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J. Biol. Chem., Vol. 275, Issue 35, 26720-26726, September 1, 2000

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Severe Impairment of Salivation in Na⁺/K⁺/2Cl Cotransporter (NKCC1)-deficient Mice^*

Richard L. Evans^§¶**, Keerang Park^§, R. James Turner, Gene E. Watson^**, Ha-Van Nguyen, Matthew R. Dennett, Arthur R. Hand, Michael Flagella^§§, Gary E. Shull^§§, and James E. Melvin^**¶¶

From the  Center for Oral Biology, Aab Institute of Biomedical Sciences and the ^** Eastman Department of Dentistry, University of Rochester Medical Center, Rochester, New York 14642, the  NIDCR, Gene Therapy & Therapeutics Branch, National Institutes of Health, Bethesda, Maryland 20892, the  Department of Pediatric Dentistry, University of Connecticut, Farmington, Connecticut 06030, and the ^§§ Department of Molecular Genetics, Biochemistry & Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

Received for publication, May 3, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The salivary fluid secretory mechanism is thought to require Na⁺/K⁺/2Cl cotransporter-mediated Cl uptake. To directly test this possibility we studied the in vivo and in vitro functioning of acinar cells from the parotid glands of mice with targeted disruption of Na⁺/K⁺/2Cl cotransporter isoform 1 (Nkcc1), the gene encoding the salivary Na⁺/K⁺/2Cl cotransporter. In wild-type mice NKCC1 was localized to the basolateral membranes of parotid acinar cells, whereas expression was not detected in duct cells. The lack of functional NKCC1 resulted in a dramatic reduction (>60%) in the volume of saliva secreted in response to a muscarinic agonist, the primary in situ salivation signal. Consistent with defective Cl uptake, a loss of bumetanide-sensitive Cl influx was observed in parotid acinar cells from mice lacking NKCC1. Cl/ HCO₃ exchanger activity was increased in parotid acinar cells isolated from knockout mice suggesting that the residual saliva secreted by mice lacking NKCC1 is associated with anion exchanger-dependent Cl uptake. Indeed, expression of the Cl/ HCO₃ exchanger AE2 was enhanced suggesting that this transporter compensates for the loss of functional Na⁺/K⁺/2Cl cotransporter. Furthermore, the ability of the parotid gland to conserve NaCl was abolished in NKCC1-deficient mice. This deficit was not associated with changes in the morphology of the ducts, but transcript levels for the -, -, and -subunits of the epithelial Na⁺ channel were reduced. These data directly demonstrate that NKCC1 is the major Cl uptake mechanism across the basolateral membrane of acinar cells and is critical for driving saliva secretion in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Saliva is initially secreted as an isotonic, plasma-like fluid, the production of which is dependent on the concerted activity of a number of membrane transport proteins to drive transepithelial Cl movement across acinar cells (see reviews in Refs. 1 and 2). According to the current model, Na⁺/K⁺/2Cl cotransport and coupled Na⁺/H⁺ and Cl/ HCO₃ exchange utilize the inwardly directed Na⁺ chemical gradient generated by the Na⁺ pump to mediate Cl influx across the basolateral membrane of acinar cells, resulting in a 4-5-fold intracellular accumulation of Cl above electrochemical equilibrium (3). Stimulation via Ca²⁺-mobilizing agonists triggers secretion by enhancing the K⁺ and Cl permeability of the basolateral and apical membranes, respectively, and thereby initiating lumenal Cl accumulation (4, 5). These same agonists dramatically up-regulate acinar Na⁺/K⁺/2Cl cotransport (6) and Na⁺/H⁺ exchange activity (7, 8) consistent with the proposed role of these transporters in driving acinar Cl entry (9-14). Na⁺ is then thought to enter the acinar lumen through the acinar tight junctions to neutralize the lumen-negative transepithelial potential created by the accumulation of Cl, and water follows the resulting NaCl osmotic gradient.

The salivary Na⁺/K⁺/2Cl cotransporter has been identified as NKCC1¹ (15), a member of the mammalian, cation-chloride cotransporter gene family (16, 17). Other members of this family include at least four distinct K⁺/Cl cotransporters (18), a Na⁺/Cl cotransporter (19), and a renal Na⁺/K⁺/2Cl cotransporter isoform (20, 21). NKCC1 is referred to as the "secretory" cotransporter isoform, although it is not necessarily involved in secretion in all secretory epithelia in which it is expressed. Surprisingly, for example, in NKCC1 knockout mice, gastric acid secretion in adults was not impaired nor was intestinal fluid secretion in suckling pups (22); two tissues where significant levels of this transporter have been detected (20, 23).

Considerable evidence supporting the involvement of Na⁺/K⁺/2Cl cotransport in the secretion of saliva has been generated over the past two decades (9-14). However, this relationship is primarily based on indirect evidence derived from experiments that (necessarily) employed nonphysiological ion substitutions and/or, in some cases, relatively nonselective inhibitors to infer the role of NKCC1 in acinar function. Therefore, to directly explore the relationship of NKCC1 expression to Cl uptake and in vivo salivation, we examined the effects of Nkcc1 gene disruption (see Ref. 22) on mouse parotid gland function. Our results demonstrate that NKCC1 is located in the basolateral membranes of the wild-type mouse parotid acinar cells, consistent with its proposed role in salivary secretion. Relative to wild-type mice, in NKCC1 knockout mice there is a dramatic reduction in acinar Cl influx in vitro and a severe deficit in the secretion of saliva measured in vivo. This reduced flow is also associated with an inability to conserve NaCl, apparently because of decreased expression of the epithelial Na⁺ channel ENaC in the salivary ducts of Nkcc1 knockout mice.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials and Null Mutant Animals-- All chemicals were from Sigma. Targeted disruption of the murine Nkcc1 gene was performed as described by Flagella (22). Heterozygous offspring were used to establish breeding colonies in the University of Rochester vivarium. All animals were housed in micro-isolator cages with access to laboratory chow and water ad libitum with a 12-h light/dark cycle. Experiments were carried out on animals aged between 2 and 4 months. Body and parotid gland weights were recorded for each animal used.

Immunohistochemistry-- The antibody -wNT, raised in rabbits against a 6xHis fusion protein corresponding to amino acids 3-202 of rat NKCC1 (25), was used for immunolocalization experiments. Parotid glands from wild-type and Nkcc1^/ (negative control) animals were removed, dispersed as described below, and fixed for 30 min with 3% paraformaldehyde. Cells were permeabilized, and nonspecific binding sites were blocked as described previously (26). Cells were incubated overnight at 4 °C with 150 µl of blocking serum containing a 1:5000 dilution of -wNT and then treated with fluorescein isothiocyanate-labeled secondary antibody (1:50 goat anti-rabbit, Jackson ImmunoResearch Laboratory, West Grove, PA) for 1 h at room temperature. Images were recorded and analyzed using a Leica confocal microscope.

Morphological Analyses-- For light and electron microscopic studies of the parotid gland, mice were anesthetized with Ketamine/Xylazine (100 mg/10 mg, intraperitoneal) and perfused intracardially with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4. The glands were excised, immersed in fixative for an additional 3-4 h, trimmed into small pieces, and rinsed in 0.1 M cacodylate buffer. The tissues were postfixed in 1% osmium tetroxide, 0.8% potassium ferricyanide in cacodylate buffer and stained in block with 0.5% aqueous uranyl acetate. After dehydration in graded ethanol solutions and substitution with propylene oxide, the tissues were embedded in Polybed epoxy resin (Polysciences). For light microscopy, 1-µm sections were stained with methylene blue-Azure II and examined in a Leitz Orthoplan microscope. Thin sections were stained with uranyl acetate and lead citrate and examined in a Philips CM10 transmission electron microscope.

Acinar Cell Preparation and in Vitro Intracellular Cl and pH Measurements-- Microfluorimetric experiments to measure intracellular Cl concentration were carried out in a physiological salt solution containing: 135 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl₂, 0.8 mM MgSO₄, 0.33 mM NaH₂PO₄, 0.4 mM KH₂PO₄, 10 mM glucose, 20 mM Hepes (pH 7.4 with NaOH), and 2 mM glutamine. Measurements of Cl/HCO₃ exchanger activity were performed in HCO₃-replete physiological salt solution in which 25 mM NaCl was substituted with 25 mM NaHCO₃. Chloride salts were replaced with gluconate salts in the Cl-free, HCO₃-containing solution. In this latter solution, the concentration of calcium gluconate was increased to 5.2 mM to compensate for chelation of calcium by gluconate. HCO₃-free and HCO₃-containing solutions were continuously gassed with 100% O₂ or 95% O₂, 5% CO₂, respectively.

Parotid acini (5-20 cells) were prepared from Nkcc1 littermates of the wild-type (+/+) and knockout (/) genotype by collagenase digestion (7, 27). In brief, glands removed from male and female animals were minced in ice-cold Earle's minimum essential medium (Biofluids, Rockville, MD) supplemented with 0.075 units/ml collagenase P, 2 mM glutamine, and 0.1% bovine serum albumin and incubated in the same medium at 37 °C for 75 min. The final acinar preparation was either: 1) resuspended in physiological salt solution containing NaHCO₃ and loaded with the pH-sensitive fluorescent indicator BCECF by incubation with BCECF/AM (2 µM) for 30 min; or 2) resuspended in HCO₃-free physiological salt solution, and loaded with the halide-sensitive fluorescent indicator by incubation with 500 µM SPQ for 15 min.

Intracellular BCECF fluorescence was monitored in ratio mode from single acinar clumps adhering to the base of a superfusion chamber mounted on a Nikon Diaphot microscope interfaced with a Spex ARCM microfluorimeter (Edison, NJ). Cells were excited at 495 and 433 nm using monochromators (0.5 µm slit width), and emitted fluorescence was measured at 530 nm. Intracellular pH was estimated by in situ calibration of the excitation ratio using the high K⁺/nigericin protocol as described previously (7). Cl/ HCO₃ exchanger activity was determined in BCECF-loaded acinar cells by switching the superfusate to a HCO₃-containing, Cl-free salt solution. This maneuver results in an intracellular alkaline load in cells with functional anion exchanger activity (28).

Intracellular fluorescence of the Cl-sensitive dye SPQ was monitored in cells excited with the UV bands generated by an Enterprise argon laser (Ultima confocal microscope, Genomic Solutions, Ann Arbor, MI), and emitted fluorescence was measured at >400 nm. SPQ fluorescence was normalized to that observed from resting (unstimulated) acini, and Cl concentration is expressed as 1/normalized SPQ fluorescence.

Measurement of Parotid Gland Fluid Secretion in Vivo-- To avoid contamination of saliva by other body fluids (e.g. tracheal and nasal secretions), saliva was collected directly from isolated parotid gland ducts. Wild-type and null mutant animals of either sex were anesthetized with chloral hydrate (500 mg/kg body weight, intraperitoneal), and the main excretory ducts of the right and left parotid glands were isolated using a dissecting microscope. Prior to saliva collection a tracheal tube was placed to prevent blockage of the windpipe and asphyxiation via excessive mucous production during secretory stimulation. Secretion was initiated by the injection of the muscarinic agonist pilocarpine HCl (10 mg/kg, intraperitoneal), and saliva was collected from the cut end of each duct in a calibrated glass micropipette (Sigma) by capillary flow. The rate of fluid production was measured by marking the position of the fluid front on the micropipette wall every 5 min. Each animal was weighed prior to the experiment and parotid glands were subsequently dissected, trimmed free of connective tissue, and weighed. For data presentation, the volume of saliva secreted and the rate of parotid saliva flow were normalized to parotid gland weight. Results are expressed as mean ± S.E. of the saliva flow from both the right and left glands from n animals measured at each time point.

Collected saliva samples were analyzed for total sodium and potassium content by atomic absorption using a Perkin-Elmer 3030 spectrophotometer. Sample osmolality was measured using a Wescor 5500 Vapor Pressure Osmometer, and Cl activity was determined using an Orion Research Model EA 940 Expandable Ion Analyzer.

Northern Blot Analysis-- Total RNA was isolated from parotid glands of mice using Trizol reagent (Life Technologies, Inc.), followed by poly(A) selection on an oligo(dT) cellulose column (Life Technologies, Inc.). Each gland sample was pooled from three mice. Northern blots were prepared and hybridized as described previously (28) using cDNA probes for NKCC1 (rat nt 3368-3563, accession number AF051561), AE2 (mouse nt 1300-1776, accession number J04036), NHE3 (rat nt 1857-2378, accession number M85300), ENaC (mouse nt 931-1185, accession number AF112185), ENaC (mouse, accession number AA240885), and ENaC (mouse nt 1882-2184, accession number AF112187). A cDNA for mouse ribosomal messenger RNA L32 (mouse nt 3078-3244, accession number K02060) was used to normalize expression between preparations. Quantitation of the autoradiographs was performed by densitometry using the Alpha Imager system (Alpha Innotech Corp., San Leandro, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Localization of NKCC1 Protein in Mouse Parotid Gland-- To better understand the function of NKCC1 expressed in mouse parotid glands we first documented its distribution by immunohistochemistry. NKCC1 has been observed in the basolateral membrane of acinar (15), but not duct cells in the rat parotid gland,² whereas both acinar and some duct cells were labeled in the rat submandibular gland (29). Fig. 1A shows that NKCC1 is localized to the basolateral membrane of acinar cells from the parotid gland of wild-type mice. Fig. 1B is a Nomarski image of the same field. In contrast, duct cells failed to stain with the anti-NKCC1 antibody (Fig. 1, C and D). Furthermore, verifying the specificity of the antibody, no staining was detected in parotid acini from Nkcc1 null mutant (/) mice (Fig. 1, E and F). Taken together, these experiments confirmed the localization of the NKCC1 isoform to the basolateral membrane of mouse parotid acinar cells.

View larger version (98K): [in this window] [in a new window]   Fig. 1.   Immunolocalization of NKCC1 to the basolateral membrane of acinar cells in mouse parotid gland. Dispersed cells from wild-type (+/+) and Nkcc1 null mutant (/) animals were treated as described under "Experimental Procedures," incubated overnight with polyclonal anti-NKCC1 antibody, and then treated with fluorescein isothiocyanate-labeled secondary antibody. A, wild-type parotid gland acinar cells treated with anti-NKCC1 antibody show specific staining of the basolateral membrane. B, Nomarski image of cells shown in A. C, wild-type parotid gland duct cells treated with anti-NKCC1 antibody show no specific staining. D, Nomarski image of duct shown in C. E, anti-NKCC1 antibody shows no staining in parotid acinar cells prepared from Nkcc1 null mutant animals. F, Nomarski image of cells shown in E.

Loss of Bumetanide-sensitive Cl Uptake in Acinar Cells from NKCC1-deficient Mice-- In rat parotid acinar cells, the primary mechanism for Cl re-uptake during fluid secretion appears to be a bumetanide-sensitive Na⁺/K⁺/2Cl cotransporter (11, 12). To determine whether a similar mechanism is present in mouse parotid acini, cells were loaded with the Cl-sensitive dye SPQ and stimulated with the muscarinic agonist carbachol. A of Fig. 2 shows a rapid loss of intracellular Cl following stimulation of acinar cells isolated from wild-type mice (+/+), presumably mediated by Ca²⁺-activated Cl channels (4). This Cl efflux is followed by a slower, bumetanide-sensitive Cl re-uptake, similar to previous observations in the rat parotid gland (11, 12). In acinar cells isolated from mice lacking expression of NKCC1 (/), the initial channel mediated, Cl loss in response to muscarinic stimulation was intact; however, Cl re-uptake was virtually absent (Fig. 2B).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Loss of bumetanide-sensitive Cl uptake in acinar cells from mice lacking NKCC1. Intracellular fluorescence of the Cl-sensitive dye SPQ was monitored in parotid acini cells prepared from Nkcc1 littermates of wild-type (+/+) and knockout (/) genotype. A, SPQ-loaded acinar cells from wild-type mice were stimulated with 10 µM carbarchol during the time period indicated (bar) in the presence (n = 12) or absence of 10 µM bumetanide (n = 15). B, SPQ-loaded acinar cells from mice lacking NKCC1 were stimulated with 10 µM carbarchol during the time period indicated by the bar (n = 20). Data are mean ± S.E. for the number of experiments indicated above.

NKCC1 Expression Is Required for Muscarinic Agonist-induced in Vivo Salivation-- To determine whether decreased in vitro Cl uptake translates into decreased in vivo salivation, pilocarpine-stimulated parotid saliva was collected from Nkcc1 wild-type and null mutant mice over a 50-min time period. Fig. 3A shows that targeted disruption of Nkcc1 (/, open circles) reduced the total volume of pilocarpine-stimulated saliva secreted during the 50-min collection period by 63% compared with +/+ animals (filled circles). The magnitude of the decrease in flow rate (Fig. 3B) was greatest during the first 5-min period (greater than 85% decrease), inhibiting secretion by less than 55% at the end of the 50-min collection period.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Effect of targeted disruption of the Nkcc1 gene on muscarinic-induced saliva secretion in vivo. Parotid gland salivation was induced and measured in anethestized littermate wild-type and null mutant Nkcc1 mice as described under "Experimental Procedures." The volume of saliva secreted (A) and the saliva flow rate (B) have been normalized to gland wet weight. A, targeted disruption of the Nkcc1 gene (/; open circles) significantly reduces the volume of stimulated saliva produced over a 50-min time course compared with +/+ animals (solid circles). B, deletion of NKCC1 expression induces a marked reduction in the rate of saliva secretion. Data are mean ± S.E. for 12 parotid glands from 7 individual animals (+/+) and 14 glands from 7 animals (/). All data for / glands are significantly less than littermate +/+ glands, p < 0.05.

Cl/HCO₃ Exchanger Activity in Mouse Parotid Acinar Cells-- The above results, showing loss of Cl re-uptake in vitro (Fig. 2) and severe hyposalivation in vivo from the parotid glands of NKCC1^/ mice (Fig. 3), directly demonstrate that NKCC1 is a major ion transport pathway involved in the fluid secretion process. However, another Cl uptake mechanism must be present because 1) parotid acinar cells from knockout mice efflux Cl upon stimulation, demonstrating the ability to concentrate intracellular Cl above electrochemical equilibrium, and 2) these glands continue to secrete saliva, although at a dramatically reduced rate. Some exocrine glands employ Cl/ HCO₃ exchangers, coupled with Na⁺/H⁺ exchangers, to drive fluid secretion (13, 30), whereas, others do not (31, 32). Fig. 4 shows that acinar cells isolated from both wild-type (A) and NKCC1-deficient (B) mice express such a Cl/ HCO₃ exchange mechanism. Here, Cl/ HCO₃ exchanger activity was detected in acinar cells by switching the superfusate to a HCO₃-containing, Cl-free salt solution, a maneuver that raises the intracellular pH in cells with functional anion exchanger activity (13, 28). Comparison of the initial rates of the Cl/ HCO₃ exchanger-mediated alkalinization suggests that acinar cells isolated from NKCC1-deficient mice express more activity than cells from wild-type animals (Fig. 4C). The initial rate of the alkalinization upon extracellular Cl removal for knockout mice was about 50% faster than controls (Fig. 4D).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Cl/HCO3 exchanger activity in acinar cells from NKCC1+/+ and NKCC1/ mice. Intracellular pH was monitored in parotid acini cells prepared from Nkcc1 littermates of wild-type (+/+) and knockout (/) genotype as described under "Experimental Procedures." Cl/ HCO3 exchanger activity was monitored by switching the superfusate to a HCO3-containing, Cl-free salt solution during the time period indicated. The resulting alkaline load is a measure of functional anion exchanger activity (13, 28). A, Cl/ HCO3 exchanger activity in acinar cells from NKCC1+/+ mice (n = 15). B, Cl/ HCO3 exchanger activity in acinar cells from NKCC1/ mice (n = 17). C, direct comparison of the initial rates of alkalinization for the boxed areas in A and B. D, summary of the effects of knocking out the Nkcc1 gene (0.053 ± 0.002 pH units/min for NKCC1+/+ mice versus 0.080 ± 0.004. For NKCC1/ mice, p < 0.01).

Enhanced Expression of AE2 Transcripts-- Although the enhanced alkalinization shown in Fig. 4 may be because of several factors, one potential mechanism for increasing Cl/ HCO₃ exchanger activity in acinar cells from NKCC1-deficient mice is to increase AE2 expression, the Cl / HCO₃ exchanger isoform expressed in this cell type (29). Northern analysis of poly(A)-selected RNA using a 3'-cDNA probe for NKCC1 verified that expression of a 7.4-kilobase mRNA is eliminated in parotid glands isolated from Nkcc1 knockout mice (Fig. 5, left panel). The right panel of Fig. 5 demonstrates that the level of transcripts for AE2 in the parotid gland was enhanced by about 30% in null mutant mice relative to wild-type controls. Although the mechanism for this enhanced expression is unclear, these results suggest that Cl/ HCO₃ exchanger expression is up-regulated in acinar cells in an attempt to compensate for the loss of NKCC1.

View larger version (59K): [in this window] [in a new window]   Fig. 5.   Northern hybridizatioin analysis of anion exchangers involved in fluid secretion in parotid salivary glands. Northern blots of poly(A)-selected RNA (5 µg/lane) from parotid glands of wild-type (+/+) and homozygous mutant (/) mice were hybridized with cDNA probes for NKCC1 (left panel) and AE2 (right panel). mRNA sizes are shown on the right.

Targeted Disruption of Nkcc1 Inhibits NaCl Reabsorption-- According to the currently accepted model for salivary secretion (1, 2), the initial step in the formation of saliva is the secretion of a plasma-like primary fluid from the acinar cells. Subsequently, duct cells are thought to reabsorb much of the secreted NaCl in a flow rate-dependent fashion, with little or no reabsorption of water, to produce a final hypotonic saliva. To examine the effects of Nkcc1 disruption on the final salivary fluid, parotid saliva was collected from wild-type and null mutant mice, and the sodium and potassium content, Cl activity, and osmolality were determined. Our prediction, based on the reduced flow in Nkcc1 null mice, was that the osmolality of their saliva would be reduced compared with wild-type mice owing to its increased transit time in the ducts. However, Fig. 6 shows that the sodium content, Cl activity, and osmolality of saliva collected from NKCC1-deficient mice were significantly elevated compared with that secreted by littermate, wild-type mice, whereas, no change in K⁺ content was detected.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Targeted disruption of the Nkcc1 gene inhibits NaCl reabsorption. Stimulated saliva was collected for 50 min from mouse parotid glands and the osmolality (A), Cl activity (B), sodium content (C), and potassium content (D) were determined. Osmolality was measured using a vapor pressure osmometer. Sodium and potassium content were analyzed by atomic absorption (see "Experimental Procedures"). Cl activity was determined using an expandable ion analyzer. Significantly different from +/+: *, p < 0.05 (Student's t test).

Morphology of the Parotid Gland in NKCC1-deficient Mice-- It is hard to account for the increased sodium and Cl concentrations, and consequently the higher osmolality of the saliva, from NKCC1 knockout mice within the two-stage secretion hypothesis described above without suggesting that the reabsorption of salt by the ducts, is reduced. It is often possible to predict functional changes induced by gene disruption by examining the morphology of affected organs (33, 34). Thus, morphological changes in the ducts of NKCC1-deficient mice may reflect some sort of compensatory mechanism associated with chronically low saliva output. To test this hypothesis, parotid glands of NKCC1^+/+ and NKCC1^/ mice were examined by light and electron microscopy.

As described previously (22), homozygous Nkcc1 mutants exhibited decreased rates of postnatal growth resulting in significantly lower body weights than their wild-type littermates. In the present experiments, mean body weights for wild-type animals were 31.3 ± 1.2 g (+/+, n = 24) and 24.2 ± 1.2 g for Nkcc1 mutant mice (/, n = 15; p < 0.003 compared with +/+, unpaired Student's t test). Loss of body weight did not correlate with a decrease in parotid gland weight. In fact, the parotid glands were larger in knockout mice; parotid weights (mg) were 38.2 ± 1.9 (+/+, n = 24) and 49.1 ± 4.0 (/, n = 13; p < 0.022 compared with +/+). No obvious differences between +/+ and / mice were observed with regard to acinar and duct cell morphology (Fig. 7). In particular, the apical cell surface and junctional complexes, extent of basolateral membrane infolding, and the size and number of mitochondria in the striated duct cells appeared similar in wild-type and knockout mice (Fig. 7, C and D, respectively). The size of the acinar and duct cells, and the ratio of acinar to ductal elements in the glands appeared comparable between the +/+ and / mice, although detailed morphometric analyses to confirm these observations were not performed.

View larger version (167K): [in this window] [in a new window]   Fig. 7.   Morphology of the parotid gland in Nkcc1+/+ and Nkcc1/ mice. Electron micrographs of NKCC1+/+ (A and C) and NKCC1/ (B and D) parotid glands. Acinar cell structure in +/+ (A) and / (B) glands appears similar. Secretory granule density is variable in both +/+ (note cell in upper right of A) and / glands (note cell in lower right of B). The structure of striated duct cells in +/+ (C) and / (D) glands also appears similar. Basal infoldings are well developed and mitochondria are numerous in cells of both +/+ and / glands. N, nucleus; L, lumen. Scale bars, 2 µm.

Decreased Expression of -, -, and ENaC Transcripts in NKCC1-deficient Mice-- NaCl reabsorption by salivary ducts is likely to be driven by Na⁺ uptake via the epithelial Na⁺ channel ENaC and/or Na⁺/H⁺ exchange (both NHE2 and NHE3 are expressed in the apical membrane of duct cells; see Refs. 26, 29, 35). Northern blot analysis was performed to determine whether the expression of transcripts for these proteins was affected by disrupting the expression of NKCC1. Active ENaC Na⁺ channels require the co-expression of -, -, and -subunits. The left panels of Fig. 8 demonstrate that transcripts for the -, -, and -subunits were decreased ~10, 40, and 80%, respectively, when normalized to L32 ribosomal mRNA. In contrast, NHE3 expression was unchanged (right panel of Fig. 8). Taken together, these results indicate that the inability of ducts in NKCC1-deficient mice to reabsorb NaCl correlates with decreased expression of the Na⁺ channel ENaC.

View larger version (50K): [in this window] [in a new window]   Fig. 8.   Northern hybridization analysis of ion transporters involved in NaCl absorption in parotid duct cells. Northern blots of poly(A)-selected RNA (5 µg/lane) from parotid glands of wild-type (+/+) and homozygous mutant (/) mice were hybridized with cDNA probes for ENaC, ENaC, ENaC (left panels), and NHE3 (right panel). mRNA sizes are shown on the right.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Most reports of oral dryness correlate with side effects of medications, immunological diseases such as Sjögren's syndrome, radiation treatment for head and neck cancers, or ductal obstructions (36-38). However, in a significant subpopulation of individuals (up to 20%), the cause of dry mouth is unknown (39, 40). It is likely that genetic defects make up a subset of this idiopathic patient population, particularly defects involving critical components of the fluid secretion process. Indirect evidence suggests that the Na⁺/K⁺/2Cl cotransport mechanism plays an important role in the production and regulation of fluid secretion by salivary glands (3, 6, 9-12). Up-regulation of the Na⁺/K⁺/2Cl cotransport activity occurs in response to agonists that mobilize intracellular Ca²⁺ (6), the primary in vivo secretion signal, as well as agonists that potentiate secretion by increasing cAMP (41). The objective of the current study was to determine directly, using knockout mice, the involvement of the NKCC1 isoform in salivary gland fluid secretion, and thus provide insight into potential mechanisms of "idiopathic" dry mouth disease. Other functional effects of disrupting the expression of the murine Nkcc1 gene have been previously reported (22, 42). NKCC1 appears to be critical for secretion, most notably in organs such as the trachea, jejunum, cecum, and those involved in hearing and balance (22, 42). However, NKCC1 is not the only transport mechanism driving secretion because acid secretion by the adult stomach and fluid secretion by the intestine of young mice was normal in animals lacking expression of NKCC1 (22).

In the present study, NKCC1 was localized to the basolateral membrane of mouse parotid acinar cells but was not detected in duct cells. The distribution of this labeling was confirmed using "negative control" parotid glands from Nkcc1 null mutant animals, in which staining was absent in acinar cells. The presence of NKCC1 in rat submandibular duct cells (29), but not mouse (Fig. 1) and rat parotid ducts (15), suggests a gland-specific difference. Nevertheless, functional cotransporter activity has not been reported in duct cells for any salivary gland, including the rat parotid (43) and submandibular glands (44).

In vitro studies revealed that the bumetanide-sensitive uptake of Cl was absent in NKCC1-deficient mice. Consistent with this observation, in vivo functional studies demonstrated that knockout of this gene dramatically reduced the total volume of saliva secreted by more than 60%. Thus, our data demonstrate that NKCC1 controls the rate of salivary gland secretion by regulating acinar Cl entry. These results are the first to directly establish that NKCC1 is a major transport protein involved in modulating the rate of saliva secretion. It is interesting to note that the magnitude of the inhibition of the salivary flow rate in mice lacking NKCC1 decreased over time, from greater than 85% inhibition during the first 5 min to less than 55% after a 50-min stimulation. These results are in agreement with earlier studies where Na⁺/K⁺/2Cl cotransport was shown to initially be the dominant Cl uptake mechanism driving fluid secretion (9-12), whereas Cl/ HCO₃ exchange contributed more to Cl influx during sustained muscarinic stimulation (13), as the intracellular pH increased because of enhanced Na⁺/H⁺ exchanger activity (45, 46). Thus, the saliva secreted by Nkcc1 null mice is likely dependent on the Cl/ HCO₃ exchanger activity detected in acinar cells isolated from these animals. Consistent with this possibility, transcripts for the AE2 Cl/ HCO₃ exchanger were slightly increased in / mice relative to wild-type littermates.

Duct cells modify acinar cell secretions primarily by absorbing NaCl to produce a final hypotonic saliva. This process is dependent on the salivary flow rate because the ductal reabsorption mechanisms saturate at higher flows (see Refs. 1 and 2). Duct cells possess two potential Na⁺ uptake mechanisms in their lumenal membrane. The first of these is Na⁺/H⁺ exchange, although functional studies have not indicated a major role for this apical pathway. The second mechanism is an amiloride-sensitive Na⁺ channel (47), likely the epithelial Na⁺ channel ENaC (48). Therefore, because of the reduced flow rate, it was surprising to find that saliva from NKCC1-deficient mice had elevated osmolality compared with that measured in saliva collected from wild-type littermates (Fig. 6). The interpretation of these results is difficult to understand in terms of the two-stage secretion model, because parotid duct cells do not express detectable levels of NKCC1. The increased osmolality of saliva from mice lacking NKCC1 is because of an increase in NaCl content (see Fig. 6) and not an increase in the protein concentration (data not shown).

One potential mechanism for the reduction in the reabsorption of NaCl by parotid ducts may be that the chronically low flow of saliva in NKCC1-deficient mice induced changes in ion transporter capacity; e.g. as has been noted in the colon of NHE3-deficient mice (24, 28). These changes might be associated with altered duct cell morphology. However, we were unable to detect any morphological changes in duct cells consistent with this hypothesis, although we cannot rule out the possibility that functional changes have occurred that are unapparent morphologically. Another possibility is that this is an adaptive mechanism to increase the final salivary flow. Although little fluid reabsorption is thought to occur in the ducts, any that does occur is presumably a consequence of ductal salt reabsorption. Thus, down-regulating this process would spare whatever fluid is lost in the ducts. In any case, the expression of the -, -, and -subunits of ENaC was reduced. These results strongly suggest that the inability of the ducts to produce hypotonic saliva in NKCC1-deficient mice is linked to a decreased ability of these cells to uptake Na⁺ mediated by ENaC.

In conclusion, the Nkcc1 knockout mouse presents an excellent system for in vivo and in vitro studies of Cl-dependent fluid secretion, as well as providing a potential animal model for human genetic defects that produce idiopathic dry mouth disease. The present study demonstrates, utilizing an NKCC1-deficient mouse model, that the basolateral Na⁺/K⁺/2Cl cotransporter isoform 1 is the major Cl uptake pathway involved in the fluid secretion process in vivo. By regulating intracellular Cl uptake, this isoform plays a key role in driving and maintaining the transepithelial movement of Cl ions across acinar cells.

	ACKNOWLEDGEMENT

We thank L. Richardson for technical assistance with genotyping animals.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants DK50594 (to G. E. S.) and DE13539, DE08921, and DE09692 (to J. E. M.).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.

^§ These authors contributed equally to this project.

^¶ Present address: Unilever Research, Port Sunlight Laboratory, Quarry Road East, Bebington, Wirral CH63 3JW, UK. E-mail: Richard.Evans@Unilever.com.

^¶¶ To whom correspondence should be addressed: Center for Oral Biology, University of Rochester, Medical Center Box 611, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-3444; Fax: 716-473-2679; E-mail: james_melvin@urmc.rochester.edu.

Published, JBC Papers in Press, May 30, 2000, DOI 10.1074/jbc.M003753200

² M. L. Moore-Hoon and R. J. Turner, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: NKCC1, Na⁺/K⁺/2Cl cotransporter isoform 1; -, -, and ENaC, -, -, and -subunits of the epithelial Na⁺ channel; BCECF/AM, 2',7'-bis(carboxyethyl)-5-carboxyfluorescein-pentaacetoxymethyl ester; nt, nucleotide(s); AE2, Cl / HCO₃ exchanger isoform 2; NHE2 and NHE3, Na⁺/H⁺ exchanger isoforms 2 and 3; SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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