Chem. Senses 27: 13-21,
2002
© Oxford University Press 2002
Glucocorticoid Enhances Na+/K+ ATPase mRNA Expression in Rat Olfactory Mucosa during Regeneration
A Possible Mechanism for Recovery from Olfactory Disturbance
Department of Otorhinolaryngology, Kanazawa University School of Medicine, Kanazawa, Japan 1 Department of Pathology, Kanazawa University School of Medicine, Kanazawa, Japan
Correspondence to be sent to: Toshiro Nishimura, Department of Otorhinolaryngology, Kanazawa University School of Medicine, 13-1 Takaramachi, Kanazawa, Ishikawa, 920-8641, Japan. e-mail: nishimut{at}orl.m.kanazawa-u.ac.jp
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Abstract |
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Systemic or topical application of glucocorticoid is the treatment of choice for olfactory disturbance. Recently, Na+/K+ ATPase and glucocorticoid receptor immunoreactivity in the olfactory mucosa was reported. To elucidate a glucocorticoid action on Na+/K+ ATPase production, an animal model was produced by an intra-nasal application of 5% ZnSO4 solution to Wistar rats. Dexamethasone was injected i.p. (0.01 mg/100 g) for 14 days after the insult. Histologically, the regeneration process was completed on day 14 in both dexamethasone- and saline-injected control rats. We used a quantitative polymerase chain reaction (PCR) method to evaluate mRNA production of Na+/K+ ATPase and glucocorticoid receptor. In dexamethasone-injected rats, up-regulation of glucocorticoid receptor mRNA (95% more than control rats, P = 0.00068, unpaired t-test) and of Na+/K+ ATPase mRNA expression (76% more than control rats, P = 0.0042) was observed on day 14. The increased Na+/K+ ATPase expression in the regenerated olfactory mucosa is thought to be beneficial for an active uptake of K+, which is released during excitation, around olfactory neurons and for the transepithelial absorption of Na+ from olfactory mucus. Dexamethasone may thus contribute to the recovery of function after the morphological regeneration in part, at least, through its receptor by regulation of the ionic concentration in the olfactory mucosal microenvironment.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Systemic or topical application of glucocorticoids is the treatment of choice for olfactory disturbance (Robinson et al., 1998
; Fong
et al., 1999). Although the glucocorticoids' mechanism of
action remains unclear, it is supposed that they suppress the production of
inflammatory cytokines and resolve inflammatory reaction in the olfactory
mucosa. Glucocorticoids are thus thought to improve the airflow in the nasal
cavity, allowing the odorant-containing airflow to reach the olfactory sensory
mucosa (Robinson et al.,
1998).
Recently, the presence and localization of glucocorticoid receptors (GR)
(Kern et al., 1997a,
b;
Robinson et al.,
1998) and Na+/K+ ATPase in olfactory mucosa
of guinea pigs (Kern et al.,
1991; Fong et al.,
1999) have been reported. Within the olfactory epithelium,
Na+/K+ ATPase immunoreactivity has been reported at the
supranuclear region of sustentacular cells and/or dendrites of olfactory
receptor neurons (Fong et al.,
1999). Moreover, glucocorticoid immunoreactivity has been detected
at the apical surface including the dendrites, knobs and cilia of olfactory
receptor neurons and the supranuclear region of sustentacular cells
(Robinson et al.,
1998).
Na+/K+ ATPase is an ubiquitous transmembrane
hetero-dimeric protein complex, consisting of a catalytic 
and a
glycosylated ß subunit, that couples the hydrolysis of ATP to the
vectorial transport of Na+ outward and K+ inward across
the cell plasma membrane (Ewart et
al., 1995; Verrey et
al., 1996; Derfoul et
al., 1998).
Na+/K+ ATPase expressed in olfactory receptor and
sustentacular cells may participate in the maintenance and restoration of the
receptor cells' resting potential, because Na+/K+ ATPase
along the basolateral sustentacular cell membrane may buffer high
extracellular K+ concentrations released from adjacent olfactory
neurons during excitation through the active uptake of K+
(Kern et al.,
1991).
It has also been suggested that Na+/K+ ATPase may
maintain the ionic balance of olfactory mucus through transepithelial
Na+ absorption from the olfactory mucus
(Fong et al., 1999).
Na+ in olfactory mucus is originally secreted from Bowman's gland
and Na+ is supposed to be removed by cotransport or exchange from
olfactory mucus into sustentacular cells. Then Na+ would be
actively pumped from sustentacular cells into the interstitial space of
olfactory mucosa. Since the sodium concentration of olfactory mucus is
reportedly lower than the mucus that covers the respiratory mucosa
(Joshi et al., 1987),
Na+/K+ ATPase is thought to play an important role in
the maintenance of the proper ionic microenvironment necessary for olfactory
transduction.
We produced animal models of olfactory mucosa injury, which were given i.p. glucocorticoid (dexamethasone) in order to clarify its effect on the olfactory mucosa during regeneration. The mRNA expressions of GR and Na+/K+ ATPase were assessed to determine whether they may indicate a direct action of glucocorticoid on the olfactory mucosa during regeneration following injury.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Animal models
Sixty-one male Wistar rats (150-180 g) were obtained from Charles River Japan (Atsugi, Japan) and maintained on a 12 h light—dark cycle in a temperature-controlled room. All received similar amounts of a standard solid diet (CRF-1, Charles River Japan) and water ad libitum throughout the study. The experiment was in accordance with the protocol approved by the Animal Care and Use Committee of Kanazawa University (Kanazawa, Japan). Thirty-three rats were used for histologic (n = 3 for each observation) and 28 rats (n = 4 for each observation) for mRNA assessments.
Olfactory mucosa injury was produced by means of topical application of a
5% ZnSO4 solution (Kimura
et al., 1991). The animals were anesthetized with an i.p.
pentobarbital sodium injection (1 mg/kg) and 0.2 ml of the ZnSO4
solution was applied to the nasal cavity of each nostril with a plastic 20
gauge needle, after which the animals were kept immobile for 10 min.
A therapeutic dose of dexamethasone phosphate (Banyu Seiyaku, Tokyo, Japan) was injected i.p. (0.01 mg/100 g body wt) every day for 14 days, starting immediately after the olfactory mucosa insult. Control rats were injected with the same volume of physiological saline.
Sample preparations
For histological studies, rats were deeply anesthetized with pentobarbital sodium (30 mg/100 g) and transcardially perfused with ice-cold physiological saline followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The rats were then decapitated and the head post-fixed for 24 h in the same fixative, decalcified in 10% ethylene-diamine tetra-acetic acid (EDTA) solution for 14 days, dehydrated and embedded in paraffin. Finally, 4 µm sections of the head were made in a coronal direction.
For RNA extraction, the nasal mucosa was quickly microdissected and frozen in liquid nitrogen after the rats had been anesthetized. The specimens were stored at -70°C until the subsequent experiments.
Immunohistochemistry
Immunohistochemical studies used the avidin biotin peroxidase complex (ABC)
method (Hsu et al.,
1981). After blocking of the endogenous peroxidase activity in a
methanol solution containing 0.3% hydrogen peroxide, the sections were
incubated in either 10% normal goat serum (for the polyclonal primary
antibody) or horse serum (for the monoclonal antibody) for 30 min at room
temperature to reduce non-specific antibody binding. The primary antibodies
used were rabbit polyclonal antibody specific for the ß1
subunit of Na+/K+ ATPase (Upstate Biotechnology, Lake
Placid, NY) at a 1:100 dilution, and mouse monoclonal antibody for
glucocorticoid receptors (Oncogene Research Products, Cambridge, MA) at a
1:200 dilution. Specimens were incubated with a diluted primary antibody
overnight at 4°C. Sections were then incubated with either biotinylated
goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) or biotinylated
horse anti-mouse IgG (Vector Laboratories) at a 1:200 dilution for 30 min at
room temperature. Reaction with the strept-avidin biotin peroxidase complex
reagent (DAKO Japan, Tokyo, Japan) was performed at room temperature for 30
min. After color development, nuclear staining was performed with hematoxylin.
For positive controls, rat kidneys were stained for
Na+/K+ ATPase and glucocorticoid receptor. A negative
control was included for each specimen by substituting a primary antibody for
either normal goat serum or normal horse serum.
Real-time quantitative reverse transcription PCR procedure
Total RNA was extracted with the aid of RNeasy mini-kits (Qiagen, Tokyo, Japan) from 5 mg of olfactory mucosa from each rat. Tissue was manually homogenized with a disposable plastic micropestle in a microcentrifuge tube containing 350 µl of RLT lysis buffer. The homogenate was then passed through a QIAshredder microcolumn (Qiagen) by centrifugation. The resulting cell lysate was mixed with an equal volume of 70% ethanol in diethyl pyrocarbonate (DEPC)-treated water and loaded onto RNeasy spin columns and centrifuged. The columns were washed with 700 µl RW1 buffer and centrifuged for 15 s. A mixture of 70 µl of RDD buffer and 10 µl of 3 U/µl of deoxyribonuclease I solution (Qiagen) was then applied to the column for 15 min at 30°C. The column was centrifuged and serially washed with 700 µl of RW1 buffer and 500 µl of RPE buffer and centrifuged for 3 min. Total RNA was eluted by the addition of 50 µl of DEPC-treated water to the column followed by centrifugation. RNA concentration was determined with a spectrophotometer to measure absorbance at 260 nm.
One microgram of total RNA was reverse-transcribed with a First-Strand cDNA synthesis kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ) in a total reaction volume of 15 µl containing a bulk first-strand reaction mix consisting of moloney murine leukemia virus reverse transcriptase, ribonuclease inhibitor, 0.08 mg/ml bovine serum albumin, 1.8 mM of dATP, 1.8 mM of dCTP, 1.8 mM of dTTP, 45 mM Tris (pH 8.3), 68 mM potassium chloride, 15 mM DTT, 9 mM magnesium chloride and 40 pmol of random hexadeoxynucleotides. The reaction was performed at 37°C for 60 min. A preliminary standard 40 cycle PCR, using the ß-actin primer (Promega, Madison, WI) spanning one intron, was performed on 0.5 µl of each cDNA sample to confirm the quality of the sample to prevent contamination of genomic DNA (data not shown).
Real-time quantitative PCR (Gibson
et al., 1996) was performed with an ABI Prism 7700
sequence detection system (Perkin-Elmer Applied Biosystems, Foster City, CA).
The expression of 18S ribosomal RNA was used as the internal control
(Bhatia et al., 1994).
For the ß1 subunit of Na+/K+ ATPase and
GR mRNA quantification, primers and probes
(Table 1) were chosen with the
aid of Primer Express (Perkin-Elmer Applied Biosystems). Primers were
purchased from Takara-Syuzo (Kusatsu, Japan) and probes from Perkin-Elmer
Japan (Urayasu, Japan). Amplification mixes (50 µl) contained the sample
cDNA (
30 ng), 10 TaqMan buffer (5 µl), 200 mM dATP, dCTP, dGTP and 400
mM dUTP, 5 mM MgCl2, 1.25 units of AmpliTaq Gold, 0.5 units of
AmpErase uracil N-glycosylate (UNG), 300 nM of each primer and a 200 nM probe.
Thermal cycling was performed for 2 min at 50°C and for 10 min at
95°C, and consisted of 40 cycles at 95°C for 15 s and at 65°C for
1 min.
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Each assay included samples for a standard curve consisting of 1 µml of
kidney cDNA solution (50 ng/µl) made from kidney total RNA serially diluted
1, 4, 16 and 64 times in duplicate, a non-template control and 30 ng of
the sample cDNA in quadruplicate.
All samples with a coefficient of variation >10% were re-tested. Authenticity of PCR products was confirmed by standard 4% agarose gel electrophoresis with ethidium bromide staining and direct sequencing of representative samples.
All measurements were done in triplicate and the mean values and standard deviations were calculated. The sample values were divided by the value of 18 S ribosomal RNA expression and the value of olfactory mucosa before injury was standardized at 1.0 to facilitate comparisons among samples.
Data analysis
The statistical significance of differences between groups with numerically defined variables was established by means of ANOVA and the Student's t-test using a level of significance of P < 0.05 (StatView, SAS Institute Inc., NC).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Na+/K+ ATPase and GCR immunohistochemistry in normal olfactory mucosa
Figure 1A shows that the most intense Na+/K+ ATPase immunoreactivity was observed at the supranuclear region of the sustentacular cells and the dendrites of olfactory receptor neurons. The supranuclear region of several sustentacular cells was stained very intensely and showed a wide staining pattern. Most of all, the dendrites of olfactory receptor neurons exhibited a clear linear staining pattern between sustentacular cells. Less intense activity was observed at the vascular wall and some of the acinar cells of Bowman's gland.
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These results were in agreement with a previous report using guinea pigs
(Fong et al., 1999),
but that report showed more intense immunoreactivity in acinar and ductal
cells of Bowman's gland. In our specimen, it was difficult to find ductal
cells of Bowman's gland and the difference seemed to depend on the animal
species used.
In positive control sections of rat kidney (Figure 1B), cells in collecting tubules were strongly positive. In negative control sections (Figure 1C), in which the primary antibody was omitted, there were no positive cells in the olfactory mucosa.
Figure 2 demonstrates that
the immunoreactivity of GR was most intense at the surface layer of the
olfactory mucosa. This area corresponds to the apical knobs, cilia and
dendrites of olfactory receptor neurons and the supra-nuclear region of
sustentacular cells. Almost all the sustentacular cells exhibited strong to
moderate staining. Some linear pattern stains were observed between
sustentacular cells and those were considered to be the dendrites of olfactory
neurons. Although a previous report
(Robinson et al.,
1998) demonstrated an intense immunoreactivity in the acinar cells
of the Bowman's gland and nerve bundles of olfactory nerve processes, neither
structure exhibited marked staining in our specimens. Again, the difference
seemed to depend on the animal species and staining method used (the previous
report used immunofluorescence method).
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In positive control sections of rat kidney (Figure 2B), cells in renal tubules were strongly positive. In negative control sections (Figure 2C), in which the primary antibody was omitted, there were no positive cells in the olfactory mucosa.
Histological findings of regeneration of olfactory mucosa
Figure 3 shows that the olfactory mucosa was severely damaged after the application of a 5% zinc sulfate solution. In rats without dexamethasone injection, only a few cell layers of olfactory neurons remained on day 2. The number of layers had increased on days 5 and 8. On day 8, the sustentacular cells had regenerated and matured and eight or nine layers of regenerated olfactory neurons were observed on day 14.
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In rats with dexamethasone injection, the sustentacular cells seemed more mature on day 8, but the number of layers of olfactory neurons was the same as that of rats without dexamethasone injection (six to seven layers). On day 14, eight or nine layers of the olfactory neurons were observed and the regeneration of the olfactory mucosa had morphologically been completed.
Na+/K+ ATPase and GR mRNA expression during regeneration of olfactory mucosa
Figure 4A demonstrates the
kinetics of the reaction with change in fluorescent signal
(
Rn) on the y-axis and the cycle of PCR on
the x-axis, that was plotted with serial dilutions of kidney cDNA.
The estimation of target sequence is related to the threshold cycle (the cycle
at which statistically significant increase in Rn is first detected)
and threshold is defined as the average standard deviation of normalized
reporter emission intensity for the early cycles, multiplied by an adjustable
factor (Gibson et al.,
1996).
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A plot of the threshold cycle at the various kidney control cDNA dilutions (black dots) is shown in Figure 1B that demonstrates a linear relationship that was noted over the range tested (0.78-50 ng of kidney cDNA) with a correlation efficient of 0.999. Unknown samples from olfactory mucosa were also plotted for the estimation of mRNA expression.
In Figure 5A, Na+/K+ ATPase and GR mRNA expression was measured on days 0, 3, 7 and 14 until the damaged olfactory mucosa had morphologically been completely regenerated. Figure 5A shows that Na+/K+ ATPase mRNA expression in the olfactory mucosa gradually increased after the 5% zinc sulfate solution insult. In glucocorticoid-treated rats, there was a statistically significant increase of 76% in Na+/K+ ATPase expression compared to that in control rats on day 14 (P = 0.0042, unpaired t-test).
Figure 5B demonstrates that GR mRNA expression of the olfactory mucosa was similar to Na+/K+ ATPase mRNA expression and this expression gradually increased after the insult. On day 14, GR expression of glucocorticoid injected rats was 95% higher than that of control rats (P = 0.00068, unpaired t-test).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Our study indicates that glucocorticoid could have a direct effect on Na+/K+ ATPase expression in rat olfactory mucosa, although glucocorticoid was of little benefit for the morphological regeneration of the olfactory mucosa after the injury.
In renal and cardiac cells, aldosterone but not dexamethasone activates
Na+/K+ ATPase gene expression, while the reverse is
observed in colonic surface cells. This suggests that both mineralocorticoid
and glucocorticoid receptors (MR and GR) are involved in the regulation of the
enzyme (Orlowski et al.,
1990; Ikeda et al.,
1991; Derfoul et al.,
1998;
Zemanoveá
et al., 1998). Our result offers support for the notion
that glucocorticoid (dexamethasone) can induce Na+/K+
ATPase gene expression in the olfactory mucosa.
The immunoreactivities of regenerated olfactory mucosa in
dexamethasone-injected rats and control rats were not directly compared in
this study. Because there is a possibility of the post-transcriptional
down-regulation of both Na+/K+ ATPase and GR mRNA
(Orlowski et al.,
1990; Ikeda et al.,
1991; Derfoul et al.,
1998), this possibility should be further examined from the
viewpoint of protein production using the Western blot technique.
In our experiment, the most significant increase in both GR and Na+/K+ ATPase mRNA production occurred after day 7, when the sustentacular cells had completed their regeneration. In the early regeneration period, an increase in the number of cell is thought to be mainly responsible for to the total increase of mRNA production. But in the later regeneration period after day 7, an increase in the mRNA production of each cell due to dexamethasone administration is considered to be the main contributory factor in the overall increase in mRNA production in the olfactory mucosa.
Glucocorticoids enter most cells by diffusion and bind to glucocorticoid
receptors. The receptor—hormone complex then becomes dimerized and the
dimers bind to glucocorticoid response elements (GRE) in target DNA enhancer
sequences. This usually results in gene activation
(Bookstein et al.,
1992). A functional GRE at position -650 of the
Na+/K+ ATPase ß1 gene promotor for both
glucocorticoids and mineralocorticoids has been reported
(Derfoul et al.,
1998).
Glucocorticoids usually down-regulate GR in an intact organ
(Bookstein et al.,
1992), but their regulatory activities in various organs during
regeneration remain to be solved. In rats, glucocorticoid receptor expression
was found to be elevated during liver regeneration and dexamethasone induced
hepatic tyrosine aminotransferase and tryptophan oxygenase in the regenerating
rat liver after partial hepatectomy
(Karabélyos
et al., 1999). Our results represent supportive evidence
that olfactory mucosa shows a similar up-regulation of GR during the
regeneration period.
The significance of an increased expression of Na+/K+
ATPase in the olfactory mucosa during regeneration is twofold. One is that
this increase maintains and restores the receptor cells' resting potential and
the other is that it maintains the ionic balance of the olfactory mucus
through transepithelial Na+ absorption
(Kern et al., 1991;
Fong et al., 1999).
These speculations are mainly based on indirect evidence of morphological
studies. Since no direct evidence of electrophysiological data has been
reported, direct measurements of the resting potential of the olfactory
receptor neurons and the concentrations of sodium and potassium ions in the
olfactory mucus during regeneration are required.
In the olfactory mucosa, both GR and Na+/K+ ATPase were up-regulated during regeneration, and glucocorticoid further enhanced these expressions. Our results suggest that the clinical implication of glucocorticoid administration is that it induces Na+/K+ ATPase expression during regeneration and may contribute to recovery from olfactory disturbance in part, at least, through its receptor by means of regulation of the ionic concentration in the olfactory mucosal microenvironment.
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Acknowledgments |
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This work was supported by a Grant-in-Aid for Scientic Research (C12671655 to T.N.) from the Ministry of Education, Science and Culture of Japan.
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Accepted September 4, 2001
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