NKCC1 and NHE1 are abundantly expressed in the basolateral plasma membrane of secretory coil cells in rat, mouse, and human sweat glands

Lene N. Nejsum, Jeppe Praetorius, and Søren Nielsen

The Water and Salt Research Center, University of Aarhus, Aarhus, Denmark

Submitted 7 May 2004 ; accepted in final form 5 March 2005


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In isolated sweat glands, bumetanide inhibits sweat secretion. The mRNA encoding bumetanide-sensitive Na+-K+-Cl cotransporter (NKCC) isoform 1 (NKCC1) has been detected in sweat glands; however, the cellular and subcellular protein localization is unknown. Na+/H+ exchanger (NHE) isoform 1 (NHE1) protein has been localized to both the duct and secretory coil of human sweat duct; however, the NHE1 abundance in the duct was not compared with that in the secretory coil. The aim of this study was to test whether mRNA encoding NKCC1, NKCC2, and Na+-coupled acid-base transporters and the corresponding proteins are expressed in rodent sweat glands and, if expressed, to determine the cellular and subcellular localization in rat, mouse, and human eccrine sweat glands. NKCC1 mRNA was demonstrated in rat palmar tissue, including sweat glands, using RT-PCR, whereas NKCC2 mRNA was absent. Also, NHE1 mRNA was demonstrated in rat palmar tissue, whereas NHE2, NHE3, NHE4, electrogenic Na+-HCO3 cotransporter 1 NBCe1, NBCe2, electroneutral Na+-HCO3 cotransporter NBCn1, and Na+-dependent Cl/HCO3 exchanger NCBE mRNA were not detected. The expression of NKCC1 and NHE1 proteins was confirmed in rat palmar skin by immunoblotting, whereas NKCC2, NHE2, and NHE3 proteins were not detected. Immunohistochemistry was performed using sections from rat, mouse, and human palmar tissue. Immunoperoxidase labeling revealed abundant expression of NKCC1 and NHE1 in the basolateral domain of secretory coils of rat, mouse, and human sweat glands and low expression was found in the coiled part of the ducts. In contrast, NKCC1 and NHE1 labeling was absent from rat, mouse, and human epidermis. Immunoelectron microscopy demonstrated abundant NKCC1 and NHE1 labeling of the basolateral plasma membrane of mouse sweat glands, with no labeling of the apical plasma membranes or intracellular structures. The basolateral NKCC1 of the secretory coils of sweat glands would most likely account for the observed bumetanide-sensitive NaCl secretion in the secretory coils, and the basolateral NHE1 is likely to be involved in Na+-coupled acid-base transport.

bumetanide; eccrine glands; immunohistochemistry; immunoblotting


ECCRINE SWEAT GLANDS ARE COILED tubular glands situated on the digits and footpads of the paws of rodents (11). In contrast, human eccrine sweat glands are found both in the histologically thick skin of the palmar and plantar surfaces and in the thin skin that covers most of the rest of the body (for review, see Ref. 19). The palmar and plantar eccrine glands are thought to provide enhanced tactile sensitivity rather than serving the thermoregulatory role ascribed to eccrine glands of the thin skin in humans (25). The epithelium of the human duct is reabsorptive and contains two layers of cells, while ion and water transport are minimal across the single layer of rodent ducts. Sweat produced under basal conditions is hypotonic in humans and hypertonic in rodents (19).

The bumetanide-sensitive cotransporters mediate the coupled electroneutral movement of Na+, K+, and 2Cl ions into cells and play important roles in transepithelial salt transport and maintenance of cell volume (6). Two genes have been identified for bumetanide-sensitive Na+-K+-Cl cotransporters (NKCC). The first gene, encoding NKCC isoform 1 (NKCC1, or bumetanide-sensitive cotransporter BSC-2), is localized basolaterally in multiple secretory epithelia such as the salivary glands (13) and the pancreas (12). The other gene, encoding NKCC isoform 2 (NKCC2, or BSC-1), is localized exclusively in the kidney to the apical plasma membranes of the thick ascending limb of Henle (10), where it is involved in Na+-K+-Cl reabsorption.

Application of bumetanide to isolated monkey palm sweat glands has been shown to inhibit sweat secretion and influx of ions (18). The presence of bumetanide-sensitive K+ and Cl influx into this cell type further evidenced the expression of a NKCC transporter (20). Furthermore, NKCC1 mRNA has been demonstrated in rhesus monkey eccrine clear cells, which display bumetanide Na+-dependent cell volume recovery after shrinkage (23). The experiments did not discriminate between apical or basolateral transport, but from homology to other exocrine glands, such as salivary glands, NKCC1 is most likely expressed at the basolateral domain. In summary, the cellular and subcellular localization of bumetanide-sensitive cotransport proteins have not been established in sweat glands.

The transport of Na+ and Cl into glandular cells alternatively can occur by the combined action of Na+/H+ exchange and Cl/HCO3 exchange (by NHE1 and anion exchanger AE2, respectively; Ref. 9) or by Na+-dependent HCO3 transporters. The presence of basolateral Na+/H+ exchange has previously been reported in human sweat gland ducts (1), and recently the Na+/H+ exchanger NHE1 has been immunolocalized to the duct cells of human sweat glands (5). However, it was not reported whether NHE1 was expressed to the same extent in the secretory coil. Thus the purpose of the present study was 1) to define the cellular and subcellular localization of NKCC1 and NHE1 in rat, mouse, and human sweat glands; and 2) to determine whether NKCC2, NHE2, NHE3, NHE4, or Na+-dependent HCO3 transporter mRNA are expressed in sweat glands and, if expressed, to localize the proteins. This was achieved by performing RT-PCR, immunoblotting, immunohistochemistry, and immunoelectron microscopy.


    METHODS
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 METHODS
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Experimental animals. The animal protocols were approved by the Institutional Animal Care and Use Committee and by the boards of the Institute of Anatomy, University of Aarhus, in accordance with the licenses for the care and use of experimental animals issued by the Danish Ministry of Justice. Male Wistar rats (180–260 g) and male NMRI mice (20–30 g; M & B, Ry, Denmark) were maintained on a standard rodent diet (Altromin, Lage, Germany) with free access to water. The animals were anesthetized by halothane inhalation before being killed.

Human skin. Human palm skin was obtained postmortem after we obtained informed consent from the relatives of the deceased according to the Danish guidelines for the use of human material.

RNA extraction and RT-PCR. Total RNA was extracted from rat tissues using the RNeasy Mini kit (Qiagen, VWR, Denmark). After DNAse treatment (RQ DNase I; Promega, Ramcon, Denmark), total RNA was reverse transcribed using SuperScript II (Life Technologies, Tåstrup, Denmark). PCR was performed for 30 cycles using sequence-specific sense and antisense oligonucleotide primers (Table 1) with HotStar Taq Master Mix (Qiagen). Negative PCR controls included omission of reverse transcriptase or omission of cDNA. PCR products were subjected to gel electrophoretic separation (2% agarose) and photographed under ultraviolet illumination. PCR products were sequenced to confirm specificity (Lark Technologies, Essex, UK).


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Table 1. Primers used for RT-PCR

 
Primary antibodies. Previously characterized rabbit anti-rat immunosera and affinity-purified antibodies included anti-NH2-terminal and anti-COOH-terminal NKCC1 (13), NKCC2 (2), NHE1 (8), NHE2 (24), and NHE3 (3).

Membrane fractionation and immunoblotting. Tissues were homogenized for 20–30 s and centrifuged at 4,000 g for 15 min at 4°C to remove whole cells and nuclei. Gel samples were made from the supernatant [2% wt/vol SDS, 40.0 mM 1,4-dithiothreitol, 6% glycerol (vol/vol), and 10 mM Tris-(hydroxymethyl)aminomethane (Tris), pH 6.8, with bromophenol blue] and were run on 9% SDS-PAGE minigels. Proteins were transferred to nitrocellulose membranes, which were blocked by incubation in 5% nonfat dry milk followed by overnight incubation at 4°C with primary antibodies. Bound antibody was visualized using horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Dako, Glostrup, Denmark) and enhanced chemiluminescence (ECL; Amersham Pharmacia, Denmark).

Immunohistochemistry and light microscopy. Animals were fixed by performing cardiac perfusion with 3% paraformaldehyde in 0.1 M cacodylate buffer, and human skin was fixed by immersion in the same buffer. The tissues were dehydrated and embedded in paraffin wax, and 2-µm sections were cut using a rotary microtome (Leica, Heidelberg, Germany). The sections were dewaxed in xylene and rehydrated gradually through 99%, 96%, and 70% ethanol, and endogenous peroxidase was blocked using 0.5% H2O2 in absolute methanol. The sections were boiled in 1 mM Tris, pH 9, with 0.5 mM EGTA to reveal antigens and then quenched in 50 mM NH4Cl and blocked in phosphate-buffered saline (PBS) solution with 1% bovine serum albumin (BSA), 0.05% saponin, and 0.2% gelatin. The sections were incubated overnight at 4°C with primary antibodies diluted in PBS with 0.1% BSA and 0.3% Triton X-100 and then with horseradish peroxidase-conjugated goat anti-rabbit IgG (Dako) diluted in PBS with BSA and Triton X-100. The peroxidase stain was visualized using 0.05% 3,3'-diaminobenzidine tetrahydrochloride dissolved in PBS with 0.1% H2O2. Mayer's hematoxylin was used for counterstaining, and the sections were dehydrated and mounted in hydrophobic Eukitt mounting medium (O. Kindler, Freiburg, Germany). Microscopy was performed using a Leica DMRE bright-field microscope equipped with a Leica DM300 digital camera.

Immunogold labeling and electron microscopy. Tissue blocks prepared from mouse palmar skin were cryoprotected with 2.3 M sucrose and 2% paraformaldehyde and rapidly frozen in liquid nitrogen. The samples were first freeze substituted using sequential equilibration in methanol containing 0.5% uranyl acetate at temperatures raised gradually from –80 to –70°C; then rinsed in pure methanol while the temperature was increased from –70 to –45°C; infiltrated with Lowicryl HM20 and methanol 1:1, 2:1, and finally pure Lowicryl HM20; and then ultraviolet polymerization was performed at –45 and 0°C. Immunolabeling was performed on ultrathin Lowicryl HM20 sections (40–80 nm) pretreated with a saturated solution of NaOH in absolute ethanol and preincubated with 0.1% sodium borohydride and 50 mM glycine in 0.05 M Tris, pH 7.4, containing 0.1% Triton X-100. Sections were incubated overnight at 4°C with the primary antibody diluted in 0.05 M Tris, pH 7.4, containing 0.1% Triton X-100 with 0.2% milk. Sections were then incubated with goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles (GAR.EM10; BioCell Research Laboratories, Cardiff, UK). The sections were stained with uranyl acetate and lead citrate before examination under a Philips Morgagni 268D electron microscope.


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RT-PCR detection of NKCC, NHE, and NBC mRNA. RT-PCR was performed on total RNA from rat palmar skin using specific primers (Figs. 1 and 2). Rat kidney, duodenum, and pylorus RNA were used as positive controls. PCR with {beta}-actin primers confirmed the presence of cDNA in all samples (data not shown). NKCC1 primers produced a 566-bp band in samples from both rat palmar skin and kidney (Fig. 1). NKCC2 primers failed to amplify mRNA from rat palmar skin, but 272-bp bands were produced from positive controls (Fig. 1). Thus only NKCC1 mRNA was detected in rat palmar skin.



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Fig. 1. RT-PCR analysis of rat palmar skin for Na+-K+-Cl cotransporter (NKCC) mRNA. RT-PCR using NKCC isoform 1 (NKCC1) primers amplified a 566-bp product from rat palmar skin and kidney. NKCC2 primers amplified a 272-bp product from rat kidney but failed to produce a product from rat palmar skin. Negative controls were omission of reverse transcriptase (RT–) and omission of cDNA (H2O). A 100-bp DNA ladder was used as a molecular size marker, and the arrows indicate 500 bp.

 


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Fig. 2. RT-PCR analysis of rat palmar skin for Na+/H+ exchanger (NHE) mRNA. RT-PCR using NHE isoform 1 (NHE1)-specific primers amplified 247-bp products from rat palmar skin and kidney. NHE2, NHE3, NHE4, anion exchanger AE2, electroneutral Na+-HCO3 cotransporter NBCn1, electrogenic Na+-HCO3 cotransporter 1 (NBCe1), NBCe2, and Na+-dependent Cl/HCO3 exchanger NCBE primers all produced products from positive control tissues but failed to produce products using rat palmar skin RNA. Negative controls were omission of reverse transcriptase (RT–) and omission of cDNA (H2O). A 100-bp DNA ladder was used as a molecular size marker, and the arrows indicate 500 bp.

 
NHE1 primers produced a 247-bp band in samples from rat palmar skin, duodenum, and kidney (Fig. 2). The >500-bp product observed with kidney RNA was sequenced and identified as an alternatively spliced variant of NHE1. The NHE2, NHE3, and NHE4 primers failed to amplify mRNA from rat palmar skin, but bands were produced from positive controls (Fig. 2). Furthermore, RT-PCR failed to detect mRNA encoding the HCO3 transporters AE2, NBCe1, NBCe2, NBCn1, and NCBE (Fig. 2). Thus the only Na+-coupled acid-base transporter expressed in rat palmar skin was NHE1.

Immunoblotting for NKCC and NHE protein expression. Immunoblots prepared from rat palmar skin were probed with specific antibodies to NKCC1 and NKCC2. Anti-NKCC1 antibodies specifically reacted with an ~200-kDa protein in rat palmar skin (Fig. 3) corresponding to a NKCC1 monomer (14). Anti-NKCC2 antibodies failed to react with samples of rat palmar skin but reacted with a sample of inner stripe of outer medulla of rat kidney generating a band of ~200 kDa (Fig. 3).



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Fig. 3. Immunoblot analysis of rat palmar skin for NKCC and NHE proteins. Anti-NKCC1 immunoblot of rat palmar skin showed an ~200-kDa protein in rat palmar skin. Anti-NKCC2 immunoblot analysis of rat palmar skin and the inner stripe of outer renal medulla (ISOM) showed an ~200-kDa protein in ISOM, whereas the NKCC2 antibody failed to react with membranes from rat palmar skin. Anti-NHE1 immunoblot analysis showed an ~85-kDa protein in rat palmar skin and renal inner medulla (IM). Anti-NHE2 and anti-NHE3 antibodies failed to produce a band from rat palmar skin but produced bands of ~85 kDa from rat renal cortex (Ctx) and whole kidney (Kidney), respectively.

 
Immunoblots prepared from rat palmar skin were probed with antibodies to NHE1, NHE2, and NHE3. Membrane samples prepared from rat kidney served as positive controls. Anti-NHE1 antibodies specifically reacted with an ~85-kDa protein in rat palmar skin and renal inner medulla (Fig. 3). Anti-NHE2 and anti-NHE3 antibodies failed to react with rat palmar skin samples but reacted with rat kidney control samples generating bands of ~85 kDa (Fig. 3). Because we did not have access to a specific NHE4 antibody, investigations of NHE4 were not conducted at the protein level. Thus the expression of NKCC1 and NHE1 protein was demonstrated in rat palmar skin.

Immunohistochemical and immunoelectron microscopic localization of NKCC1. Cellular and subcellular localization of NKCC1 was defined using immunoperoxidase labeling of 2-µm paraffin sections from rat palmar skin (Fig. 4). Immunohistochemical analysis revealed strong NKCC1 labeling of the basolateral plasma membrane domains of the secretory coils of rat sweat glands but no labeling of the apical plasma membrane domain (Fig. 4, A and B). The labeling intensity was reduced in the ducts compared with the secretory coils (Fig. 4, C and D). The figures exemplify the labeling observed using the anti-NH2-terminal NKCC1 antibody. An anti-COOH-terminal antibody labeled sweat glands and kidneys with an identical staining pattern (data not shown). Anti-NKCC1 antibodies did not label the epidermis (Fig. 4E). An identical labeling pattern was found in human (data not shown) and mouse sweat glands (data not shown). Anti-NKCC2 antibodies did not label rat sweat glands (Fig. 4F) but labeled control kidney sections (Fig. 4F, inset). Peptide preabsorption tests were not performed, because the immunizing peptides were not available. The specific staining patterns for each of the two NKCC antibodies never overlapped and thus served as negative controls for each other.



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Fig. 4. Immunoperoxidase localization of NKCC1 in paraffin-embedded sections of rat palmar skin. A and B: anti-NKCC1 antibody labeling revealed immunoreactivity corresponding to the basolateral plasma membranes (arrows) of the secretory coils (SC) of the sweat glands, with no labeling of the apical plasma membranes (arrowheads). C and D: NKCC1 staining was reduced in the duct (Dt; arrowheads) compared with the secretory coils (arrows). E: no anti-NKCC1 labeling was observed in epidermis (arrowheads), including the terminal duct (arrows). F: no anti-NKCC2 labeling was observed in secretory coils (arrowheads) or in ducts. Inset shows positive apical anti-NKCC2 labeling in renal medullary thick ascending limbs (T), while the NKCC1-positive collecting ducts (D) did not stain.

 
Immunogold electron microscopy was performed to test whether the NKCC1 labeling of the secretory cells in sweat glands was associated with the plasma membrane. The anti-NKCC1 labeling was abundantly and exclusively associated with the basolateral plasma membranes of secretory cells of mouse sweat glands (Fig. 5). The apical plasma membranes did not label with the anti-NKCC1 antibody (Fig. 5, inset). Intracellular structures were unlabeled.



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Fig. 5. NKCC1 immunogold electron microscopy of sweat gland secretory cells in NMRI mouse palmar skin. Anti-NKCC1 gold labeling was observed in the basolateral plasma membranes (arrows); intracellular compartments were not labeled. Anti-NKCC1 antibodies did not label the apical plasma membrane (inset). The following structures are indicated: mitochondria (M), cytosol (Cy), basement membrane (BM), microvilli (MV), lumen (Lu), and connective tissue (CT).

 
Immunohistochemical and immunoelectron microscopic localization of NHE1. Cellular and subcellular localization of NHE1 was determined using immunoperoxidase labeling of 2-µm paraffin sections from rat palmar skin (Fig. 6). Immunohistochemical analysis revealed strong NHE1 labeling of the basolateral plasma membrane domains of the secretory coils of rat sweat glands but no labeling of the apical plasma membrane (Fig. 6, A and B, respectively). Interestingly, the labeling intensity was reduced in the ducts compared with the secretory coils (Fig. 6, C and D). Anti-NHE1 antibodies did not label the epidermis (data not shown). An identical labeling pattern was found in human sweat glands (data not shown) and in mouse sweat glands (data not shown). Anti-NHE2 (Fig. 6E) and anti-NHE3 (Fig. 6F) did not label sweat glands but labeled control kidney sections. To verify antibody specificity, rat kidney sections were labeled. This demonstrated basolateral NHE1 (Fig. 6D, inset) staining of renal inner medullary collecting ducts as well as apical NHE2 (Fig. 6E, inset) and NHE3 (Fig. 6F, inset) labeling of renal proximal tubule. Peptide preabsorption tests were not performed, because the immunizing peptides were not available.



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Fig. 6. Immunoperoxidase localization of NHE1, NHE2, and NHE3 in paraffin-embedded sections of rat palmar skin. A and B: anti-NHE1 antibody labeling of a rat palmar skin revealed immunoreactivity corresponding to the basolateral plasma membrane domains of the secretory coils of the sweat glands (arrows) with no labeling of the apical plasma membrane domains (arrowheads). C: anti-NHE1 immunolabeling was reduced in the ducts (arrowhead) compared with the secretory coils (arrow). D: anti-NHE1 immunolabeling decreases corresponding to the transition from secretory coil (arrowheads) to duct. Inset shows positive NHE1 labeling of the basolateral membrane domains of renal inner medullary collecting ducts. E and F: no anti-NHE2 or anti-NHE3 immunolabeling was observed in secretory coils of sweat glands (arrowheads). Insets show positive NHE2 and NHE3 labeling, respectively, of the apical membrane domains of renal proximal tubules.

 
Immunogold electron microscopy was used to test whether NHE1 labeling of the basolateral plasma membrane domain of secretory cells in sweat glands was associated with the plasma membrane. The anti-NHE1 labeling was indeed confined to the basolateral plasma membranes of secretory cells of mouse sweat glands (Fig. 7). The apical plasma membranes did not label with the anti-NHE1 antibody (Fig. 7, inset). No intracellular structures were labeled.



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Fig. 7. NHE1 immunogold electron microscopy of sweat gland secretory cells in NMRI mouse palmar skin. Anti-NHE1 labeling was observed in the basolateral plasma membranes (arrows); intracellular compartments were not labeled. Anti-NHE1 antibodies did not label the apical plasma membrane (inset). The following structures are indicated: mitochondria (M), cytosol (Cy), basement membrane (BM), microvilli (MV), lumen (Lu), and connective tissue (CT).

 

    DISCUSSION
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 METHODS
 RESULTS
 DISCUSSION
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Rodent sweat glands are exclusively present on the palms of the paws, while in humans they are found both in the thick skin of the palms and on the thin skin covering most of the remaining body surface (for review, see Ref. 19). Rodent sweat glands are thought to provide enhanced tactile sensitivity like they do in human palms. The palmar sweat glands do not participate significantly in thermoregulation as do the sweat glands in human thin skin (25). The eccrine glands consist of two different functional units: the secretory coil and the duct. In the secretory coil, the sweat is secreted as an almost isotonic fluid in humans and as a hypertonic fluid in rodents (19). In humans, the primary sweat is modified in the duct, where Na+ and Cl are reabsorbed by the ionic epithelial Na+ channels and CFTR channels, respectively (16). This normally results in the generation of a hypotonic sweat in humans (19). The rodents are not as prone to significant losses of NaCl, owing to the limited surface area containing eccrine sweat glands, and hence the ducts are not equipped to reabsorb solutes. Sweat secretion is elicited by sympathetic innervation of the sweat glands. Upon stimulation, human sweat becomes more isotonic and less acidic because ductal NaCl reabsorption and H+ secretion do not have the capacity to fully compensate for the rise in demand during increased sweat secretion (4).

In this study, we localized NKCC1 to the basolateral plasma membrane of secretory cells of sweat glands in rat, mouse, and human skin and also observed NKCC1 in the basolateral plasma membrane of the cells in the coiled part of the ducts, although at a much lower expression level. These findings support the physiological data from isolated monkey palm sweat glands, in which sweat secretion has been shown to be inhibited by bumetanide, a selective inhibitor of the NKCC/BSC proteins (18). The recovery of methacholine-induced shrinkage of simian eccrine clear cells (21, 22) was also shown to be blocked completely by bumetanide (23), demonstrating that bumetanide effectively blocked entry of Na+, K+, and Cl ions into the clear cells. These physiological data indicate that a NKCC protein is involved in sweat formation and in the recovery of the shrinkage observed in eccrine clear cells after sweat induction. This was further supported by the finding of mRNA encoding NKCC1 in simian eccrine clear cells (23). Despite the demonstration that NKCC1 mRNA is expressed in rhesus sweat glands, the exact localization (cellular and subcellular) of NKCC1 protein was not defined directly in sweat glands before the present study. No NKCC2 mRNA or protein was found in rodent sweat glands. It is thought that NKCC1 is involved in the Cl accumulation in the gland cells against the electrochemical gradient, which may be involved in creating the gradient for Cl to the luminal space (17). The subcellular localization observed in the present study is consistent with such a role for NKCC1. The apical Cl secretion then drives Na+ (by a paracellular pathway) and H2O secretion in sweat glands as it does in other exocrine glands (19).

The NKCC1 in eccrine glands was shown to be activated by cell shrinkage (6). Cell shrinkage is thought to be an early event in secretion because apical Cl and K+ loss would drag water out of the cells. Thus basolateral NKCC1 would likely both supply the cells with Cl and compensate for the volume change. NKCC1 may also serve as a volume regulator in the coiled part of the duct, and the lower expression level could reflect a smaller capacity and need for volume regulation in these cells compared with secretory coil cells. Cell shrinkage could also be counteracted by the combined action that basolateral NHE and AE proteins provide, which is shown to be the route for NaCl entry into other glandular epithelia (7, 9). NHE1 has previously been localized to the basolateral domain of human eccrine duct and secretory coil (5); however, these previous authors did not report whether there were any differences in the expression of NHE1 between the duct and the secretory part. We also localized NHE1 to the basolateral plasma membrane of the secretory coil and coiled duct of rodent and human sweat glands. NHE1 expression was much higher in the secretory coil compared with the coiled duct in both rodent and human skin. Nevertheless, the involvement of NHE1 in volume regulation and hence in sweat secretion is unlikely, because AE2 mRNA and protein are absent from the rodent sweat gland (immunohistochemical data not shown). Furthermore, basolateral administration of bumetanide to human sweat glands did not inhibit secretion (15). The role of NHE1 in the basolateral plasma membrane is likely to provide an exit pathway for excess H+ generated by oxidative phosphorylation. No other known Na+-dependent acid-base transporter seems to be expressed in sweat glands, because mRNA, and also protein for NHE2 and NHE3, was absent from the skin, including both the secretory coils and the ducts of sweat glands. The RT-PCR analysis included the Cl-transporting forms NDCBE1 (Na+-driven Cl/HCO3 exchanger isoform 1) and NCBE, which were not detected.

In conclusion, we have found that eccrine sweat gland NKCC1 and NHE1 are exclusively present in the basolateral plasma membrane of rat, human, and mouse secretory coils and to a lesser degree in the coiled parts of the ducts.


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The Water and Salt Research Center at the University of Aarhus is established and supported by the Danish National Research Foundation (Danmarks Grundforskningsfond). This work was supported by the Karen Elise Jensen Foundation, the European Union Commission (QRLT 2000 00778 and QRLT 2000 00987), Novo Nordisk Foundation, Danish Medical Research Council, Helen and Ejnar Bjørnows Foundation, A. P. Møller and Spouse Chastine McKinney Møllers Foundation, Ruth T. E. Konig-Petersens Research Foundation for Kidney Diseases, and The Research Foundation of the Danish Kidney Association.


    ACKNOWLEDGMENTS
 
We thank Ida Maria Jalk, Mette Frank Vistisen, Zhila Nikrozi, Lotte V. Holbech, Inger Merete Paulsen, Helle Høyer, Dorte Wulff, and Gitte Kall for excellent technical assistance. Antibodies were generously provided by Dr. R. J. Turner (NKCC1), Dr. M. Donowitz (NHE1 and NHE2), Dr. M. Knepper (NHE3), and Dr. S. L. Alper (AE2).


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. Nielsen, The Water and Salt Research Center, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus, Denmark (e-mail: sn{at}ana.au.dk)

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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