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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ABSTRACT |
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bumetanide; eccrine glands; immunohistochemistry; immunoblotting
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.
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METHODS |
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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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Membrane fractionation and immunoblotting. Tissues were homogenized for 2030 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 (4080 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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RESULTS |
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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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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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DISCUSSION |
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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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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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