Co-expression of Pendrin, Vacuolar H+-ATPase 4-Subunit and Carbonic Anhydrase II in Epithelial Cells of the Murine Endolymphatic Sac
Center for Hearing and Deafness Research, Department of Pediatric Otolaryngology, Children's Hospital Medical Center, Cincinnati, Ohio (HD,JHG,DC); Department of Medicine, University of Cincinnati, Cincinnati, Ohio (JX,ZW,MS); Veterans Affairs Medical Center at Cincinnati, Cincinnati, Ohio (MS); and Department of Medical Genetics (ANS,FEK) and Division of Nephrology (FEK), Cambridge Institute for Medical Research, Cambridge, United Kingdom
Correspondence to: Daniel Choo, Center for Hearing and Deafness Research, Dept. of Pediatric Otolaryngology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail: Daniel.Choo{at}cchmc.org
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Summary |
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(J Histochem Cytochem 52:13771384, 2004)
Key Words: endolymphatic sac acidbase regulation pendrin proton pump carbonic anhydrase II
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Introduction |
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The functional significance of an acidic ES lumen has been postulated to be related to endolymph homeostasis, which is essential for the normal function of the inner ear (Couloigner et al. 2000). Mechanisms for generation and maintenance of this acidic lumen are poorly understood. Carbonic anhydrase (CA), an enzyme that catalyzes the hydration of CO2 to ultimately yield bicarbonate and protons, was initially proposed as being involved in the acidification, based on the observations that the ES epithelium contained high levels of cytosolic CA and that the CA inhibitor acetazolamide caused a decrease in luminal pH and bicarbonate concentration (Yamashita et al. 1992
; Tsujikawa et al. 1993
). More recently, mRNA for the vacuolar (v) H+-ATPase ß1-subunit was detected in specific cells lining the mouse ES lumen (Karet et al. 1999
; Dou et al. 2003
). Immunocytochemical experiments also showed vH+-ATPase subunit E in the apical membrane of ES epithelial cells in guinea pig (Stankovic et al. 1997
). The vH+-ATPases are a family of multisubunit ATP-dependent proton pumps responsible for intracellular as well as luminal or interstitial space acidification (Nelson and Harvey 1999
; Alper et al. 2002
). Identification of proton pumps in ES cells suggests their role in ES pH regulation. Further supporting evidence is provided by pharmacological studies in guinea pig demonstrating that luminal administration of a specific inhibitor of the proton pump, bafilomycin, resulted in significant increase of the ES luminal pH (Couloigner et al. 2000
).
In addition to CA and vH+-ATPase, other acidbase regulatory proteins, including anion Cl/HCO3 exchangers (AEs) and pendrin, also may be involved in the regulation of acidbase balance in the ES. AEs are a family of variably expressed membrane proteins that exchange Cl for HCO3 across the plasma membrane. They are the products of at least three homologous genes of the SLC4 family encoding Na+-independent Cl/HCO3 exchangers that are designated as AE1, AE2, and AE3. These polypeptides contribute to regulation of intracellular pH or cellular electrochemical equilibrium potentials for Cl, HCO3, and H+ (Alper et al. 1997,2002
). Conversely, pendrin is a membrane protein encoded by the Slc26A4 gene (Pds), which belongs to a superfamily of anion exchangers (SLC26) (Everett et al. 1997
). Mutations in Pds cause Pendred syndrome, a genetic disorder characterized by sensorineural deafness and goiter (Everett et al. 1997
,1999
). Recent data have shown that pendrin mediates Cl/HCO3 exchange (Royaux et al. 2001
; Soleimani et al. 2001
) and is downregulated in response to metabolic acidosis (Wagner et al. 2002
; Frische et al. 2003
; Petrovic et al. 2003
), suggesting that pendrin may function as an acidbase regulator. Immunoreactive signals to antibodies that can recognize both AE1 and AE2 have been identified at the basolateral membrane of ES epithelial cells in guinea pig (Stankovic et al. 1997
). Pendrin mRNA and protein have also been detected in a subpopulation of murine ES cells (Everett et al. 1999
; Royaux et al. 2003
). These data suggest that AE and pendrin may participate in acidification processes in the ES.
Although different acidbase regulatory proteins have been localized in mammalian ES cells (Yamashita et al. 1992; Stankovic et al. 1997
; Royaux et al. 2003
), there is lack of information on the spatial relationship of their expression patterns, which is important for the understanding of the function of these acidbase regulators. The purpose of this study was to determine the cellular expression patterns of pendrin, vH+-ATPase, and CA II in the murine ES epithelium by IHC. An additional objective was to compare their expression patterns by double immunostaining. Proteins that participate in pH regulation of the ES endolymph have not been extensively studied. The information from this study would provide direct in situ morphological information on the distribution patterns of these molecules in the ES, which would improve our understanding of ES acidbase regulatory mechanisms.
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Materials and Methods |
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Antibodies
An affinity-purified rabbit polyclonal antiserum raised against a synthetic pendrin peptide corresponding to amino acids 734752 (CKSREGQD-SLLETVARIRDC) was used to detect pendrin. The sequence of the synthetic peptide used for antibody generation was identical for rat, mouse, and human pendrin and was previously reported (Petrovic et al. 2003). The generation and characterization of rabbit polyclonal antibody against the
4-subunit of vH+-ATPase (
4) also has been previously described (Herak-Kramberger et al. 2000
). An affinity-purified goat antibody against a peptide mapping near the N-terminus of CA II of human origin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Biotinylated goat anti-rabbit IgG, biotinylated rabbit anti-goat IgG (Vector Laboratories; Burlingame, CA), fluorescent isothiocyanate (FITC)-conjugated donkey anti-goat IgG, fluorescent FITC-conjugated Fab fragment of goat anti-rabbit IgG (H+L), and lissaminerhodamine-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch; West Grove, PA) were used as secondary antibodies.
Tissue Preparation
Five postnatal-day (P) 5 and five 5-week-old (adult) mice were sacrificed by CO2 inhalation and the inner ears were removed and fixed in 4% paraformaldehyde dissolved in 0.1 M PBS overnight. Ears from P5 mice were decalcified in 0.12 M EDTA at room temperature (RT) for 3 days, while ears from adult mice were decalcified in 0.12 M EDTA at RT for 12 day. Then the ears were cryoprotected in 30% sucrose. After embedding in OCT (Tissue Tek; Miles, Elkhart, IN), 10-µm-thick sections were cryostat-cut, placed on Superfrost Plus slides (Fisher Scientific; Pittsburgh, PA) and stored at 20C until use.
Immunohistochemistry
Localization of acidbase regulators in the ES was performed using an immunoperoxidase procedure. Sections were washed in PBS, then incubated with 0.5% H2O2 in methanol (Fisher Scientific) for 15 min, followed by 5% normal rabbit serum (Sigma Chemical; St Louis, MO) for anti-CA II or 5% normal goat serum (Sigma) for anti-4 and anti-pendrin primary antibodies for 30 min. After removal of the excess serum, the sections were incubated with the primary antibodies for 3 hr at 4C. Antibody dilutions for CA II,
4, and pendrin were 1:1000, 1:3000, and 1:300, respectively. Negative controls were performed by incubating slides with normal serum in place of primary antibodies. After three washes in PBS, sections were incubated with secondary antibodies (biotinylated goat anti-rabbit IgG or rabbit anti-goat IgG; Vector) diluted 1:200 in PBS for 30 min at RT. Avidinbiotin complex was then applied (Vector). Color was developed with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma).
Immunofluorescence Double Labeling
Double staining of pendrin and 4 with CA II (antisera raised in different species) was performed sequentially. For the first immunoreaction, sections were washed in PBS, then incubated with 0.5% H2O2 in methanol (Fisher Scientific) for 15 min followed by 5% normal goat serum for 30 min. After removal of the excess serum, the sections were incubated with the primary antibodies of either rabbit anti-
4 (1:2000) or rabbit anti-pendrin (1:100) overnight at 4C. Then the sections were washed three times in PBS and incubated with a secondary rhodamine-conjugated donkey anti-rabbit IgG (red color) (1:200) for 1 hr at RT. Sections were washed in PBS five times (10 min each time), then processed for CA II immunolabeling.
The slides were similarly incubated in 5% normal rabbit serum for 30 min, then incubated with anti-CA II antibody (1:500) overnight at 4C. After washing in PBS three times, sections were incubated with an FITC-conjugated donkey anti-goat IgG (green color) (1:200) for 1 hr. Thereafter, sections were rinsed in PBS three times (10 min each time) and mounted using Vectashield mounting medium (Vector).
Double staining of pendrin and 4 (antisera raised in the same species) was performed according to the method described by Negoescu et al. (1994)
. The cryostat sections were incubated first with rabbit polyclonal anti-
4 antibody (diluted 1:2000) overnight at 4C, followed by an FITC-conjugated Fab fragment of goat anti-rabbit IgG (H+L) (diluted 1:200) at RT for 1 hr After rinsing, sections were incubated with unlabeled Fab fragments of goat anti-rabbit IgG (H+L) (diluted 1:100) for 3 hr (Jackson) to block all possible remaining binding sites of the first primary antibody. Afterwards, sections were incubated with a second rabbit polyclonal antiserum against pendrin (diluted 1:200). This primary antibody was detected with rhodamine-conjugated donkey anti-rabbit IgG (1:200). After a final wash, sections were mounted in Vectashield. Controls for crossreaction of the first and second immunolabeling were performed by omitting the primary antibody in the first immunolabeling step.
All immunofluorescence labeling slides were examined with a Nikon epifluorescence microscope with fluorescein filters.
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Results |
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Discussion |
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In this study we used IHC to identify the cellular expression patterns of pendrin, vH+-ATPase, and CA II in ES epithelial cells. We show that pendrin is co-expressed with the 4-subunit of vH+-ATPase and CA II in the same subgroup of ES cells. The role of pendrin in the ES is unclear. However, on the basis of our data and the observation that pendrin functions as an acidbase regulator in the kidney (Wagner et al. 2002
; Frische et al. 2003
; Petrovic et al. 2003
), we propose that pendrin may function as an acidbase transporter in the ES by mediating Cl/HCO3 exchange across the apical membrane of the ES cells. Loss of function of pendrin may cause pH changes in the ES endolymph, which would affect normal absorptive function of the ES (Lundquist et al. 1984
) and result in enlargement of endolymphatic duct and sac, the pathology often seen in patients with Pendred syndrome as well as in knockout mice (Everett et al. 1997
,1999
,2001
).
Incorporating our results and previous findings of AE on the basolateral site of ES cells (Stankovic et al. 1997), we have employed a working model of acidbase regulation in the ES epithelium (Figure 6)
. We hypothesize that polarized distribution of acidbase transporters as well as cellular distribution of an acidbase buffering system in the specific type of the ES cells are involved in the ES endolymph acidbase regulation. Protons are secreted into the ES lumen through apically located proton pumps coupled with transepithelial reabsorption of HCO3 and secretion of Cl through apically located pendrin. Parallel, basolaterally located anion Cl/HCO3 exchangers (AE) facilitate H+ and Cl secretion by transporting HCO3 out of the cells across the basolateral membrane. Cytoplasmic CA II participates in H+ secretion process by catalyzing the formation of HCO3 and H+ in the presence of H2O and CO2.
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In summary, the ES endolymph is acidic and the mechanisms involved in the acidbase balance processes have not been extensively studied. The cellular expression patterns of pendrin, vH+-ATPase, and CA II demonstrated in this study suggest that a specific subset of cells in the ES epithelium are responsible for ES acidbase homeostasis and that coordinated activities of pendrin, vH+-ATPase, and CA II are involved in the processes.
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Acknowledgments |
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We thank Mr Joseph Alward for assistance in the preparation of this manuscript.
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Footnotes |
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