FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2107017864> ?p ?o ?g. }

• W2107017864 endingPage "27050" @default.
• W2107017864 startingPage "27042" @default.
• W2107017864 abstract "Multiple Na+/H+exchangers (NHEs) are expressed in salivary gland cells; however, their functions in the secretion of saliva by acinar cells and the subsequent modification of the ionic composition of this fluid by the ducts are unclear. Mice with targeted disruptions of the Nhe1,Nhe2, and Nhe3 genes were used to study the in vivo functions of these exchangers in parotid glands. Immunohistochemistry indicated that NHE1 was localized to the basolateral and NHE2 to apical membranes of both acinar and duct cells, whereas NHE3 was restricted to the apical region of duct cells. Na+/H+ exchange was reduced more than 95% in acinar cells and greater than 80% in duct cells of NHE1-deficient mice (Nhe1 −/−). Salivation in response to pilocarpine stimulation was reduced significantly in bothNhe1 −/− and Nhe2 −/−mice, particularly during prolonged stimulation, whereas the loss of NHE3 had no effect on secretion. Expression of Na+/K+/2Cl− cotransporter mRNA increased dramatically in Nhe1 −/− parotid glands but not in those of Nhe2 −/− orNhe3 −/− mice, suggesting that compensation occurs for the loss of NHE1. The sodium content, chloride activity and osmolality of saliva in Nhe2 −/− orNhe3 −/− mice were comparable with those of wild-type mice. In contrast, Nhe1 −/− mice displayed impaired NaCl absorption. These results suggest that in parotid duct cells apical NHE2 and NHE3 do not play a major role in Na+ absorption. These results also demonstrate that basolateral NHE1 and apical NHE2 modulate saliva secretion in vivo, especially during sustained stimulation when secretion depends less on Na+/K+/2Cl−cotransporter activity. Multiple Na+/H+exchangers (NHEs) are expressed in salivary gland cells; however, their functions in the secretion of saliva by acinar cells and the subsequent modification of the ionic composition of this fluid by the ducts are unclear. Mice with targeted disruptions of the Nhe1,Nhe2, and Nhe3 genes were used to study the in vivo functions of these exchangers in parotid glands. Immunohistochemistry indicated that NHE1 was localized to the basolateral and NHE2 to apical membranes of both acinar and duct cells, whereas NHE3 was restricted to the apical region of duct cells. Na+/H+ exchange was reduced more than 95% in acinar cells and greater than 80% in duct cells of NHE1-deficient mice (Nhe1 −/−). Salivation in response to pilocarpine stimulation was reduced significantly in bothNhe1 −/− and Nhe2 −/−mice, particularly during prolonged stimulation, whereas the loss of NHE3 had no effect on secretion. Expression of Na+/K+/2Cl− cotransporter mRNA increased dramatically in Nhe1 −/− parotid glands but not in those of Nhe2 −/− orNhe3 −/− mice, suggesting that compensation occurs for the loss of NHE1. The sodium content, chloride activity and osmolality of saliva in Nhe2 −/− orNhe3 −/− mice were comparable with those of wild-type mice. In contrast, Nhe1 −/− mice displayed impaired NaCl absorption. These results suggest that in parotid duct cells apical NHE2 and NHE3 do not play a major role in Na+ absorption. These results also demonstrate that basolateral NHE1 and apical NHE2 modulate saliva secretion in vivo, especially during sustained stimulation when secretion depends less on Na+/K+/2Cl−cotransporter activity. Na+/K+/2Cl− cotransporter isoform 1 Na+/H+ exchanger isoforms 1–4 epithelial Na+ channel β-, and γ-ENaC, α, β, and γ subunits of ENaC nucleotides Saliva formation is thought to involve a two-stage process (1Martinez J.R. Holzgreve H. Frick A. Pflugers Arch. Gesamte Physiol. Menschen Tiere. 1966; 290: 124-133Crossref PubMed Scopus (70) Google Scholar, 2Thaysen J.H. Thorn N.A. Schwartz I.L. Am. J. Physiol. 1954; 178: 155-159Crossref PubMed Scopus (195) Google Scholar, 3Young J.A. Schogel E. Pflugers Arch. Gesamte Physiol. Menschen Tiere. 1966; 291: 85-98Crossref PubMed Scopus (93) Google Scholar). Initially, acinar cells secrete an isotonic plasma-like fluid, the generation of which depends on the coordinated activity of a number of membrane transport proteins that drive net transepithelial Cl− movement and significant HCO 3− efflux (see Refs. 4Melvin J.E. Crit. Rev. Oral Biol. Med. 1999; 10: 199-209Crossref PubMed Scopus (55) Google Scholar and 5Cook D.I. Van Lennep E.W. Roberts M.L. Young J.A. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, Ltd., New York1994: 1061-1117Google Scholar). The available evidence suggests that Cl− uptake across the basolateral membrane of acinar cells is primarily mediated via the electroneutral Na+/K+/2Cl−cotransporter. This has been demonstrated clearly in mice lacking expression of theNkcc1 1 gene, in which pilocarpine-stimulated secretion is greatly reduced but not eliminated (6Evans R.L. Park K. Turner R.J. Watson G.E. Nguyen H.V. Dennnett M.R. Hand A.R. Flagella M. Shull G.E. Melvin J.E. J. Biol. Chem. 2000; 275: 26720-26726Abstract Full Text Full Text PDF PubMed Google Scholar). In situ, the main fluid and electrolyte agonist acetylcholine triggers secretion by increasing the Cl− and HCO 3− permeability of the apical membrane. HCO 3− efflux via the apical anion channel produces an intracellular acid load that is rapidly buffered by an increase in Na+/H+exchanger activity (7Lau K.R. Elliott A.C. Brown P.D. Am. J. Physiol. 1989; 256: C288-C295Crossref PubMed Google Scholar, 8Melvin J.E. Moran A. Turner R.J. J. Biol. Chem. 1988; 263: 19564-19569Abstract Full Text PDF PubMed Google Scholar, 9Soltoff S.P. McMillian M.K. Cantley L.C. Cragoe Jr., E.J. Talamo B.R. J. Gen. Physiol. 1989; 93: 285-319Crossref PubMed Scopus (69) Google Scholar). Therefore, the residual secretion from NKCC1-deficient mice is likely mediated by enhanced Na+/H+ exchange, which drives Cl−uptake via the coupled operation of Na+/H+ and Cl−/HCO 3− exchangers as well as carbonic anhydrase- and Na+/H+exchanger-dependent HCO 3−efflux (5Cook D.I. Van Lennep E.W. Roberts M.L. Young J.A. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, Ltd., New York1994: 1061-1117Google Scholar). During the second stage of secretion, ductal cells modify acinar secretions primarily by conserving NaCl in a flow rate-dependent fashion; and because the apical surfaces of salivary ducts are relatively impermeant to water, saliva is generally hypotonic (see Refs. 4Melvin J.E. Crit. Rev. Oral Biol. Med. 1999; 10: 199-209Crossref PubMed Scopus (55) Google Scholar and 5Cook D.I. Van Lennep E.W. Roberts M.L. Young J.A. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, Ltd., New York1994: 1061-1117Google Scholar). A “typical” NaCl-conserving duct cell is thought to possess at least two Na+ uptake mechanisms. The first of these is Na+/H+exchange located in the lumenal membrane (10Park K. Olschowka J.A. Richardson L.A. Bookstein C. Chang E.B. Melvin J.E. Am. J. Physiol. 1999; 276: G470-G478PubMed Google Scholar, 11Lee M.G. Schultheis P.J. Yan M. Shull G.E. Bookstein C. Chang E. Tse M. Donowitz M. Park K. Muallem S. J. Physiol. (Lond.). 1998; 513: 341-357Crossref Scopus (64) Google Scholar, 12He X. Tse C.M. Donowitz M. Alper S.L. Gabriel S.E. Baum B.J. Pflugers Arch. Eur. J. Physiol. 1997; 433: 260-268Crossref PubMed Scopus (145) Google Scholar). Of the different Na+/H+ exchanger (NHE) isoforms expressed in salivary gland duct cells, NHE2 and NHE3 are thought to be associated with Na+ absorption in other epithelial tissues (see Refs.13Counillon L. Pouyssegur J. J. Biol. Chem. 2000; 275: 1-4Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar and 14Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar). A second mechanism for Na+ uptake by salivary gland duct cells is an amiloride-sensitive Na+ channel (15Dinudom A. Young J.A. Cook D.I. Pflugers Arch. Eur. J. Physiol. 1993; 423: 164-166Crossref PubMed Scopus (35) Google Scholar). This channel has properties comparable with the cloned epithelial Na+ channel ENaC (16Ishikawa T. Marunaka Y. Rotin D. J. Gen. Physiol. 1998; 111: 825-846Crossref PubMed Scopus (118) Google Scholar), which is involved in Na+absorption in the kidney and lungs (see Ref. 17Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1035) Google Scholar). Consistent with the two-stage secretion model, inhibitor studies suggest that Na+/H+ exchangers may contribute both to fluid and electrolyte secretion from acinar cells and to reabsorption of NaCl by duct cells (18Dissing S. Nauntofte B. Am. J. Physiol. 1990; 259: G1044-G1055PubMed Google Scholar, 19Lau K.R. Howorth A.J. Case R.M. J. Physiol. (Lond.). 1990; 425: 407-427Crossref Scopus (24) Google Scholar, 20Martinez J.R. Cassity N. Arch. Oral Biol. 1985; 30: 797-803Crossref PubMed Scopus (13) Google Scholar, 21Pirani D. Evans L.A. Cook D.I. Young J.A. Pflugers Arch. Eur. J. Physiol. 1987; 408: 178-184Crossref PubMed Scopus (63) Google Scholar, 22Robertson M.A. Foskett J.K. Am. J. Physiol. 1994; 267: C146-C156Crossref PubMed Google Scholar). The relative lack of specificity of these inhibitors makes it unclear as to which NHE isoforms are involved. The mammalian NHE gene family consists of six isoforms (13Counillon L. Pouyssegur J. J. Biol. Chem. 2000; 275: 1-4Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 14Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar). Of these, NHE1, NHE2, NHE3, and NHE4 are expressed in the plasma membrane of epithelial tissues including salivary glands (10Park K. Olschowka J.A. Richardson L.A. Bookstein C. Chang E.B. Melvin J.E. Am. J. Physiol. 1999; 276: G470-G478PubMed Google Scholar, 11Lee M.G. Schultheis P.J. Yan M. Shull G.E. Bookstein C. Chang E. Tse M. Donowitz M. Park K. Muallem S. J. Physiol. (Lond.). 1998; 513: 341-357Crossref Scopus (64) Google Scholar, 12He X. Tse C.M. Donowitz M. Alper S.L. Gabriel S.E. Baum B.J. Pflugers Arch. Eur. J. Physiol. 1997; 433: 260-268Crossref PubMed Scopus (145) Google Scholar, 23Robertson M.A. Woodside M. Foskett J.K. Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 287-294Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). However, little is known about the specific functions of the individual NHE isoforms in salivary glands. To determine the molecular identity of the Na+/H+ exchangers involved in the above processes, we examined the effects ofNhe1, Nhe2, and Nhe3 gene disruptions on mouse parotid gland function. Our results demonstrate that Na+/H+ exchanger isoforms NHE1 and NHE2, which are located in the basolateral and apical membranes of acinar cells, respectively, are important regulators of saliva secretion in vivo. Furthermore, the loss of NHE2 or NHE3 does not inhibit NaCl absorption by duct cells, whereas NaCl absorption was blunted in NHE1-deficient mice. Unexpectedly, these results suggest that neither NHE2 nor NHE3 are primary Na+ absorption mechanisms in mouse parotid glands. All chemicals were from Sigma. Targeted disruptions of the murine Nhe1,Nhe2, and Nhe3 genes were performed previously (24Bell S.M. Schreiner C.M. Schultheis P.J. Miller M.L. Evans R.L. Vorhees C.V. Shull G.E. Scott W.J. Am. J. Physiol. 1999; 276: C788-C795Crossref PubMed Google Scholar, 25Schultheis P.J. Clarke L.L. Meneton P. Harline M. Boivin G.P. Stemmermann G. Duffy J.J. Doetschman T. Miller M.L. Shull G.E. J. Clin. Invest. 1998; 101: 1243-1253Crossref PubMed Scopus (220) Google Scholar, 26Schultheis P.J. Clarke L.L. Meneton P. Miller M.L. Soleimani M. Gawenis L.R. Riddle T.M. Duffy J.J. Doetschman T. Wang T. Giebisch G. Aronson P.S. Lorenz J.N. Shull G.E. Nat. Genet. 1998; 19: 282-285Crossref PubMed Scopus (698) Google Scholar), and 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 waterad libitum with a 12-hour light/dark cycle. Experiments were carried out on animals aged between 1.5 and 4 months. Body and parotid gland weights were recorded for each animal used. HomozygousNhe1 −/− mutants exhibited decreased rates of postnatal growth resulting in significantly lower body weights than their wild-type or heterozygous littermates, exhibited an ataxic gait, and became prone to epileptic seizures that ended in a catatonic-like state, from which the animal usually recovered (24Bell S.M. Schreiner C.M. Schultheis P.J. Miller M.L. Evans R.L. Vorhees C.V. Shull G.E. Scott W.J. Am. J. Physiol. 1999; 276: C788-C795Crossref PubMed Google Scholar). In our experiments, mean body weights (g) for NHE1 animals were 30.8 ± 2.2 (+/+, n = 11) and 16.4 ± 2.2 (−/−,n = 12, p < 0.01 compared with +/+, Student's t test). The magnitude of the body weight loss (>45%) did not correlate with a comparable decrease in parotid gland weight (∼7%); parotid weights (mg) were 33.9 ± 2.4 (+/+,n = 12) and 31.5 ± 1.9 (−/−, n= 14). Nhe2 and Nhe3 mutant mice grew normally and exhibited the same appearance and behavior as wild-type animals (25Schultheis P.J. Clarke L.L. Meneton P. Harline M. Boivin G.P. Stemmermann G. Duffy J.J. Doetschman T. Miller M.L. Shull G.E. J. Clin. Invest. 1998; 101: 1243-1253Crossref PubMed Scopus (220) Google Scholar, 26Schultheis P.J. Clarke L.L. Meneton P. Miller M.L. Soleimani M. Gawenis L.R. Riddle T.M. Duffy J.J. Doetschman T. Wang T. Giebisch G. Aronson P.S. Lorenz J.N. Shull G.E. Nat. Genet. 1998; 19: 282-285Crossref PubMed Scopus (698) Google Scholar). Nhe2 body weights were 30.3 ± 0.9 (+/+,n = 30) and 29.2 ± 1.0 (−/−, n= 23), and parotid weights were 33.4 ± 1.8 (+/+,n = 20) and 32.5 ± 2.0 (−/−, n= 8). Nhe3 body weights were 32.4 ± 1.6 (+/+,n = 26) and 31.3 ± 1.1 (−/−, n= 22), and parotid weights were 45.3 ± 3.1 (+/+,n = 14) and 41.7 ± 2.8 (−/−, n= 10). For immunolocalization experiments, Na+/H+ exchanger isoform NHE1 was detected using a polyclonal antibody kindly provided by Dr. J. Noel. Specific antisera for NHE2 (antibody 2M5) and NHE3 (1314) were used as described previously (27Bookstein C. Xie Y. Rabenau K. Musch M.W. McSwine R.L. Rao M.C. Chang E.B. Am. J. Physiol. 1997; 273: C1496-C1505Crossref PubMed Google Scholar, 28Amemiya M. Loffing J. Lotscher M. Kaissling B. Alpern R.J. Moe O.W. Kidney Int. 1995; 48: 1206-1215Abstract Full Text PDF PubMed Scopus (352) Google Scholar). Parotid glands fromNhe1 −/−, Nhe2 −/−,Nhe3 −/− (negative control), and wild-type animals were removed and immediately placed in 4% paraformaldehyde (NHE1) or frozen in 2-methylbutane on dry ice (NHE2 and NHE3). Paraformaldehyde-treated tissue was paraffin-embedded and sectioned at 4 µm. Frozen sections (10 µm) were fixed and permeabilized, and nonspecific binding sites were blocked as described previously (11Lee M.G. Schultheis P.J. Yan M. Shull G.E. Bookstein C. Chang E. Tse M. Donowitz M. Park K. Muallem S. J. Physiol. (Lond.). 1998; 513: 341-357Crossref Scopus (64) Google Scholar). Sections were incubated overnight at 4 °C in PBS, O.8% bovine serum albumin, 0.1% gelatin, and 0.1% Triton X-100 containing 1:500 (NHE1) or 1:200 (NHE2 and NHE3) dilutions of antibody and then treated with 1:1000 Alexa 594 fluor-conjugated goat anti-rabbit (Molecular Probes, Eugene, OR) in the above buffer (NHE1) or 1:500 fluorescein isothiocyanate-labeled secondary antibody (NHE2 and NHE3, goat anti-rabbit, Jackson ImmunoResearch Laboratory, West Grove, PA) for 1 h at room temperature. Images were recorded and analyzed using a Zeiss Axioplan microscope or a Leica confocal microscope. 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, and the main excretory duct of the right and left parotid glands were isolated using a dissecting microscope. Prior to saliva collection, a tracheotomy was performed to prevent asphyxiation. Secretion was initiated by the injection of the cholinergic agonist pilocarpine HCl (10 mg/kg, intraperitoneal), and saliva was collected from 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 an experiment, and parotid glands were subsequently dissected, trimmed free of connective tissue, and weighed. For data presentation, the volume of saliva secreted (µl) and the rate of parotid saliva flow in µl/min were normalized to 100 mg parotid gland weight. Results are expressed as mean ± S.E. of the saliva flow from both the right and left glands fromn 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 chloride activity was estimated using an Orion EA 940 expandable ion analyzer. Total RNA was isolated from the parotid glands of mice using Trizol reagent (Life Technologies, Inc.) followed by poly(A)+ mRNA selection using an Oligotex mRNA kit from Quiagen. For Northern analysis, each gland sample was pooled from three or more mice. Northern blots were prepared and hybridized as described previously (6Evans R.L. Park K. Turner R.J. Watson G.E. Nguyen H.V. Dennnett M.R. Hand A.R. Flagella M. Shull G.E. Melvin J.E. J. Biol. Chem. 2000; 275: 26720-26726Abstract Full Text Full Text PDF PubMed Google Scholar) using32P-labeled cDNA probes for NKCC1 (rat nts 3368–3563, GenBankTM accession number AF051561), Cl−/HCO 3− exchanger isoform 2 (mouse nts 1300–1776, GenBankTM accession numberJ04036), NHE3 (rat nts 1857–2378, GenBankTM accession number M85300), α-ENaC (mouse nts 931–1185, GenBankTMaccession number AF112185), β-ENaC (mouse nts 160–380, GenBankTM accession number U16023), and γ-ENaC (mouse nts 1882–2184, GenBankTM accession number AF112187). A cDNA for mouse ribosomal messenger RNA L32 (mouse nts 3072–3244, GenBankTM accession number K02060) was used to normalize the expression between preparations. For dot blots, mRNA was isolated from the parotid glands of individual animals. Ten separate replicate dot blots were prepared using each mRNA sample. Each blot was hybridized with both a specific probe (as described above) then stripped and probed with L32 cDNA. Quantitation was performed by PhosphorImager analysis (Bio-Rad). Parotid acini (5–20 cells) were prepared from wild-type and knockout littermates by collagenase digestion (29Evans R.L. Bell S.M. Schultheis P.J. Shull G.E. Melvin J.E. J. Biol. Chem. 1999; 274: 29025-29030Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In brief, glands were minced in 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 loaded with a pH-sensitive fluorescent indicator by incubation with 2 µm carboxy SNARF1-acetoxymethyl ester (Molecular Probes) for 30 min in a physiological salt solution. Experiments to measure intracellular pH were carried out in physiological salt solution containing (in mm): 135 NaCl, 5.4 KCl, 1.2 CaCl2, 0.8 MgSO4, 0.33 NaH2PO4, 0.4 KH2PO4, 10 glucose, and 20 Hepes (pH 7.4 with NaOH). NH4Cl-containing physiological salt solution was made by substituting 30 mmNaCl with 30 mm NH4Cl. Intracellular fluorescence was monitored in ratio mode from acini and ducts adhering to the base of a superfusion chamber mounted on an UltimaTMconfocal microscope (Genomic Solutions, Ann Arbor, MI). Cells were excited at 514 nm and emitted fluorescence measured at 570 and ≥630 nm. Intracellular pH was estimated by in situ calibration of the excitation ratio using the high K+/nigericin protocol as described previously (30Thomas J.A. Buchsbaum R.N. Zimniak A. Racker E. Biochemistry. 1979; 18: 2210-2218Crossref PubMed Scopus (1764) Google Scholar). Na+/H+ exchanger activity was monitored after an NH4Cl-induced acid load (31Roos A. Boron W.F. Physiol. Rev. 1981; 61: 296-434Crossref PubMed Scopus (2283) Google Scholar). 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, then trimmed into small pieces, and rinsed in 0.1 m cacodylate buffer. The tissues were post-fixed in 1% osmium tetroxide/0.8% potassium ferricyanide in cacodylate buffer and then 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. Previous reports have demonstrated that the distribution of the different NHE isoforms is species- and salivary gland type-specific. Thus, to better understand the precise function(s) of the various NHE isoforms expressed in mouse parotid glands we first documented their distribution by immunohistochemistry. NHE1 has been localized to the basolateral membrane of acinar and duct cells in rat parotid (10Park K. Olschowka J.A. Richardson L.A. Bookstein C. Chang E.B. Melvin J.E. Am. J. Physiol. 1999; 276: G470-G478PubMed Google Scholar, 23Robertson M.A. Woodside M. Foskett J.K. Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 287-294Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and submandibular glands (11Lee M.G. Schultheis P.J. Yan M. Shull G.E. Bookstein C. Chang E. Tse M. Donowitz M. Park K. Muallem S. J. Physiol. (Lond.). 1998; 513: 341-357Crossref Scopus (64) Google Scholar, 12He X. Tse C.M. Donowitz M. Alper S.L. Gabriel S.E. Baum B.J. Pflugers Arch. Eur. J. Physiol. 1997; 433: 260-268Crossref PubMed Scopus (145) Google Scholar). NHE2 and NHE3 are located in the apical membranes of submandibular ducts and acini, whereas only NHE3 was detected in the ductal apical membrane of the rat parotid gland (10Park K. Olschowka J.A. Richardson L.A. Bookstein C. Chang E.B. Melvin J.E. Am. J. Physiol. 1999; 276: G470-G478PubMed Google Scholar). The top left panel of Fig.1 shows that NHE1 is localized to the basolateral membrane of acinar cells in wild-type mice. No labeling was detected in parotid sections from Nhe1 −/− null mutant mice (top right panel), verifying the specificity of the antibody. Much more intense staining was seen in the ducts of parotid gland. An image of a duct in cross-section is shown in themiddle left panel of Fig. 1, where the intensity of the illumination was reduced relative to that used in the top left panel to prevent overexposure. The middle right panelof Fig. 1 is a Nomarski image of the duct shown in the middle left panel. To more clearly demonstrate the acinar localization of NHE1, parotid cells were dispersed by treatment with collagenase. Consistent with the staining observed in tissue sections (top left panel), NHE1 is expressed in the basolateral membrane of isolated acinar cells (bottom left panel). A Nomarski image of this acinus is provided in the bottom right panel of Fig.1. These data confirm the targeting of NHE1 to the basolateral membranes of mouse parotid acinar and duct cells. In contrast to NHE1 staining, the top left panel of Fig.2 shows that NHE2 protein was distributed primarily to the apical membranes of duct cells (strong specific apical staining is indicated by arrows; note that the basal borders of the duct are overlaid by dashed lines). Much less intense staining of the apical membrane of acinar cells was detected; in fact, in some acini, expression of NHE2 protein was either absent or too low to be detected. The antibody used was specific because parotid sections from Nhe2 null mutants showed an absence of staining (top right panel). Parotid glands were dispersed by treatment with collagenase to more clearly demonstrate the localization of NHE2 in acinar cells. In agreement with the staining observed in tissue sections (top left panel), NHE2 was primarily expressed in the apical region of isolated acinar cells (middle left panel; the outline of the acinus is represented by thedashed line). A Nomarski image of this acinus is provided in the middle right panel of Fig. 2. NHE3 protein was not detected in parotid acinar cells but only in the apical region of duct cells (arrow in the bottom left panel of Fig. 2; the basal border of the duct cut in cross-section is overlaid by adashed line). No staining was detected in NHE3-deficient mice (bottom right panel of Fig. 2). Nhe1,Nhe2, and Nhe3 null mutant animals were used to examine the contribution of each isoform to pH regulation in parotid acinar and duct cells. The NH4+ pulse method was used to acid-load cells to monitor Na+/H+exchanger activity. The average intracellular pH responses to this manipulation in acinar cells isolated from wild-type (+/+,dotted line) and NHE1-deficient animals (−/−,solid line) are shown in Fig.3 A. Removal of NH4Cl led to an intracellular acidification followed by an intracellular pH recovery, and this recovery was inhibited by more than 95% in NHE1-deficient mice, whereas disruption of the Nhe2and Nhe3 genes (data not shown) had little or no effect on recovery rates (see also Ref. 29Evans R.L. Bell S.M. Schultheis P.J. Shull G.E. Melvin J.E. J. Biol. Chem. 1999; 274: 29025-29030Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Duct cells isolated fromNhe1+/+ animals (+/+, dotted line) also recovered their intracellular pH (Fig.3 B) but at an initial rate nearly 3-fold faster than that of acinar cells. The more robust Na+/H+ exchanger activity in duct cells likely reflects the higher expression of Na+/H+ exchangers in this cell type (see Figs.1 and 2). The magnitude of the inhibition of pH recovery caused by disruption of the Nhe1 gene was less dramatic in duct cells (∼80%, −/−, solid line). This result correlates with the abundance of NHE2 and NHE3 in ducts (see Fig. 2). Moreover, the residual pH recovery in duct cells from NHE1-deficient mice was about an order of magnitude more resistant to the amiloride derivative ethylisopropyl amiloride than ducts from wild-type mice (data not shown), in agreement with immunological staining suggesting that NHE3 is strongly expressed in this cell type. These results confirm that NHE1 is the major regulator of intracellular pH in mouse parotid acinar cells and also demonstrate that NHE1 contributes to pH regulation in duct cells, albeit less significantly. Earlier studies using the Na+/H+exchange inhibitor amiloride and its analogs suggested that Na+/H+ exchangers may be involved in salivation. Localization of NHE1 and NHE2 (Figs. 1 and 2, respectively) to acinar cells suggests that one or both of these isoforms might contribute to secretion. To directly test the role of each NHE isoform, parotid saliva was collected from Nhe1, Nhe2, orNhe3 wild-type and null mutant mice over a 50-min time period. Fig. 4 A shows that targeted disruption of Nhe1 (open circles) reduced the total volume of pilocarpine-stimulated saliva secreted during the 50-min collection period by 34% compared with wild-type animals (solid circles). The magnitude of the decrease in flow rate increased over time. The flow rate was reduced by 16% during the first 5 min and reached 42% inhibition at the end of the 50-min collection period (Fig. 4 B). Likewise, disruption of NHE2 expression reduced the total volume of saliva secreted by 29% (Fig.5 A), and the effect on the flow rate increased during prolonged stimulation from 18% during the first 5 min to 46% inhibition at 50 min (Fig. 5 B). In contrast, normal salivation was observed in NHE3-deficient mice (Fig.5, C and D). For all genotypes, note the high initial flow rate seen at the commencement of secretion, which declines to a lower relatively constant rate thereafter. The secretion kinetics and flow rates are similar to those reported previously (6Evans R.L. Park K. Turner R.J. Watson G.E. Nguyen H.V. Dennnett M.R. Hand A.R. Flagella M. Shull G.E. Melvin J.E. J. Biol. Chem. 2000; 275: 26720-26726Abstract Full Text Full Text PDF PubMed Google Scholar, 32Marmary Y. Fox P.C. Baum B.J. Comp. Biochem. Physiol. A. 1987; 88: 307-310Crossref PubMed Scopus (20) Google Scholar). The initial step in the formation of saliva is the secretion of a plasma-like NaCl-rich fluid from acinar cells. Subsequently, duct cells reabsorb much of the secreted NaCl to produce a hypotonic NaCl-poor saliva. Na+/H+ exchangers play a major role in NaCl absorption in other epithelia, although it is unknown whether they serve a similar function in salivary glands. To examine this possibility, parotid saliva was collected from Nhe1,Nhe2, or Nhe3 wild-type and null mutant mice, and the sodium and potassium content, Cl− activity, and osmolality were determined. Table I shows that the ion content and the osmolality of saliva collected from NHE2- and NHE3-deficient mice were comparable with secretions from littermate wild-type mice, suggesting that these Na+/H+exchangers do not play a major role in NaCl reabsorption in this tissue. In contrast, the osmolality and sodium, potassium, and chloride content increased significantly in saliva fromNhe1 −/− mice.Table ITargeted disruption of murine Nhe1, Nhe2, and Nhe3 genes: effects on osmolality, sodium content, potassium content, and chloride activity of pilocarpine-stimulated salivaNHE" @default.
• W2107017864 created "2016-06-24" @default.
• W2107017864 creator A5006961977 @default.
• W2107017864 creator A5010967815 @default.
• W2107017864 creator A5024094891 @default.
• W2107017864 creator A5027765981 @default.
• W2107017864 creator A5029194204 @default.
• W2107017864 creator A5041694844 @default.
• W2107017864 creator A5058665792 @default.
• W2107017864 creator A5072990295 @default.
• W2107017864 creator A5073090713 @default.
• W2107017864 creator A5077140385 @default.
• W2107017864 date "2001-07-01" @default.
• W2107017864 modified "2023-10-15" @default.
• W2107017864 title "Defective Fluid Secretion and NaCl Absorption in the Parotid Glands of Na+/H+ Exchanger-deficient Mice" @default.
• W2107017864 cites W1560422398 @default.
• W2107017864 cites W1578170165 @default.
• W2107017864 cites W1589657989 @default.
• W2107017864 cites W1974615597 @default.
• W2107017864 cites W1974953275 @default.
• W2107017864 cites W1988956665 @default.
• W2107017864 cites W1990752157 @default.
• W2107017864 cites W1993023006 @default.
• W2107017864 cites W1995804648 @default.
• W2107017864 cites W2004347894 @default.
• W2107017864 cites W2010506174 @default.
• W2107017864 cites W2022597663 @default.
• W2107017864 cites W2023329018 @default.
• W2107017864 cites W2027071672 @default.
• W2107017864 cites W2030192076 @default.
• W2107017864 cites W2031459917 @default.
• W2107017864 cites W2033936610 @default.
• W2107017864 cites W2040394607 @default.
• W2107017864 cites W2040682991 @default.
• W2107017864 cites W2063571623 @default.
• W2107017864 cites W2063977457 @default.
• W2107017864 cites W2064094026 @default.
• W2107017864 cites W2066940775 @default.
• W2107017864 cites W2069939390 @default.
• W2107017864 cites W2074750856 @default.
• W2107017864 cites W2080021435 @default.
• W2107017864 cites W2081230957 @default.
• W2107017864 cites W2118044213 @default.
• W2107017864 cites W2120245762 @default.
• W2107017864 cites W2129055394 @default.
• W2107017864 cites W2151783154 @default.
• W2107017864 cites W2155753674 @default.
• W2107017864 cites W2185156651 @default.
• W2107017864 cites W2186437658 @default.
• W2107017864 cites W2228553450 @default.
• W2107017864 cites W2275660421 @default.
• W2107017864 cites W2332464156 @default.
• W2107017864 cites W2337373837 @default.
• W2107017864 cites W2338118010 @default.
• W2107017864 cites W2342135985 @default.
• W2107017864 cites W4238605098 @default.
• W2107017864 cites W4248035914 @default.
• W2107017864 doi "https://doi.org/10.1074/jbc.m102901200" @default.
• W2107017864 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11358967" @default.
• W2107017864 hasPublicationYear "2001" @default.
• W2107017864 type Work @default.
• W2107017864 sameAs 2107017864 @default.
• W2107017864 citedByCount "73" @default.
• W2107017864 countsByYear W21070178642012 @default.
• W2107017864 countsByYear W21070178642013 @default.
• W2107017864 countsByYear W21070178642014 @default.
• W2107017864 countsByYear W21070178642015 @default.
• W2107017864 countsByYear W21070178642016 @default.
• W2107017864 countsByYear W21070178642017 @default.
• W2107017864 countsByYear W21070178642018 @default.
• W2107017864 countsByYear W21070178642019 @default.
• W2107017864 countsByYear W21070178642020 @default.
• W2107017864 countsByYear W21070178642021 @default.
• W2107017864 countsByYear W21070178642022 @default.
• W2107017864 crossrefType "journal-article" @default.
• W2107017864 hasAuthorship W2107017864A5006961977 @default.
• W2107017864 hasAuthorship W2107017864A5010967815 @default.
• W2107017864 hasAuthorship W2107017864A5024094891 @default.
• W2107017864 hasAuthorship W2107017864A5027765981 @default.
• W2107017864 hasAuthorship W2107017864A5029194204 @default.
• W2107017864 hasAuthorship W2107017864A5041694844 @default.
• W2107017864 hasAuthorship W2107017864A5058665792 @default.
• W2107017864 hasAuthorship W2107017864A5072990295 @default.
• W2107017864 hasAuthorship W2107017864A5073090713 @default.
• W2107017864 hasAuthorship W2107017864A5077140385 @default.
• W2107017864 hasBestOaLocation W21070178641 @default.
• W2107017864 hasConcept C120665830 @default.
• W2107017864 hasConcept C121332964 @default.
• W2107017864 hasConcept C125287762 @default.
• W2107017864 hasConcept C12554922 @default.
• W2107017864 hasConcept C126322002 @default.
• W2107017864 hasConcept C134018914 @default.
• W2107017864 hasConcept C142724271 @default.
• W2107017864 hasConcept C185592680 @default.
• W2107017864 hasConcept C2780395140 @default.
• W2107017864 hasConcept C49039625 @default.
• W2107017864 hasConcept C55493867 @default.
• W2107017864 hasConcept C71924100 @default.

• next