle-associated membrane protein

THE THICK ASCENDING LIMB OF Henle’s loop (TAL) plays an important role in the maintenance of salt and fluid homeostasis. The TAL absorbs 20–30% of NaCl filtered through the glomeruli and helps maintain the corticomedullary osmotic gradient necessary for urine concentration (26). Absorption of NaCl by the TAL is a two-step process in which 1) Na, K, and Cl enter the cell across the apical membrane via electroneutral Na-K-2Cl cotransport; and 2) Na is transported out across the basolateral membrane via Na-K-ATPase and Cl exits via Cl channels or K-Cl cotransport (11, 19, 20, 22, 41). The electroneutral Na-K-2Cl cotransporter that mediates apical Na and Cl entry into the TAL has been cloned and named NKCC2 and is also called bumetanide-sensitive cotransporter 1 (BSC-1) (14, 29).

NaCl absorption by the TAL is regulated by various hormones and autacoids. Arginine vasopressin (AVP), parathyroid hormone (PTH), glucagon and beta-adrenergic agonists are known to stimulate TAL NaCl absorption by increasing cAMP production (19, 22). cAMP then stimulates NaCl absorption by increasing apical NaCl entry due to direct activation of NKCC2 (23, 25, 42, 56). However, the mechanism by which cAMP stimulates NKCC2-dependent NaCl entry into the TAL is not understood. cAMP may stimulate Na and Cl entry via NKCC2 by increasing steady-state levels of NKCC2 in the apical surface, the transport capacity of NKCC2 already present in the apical surface (by altering the affinity for transported ions), or both. In Xenopus laevis oocytes coexpressing the full-length mouse NKCC2 and its short splice variant (S-mNKCC2), cAMP increases NKCC2 activity and membrane levels by altering NKCC2 trafficking to the plasma membrane (40). In mice, short-term administration of vasopressin increases NKCC2 immunolabeling in the apical membrane and subapical space of the TAL (16). Taken together, these data indicate that cAMP regulates NKCC2 trafficking to the plasma membrane. However, it is not known whether cAMP stimulates NaCl absorption in TALs by increasing NKCC2 levels in the apical membrane or which molecular mechanisms are involved.

In renal and nonrenal epithelial cells, cAMP increases surface levels of transport proteins by stimulating trafficking and fusion of transporter-containing vesicles with the apical membrane (3, 9, 43, 44, 50, 62). In most cases studied, fusion of vesicles with the plasma membrane is mediated by proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family (10). SNARE proteins present in vesicles (vesicle-associated membrane proteins; VAMPs) interact with protein receptors present in the target membrane [synaptosome-associated proteins (SNAP) and syntaxins], allowing vesicle docking and fusion with the membrane (10). In the mTAL, NKCC2 is located in the apical membrane and also in abundant subapical intracellular vesicles (45). However, it is not known whether VAMPs are present in the TAL, or whether they are involved in the regulation of NKCC2 or other transporters.

We hypothesized that cAMP increases NKCC2 levels in the apical membrane and NaCl absorption and that VAMPs are involved in this process. By studying surface NKCC2 expression, NaCl absorption, and VAMP localization in rat mTALs, we found that cAMP increased NKCC2 surface expression and NaCl absorption. We also found that VAMP-2 and -3 are present in TALs, where they seem to be involved in NKCC2 trafficking and NaCl absorption.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Suspensions of medullary TALs. Male Sprague-Dawley rats, weighing 200–250 g (Charles River Breeding Laboratories, Wilmington, MA), were fed 0.22% sodium and 1.1% potassium (Purina, Richmond, IN) for at least 5 days. All protocols were approved and conducted in accordance with Institutional Animal Care and Use Committee guidelines. On the day of the experiment, rats were anesthetized with ketamine (100 mg/kg body wt ip) and xylazine (20 mg/kg body wt ip). Suspensions of medullary TALs (mTAL) were prepared according to a modified protocol as described previously (46, 47). Briefly, kidneys were perfused retrograde via the aorta with a solution containing 0.1% collagenase (Sigma, St. Louis, MO) and 100 U heparin. The inner stripe of the outer medulla was cut from coronal slices, minced, and incubated at 37°C for 30 min in 0.1% collagenase. The tissue was pelleted by centrifugation at 120 g for 5 min, resuspended in cold perfusion solution, and stirred on ice for 30 min to release the tubules. The suspension was filtered through 250-µm nylon mesh and centrifuged at 120 g. The pellet was washed, centrifuged again, and finally resuspended in 0.1 ml cold perfusion solution. The composition of the perfusion solution was (in mM) 130 NaCl, 2.5 NaH2PO4, 4.0 KCl, 1.2 MgSO4, 6 L-alanine, 1.0 Na2citrate, 5.5 glucose, 2.0 Ca lactate, and 10 HEPES (pH 7.40).

Surface biotinylation of mTAL suspensions. Biotinylation of cell surface proteins was performed using a modification of Gottardi’s protocol (17). mTAL suspensions from the same animal were aliquoted into two or three samples of equal volume (200 µl) in cold perfusion solution. TALs were equilibrated at 37°C for 20 min and oxygenated every 5 min with 95% O2-5% CO2. Then, TALs were treated with vehicle or dibutyryl cAMP (db-cAMP; Sigma) for 20 min, gassing every 5 min. In a different set of suspensions, tetanus toxin (Calbiochem, San Diego, CA) was present in the bath throughout the experiment. Suspensions were rapidly cooled to 4°C, washed twice with the chilled perfusion solution, and centrifuged at 120 g for 2 min. TALs were incubated with 0.75 ml of chilled biotinylation solution (perfusion solution without L-alanine or Na2citrate; pH 7.5) containing the cell-impermeant reagent NHS-SS-biotin (Pierce) at a concentration of 1.2 mg/ml in a rocker platform at 4°C. After 15 min, 0.75 ml freshly prepared NHS-SS-biotin (1.2 mg/ml) was added on top and the samples were incubated for 15 min (total, 30-min incubation with NHS-SS). After biotinylation, TALs were washed once with the perfusion solution at 4°C and twice with the same solution containing 100 mM glycine to remove excess NHS-SS-biotin. TALs were pelleted by centrifugation at 120 g for 10 min and lysed at 4°C in buffer containing 150 mM NaCl, 50 mM HEPES (pH 7.5), 5 mM EDTA, 1% Triton X-100, 0.1% SDS, and protease inhibitors [10 µg/ml aprotinin, 5 µg/ml leupeptin, 4 mmol/l benzamidine, 5 µg/ml chymostatin, 5 µg/ml pepstatin A (Sigma)]. Lysates were centrifuged at 15,000 g, and the pellet was resuspended in the same buffer by pipetting up and down without bubble formation. (We have compared this buffer with several other lysis buffers by Western blotting and found that it extracts 99% of total NKCC2 from mTAL suspensions.) Total lysates were cleared of insoluble proteins by centrifugation at 15,000 g for 10 min. Total protein content in each sample was measured in duplicate. An aliquot of total lysate was saved and used to examine total NKCC2 levels in TALs treated with cAMP analogs and tetanus toxin. Equal amounts of total protein (200 µg) were incubated overnight at 4°C with streptavidin-coated agarose beads (Pierce) at a final concentration of 12.5% in lysis buffer. Beads were precipitated by centrifugation (5,000 g); the supernatant was recovered and reincubated with 12.5% streptavidin-coated beads for 2 h at 4°C for a second round of precipitation. Beads from the first and second precipitation were pooled, washed twice with lysis buffer, twice with high-salt buffer (500 mM NaCl, 0.1% Triton X-100, 50 mM HEPES, pH 7.5, 0.1% SDS) and twice with no-salt buffer (50 mM HEPES, pH 7.5, 0.1% SDS) at 4°C. Proteins were extracted from beads by boiling for 10 min in 50 µl of SDS loading buffer containing 50 mM DTT and 10% beta-mercaptoethanol to cleave the disulfide bridge in NHS-SS-biotin. The entire supernatant (50 µl) from each sample was loaded into 6% SDS-polyacrylamide, and NKCC2 in the biotinylated fraction was detected by Western blotting as described below. In control experiments, we observed that all biotinylated NKCC2 was recovered after the second round of precipitation, because no NKCC2 was detected after a third round. We also found no biotinylation of the intracellular proteins GAPDH and transcription factor IIB (data not shown) and determined that all biotinylated NKCC2 is eluted from the beads in one round of boiling in 50 mM DTT/ beta-mercaptoethanol (10%).

Western blot analysis. Boiled samples were centrifuged (1 min at 10,000 g), loaded into each lane, separated by electrophoresis, and transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore). The membrane was incubated in blocking buffer containing 50 mM Tris, 150 mM NaCl, 5% nonfat dry milk, and 0.1% Tween-20 for 60 min and then with primary antibodies in blocking buffer for 120 min at room temperature. Rabbit anti-rat NKCC2 L320 antibody (NH2 terminus) was a kind gift of Dr. Mark Knepper (Laboratory of Kidney and Electrolyte Metabolism, National Institutes of Health) (34) and was used at 1:2,000. Rabbit anti-rat NKCC2 (COOH terminus, directed toward a peptide sequence corresponding to amino acids 859–873 in rat NKCC2) from Chemicon (Temecula, CA) was used at 1:1,000. The COOH-terminus-directed antibody from Chemicon recognizes a predominant 155 ± 10-kDa band that is not observed when the antibody is preadsorbed by incubation with the purified antigenic peptide (50 µg/ml). Experiments measuring the surface-to-total NKCC2 ratio (protocol 1) were performed with the Chemicon NKCC2 antibody. For protocols studying the effect of cAMP and tetanus toxin, we used the L320 NH2-terminal antibody. Monoclonal anti-GAPDH (1:10,000) was obtained from Chemicon, monoclonal anti-VAMP-2 (1:1,000) was from Synaptic Systems, and rabbit polyclonal anti-VAMP-3 (1:1,000) was from AbCam (Cambridge, UK). These antibodies have been used to recognize VAMP-2 and -3 isoforms (15, 49, 52, 61). Primary antibodies were washed twice for 15 min in TBS-T buffer and incubated with a 1:1,000 dilution of goat anti-rabbit or anti-mouse secondary antibody conjugated to horseradish peroxidase (Amersham). The reaction products were detected with a chemiluminescence kit (Amersham) by exposure to Fuji RX film and quantified by densitometry as follows. Films were scanned at 1,200-dpi resolution, 16-bit grayscale, with an Epson 1680 Expression Pro scanner on positive film mode and saved as uncompressed TIFF. Optical density software specifically written to quantify band intensity was produced by software engineers at Henry Ford Health System. Software was calibrated on transmittance mode and used to obtain the mean optical band densities. Exposure time and amount of protein loaded were optimized so that optical densities were in the linear range of the film.

Surface-to-total NKCC2 ratio. To estimate surface and total NKCC2, mTALs were biotinylated as described above. Surface proteins from 200 µg of total protein from TAL lysates were precipitated with streptavidin-coated beads, eluted from the beads in 50 µl of loading buffer, and loaded on 6% SDS-polyacrylamide gels. A one-tenth fraction of the supernatant (20 µg total protein) containing intracellular nonbiotinylated proteins was loaded into the same gels, resolved, and NKCC2 was measured by Western blotting. Optical densities from surface and intracellular NKCC2 bands were used to calculate total NKCC2 and per cent surface NKCC2. Given the small percentage of NKCC2 at the surface, we used intracellular NKCC2 rather than total NKCC2 to calculate the surface fraction to decrease variability of the measurement. To make sure that streptavidin-coated beads were not saturated, we generated standard curves with increasing concentrations of total protein from TAL lysates (100–600 µg) and measured surface NKCC2 levels while keeping the concentration of beads constant (12.5%). Because we observed that beads were saturated with 400–600 µg of total protein, we used 200 µg of total protein lysate to measure surface NKCC2 in all experiments.

TAL isolation and perfusion. Male Sprague-Dawley rats, weighing 120–150 g (Charles River), were used for TAL perfusion. After anesthesia, the abdominal cavity was opened; and the left kidney was bathed in ice-cold saline and removed. Coronal slices were placed in oxygenated physiological saline. mTALs were dissected from the medullary rays under a stereomicroscope at 4–10°C. TALs ranging from 0.5 to 1.0 mm were transferred to a temperature-regulated chamber and perfused using concentric glass pipettes at 37 ± 1°C as described previously (47). The flow rate of the basolateral bath was 0.5 ml/min.

Immunofluorescence and confocal microscopy for detection of NKCC2, VAMP-2, and VAMP-3 in isolated mTALs. Microdissected mTALs were perfused for 30 min at 37°C and then fixed for 60 min at 37°C with 4% paraformaldehyde in PBS, pH 7.4. Fixed cells were blocked for 30 min with 1% BSA in TBS-T perfused into the lumen, followed by a 120-min incubation with primary antibodies diluted in 1% BSA/TBS-T (NKCC2 1:500, VAMP-2, -3, 1:100) in the lumen. Rabbit anti-NKCC2 (NH2 terminus) L320, a gift of Dr. Mark Knepper (34) was used for these experiments. Monoclonal VAMP-2 antibodies were obtained from Synaptic Systems, and rabbit polyclonal VAMP-2 and VAMP-3 were from AbCam. Cells were washed with TBS-T for 10 min in both lumen and bath and then incubated for 60 min with the corresponding secondary antibody cross-adsorbed against IgGs from other species (Alexa Fluor 568 goat anti rabbit/Alexa Fluor 488 goat anti-mouse, Alexa Fluor 488 goat anti-rabbit IgG, diluted 1:200; Molecular Probes, Eugene, OR) in 1% BSA/TBS-T. Cells were washed for 10 min with TBS-T in the lumen and bath, and fluorescent images were acquired using a confocal microscope. For colocalization experiments, two rounds of labeling were performed, one for each protein. An Intervision confocal laser-scanning system was used (Noran Instruments, Middleton, WI). Alexa dyes were excited at 488 or 568 nm, and fluorescence was observed with 525/55-nm BP or 590-nm LP filters, respectively [10% laser power, 15- to 25-µm slit, with 1 image acquired for 64-s duration, slow scan, high resolution (1,024 x 1,024)]. Two-dimensional image analysis was performed with Intervision software. For colocalization experiments, we first stained NKCC2 with Alexa 568 and made sure no fluorescence was detected in the 488-nm channel. We then stained for VAMP-2 and obtained images at 488 and 568 nm. Image files were converted to TIFF via SGI imaging software, pseudocolored, and overlapped with transmitted light images using Adobe Photoshop software.

Measurement of Cl absorption. TALs were mounted on concentric glass pipettes and perfused. Luminal perfusion rate was set at 5–10 nl·min–1·mm–1. Perfusion solution gassed with air (pH = 7.40) was used for the bath and perfusate. The composition of the solution was (in mM) 130 NaCl, 2.5 NaH2PO4, 4.0 KCl, 1.2 MgSO4, 6 L-alanine, 1.0 Na2citrate, 5.5 glucose, 2.0 Ca lactate, and 10 HEPES. After initial perfusion, TALs were equilibrated for 20 min and four measurements were made to calculate basal Cl absorption rate. Then, compounds of interest were added to either the bath or lumen as indicated in RESULTS. After a 20-min reequilibration period, four additional collections were made. Cl concentration in the perfusate and collected fluid was measured by microfluorometry. All data were recorded and stored using data-acquisition software (DATAQ Instruments, Akron, OH). Data analysis was performed with software for voltage-spike analysis. Because water is not reabsorbed by the TAL, Cl absorption (JCl–) was calculated as follows:

Formula
where CR is the collection rate normalized per tubule length, CoCl– is the Cl concentration in the perfusion solution, and ClCl– is the Cl concentration in the collected fluid.

Statistics. Results are expressed as means ± SE. One-way ANOVA was used to determine statistical differences between means in different treatment groups when surface and total NKCC2 were measured by Western blotting. Student’s paired t-test was used to determine statistical differences between means before and after treatment in the same group of tubules (Cl absorption protocols). P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Electron microscopy showed that NKCC2 is located in the apical membrane of TALs. However, a significant amount of intracellular NKCC2 labeling was observed in subapical vesicles (45), indicating that a substantial fraction of NKCC2 is intracellular. Using surface biotinylation of mTAL suspensions and Western blotting, we examined surface and intracellular NKCC2 in the same mTAL suspension to estimate the ratio of surface to total NKCC2. As described in MATERIALS AND METHODS, we resolved samples from the biotinylated fraction (containing surface NKCC2) and from the remaining nonbiotinylated fraction (containing intracellular NKCC2) in the same gels. Surface and intracellular NKCC2 signals were quantified by densitometry and used to calculate total NKCC2 and percent NKCC2 in the plasma membrane. We calculated that 2.6 ± 0.5% of total NKCC2 was present on the surface under basal conditions (n = 9) (Fig. 1A). Control experiments showed that the intracellular protein GAPDH was not found in the biotinylated fraction but was abundant in samples from the nonbiotinylated fraction, indicating that the biotinylated fraction contains only surface proteins (Fig. 1B). Our protocol recovered 99% of biotinylated NKCC2. Thus our data indicate that, as with other Na transporters (35, 58, 60), only a small fraction of total NKCC2 is present on the apical surface under basal conditions.


Figure 1
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  		Fig. 1. Surface-to-total Na-K-2Cl cotransporter (NKCC2) ratio in medullary thick ascending limbs of Henle’s loop (mTALs) under basal conditions. A: representative Western blot (WB) showing biotinylated surface NKCC2 obtained from 100 (lane 1) and 200 µg (lane 2) of mTAL protein lysates compared with 1/10 fractions of intracellular NKCC2 in samples 1 (lane 3) and 2 (lane 4). We found that 2.6 ± 0.5% of total NKCC2 was at the cell surface (n = 9). B: representative WB showing that the intracellular protein GAPDH is not present in biotinylated surface fractions (lane 1), whereas it is strongly expressed in the nonbiotinylated intracellular fraction (lane 2).

 
cAMP stimulates NKCC2 in TALs. In oocytes expressing mouse NKCC2, cAMP increases bumetanide-sensitive Rb uptake by regulation of NKCC2 trafficking to the plasma membrane (40). Therefore, we examined whether the cell-permeant cAMP analog db-cAMP and stimulation of endogenous cAMP with forskolin/IBMX would increase surface NKCC2 levels in mTALs. Freshly obtained mTAL suspensions were aliquoted into three samples, equilibrated for 20 min at 37°C, and then vehicle, db-cAMP (final concentration 10–3 M), or a combination of forskolin (10 µM) and IBMX (1 mM) was added. After 20 min at 37°C, TALs were rapidly cooled, and surface and total NKCC2 from control and treated TALs were measured by Western blotting. We found that db-cAMP increased surface NKCC2 levels by 126 ± 23% compared with basal levels (n = 7, P < 0.01). Similarly, forskolin/IBMX increased surface NKCC2 levels by 92 ± 17% compared with basal (P < 0.02; Fig. 2A). NKCC2 expression in total lysates was not changed by db-cAMP or forskolin/IBMX (Fig. 2B). In a different set of experiments, a 20-min incubation with a higher concentration of db-cAMP (10–2 M) increased surface NKCC2 by 260 ± 81% compared with basal (n = 7, P < 0.01) (data not shown). These data indicate that cAMP increases NKCC2 trafficking into the surface of TALs.


Figure 2
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  		Fig. 2. cAMP increases surface NKCC2 expression in mTALs. A: cumulative data showing the effect of dibutyryl-cAMP (db-cAMP; 10–3 M) and forskolin/IBMX on surface NKCC2 expression compared with basal levels (n = 7). *P < 0.03 vs. basal. B: representative WB showing surface NKCC2 in basal (lane 1), db-cAMP (lane 2)-, and forskolin/IBMX-treated mTALs (lane 3) compared with total NKCC2 in the same samples (lane 4: basal; lane 5: db-cAMP; lane 6: forskolin/IBMX). C: tetanus toxin blocks cAMP-induced increase in surface NKCC2 in mTALs. Cumulative data show the effect of db-cAMP (10–3 M, 20 min) on surface NKCC2 expression in mTALs pretreated with tetanus toxin (30 nM) for 20 min compared with basal levels in the presence of tetanus toxin alone (n = 6). D: representative WB showing surface NKCC2 in the presence of tetanus toxin alone (lane 1) or after treatment with db-cAMP for 20 min (lane 2) and total NKCC2 (lanes 3 and 4) from the same lysates. Bottom: GAPDH expression only in total lysates but not in the surface fraction.

 
In other nephron segments, increased trafficking of transporters to the plasma membrane depends on the SNARE protein VAMP (1, 31, 54). To examine a possible role of VAMP in stimulation of surface NKCC2 expression, we used tetanus toxin, a clostridial toxin that blocks vesicle trafficking and exocytosis by inactivating VAMP-2 and -3 (28, 39, 55, 59). We studied whether tetanus toxin could block the cAMP-induced increase in surface NKCC2 levels. Freshly obtained mTAL suspensions were aliquoted into two samples, equilibrated for 20 min at 37°C in the presence of tetanus toxin (30 nM final concentration), and then vehicle or db-cAMP (10–3 M) was added. These experiments were carried out in parallel with those examining the effect of cAMP alone (described above). After 20 min at 37°C, TALs were rapidly cooled and biotinylated, and surface NKCC2 was measured. We found that in TALs pretreated with tetanus toxin, adding db-cAMP to the bath did not increase surface NKCC2 levels (tetanus toxin = 100%, tetanus toxin + db-cAMP = 102 ± 21%; n = 6; not significant) (Fig. 2C). No changes in total NKCC2 were detected during treatment with tetanus toxin. GAPDH was detected in total lysates but not in the surface fraction. These data indicate that tetanus toxin completely blocks the stimulatory effect of cAMP on translocation of NKCC2 to the apical surface.

Because tetanus toxin blocks exocytosis and vesicle trafficking by selectively cleaving the SNARE proteins VAMP-2 and -3, we studied expression of VAMP-2 and VAMP-3 in mTALs by immunofluorescence and Western blotting. TALs were isolated, perfused, and equilibrated for 20 min at 37°C before fixation with paraformaldehyde. Using monoclonal antibodies against VAMP-2, we observed fluorescent staining in all TAL cells, with a predominant subapical localization (n = 3; Fig. 3A). Although most cells showed strong subapical immunolabeling, a few cells were weakly labeled, showing more diffuse staining. VAMP-2 localization to the subapical space of TALs was confirmed with a different antibody against VAMP-2 (n = 3). We also examined VAMP-3 expression and found strong staining in all TAL cells along tubules. Similar to VAMP-2, staining for VAMP-3 was primarily localized to the subapical space (n = 3; Fig. 3B). Nonspecific fluorescent staining was not observed in control TALs exposed only to labeled secondary antibodies (Fig. 3C). Because NKCC2 is primarily localized to the apical membrane and subapical vesicles of rat TALs, we examined whether NKCC2 and VAMP-2 were colocalized in TAL cells by double-labeling studies. In separate TALs, we observed that NKCC2 and VAMP-2 were colocalized in the subapical space of TAL cells (Fig. 3, D and E). We next examined VAMP-2 and -3 expression by Western blotting in total lysates from freshly prepared mTAL suspensions. Twenty micrograms of TAL lysates were resolved in 15% SDS-polyacrylamide gels. As shown in Fig. 3F (left), VAMP-2 monoclonal antibodies detected a protein at the predicted molecular weight of 18 kDa (n = 4). In a different set of experiments, VAMP-3 antibodies (right) reacted with a protein at the predicted molecular weight of 12–13 kDa (n = 4). Taken together, these data indicate that VAMP-2 and -3 are expressed in rat mTALs where they could be involved in NKCC2 trafficking to the apical membrane.


Figure 3
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  		Fig. 3. Vesicle-associated membrane protein (VAMP)-2 and VAMP-3 are expressed in the subapical space of mTALs. Representative confocal micrographs show localization of VAMP-2 (red; A) and VAMP-3 (yellow; B) in isolated perfused mTALs. C: control (ctrl) experiment showing no nonspecific fluorescence labeling in a TAL incubated with secondary antibody alone. D: representative confocal micrograph showing localization of NKCC2 (green). E: colocalization of NKCC2 (green) and VAMP-2 (red) in a single TAL cell. Fluorescent images were overlapped with transmitted light images to improve visualization of protein localization within the cell. F: representative WB showing expression of VAMP-2 (left) and VAMP-3 (right) in mTAL lysates (n = 4).

 
Because tetanus toxin completely blocked cAMP-induced stimulation of surface NKCC2, we next examined whether tetanus toxin could block the stimulatory effect of db-cAMP on NaCl absorption by TALs. mTALs were isolated and perfused, and net Cl absorption was measured as described in MATERIALS AND METHODS. Basal Cl absorption averaged 72.3 ± 7.0 pmol·min–1·mm–1. Twenty minutes after db-cAMP (10–3 M) was added to the bath, Cl absorption increased to 137 ± 25.2 pmol·min–1·mm–1, a 95.0 ± 34.8% increase (n = 6, P < 0.03) (Fig. 4A), confirming the stimulatory effect of cAMP in rat mTALs. In a different set of TALs incubated with tetanus toxin (30 nM) in the lumen, Cl absorption was normal and averaged 110.9 ± 15.9 pmol·min–1·mm–1. However, 20 min after db-cAMP (10–3 M), was added to the bath Cl absorption was 109.7 ± 16.6 pmol·min–1·mm–1, not significantly different from baseline (n = 6) (Fig. 4B). In control experiments, adding 30 nM tetanus toxin to mTALs for 20–30 min did not significantly affect basal Cl absorption (from 82.0 ± 25.0 to 73.8 ± 17.4 pmol·min–1·mm–1, n = 4; not significant) (data not shown). Time control experiments with tetanus toxin (30 nM) showed no significant change in Cl absorption over 90 min (from 85.8 ± 4.0 to 86.5 ± 10.7 pmol·min–1·mm–1; n = 4; not significant) (data not shown). Taken together, these data indicate that tetanus toxin blocks cAMP-induced stimulation of NaCl absorption by TALs. Thus we concluded that increased surface expression of NKCC2 plays an important role in the stimulation of NaCl absorption by cAMP in rat mTALs and that VAMP proteins are involved in this mechanism.


Figure 4
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  		Fig. 4. cAMP increases net Cl absorption in mTALs. A: cumulative data showing the effect of db-cAMP on net Cl absorption by isolated perfused mTALs. Basal Cl absorption averaged 72.3 ± 7.0 pmol·min–1·mm–1. Twenty minutes after db-cAMP (10–3 M) was added to the bath, Cl absorption increased to 137 ± 25.2 pmol·min–1·mm–1 (n = 6, *P < 0.03). B: tetanus toxin blocks cAMP-induced increase in net Cl absorption by mTALs. Cumulative data show the effect of db-cAMP on net Cl absorption by isolated, perfused mTALs pretreated with tetanus toxin (30 nM). In the presence of tetanus toxin (30 nM), Cl absorption averaged 110.9 ± 15.9 pmol·min–1·mm–1. Twenty minutes after db-cAMP (10–3 M) was added to the bath, Cl absorption was 109.7 ± 15.6 pmol·min–1·mm–1, not significantly different from tetanus toxin alone (n = 6).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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cAMP stimulates NaCl absorption and NKCC2 activity in the TAL (23, 25, 42, 56). Recent data from other groups suggest that cAMP controls NKCC2 activity by regulating its trafficking to the plasma membrane (16, 40). We tested the hypothesis that cAMP increases surface levels of NKCC2 in rat TALs and that VAMPs from the SNARE family are involved in NKCC2 trafficking and activity. We found that a small fraction of total NKCC2 was present on the cell surface under basal conditions and that cAMP increased surface NKCC2 levels in mTALs. We also found that tetanus toxin prevented the increase in surface NKCC2 caused by cAMP and that VAMP-2 and VAMP-3 are expressed in TALs, where they localize to the subapical space. Finally, tetanus toxin completely blocked cAMP-induced stimulation of Cl absorption by TALs. We concluded that cAMP increases surface NKCC2 levels in rat TALs and that this mechanism is important for the stimulation of NaCl absorption by cAMP. We believe our data indicate for the first time that members of the VAMP family are involved in cAMP-stimulated NKCC2 trafficking and activity in the TAL. To our knowledge, this is the first direct evidence that NKCC2 trafficking to the apical membrane is involved in cAMP-induced stimulation of NaCl absorption by TALs.

The renal Na-K-2Cl cotransporter NKCC2 is encoded by the SLC12A1 gene (53). The full-length cDNA predicts an amino acid structure with 12 transmembrane-spanning domains, a large COOH terminus (454 amino acids), and a shorter NH2 terminus (174 amino acids) (14). As with other apical transporters, NKCC2 must traffic from the endoplasmic reticulum to the apical membrane, where it transports Na, K, and Cl across the membrane. Using electron microscopy, Nielsen et al. (45) first showed that NKCC2 is located in the apical membrane and intracellular subapical vesicles of rat TAL cells, and they suggested that NKCC2 activity may be regulated by trafficking to the apical membrane. Despite these original observations, little is known about the mechanism of NKCC2 trafficking to the apical membrane. To examine the distribution of NKCC2 (surface vs. intracellular) in TALs under basal conditions, we used surface biotinylation of mTAL suspensions, adapting the technique described by Gottardi et al. (17) for cultured epithelial cells. By measuring NKCC2 in the biotinylated (surface) and nonbiotinylated (intracellular) fraction, we calculated that 2–3% of total NKCC2 is at the cell surface under basal conditions. Control experiments showed complete recovery of total and surface NKCC2 from TAL lysates, and we found that intracellular proteins were not biotinylated in mTAL suspensions. Thus our data indicate that a small fraction of NKCC2 is located in the apical membrane under basal conditions.

Recently, Gimenez and Forbush (16) used electron microscopy to study NKCC2 distribution in mouse TALs and reported that ~5–6% of total NKCC2 immunolabeling is found either within the apical membrane or 70 nm from it. They also found that up to 45% of total NKCC2 is located within 140 nm of the apical membrane (16). Because the average size of exocytotic and endocytotic vesicles ranges from 50 to 100 nm (4, 8, 12, 48), the large percentage of intracellular NKCC2 in proximity to the apical membrane (140 nm) may represent a pool of NKCC2 located in docked vesicles or undergoing recycling. Thus our results and those from other investigators indicate that a small fraction of total NKCC2 is accessible to the TAL lumen. Similar to our results for NKCC2, other renal Na transporters that are regulated by trafficking, such as the epithelial Na channel and the inorganic phosphate cotransporter (NaPi2), show a small percentage (1–5%) of the total transporter pool located in the membrane surface (35, 58, 60). A low surface-to-total NKCC2 ratio in TALs may represent a mechanism to rapidly and efficiently increase NaCl influx by exocytosis of small amounts of intracellular NKCC2.

Vasopressin and other hormones that increase cAMP in the TAL are known to stimulate NaCl absorption by enhancing NKCC2-dependent Na and Cl entry (23, 25, 42, 56). However, the mechanism by which cAMP acutely stimulates NKCC2 and NaCl absorption in TALs is poorly understood. We found that a membrane-permeant cAMP analog (db-cAMP, 10–3 M) and the adenylate cyclase activator forskolin coupled to the phosphodiesterase inhibitor IBMX increased surface NKCC2 levels by 125 and 95%, respectively. In a different set of TALs, surface NKCC2 levels were increased further by a higher concentration of db-cAMP (10–2 M). We used a 20-min incubation with cAMP because others have shown that this is the point when NaCl absorption peaks after addition of AVP to TALs (21, 24, 25). We also found that total NKCC2 levels were not affected by cAMP treatment, indicating that the increase in surface expression was not related to enhanced de novo NKCC2 synthesis by cAMP. Gimenez and Forbush (16) also studied NKCC2 distribution in mouse TALs after a bolus injection of AVP. In agreement with our data, they observed that AVP increased immunolabeling of NKCC2 in the apical membrane and subapical space (140 nm from the membrane) by ~60%. The smaller change in apical NKCC2 levels they observed may be attributable to the fact that measurements were made 1 h after AVP infusion, and it is possible that surface expression peaked before 60 min, similar to stimulation of NaCl absorption.

We used tetanus toxin to further investigate the role of VAMPs in NKCC2 surface expression in TALs. We found that tetanus toxin abolished the increase in surface NKCC2 expression caused by db-cAMP. The mechanism of action of tetanus toxin is well defined; after internalization, tetanus toxin blocks vesicle fusion with the plasma membrane by selectively cleaving a 20-amino acid sequence present in VAMP-2 and -3 (28, 39, 55, 59). In renal cells, tetanus toxin has been shown to block vesicle trafficking and stimulation of transporter trafficking to the plasma membrane. For example, in collecting duct cells, tetanus toxin blocked cAMP-induced aquaporin-2 trafficking to the plasma membrane and cleaved VAMP-2 (18), although a higher toxin concentration (500 nM) was used in this study. Sterling et al. (55) used tetanus toxin (30 nM) to examine exocytosis of the renal K channel ROMK. They found that it completely blocked the stimulation of ROMK surface expression and concluded that exocytosis of ROMK was responsible for enhanced surface expression. The stimulation of steady-state surface NKCC2 we observed may be due to increased exocytic insertion of NKCC2, decreased endocytosis from the membrane, or both. Because tetanus toxin primarily blocks fusion of vesicles with the plasma membrane and exocytosis in most cells, our data suggest that cAMP stimulates exocytic insertion of NKCC2. However, a role for NKCC2 endocytosis in mediating the effects of cAMP in TALs cannot be completely ruled out, and further studies are needed to directly address this question.

Because tetanus toxin blocked the increase in surface NKCC2, we questioned whether it could also inhibit the effect of cAMP on NaCl absorption by mTALs. We found that treating isolated TALs with db-cAMP increased NaCl absorption by 95%, similar to the 100% increase in surface NKCC2. Pretreatment of mTALs with tetanus toxin (30 nM) completely blocked the stimulatory effect of cAMP on NaCl absorption. In control experiments, we found no effect of tetanus toxin on basal NaCl absorption nor when incubated continuously for the entire protocol, suggesting that the toxin has no metabolic or nonspecific effects on TALs at this concentration. Thus our data show that blocking NKCC2 trafficking to the apical surface also inhibits stimulation of TAL NaCl transport by cAMP and provides a causal relationship between surface NKCC2 expression and NaCl absorption in TALs. In agreement with our results, Meade et al. (40) showed that in oocytes expressing mouse NKCC2, cAMP increased NKCC2 activity and relative levels at the plasma membrane. They also reported that these effects were blocked by the microtubule-disrupting agent colchicine (40), suggesting that in oocytes NKCC2 trafficking mediates the stimulation of NKCC2 activity by cAMP. Thus our data and those of others provide strong evidence that trafficking of NKCC2 to the apical membrane is part of the mechanism by which cAMP increases NKCC2 activity and NaCl absorption. However, it is likely that other protein modifications such as phosphorylation (16), glycosylation, and protein-protein interactions could be induced by cAMP to fully activate NKCC2 or regulate its trafficking. The interaction and kinetics of these potential mechanisms with regulation of NKCC2 activity and trafficking in TALs need to be studied further.

The role of SNARE proteins in the regulation of TAL transporters into the plasma membrane is unknown. Because our data with tetanus toxin suggested that VAMPs were involved in NKCC2 surface expression and NaCl absorption, we used Western blotting and immunofluorescence/confocal microscopy of isolated perfused TALs to localize VAMP-2 and VAMP-3, the primary targets of tetanus toxin. Fluorescent labeling for VAMP-2 was studied with two different antibodies and found to be present in all cells along the TAL, with a predominant subapical localization. While all TAL cells showed VAMP-2 immunolabeling, a small number of cells showed less staining. Although heterogeneous staining may be attributable to varying accessibility of antibodies due to cell fixation or differences in exposure of antigenic sites, it may also represent different levels of VAMP-2. The TAL is composed of rough- and smooth-surfaced cells (2, 45), with apparent differences in the number of intracellular vesicles; thus different VAMP-2 levels may also be due to differential expression levels of this protein in rough- or smooth-surfaced cells. VAMP-3 labeling was observed in the subapical space of all cells along the TAL, with approximately equal intensity along tubules. Both VAMP isoforms were detected in total mTAL lysates by Western blotting. VAMP-2 is reportedly involved in exocytosis (13, 32, 33), whereas VAMP-3 is thought to be primarily involved in vesicle recycling (27, 36, 51, 57). Given that VAMP-2 and VAMP-3 shared a similar subcellular localization with NKCC2 in the subapical space, our data suggest that VAMPs are likely to be involved in the regulation of NKCC2 trafficking to the apical membrane of TALs. However, it is not possible to conclude from our data which VAMP isoform is responsible for the effect of cAMP because tetanus toxin inactivates both VAMP-2 and -3.

SNARE proteins mediate trafficking of both renal and nonrenal transporters to the plasma membrane. In collecting ducts, apical syntaxin-4 and SNAP-23 are thought to be the target SNAREs for binding of VAMP-2 located in vesicles containing aquaporin-2 (7, 37, 43). Also, trafficking, surface expression, and activity of H-ATPase in inner medullary collecting ducts are reportedly mediated by the SNARE proteins VAMP, SNAP-23, and syntaxin-1 (5, 6). Despite the growing importance of SNAREs in trafficking and regulation of transporters, little is known about their role in TAL transport. Some members of the SNARE family have been found in TALs. Using immunohistochemistry, Inoue et al. (30) reported apical expression of SNAP-23 in rat TALs. In a different study, mRNA expression of syntaxin-3 was detected in isolated mTALs (38). We found polarized expression of VAMP-2 and -3 in the subapical space and apical membrane of mTALs and blockade of cAMP-stimulated NKCC2 trafficking and NaCl transport by tetanus toxin. Taken together, these data suggest that other SNAREs also play an important role in the regulation of NKCC2 trafficking to the apical membrane and NaCl absorption by the TAL. The involvement of other SNAREs in regulation of NKCC2 surface expression, trafficking, and apical targeting remains to be studied.

We concluded that cAMP increases surface expression of NKCC2 in rat TALs and that trafficking of NKCC2 to the plasma membrane is part of the mechanism by which cAMP enhances NaCl absorption by the TAL. We report for the first time the presence of VAMP-2 and -3 in mTALs. Thus our data suggest a potential role of VAMP and other SNARE proteins in regulation of NKCC2 trafficking to the plasma membrane and NaCl absorption in the TAL.


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 ABSTRACT
 MATERIALS AND METHODS
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This study was supported by a scientist development grant (0430031N) from the American Heart Association.


   ACKNOWLEDGMENTS
 
The author is grateful to Dr. Mark Knepper (Laboratory of Kidney and Electrolyte Metabolism, National Institutes of Health) for continuous support of this project.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. A. Ortiz, Hypertension and Vascular Research Div., Dept. of Internal Medicine, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202 (e-mail: portiz1{at}hfhs.org)

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.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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