Download Expression of Nitric Oxide Synthase and Guanylate Cyclase in the

Expression of Nitric Oxide Synthase and Guanylate Cyclase in the Human Ciliary Body and
Trabecular Meshwork
Purpose: To investigate the expression and distribution of nitric oxide synthase (NOS) isoforms and
guanylate cyclase (GC) in human ciliary body, trabecular meshwork and the Schlemm’s canal.
Methods: Twelve eyes after corneal transplantation were used. Expression of three NOS isoforms (i.e.
neuronal NOS, nNOS; inducible NOS, iNOS and endothelial NOS, eNOS) and GC were assessed in 10
eyes by immunohistochemical staining using monoclonal or polyclonal antibody of NOS and GC.
Ciliary bodies were dissected free and the total proteins were extracted. Western blot was performed to
confirmed the protein expression of 3 NOS isoforms and GC.
Results: Expression of 3 NOS isoforms and GC were observed in the ciliary epithelium, ciliary muscle,
trabecular meshwork and the endothelium of the Schlemm’s canal. Immunoreactivity of nNOS was
detected mainly along the apical cytoplasmic junction of the non-pigmented epithelium (NPE) and
pigmented epithelial (PE) cells. Protein expressions of 3 NOS isoforms and GC were confirmed in
isolated human ciliary body by Western blot.
Conclusions: The expression of NOS isoforms and GC in human ciliary body suggest the possible
involvement of NO and cyclic guanosine monophosphate (cyclic GMP, cGMP) signalling pathway in
the cialiary body, and may play a role in both processes of aqueous humor formation and drainage.
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Introduction
Nitric oxide (NO), a gaseous molecule, has been widely recognized as an important intercellular
messenger and vaso-regulator which is reported to be involved in many physiological and pathological
processes [1, 2]. NO is derived from L-arginine when catalyzed by NO synthase (NOS) in the presence
of oxygen and other cofactors [3]. So far, three NOS isoforms, including neuronal NOS (nNOS),
endothelial cell NOS (eNOS) and inducible NOS (iNOS) [4-6] have been identified. Under normal
physiological conditions, NO activates guanylate cyclase (GC) to increase the production of cyclic
guanosine monophosphate (cGMP), which in turn triggers downstream signal cascades and results in
numerous physiological responses including signal transduction of nervous system, vasodilatation,
platelet aggregation inhibition, reduction of chemotaxis of polymorphonuclear cells, increase of ocular
blood flow and decrease of the intraocular pressure (IOP) [1, 5-12]. However, excess NO production due
to sustained activation of iNOS may lead to the accumulation of NO2-, nitrite, peroxynitrite (ONOO-)
and free radicals in the tissue, resulting in DNA damage, cell apoptosis, neurotoxicity and inflammation
of the tissue[8,13,14].
Studies have confirmed the presence of three NOS isoforms in the ocular tissues in some animal
species such as porcine, mouse, rat and rabbit 15-18]. NO are also reported to be involved in the
pathogenesis of a wide range of ocular disorders such as glaucoma, retinopathy, age-related macular
degeneration (AMD), myopia, cataract, uveitis and wound healing process in human [14,19]. In addition,
it has been reported that NO also increases the drainage of the aqueous humor in animals [20, 21]. The
aim of this study was to investigate the expression and distribution of nNOS, iNOS, eNOS and GC in
human ciliary body, trabecular meshwork and the Schlemm’s canal.
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Methods
Tissue Preparation
Twelve fresh human eye residual (after corneal transplantation, 24 hours within enucleation) were
obtained from the eye banks of Eye Center of the 2nd Affiliated Hospital, Medical College of Zhejiang
University, and Xiamen Eye Center of Xiamen University. Eyes with history of tumor, uveitis,
glaucoma and ocular trauma were excluded. The study protocol was approved by the Institutional
Review Board of both hospitals adhering with the tenets of the Declaration of Helsinki.
Ten residual eyes were preceded for paraffin embedding after fixation in 4% paraformaldehyde
in phosphate-buffered saline (PBS, pH 7.4) for 24 hours. Five Serial paraffin sections with the
thickness of 3 µm were performed. For each eye, one section was preceded to hematoxylin and eosin
(HE) staining, other tissue preparations were used for immunohistochemical staining.
Ciliary bodies of 2 eyes were dissected free for Western blot analysis. Tissues were lysed in the
RIPA buffer with Phenylmethanesulfonyl fluoride (PMSF, at a final concentration of 1 mM) and the
total protein was extracted. The protein concentration was measured by a bicinchoninic acid（BCA）
protein assay kit.
NOS and GC Immunohistochemistry Staining
Paraffin sections were dried in a 55-60°C oven overnight, then deparaffinized in xylems and
graded alcohols to water. The sections were subsequently immersed in 0.25% potassium permanganate
for 1 hour and 1% oxalic acid for 30 minutes to remove the pigment. After PBS rinse, the preparations
were treated with 0.3% hydrogen peroxide in PBS for 10 minutes to block endogenous peroxidase
activity and rinsed with PBS again. Sections were then incubated with 10% normal goat serum for 10
minutes at room temperature and removed the serum, followed by exposure to rabbit monoclonal
antibody against nNOS and rabbit polyclonal antibody against iNOS, eNOS as well as GC for 2 hours
at room temperature. The dilution rate of the primary antibody was 1:200 for nNOS, 1:100 for iNOS,
1:25 for eNOS and 1:50 for GC. After 3 rinses in PBS, the appropriate secondary antibody which had a
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peroxidase-conjugated polymer backbone and carried secondary antibody molecules against
rabbit/mouse immunoglobulins were incubated with the tissues subsequently for 1 hour and rinsed with
PBS afterward. The peroxidase reaction was introduced using diaminobenzidine (DAB) as a substrate.
The preparations were observed under light microscope with yellow or brown staining of the cytoplasm
be considered as positive. Negative controls were used by replacing the primary antibody with PBS.
Each experiment of immunostaining was repeated at least 3 times.
Western bolt analysis
The protein extractions of the ciliary bodies were boiled for 3 min with 2% beta-mercaptoethanol.
A total of 50 µg protein was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel. After
electrophoresis, the separated protein was then electrophoretically transferred to a polyvinylidene
fluoride（PVDF）membrane in transfer buffer. After being blocked with 5% skimmed milk in
Tris-buffered saline (pH 7.4) containing 0.1% Tween-20 (TBST), the membrane was clipped into
nNOS, iNOS, eNOS and GC strips and incubated with respective NOS and GC primary antibodies in
TBST overnight at 4°C (all of the primary antibodies were used at a dilution of 1:5000). After three
rinses, the membranes were incubated for 1 hour at room temperature with a goat-anti-human IgG
secondary antibody diluted as 1:5000 in TBST. The membranes were washed again and developed with
the enhanced chemi-luminescence (ECL) system.
Antibodies and Chemical Reagents
The primary rabbit monoclonal anti-nNOS antibody (P29475) was purchased from Epitomics
Institute (Epitomics, California, USA). The primary rabbit polyclonal anti-iNOS antibody (ab3523),
anti-eNOS antibody (ab66127) and anti-GC antibody (ab53084) were purchased from Abcam Institute
(Abcam, Cambridge, UK). The second antibody and DAB (K5007) were purchased from Dako
Company (Dako, Denmark). Goat anti-human IgG antibody was purchased from Beyotime Institute of
Biotechnology (Beyotime, Shanghai, China). Bicinchoninic acid (BCA) protein assay kit was
purchased from Pierce Company (Pierce, Rockford, Illinois, USA). Enhanced chemi-luminescence
system was purchased from Millipore Company (Millipore, Beijing, China). All other biochemicals and
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reagents were bought from Maxin Biotechnology Company (Maxin, Fujian, China) and Beyotime
Institute of Biotechnology.
Results
HE staining of ciliary epithelial bilayers, muscle, trabecular meshwork and Schlemm’s canal was
shown in Figure 1.
Immunohistochemical Localization of nNOS, iNOS and eNOS
Immunoreactivity of nNOS was identified in the ciliary epithelium, muscle, trabecular meshwork
and the endothelium of the Schlemm’s canal (Figure 2). In the ciliary epithelium, immunoreactivity of
nNOS was detected predominantly along the apical cytoplasmic junction of the PE and NPE cells
(Figure 2d). In the ciliary muscle cells, trabecular meshwork and the endothelium of the Schlemm’s
canal, nNOS was evenly distributed in the cytoplasm.
Expression of iNOS and eNOS were also observed to distributed evenly in the cytoplasm of the
ciliary epithelium, muscle, trabecular meshwork and the endothelium of the Schlemm’s canal (Figure
3).
Immunohistochemical Localizations of GC
Immunostaining of GC was observed in the ciliary epithelium, muscle, trabecular meshwork and
the endothelium of the Schlemm’s canal (Figure 4). In some ciliary processes, GC was detected mainly
along the apical cytoplasmic junction of the PE and NPE cells (Figure 4c), while in other ciliary
processes, GC was shown to be evenly distributed in the cytoplasm of the PE and NPE cells (Figure
4d). In the ciliary muscle cells, trabecular meshwork and the endothelium of the Schlemm’s canal, GC
was evenly distributed in the cytoplasm.
Negative control for immunostaining (incubated with PBS instead of NOS or GC primary antibody)
in the human ciliary muscle, trabecular meshwork and the ciliary epithelium was shown in Figure 5.
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Protein Expression of NOS isoforms and and GC in the Cliliary Body
When Western blot was used, the expression of 3 NOS isoforms and GC were confirmed in
isolated human ciliary bodies (Figure 6). The molecular weight is 160kDa, 135kDa, 140KDa and
70kDa for nNOS, iNOS, eNOS and GC, respectively.
Discussion
We confirmed the expression of three NOS isoforms (nNOS, eNOS and iNOS) and GC in human
ciliary body, trabecular meshwork and the endothelium of the Schlemm’s canal. These findings suggest
that NO/cGMP signalling pathway may be involved in the aqueous humor inflow and outflow in
human eyes.
Expression of nNOS has been reported in the porcine ciliary processes, similar to the findings in
our study, specific nNOS immunoreactivity was detected mainly along the apical junctional cytoplasm
of the NPE and PE cells [21]. NO has also been reported, by activating GC which further increases the
cGMP production, to mediate the effect of some ocular hypotensive agent, such as brimonidine (a drug
that decreases intraocular pressure in part by reducing aqueous humor production) in porcine ciliary
processes [22, 23]. Actually, nNOS has been thought to be related with intercellular communication and
involved in trans-epithelial fluid transport in the kidney, respiratory airway and colon, etc [24-26]. In the
present study the distribution pattern of nNOS in the ciliary epithelium strongly suggested that nNOS
might be involved in the intercellular communication between PE and NPE, hence potentially be
involved in the process of aqueous humor production in the human eye. Further studies are needed to
confirm this, as well as the presence of NO-GC-cGMP signaling pathway in the process of aqueous
humor formation of human eyes.
In our study, the expressions of 3 NOS isoforms and GC in human ciliary muscle, trabecular
meshwork and the endothelium of Schlemm’s canal were also observed. These results were slightly
different from previous reports showing that ciliary muscle (especially in the anterior longitudinal part)
and outflow pathway (including the trabecular meshwork, the Schlemm’s canal and the collecting
channels) of normal human eyes were only enriched in eNOS but not in nNOS [21]. The discrepancy
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between our study and previous reports might reflect the difference in the experimental protocols. It has
to be noticed that the anti-serum we used was different from which were used in the study metioned
above. The methods of immunostaining were also different (immunohistochemical staining vs.
immunocytochemical staining) in these two studies as well. In fact, the presence of nNOS was further
supported by the results from Western blot experiments in our study.
The role played by NO in the aqueous humor outflow is still unclear. There exist studies showing
that patients with primary open-angle glaucoma had a decrease of NADPH-diaphorase staining (a
widely used method for the identification of NOS) in the ciliary muscle, trabecular meshwork and the
Schlemm’s canal[26]. Furthermore, in the rabbit and the bovine eyes, there are multiple sites of action
for the nitrovasodilators or NO donors including ciliary muscle, trabecular meshwork, and the
endothelial cells in the aqueous drainage system [27, 28].,As the trabecular meshwork and the Schlemm’s
canal are now considered as a major site of outflow resistance [29, 30], these findings indicate that NO
may be involved in the regulation of aqueous humor outflow by its modulation on the trabecular
meshwork and the Schlemm’s canal.
Although NO are thought to increase aqueous humor production in animals [31], there exist reports
showing that L-arginine and NO donors, such as nitroprusside, isosorbide, sodium nitrite, nitroglycerin
lowered IOP and increased ocular blood flow in animal eyes [20, 32, 33]. These findings suggest that the
effect of NO on outflow might outweigh its stimulative effect on aqueous humor production, as the
result, the IOP decreases in the tested eyes. These results further indicate that both NOS substrates and
NO donors might be used as potent drugs in the treatment of glaucoma via reduction of IOP and/or
improvement of ocular blood flow. Further clinical studies need to be carried out to confirm this.
In summary, in isolated human ocular tissues, we confirmed the presence of NOS isoforms and
GC in human ciliary body and aqueous drainage system. The results suggest possible involvement of
NO-cGMP signal pathway in the aqueous humor production and drainage. Further studies are
warranted to confirm this, as well as the clinical significance of this pathway in the pathogenesis and
treatment of glaucoma.
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Legends of Figures
Figure 1. Hematoxylin and eosin (HE) staining of human ciliary epithelial bilayer (a, ×400), muscle (b,
×400) and Schlemm’s canal (c, ×400).
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Figure 2. Immunohistochemical distribution (arrows) of nNOS in human ciliary epithelium (a, x100),
muscle (b, ×200), trabecular meshwork (long arrows, d, ×400) and the endothelium of the Schlemm’s
canal (short arrows, d, ×400). In the ciliary epithelium, nNOS staining was predominantly located at
the junctional cytoplasm between the pigmented and non-pigmented epithelium (c, ×400).
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Figure 3. Immunohistochemical labeling (arrows) of iNOS (left panels) and eNOS (right panels) in
human ciliary muscle (a, b, ×200), trabecular meshwork (long arrows, c, d, ×400), and the endothelium
of Schlemm’s canal (short arrows, c, d, ×400), and the ciliary epithelium (e, f, ×400).
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Figure 4. Immunohistochemical labeling (arrows) of guanylate cyclase (GC) in the in human ciliary
muscle (a, ×200)，trabecular meshwork (long arrows, b, ×400) and endothelium of Schlemm’s canal
(short arrows, b, ×400) and the ciliary epithelium (c, d, ×400). In some ciliary epithelial bilayers, GC
staining were mainly located in the apical junctional cytoplasm of the pigmented epithelium (PE) and
non-pigmented epithelium (NPE) (c), while in others, GC staining were evenly distributed in the
cytoplasm of both the PE and NPE cells (d).
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Figure 5. Negative control for the immunostaining (incubated with PBS instead of NOS or GC primary
antibody) in the human ciliary muscle (a, ×200), trabecular meshwork (b, ×400) and the ciliary
epithelium (c, ×400).
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Figure 6. Protein Expression of nitric oxide synthase (NOS) isoforms and guanylate cyclase (GC) in
isolated human cliliary body. The molecular weight of neuronal NOS (nNOS), inducible NOS (iNOS),
endothelial NOS (eNOS) and GC is 160 kDa, 140 kDa, 135 kDa and 70 kDa, respectively.
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