ARTICLE |
Correspondence to: Roger A. Johns, Dept. Anesthesiology and Critical Care Medicine, Blalock 1415, Johns Hopkins U. School of Medicine, 600 N. Wolfe St., Baltimore, MD 21287-4965.
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Summary |
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Airway epithelia play a crucial role in protecting the lung from the external environment. Ciliated airway epithelial cells contribute to mucociliary transport systems via ciliary beating and electrolyte transport mechanisms to defend against respiratory tract infection. Both of these activities are regulated by nitric oxide (NO)-dependent mechanisms. To better understand the role of the NOcGMP signal transduction cascade in these responses, we investigated the localization of endothelial nitric oxide synthase (eNOS), soluble guanylyl cyclase (sGC), cGMP-dependent protein kinase (PKG) I-, and PKG I-ß in the tracheas and lungs of normal rats by immunohistochemistry. Mouse anti-eNOS, rabbit anti-sGC, PKG I-
, and PKG I-ß antibodies were used. Strong immunostaining for eNOS was detected in ciliated tracheal, bronchial, and bronchiolar epithelia, in Clara cells, and in Type II alveolar cells. The pattern of sGC and PKG I-ß immunostaining showed striking parallels with that of eNOS staining. No staining was detectable in ciliated epithelium with the anti-PKG I-
antibody. Taken together, these observations suggest that PKG I-ß might transduce NOsGC signaling into biological responses in ciliated respiratory epithelia.
(J Histochem Cytochem 47:13691374, 1999)
Key Words: airway epithelium, ciliary beating frequency, nitric oxide, soluble guanylate cyclase, cGMP-dependent protein kinase, immunohistochemistry
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Introduction |
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Nitrous oxide (NO) a physiologically important activator of sGC, plays an important regulatory role in airway function and is implicated in pulmonary physiology. There is evidence that ciliary beating and electrolyte transport are regulated by NO-dependent mechanisms (, and PKG I-ß protein expression in rat trachea and lung tissues.
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Materials and Methods |
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Tissue Preparations
All rats were obtained from Hilltop Laboratory Animals (Scottdale, PA) and were treated in accordance with APS/NIH guidelines. Six normal male SpragueDawley rats were anesthetized with isoflurane. Their lungs and tracheas were rapidly removed and cut as cross-sections approximately 2 mm thick. The specimens were then immersed in fixative solution containing 4% paraformaldehyde in PBS (0.1 M, pH 7.4). After 3 hr of fixation, the specimens were dehydrated in graded ethanol for paraffin embedding. Sections of 5 µm were cut (at least six slides for each lung) and mounted on Superfrost/Plus (Fisher; Pittsburgh, PA) slides for immunostaining.
Western Blot Analysis
To examine the eNOS antibody specificity, Western blot analysis was performed. For the preparation of crude extracts, rat lung, brain, spinal cord tissues, and rat lung tissues induced with LPS (10 mg/kg body weight IP for 6 hr) were homogenized in ice-cold 50 mM Tris-HCl, pH 7.4, containing 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM leupeptin, 1 mM pepstatin, and 0.1% 2-mercaptoethanol. The homogenate was centrifuged at 15,000 x g for 30 min at 4C and the pellet was discarded. The protein of the supernatant was measured using a Bio-Rad kit according to the manufacturer's instructions (Bio-Rad; Hercules, CA). The samples of lung, brain, and spinal cord, were loaded (100 µg each) and separated on a 7.5% SDS-PAGE, followed by blotting of the proteins to nitrocellulose membrane (Bio-Rad). The blot was blocked with a buffer consisting of 50 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 room temperature (RT). The blot was then incubated with monoclonal mouse anti-eNOS antiserum (1:500 dilution; Transduction, Lexington, KY) for 1 hr at RT, followed by a secondary goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Bio-Rad), and detected with ECL blotting detection reagents (Amersham; Poole, UK). After detection, the film was quantitated with a densitometer and Imagequant software (Molecular Dynamics; Sunnyvale, CA).
Antibodies for Immunohistochemistry
A mouse monoclonal antibody against eNOS was obtained from Transduction (1:500 dilution). Rabbit polyclonal antibodies against sGC were produced using synthetic peptide from the 1- and ß1-subunits of sGC (Nakane et al. 1990) (1:500 dilution; Cayman Chemical, Ann Arbor, MI). PKG I-
was made from a 15-residue synthetic peptide based on the human PKG I-
(residues 657671) and PKG I-ß from a 14-residue synthetic peptide based on the human PKG I-ß (
and PKG I-ß; StressGen, Victoria, BC, Canada). The antibodies were used for detection of eNOS, sGC, PKG I-
, and PKG I-ß, respectively.
Immunohistochemistry
After deparaffinizing, 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 60 min, tissue sections were washed (three times for 5 min in PBS) and incubated with specific antibody at 4C overnight. After washing off unbound primary antibodies with PBS, the sections were incubated with biotinylated anti-mouse (eNOS) or anti-rabbit (sGC, PKG I-, and PKG I-ß) serum (1:200 dilution) for 1 hr. Specific binding was detected using an avidinbiotinHRP complex (1:100 dilution for 1 hr; Vector, Burlingame, CA) and a substrate solution of H2O2 and diaminobenzidine (DAB) kit (Vector) according to the manufacturer's instructions. Then the slides were counterstained with hematoxylin, dehydrated through graded alcohol and xylenes, mounted on coverslips, and examined under an Olympus VANOX AHBS3 microscope. Negative controls were carried out with normal rabbit serum and mouse IgG according to the primary antibodies used for sGC, PKG I-
, PKG I-ß, and eNOS staining.
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Results |
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Immunospecificity of eNOS Antibodies
In Western blot (Figure 1A), eNOS antibody was shown to react only with eNOS protein at 135-kD mass in rat lung (Lane 1 in Figure 1A), spinal cord (Lane 3 in Figure 1A), and LPS-induced lung (Lane 4 in Figure 1A), but not in the rat brain (Lane 2 in Figure 1A). The antibody did not crossreact with bNOS and iNOS proteins because there were no proteins at 155-kD mass and 130-kD mass (the kD mass of bNOS and iNOS). The result from Western blotting indicated that the eNOS antibody was specific for the eNOS protein and did not detect other NOS isoforms in rat tissues.
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Immunoreactivity of eNOS, sGC, PKG I-, and PKG I-ß in Lung and Trachea
To determine the distribution of eNOS, sGC, PKG I-, and PKG I-ß in lung and trachea, we performed immunohistochemical analysis in sections. With eNOS staining of tracheal and bronchial tissues, we detected a strong and specific signal in ciliated epithelium (Figure 1C, blue arrows; trachea), with prominent staining of eNOS in Clara cells of the bronchioles (Figure 1D, blue arrows) and in Type II cells of alveoli (Figure 1D, yellow arrows). Nonciliated epithelia in trachea and bronchus (Figure 1C, yellow arrows) were not stained with eNOS. In conduit vessels, the endothelial cells were strongly positive for eNOS staining (red arrows in Figure 1D). The pattern of sGC (Figure 1E and Figure 1F) and PKG I-ß (Figure 1G and Figure 1H) immunostaining showed striking parallels with that of eNOS immunostaining in ciliated epithelia in trachea and bronchus (Figure 1E and Figure 1G, blue arrows; bronchi), in the Clara cells of the bronchioles (Figure 1F and Figure 1H, blue arrows), and in Type II cells of alveoli (Figure 1F and Figure 1H, yellow arrows). Moreover, PKG I-ß was localized in the smooth muscle of larger vessels and sGC was localized in smooth muscle of both large and small vessels (red arrows in Figure 1F) as well as airway smooth muscle (green arrows in Figure 1F). Nonciliated epithelia in trachea and bronchus were stained neither with sGC (Figure 1E, yellow arrows) nor with PKG I-ß (Figure 1G, yellow arrows) antibody. Negative controls showed no background staining (Figure 1B, for eNOS control). In contrast to PKG I-ß, staining with the anti-PKG I-
antibody revealed a strong signal in the smooth muscle of airway and resistance vessels. No staining was detectable in ciliated epithelium with anti-PKG I-ß antibody (Figure 2). All the immunohistochemical staining for each antibody was performed at least three times in consecutive sections from the six animals. The staining pattern and intensity for each antibody showed very little variation among the animals and in different sections. To summarize, immunostaining for eNOS, sGC, and PKG I-ß was positive in all regions of the respiratory tract of trachea and lung, whereas PKG 1-
immunostaining was negative throughout the epithelium of the airways.
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Discussion |
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NO, derived from L-arginine by NOS, is involved in regulation of several important physiological functions. Previous studies have demonstrated that eNOS is localized in ciliated epithelium of rat lung (, and PKG I-ß immunostaining exist in trachea and lung. Specifically, sGC and PKG I-ß accompanied eNOS immunostaining in all ciliated epithelia, consistent with a role of the NOcGMP pathway in regulating ciliary beat frequency. A previous study reported by our laboratory (
1- and ß1-subunits of sGC. The polyclonal anti-sGC antiserum may be more sensitive than the monoclonal antibody and may react with the sGC
1-subunit present in the airway epithelial cells.
In vitro studies have shown that several mediators or transmitters such as isoproterenol, substance P, bradykinin, TNF-, and IL-1ß employ an NO-dependent mechanism to upregulate ciliary motility (
If cGMP acts as a second messenger in this tissue, it is likely to require the presence of a PKG together with the presence of appropriate enzymes to degrade the signal. In this context, Yang et al. made an important in vitro observation about the effect of methacholine on the CBF of human adenoid explants (
It is widely assumed that NO diffuses passively from the cytosol of its cell of origin into distant target cells to elicit a response (
The physiological actions of NOcGMP in vasculature have been very well characterized (
A special type of human affliction called Kartagener's syndrome has been shown to result from absence of dynein arms in the cilia (
In summary, the finding of strong immunostaining for eNOS, sGC, and PKG I-ß in airway ciliated epithelia suggests an important role for NOcGMP in the physiology of the airway and that PKG I-ß is available to transduce NOsGC signaling into biological responses in ciliated airway epithelium. In contrast, PKG I- was localized primarily in smooth muscle. Therefore, we speculate that each isoform of PKG I may be closely linked with its own unique function in the lung.
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Acknowledgments |
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Supported by NIH grants R01 HL 39706 and R01-GM 49111.
Received for publication February 23, 1999; accepted May 20, 1999.
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