ARTICLE |
Correspondence to: Roger A. Johns, Dept. of Anesthesiology and Critical Care Medicine, Blalock 1415, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287-4965. E-mail: rajohns@jhmi.edu
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Summary |
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Endothelial nitric oxide synthase (eNOS), originally found in the endothelium of vascular tissue, also exists in other cell types, including ciliated epithelia of airways. The eNOS is ultrastructurally localized to the basal body of the microtubules of the cilia, and nitric oxide (NO) stimulates ciliary beat frequency (CBF). We examined whether the expression of eNOS is present in ciliated cells of other organs. Western blotting analysis revealed that eNOS was expressed in the rat cerebrum, lung, trachea, testis, and oviduct. Immunohistochemical staining showed that eNOS was localized in the ciliated epithelia of airways, oviduct, testis, and ependymal cells of brain in addition to the endothelium and smooth muscle of the vasculature. To confirm the activation of eNOS in the ciliated epithelia, we examined the effect of L-arginine (L-Arg), the substrate of NOS, on the production of nitrite and nitrate (NOx) in the cultured explants of rat trachea. L-Arg (100 µM) increased NOx levels significantly (p<0.05). In explants exposed to inhibitors of NOS, the effect of L-Arg on the production of NOx was blocked. These findings suggest that epithelial NO plays an important role in signal transduction associated with ciliary functions.
(J Histochem Cytochem 51:8187, 2003)
Key Words: endothelial nitric oxide, synthase, ciliated epithelium, oviduct, testis, airway, ependyma
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Introduction |
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NITRIC OXIDE (NO) is an important signaling molecule that can regulate many physiological processes, including vasodilatation (
In mammals, ciliated epithelial cells line the respiratory tract, part of the reproductive tract, and ventricles of the central nervous system. These cilia contribute to the movement and clearance of mucus (
Recently, eNOS, soluble guanylyl cyclase (sGC), and PKG I-ß have been identified in the ciliated epithelia of airways (
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Materials and Methods |
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Reagents and Antibodies
Minimal essential medium Eagle (MEME) was from Mediatech (Herndon, VA). NG-monomethyl-L-arginine (L-NMMA) was from Alexis (San Diego, CA). ECL blotting detection reagents were from Amersham (Poole, UK). Antifoaming agent was obtained from Sievers Instruments (Boulder, CO). L-Arg and all other chemicals were obtained from Sigma (St Louis, MO).
Mouse monoclonal antibodies (MAbs) against eNOS and iNOS, and a rabbit polyclonal antibody against brain NOS (bNOS), were purchased from Transduction Laboratories (Lexington, KY). Rabbit polyclonal antibody against the 1- and ß1-subunits of sGC was obtained from Cayman Chemical (Ann Arbor, MI). Rabbit polyclonal anti-PKG I-
and PKG I-ß antibodies were purchased from StressGen (Victoria, BC, Canada). Goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (HRP) and nitrocellulose membrane were from Bio-Rad (Hercules, CA). Biotinylated anti-mouse antibody, avidinbiotinHRP complex, and diaminobenzidine (DAB) kit were from Vector Labs (Burlingame, CA). Normal mouse IgG was obtained from DAKO (Carpinteria, CA). ECL blotting detection reagents were obtained from Amersham.
Tissue Preparation
All rats were obtained from Hilltop Laboratory Animals (Scottdale, PA) and were treated in accordance with NIH guidelines. Seven female adult (six for IHC and one for Western blotting analysis) and 16 male adult (six for IHC, one for Western blotting analysis, and nine for NOx measurement) SpragueDawley rats weighing 260300 g were used. Animals were sacrificed with an overdose of isoflurane (Ohmeda PPD; Liberty Corner, NJ). Then the lung, trachea, cerebrum, testis, and oviducts were rapidly removed.
For Western blotting analysis, the tissues were homogenized in ice-cold 50 mM Tris-HCl (pH 7.4) containing 0.1 mM EDTA, 0.1 mM EGTA, 0.1 µM phenylmethylsulfonyl fluoride (PMSF), 2 µM leupeptin, 1 µM pepstatin, and 0.1% 2-mercaptoethanol. The homogenates were centrifuged at 15,000 x g for 30 min at 4C and the pellet was discarded. The protein of the supernatant was used.
For IHC, the specimens were immersed in 4% paraformaldehyde in 0.1 M PBS, pH 7.4, at room temperature (RT). After 3 hr of fixation, the specimens were dehydrated in ethanol for paraffin embedding. Serial sections 5-µm thick were cut and mounted for immunostaining.
For preparation of ciliated epithelia, rat tracheal mucosa explants were cut into small pieces (45 mm3) and rinsed several times with PBS. L-Arg and L-NMMA were dissolved in PBS in 10 mM stock solution. Then the explants were incubated in 1 ml MEME containing either PBS (control group) or 100 µM L-Arg, or 100 µM L-Arg and 100 µM L-NMMA at 37C, 5% CO2. After 1 hr of culture, the media were used for NOx measurement.
Western Blotting Analysis
Protein samples were loaded (50 µg each) and separated on 7.5% SDS-PAGE, followed by blotting of the proteins to nitrocellulose membrane. The blot was blocked with a buffer consisting of 10 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 2% nonfat milk, 2% bovine serum albumin, and 0.1% Tween-20, for 1 hr at RT. The blots were then incubated with primary antibody (1:1000 dilution) for 1 hr at RT. Then the blots were incubated with a secondary goat anti-mouse or anti-rabbit IgG conjugated with HRP and detected with ECL. After detection, the bands were quantified with a densitometer and Imagequant software (Molecular Dynamics; Sunnyvale, CA).
Immunohistochemistry
After deparaffinization, the slides were treated with 3% H2O2 in PBS for 20 min to quench endogenous peroxidase activity. After blocking of nonspecific sites with 10% goat serum in PBS for 1 hr, tissue sections were incubated with mouse anti-eNOS MAb (diluted 1:500 for sections from oviduct, airway, and testis and 1:1000 for sections from cerebrum) at 4C overnight. Unbound primary antibody was washed off with PBS and the sections were incubated with biotinylated anti-mouse antibody (1:200 dilution) for 1 hr. Specific binding was detected using an avidinbiotinHRP complex (1:100 dilution) for 1 hr and a substrate solution of H2O2 and DAB. Negative controls were carried out with normal mouse IgG.
Measurement of NOx (NO2-/NO3-) in the Media
The measurement of NO and its oxidation products (NOx) was carried out using a chemiluminescence analyzer (NOA 280; Sievers Instruments, Boulder, CO), reduction of nitrite and nitrate using vanadium (III) and hydrochloric acid at 90C. Ten µl of samples was injected into the glass purge vessel containing 5 ml of vanadium chloride (0.1 M in 1 N HCl) and 100 µl of diluted (1:30) antifoaming agent. NOx were sparged from the vessel and carried in nitrogen gas to the analyzer. The output from the analyzer was integrated to determine total peak area. The nitrite/nitrate concentrations were determined by comparison with the calibration curve (125 µM) of NaNO2 in distilled water.
Statistical Analysis
The results are expressed as means ± SE. One-factor ANOVA was used to compare the difference among groups. The value of p<0.05 was considered statistically significant.
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Results |
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Western Blotting Analysis
eNOS antibody reacted with a protein of 135-kD mass in all tissues of rat brain, lung, trachea, testis, and oviduct (Fig 1). The antibody did not crossreact with bNOS and iNOS proteins because there were no bands at 160 kD or 130 kD. bNOS antibody reacted only in cerebrum and testis; iNOS reacted weakly in cerebrum, lung, and trachea. The results indicated that only eNOS was localized in all these cilia-containing tissues. The NOcGMP signal pathway proteins sGC 1, sGC ß1, PKG I-
, and PKG I-ß were also detected in all these cilia-containing tissues (Fig 2). sGC
1 was dominant in cerebrum, testis, and oviduct; sGC ß1 was dominant in lung and trachea.
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Immunoreactivity of eNOS in Airway, Cerebrum, Oviduct, and Testis
On the basis of our previous work showing the physical association of eNOS, but not iNOS or bNOS, with the basal body of the microtubules of lung epithelial cilia (
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eNOS antibody gave a strong and specific signal in ciliated epithelia of trachea (Fig 3C, blue arrows), bronchus (Fig 3D, blue arrows), and bronchiole (Fig 3E, blue arrows), with prominent staining of eNOS in Clara cells (Fig 3E, red arrows) and in type II cells (Fig 3E, green arrows) of alveoli. Nonciliated epithelia in trachea (Fig 3C, red arrows) and bronchus (Fig 3D, red arrows) were not stained with eNOS.
Positive immunostaining for eNOS was detected in the ciliated epithelium (Fig 3F, blue arrows; ampulla) and the endothelia and smooth muscle in the vasculature of the oviduct (data not shown). Nonciliated epithelia were not stained by eNOS (Fig 3F, red arrow).
In the testis, eNOS IHC staining was positive in type B spermatogonia (SB), spermatocytes (Sc), spermatids (Sd), and spermatozoa (Sz). Leydig cells (Le) and Sertoli cells (Se) were also stained with eNOS. Type A spermatogonia (SA) did not stain with eNOS (Fig 3G).
The ependymal cells of the lateral ventricles exhibited a highly positive IHC expression of eNOS (Fig 3H). There was also a strong staining in the epithelia of the choroid plexus (data not shown).
Effect of l-arginine on NOx Concentration
In explants exposed to L-Arg (Fig 4), NOx levels increased from 6.03 ± 0.29 to 8.38 ± 0.62 µM (p<0.05). In explants treated with the inhibitor of NOS, L-NMMA, the effect of L-Arg on NOx production was blocked (Fig 4).
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Discussion |
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In mammals, ciliated epithelial cells line the respiratory tract, part of the reproductive tract, and ventricles of the central nervous system. The presence of NO in the respiratory tract was suggested by the discovery of NO in exhaled air (
Kartagener's syndrome, or primary ciliary dyskinesia, is an autosomal dominant disease in which cilia lack motility as a result of the absence of dynein arms in the cilia (T) in exon 7 of eNOS and with a bi-allelic tetranucleotide polymorphism in the promoter of the iNOS gene (
eNOS staining was present in the oviduct also. Histologically, the wall of the oviduct consists of a mucosa, a muscular layer, and an external serosa. The epithelia of the mucosa are composed of two kinds of cells, one of which contains cilia. The other cell type is devoid of cilia and is considered to be secretory. Functionally, the oviduct is the site of fertilization and early embryonic development. The role of the oviduct in reproduction is more than a passive conduit and involves active transport of ova and sperm. The infundibulum is involved in oocyte pickup; the ampulla is the site of sperm penetration and fertilization; the isthmus is responsible for the transport of embryos into the uterus. Coordinated muscular peristalsis and ciliary movement are required for successful reproduction. However, the egg reaches the site of fertilization within a normal time frame even after blocking of rapid muscle-induced egg movements with isoproterenol (
NADPH-diaphorase activity was identified in pig (
Earlier studies have addressed the localization of NOS in spermatozoa. eNOS (
However, controversial results have been reported.
We also found eNOS staining in the ependymal cells of ventricles. In vertebrate brain, ependymal cells line the ventricles and central canal of the spinal cord, forming a wall separating CSF and the neural tissues. There are also several reports concerning the distribution of NADPH diaphorase-positive epithelia in ependyma. However, the widespread distribution of NOS, as revealed by NADPH diaphorase histochemistry, makes it difficult to relate ependymal NO to any specific physiological function because NADPH-diaphorase detects all isoforms of NOS. Here we demonstrated that eNOS was localized in the ciliated epithelia of the ependyma. Our findings suggest that eNOS may be involved in ependymal ciliary functions.
sGC 1, sGC ß1, PKG I-
, and PKG I-ß were also found in the tissues of cerebrum, lung, trachea, testis, and oviduct. The sGC
1-subunit was dominant in the brain, testis, and oviduct. The sGC ß1-subunit was dominant in the lung and trachea. This suggests that NO may activate different sGC subunits in different cells. As we reported in a previous study (
To confirm if the NOS was activated in the ciliated epithelia, we examined the effect of L-Arg on NOx production in ciliated epithelia of trachea. NO is synthesized by NOS from L-Arg and molecular O2 (
In summary, eNOS is present in the ciliated epithelia of oviduct, trachea, bronchus, bronchiole, lung, ependymal cells, and testis. In addition, L-Arg increased NOx production in cultured ciliated epithelia of rat trachea via NOS. This suggests that NO produced by ciliated epithelial cells may play a role in the regulation of ciliary motility in an autocrine manner and that this may occur in cilia from all tissues.
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Acknowledgments |
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Supported by NIH grants R01 HL 39706 and R01 GM 49111.
Received for publication October 12, 2001; accepted August 6, 2002.
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