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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. 1 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. 2 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 3 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 4 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. 5 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 6 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. 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Invest Ophthalmol Vis Sci 2004; 45:3213-3222. 32: Deussen A, Sonntag M, Vogel R. L-arginine-derived nitric oxide: a major determinant of uveal blood flow. Exp Eye Res1993; 57:129-134. 33: Chiou GC, Liu SX, Li BH, et al. Ocular hypotensive effects of L-arginine and its derivatives and their actions on ocular blood flow. J Ocul Pharmacol Ther 1995; 11:1-10. 9 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). 10 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). 11 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). 12 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). 13 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). 14 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. 15