August 2000
Volume 41, Issue 9
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Physiology and Pharmacology  |   August 2000
Coexistence of C-Type Natriuretic Peptide and Atrial Natriuretic Peptide Systems in the Bovine Cornea
Author Affiliations
  • Sung Zoo Kim
    From the Department of Physiology and Ophthalmology, Medical School Institute for Medical Sciences, Jeonbug National University, Jeonju, Korea.
  • Hun Sook Kim
    From the Department of Physiology and Ophthalmology, Medical School Institute for Medical Sciences, Jeonbug National University, Jeonju, Korea.
  • Kyung Sun Lee
    From the Department of Physiology and Ophthalmology, Medical School Institute for Medical Sciences, Jeonbug National University, Jeonju, Korea.
  • Sook Jeong Lee
    From the Department of Physiology and Ophthalmology, Medical School Institute for Medical Sciences, Jeonbug National University, Jeonju, Korea.
  • Kyung Hwan Seul
    From the Department of Physiology and Ophthalmology, Medical School Institute for Medical Sciences, Jeonbug National University, Jeonju, Korea.
  • Gou Young Koh
    From the Department of Physiology and Ophthalmology, Medical School Institute for Medical Sciences, Jeonbug National University, Jeonju, Korea.
  • Kyung Woo Cho
    From the Department of Physiology and Ophthalmology, Medical School Institute for Medical Sciences, Jeonbug National University, Jeonju, Korea.
  • Suhn Hee Kim
    From the Department of Physiology and Ophthalmology, Medical School Institute for Medical Sciences, Jeonbug National University, Jeonju, Korea.
Investigative Ophthalmology & Visual Science August 2000, Vol.41, 2671-2677. doi:
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      Sung Zoo Kim, Hun Sook Kim, Kyung Sun Lee, Sook Jeong Lee, Kyung Hwan Seul, Gou Young Koh, Kyung Woo Cho, Suhn Hee Kim; Coexistence of C-Type Natriuretic Peptide and Atrial Natriuretic Peptide Systems in the Bovine Cornea. Invest. Ophthalmol. Vis. Sci. 2000;41(9):2671-2677.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine whether the cornea synthesizes natriuretic peptides and contains their receptors.

methods. The synthesis of the natriuretic peptides, C-type natriuretic peptide (CNP) and atrial natriuretic peptide (ANP), in the bovine cornea was determined by high-performance liquid chromatography (HPLC) with radioimmunoassay and Southern blot analysis. The presence of natriuretic peptide receptor (NPR)-A and -B and their localizations were measured by reverse transcription–polymerase chain reaction (RT-PCR), in vitro autoradiography, and the activation of particulate guanylyl cyclase by natriuretic peptides in the corneal membrane.

results. The serial dilution curves of corneal extracts were parallel to the standard curves of CNP and ANP. With reversed-phase HPLC, a major immunoreactive peak of CNP or ANP was observed at the elution time corresponding with synthetic CNP(1-53) or atriopeptin III (APIII), respectively. The presence of mRNAs of CNP and ANP was also detected in the cornea by RT-PCR and/or Southern blot analysis. Production of 3′,5′-cyclic guanosine monophosphate (cGMP) by the activation of particulate guanylyl cyclase in the corneal membrane was stimulated by ANP, BNP, and CNP. More cGMP was produced by CNP than by the other natriuretic peptides. Specific 125I-[Tyr0]-CNP(1-22) binding sites were localized in the endothelial cell layer of cornea. The apparent dissociation constant (K d) value of the cornea was 3.06 ± 0.73 nM and the maximum binding capacity was 3.40 ± 0.63 femtomoles/mm2. Both NPR-A and NPR-B mRNAs were detected by RT-PCR.

conclusions. The cornea synthesizes CNP and ANP and contains their receptors. These results suggest that the CNP and ANP systems coexist in the bovine cornea.

The natriuretic peptide family is composed of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). 1 2 3 ANP and BNP are circulating peptides synthesized mainly in the heart and activate the guanylyl cyclase (GC)-coupled natriuretic peptide receptor (NPR)-A. 4 ANP is secreted into the blood stream in response to atrial distension 5 and causes natriuresis, diuresis, and vasorelaxation. 1 BNP secreted from the ventricles has action similar to ANP. 4 CNP, isolated from the brain by Sudoh et al., 3 is present mainly in vascular endothelium 6 and activates GC-coupled NPR-B specifically. 7 CNP may play a role in the local regulation of vascular tone. 7 Natriuretic and diuretic effects of CNP are weaker than those of ANP. 8  
ANP is also present in many other tissues, such as brain, lung, gastrointestinal organ, reproductive organ, immune system, and eye. 9 Although the amount of extra-atrial ANP is too low for the induction of systemic effects, it may have paracrine or autocrine actions. The actions of extra-atrial ANP that are known at present are to affect mitotic rates and steroidogenesis and to decrease oviductal motility, CSF production, and intraocular pressure. 9 CNP is also present in other tissues. NPR-B is expressed in neural tissue 10 11 and in atrial myocytes. 12 The actions of CNP in these tissues are the modulation of neural transmission, other hormonal secretion, and Ca2+ channeling activity in the cell membrane. 
In the eyes, the presence of immunoreactive ANP (irANP) has been reported in anterior uvea, retina, 13 aqueous humor, and ciliary body. 14 The reports about their molecular profiles and the synthesis of ANP in the eyes, however, are still controversial. 15 ANP receptor has been observed on the epithelial side of the ciliary body 14 16 and retina. 17 ANP has been known to increase the production of 3′,5′-cyclic guanosine monophosphate (cGMP) production in ocular tissues. 18 19 Sugrue and Viader 20 have reported that the administration of ANP either topically or intracamerally causes a decrease in intraocular pressure by increasing cGMP. 20 It has also been reported that the concentration of ANP in aqueous humor of glaucomatous rabbit is higher than that in normal eyes. 21 All these findings suggest that the ANP system has an important paracrine function in the eyes. 
In this study, we focused on the natriuretic peptide systems in the cornea. There are a few reports about natriuretic peptide systems in the cornea. Walkenbach et al. 22 found ANP receptors on the corneal endothelium, which may be NPR-C. The synthesis of ANP and the characteristics of its receptor in the cornea are not well-defined. Furthermore, there is no report about the presence of a CNP system in the cornea. The purpose of this study was to define whether the cornea has its own CNP system as well as an ANP system. 
Materials and Methods
Tissue Collection
For the measurement of natriuretic peptides (NPs) and GC activity, eyes from healthy adult cattle were obtained from an abattoir within 10 minutes after death and immediately transported to the laboratory in a sterile container. Cornea was separated at 4°C and kept at −70°C until assayed. For the extraction of the mRNAs for NPs and NPRs, cornea was immediately separated at an abattoir, put into liquid nitrogen, and kept at -70°C. For in vitro receptor autoradiography, eyes were immediately snap frozen in isopentane, cooled by dry ice, and stored in sealed boxes at −70°C until sectioned. 
Extraction of NPs and HPLC
For high-performance liquid chromatography (HPLC), the extraction of CNP was performed as described previously. 23 Briefly, the cornea was boiled in three volumes of distilled water for 10 minutes and then acidified with acetic acid to a final concentration of 0.2 M. The cornea was homogenized (Polytron homogenizer; Brinkman, Westbury, NY), and centrifuged at 25,000g for 30 minutes at 4°C. The supernatant of the tissue homogenate was recentrifuged at 1500g for 40 minutes at 4°C, using a centrifugal concentrator (Centriprep; Amicon, Beverly, MA) to cut high-molecular-weight proteins (over 30 kDa), and then lyophilized. The tissue extract of CNP was reconstituted with 0.1% trifluoroacetic acid (TFA) and subjected to reversed-phase HPLC (Waters, Milford, MA) on an elution column (μBondapak; Waters). Elution was performed with a linear gradient of 40% to 70% methanol at a flow rate of 1 ml/min, and the samples were collected at 30-second intervals. The fractionated samples were dried and assayed. The column was calibrated with synthetic CNP(1-22) and CNP(1-53)
The extraction of ANP was performed as described previously. 24 The tissue extract of ANP was reconstituted with 0.1% TFA and subjected to reversed-phase HPLC on the elution column. Elution was performed with a linear gradient of 20% to 60% acetonitrile in 0.1% TFA for 40 minutes at a flow rate of 1 ml/min. The fractionated samples were dried and assayed. The column was calibrated with synthetic APIII and purified proANP. 
Iodination of CNP
The iodinated[ Tyr0]-CNP(1-22) was prepared as the same method of ANP described previously. 24 25 In brief, 5 μg of synthetic[ Tyr0]-CNP(1-22) (Peninsula, Belmont, CA) were introduced into a vial containing 25 μl of 0.5 M phosphate-buffered saline (pH 7.4) followed by the addition of 1 mCi of Na125I (Amersham, Little Chalfont, UK). Chloramine-T (10 μg per 10 μl) was added to the reaction vial and mixed gently, and 30 seconds later, the reaction was terminated by bovine serum albumin (BSA) solution (60 mg/200 μl). 
The reaction mixture was immediately applied to an elution column (Sephadex G-25; Sigma, Poole, UK) and eluted with 0.1 M acetic acid containing 0.3% BSA, 0.3% lysozyme, 0.1% glycine, and 200 Kallikrein inhibiting units per milliliter aprotinin. The iodinated[ Tyr0]-CNP(1-22) was repurified by reversed-phase HPLC on an elution (μBondapak) column with a linear gradient of 20% to 60% acetonitrile in 0.1% TFA. The specific activity of 125I-[Tyr0]-CNP(1-22), measured by radioimmunoassay (RIA), 26 was approximately 1700 Ci/mmol. 
RIA of NPs
The concentration of CNP in tissue extracts was measured by RIA, as described elsewhere. 23 25 Briefly, the lyophilized samples were reconstituted with phosphate buffer (pH 7.4) containing 50 mM NaCl, 0.1% BSA, 0.1% Triton X-100, and 0.01% sodium azide. After incubation with anti-CNP antibody (Peninsula) for 24 hours at 4°C, approximately 15,000 cpm of 125I-[Tyr0]-CNP(1-22) was added, and samples were incubated again for another 24 hours at 4°C. The separation of the unbound fraction was achieved by the addition of the second antibody. The 50% intercept was at 79.0 ± 14.2 pg/tube (n = 5). The intra- and interassay coefficients of variation were 6.9% (n = 6) and 4.4% (n = 10), respectively. Cross-reactivity with ANP was less than 0.01%, and no cross-reactivity with BNP was observed. RIA for the measurement of ANP was performed as described previously. 24  
RT-PCR of mRNAs for NPs and NPRs
Reverse transcription–polymerase chain reaction (RT-PCR) was performed as described previously. 25 Total RNA was extracted from the cornea using TRI reagent (MRC, Cincinnati, OH) according to the manufacturer’s protocol. Total RNA concentrations were quantitated by UV spectrophotometry. One microgram of mRNA was suspended in 20 μl RT buffer containing 10 mM Tris (pH 8.3); 50 mM KCl; 5 mM MgCl 2; 1 mM each of dATP, dCTP, dGTP, and dTTP; 20 U RNase inhibitor; 2.5 μM random hexamers; and 150 U Moloney leukemia virus reverse transcriptase (Perkin Elmer, Branchburg, NJ). mRNA was reverse transcribed at room temperature for 10 minutes and at 42°C for 30 minutes. The reaction was stopped by heat inactivation for 5 minutes at 99°C and then chilled on ice. cDNA products were amplified by PCR with sense and antisense primers. 
For CNP, two sets of primers were used: the first round of PCR was performed with a set of CNP primers of large size with 10 μl RT-PCR product as a template. A set of CNP primers of small size was designed within internal sites from the RT-PCR product of CNP primers of large size and was used in the second round of PCR with 5 μl of the first round PCR product. The primer sets were ANP sense 5′-ATGGGCTCCTTCTCCATCACCAAGGGCTTC-3′ (1-30) and ANP antisense 5′-AGGGCCAGCGAGCAGAGCCCTCAGTTTGCT-3′ (334-363); CNP sense (small size) 5′-CTCTCCCAGCTGATCGCCTG-3′ (7–26) and CNP antisense (small size) 5′-TAACATCCCAGACCGCTCAT-3′ (361–380); CNP sense (large size) 5′-TGGCAATCCTGCTCTGCAACCG-3′ (−88 to −67) and CNP antisense (large size) 5′-CGTTGGAGGTGTTTCCAGATGCTGG-3′ (449–473); NPR-A sense 5′-AAGAGCCTGATAATCCTGAGTACT-3′ (1159–1182) and NPR-A antisense 5′-TTGCAGGCTGGGTCCTCATTGTCA-3′ (1586–1609); and NPR-B sense 5′-AACGGGCG CATTGTGTATATCTGCGGC-3′ (730–756); and NPR-B antisense 5′-TTATCACAGGATGGGTCGTCCAAGTCA-3′ (1395–1421). 
Fifty microliters of PCR buffer contained 10 mM Tris (pH 8.3); 50 mM KCl; 2 mM MgCl2; 200 μM each of dATP, dCTP, dGTP, and dTTP; 2.5 U Taq polymerase; and 1 0.5 pM (for ANP and NPRs) or 0.5 pM (for CNP) each of sense and antisense primers. The temperature profile of amplification consisted of 30 seconds of denaturation at 95°C, 1 minute of annealing at 60°C (for ANP and NPRs) or 65°C (for CNP), and 2 minutes of extension at 72°C for 40 cycles. PCR products were separated on 3% agarose gels, and the bands were visualized by ethidium bromide staining. The gels were then photographed (665 film; Polaroid, Cambridge, MA). 
Southern Blot Analysis
To confirm the presence of CNP transcript in the cornea, PCR product was subjected to Southern blot analysis. CNP cDNA for hybridization was obtained by the purification and sequence of RT-PCR product of rat pituitary gland. PCR product was transferred into nylon membrane and cross-linked using UV cross-linker. The membrane was prehybridized with hybridization buffer solution (Amersham) at 60°C for 1 hour and incubated with 32P-labeled CNP cDNA probe at 60°C for 12 hours. The membrane was washed twice in 2× SSC with 0.1% sodium dodecyl sulfate at 60°C and then exposed to film (X-Omat; Eastman Kodak, Rochester, NY) at 70°C for 2 days. 
Particulate GC Activity
Cornea was homogenized at 4°C in 30 mM phosphate buffer (pH 7.2) containing 120 mM NaCl and 1 mM phenanthrolene by three 30-second bursts of 27,000 rpm (Polytron; Brinkman). The homogenate was centrifuged at 1,500g for 10 minutes at 4°C, and the supernatant was recentrifuged at 40,000g for 60 minutes at 4°C. The membrane pellet was washed three times with 50 mM Tris-HCl (pH 7.4) and resuspended in this solution. Protein contents were determined by a bicinchoninic acid assay kit (Sigma). Particulate GC activity was measured in protein aliquots of corneal membranes, as described previously. 25 Five-microgram protein aliquots of the suspension were incubated at 37°C for 15 minutes in 50 mM Tris-HCl (pH 7.6), containing 1 mM isobutylmethylxanthine, 1 mM guanosine triphosphate (GTP), 0.5 mM adenosine triphosphate (ATP), 15 mM creatine phosphate, 80 μg/ml creatine phosphokinase, 4 mM MgCl2, and 1 μM NP. Incubations were stopped by adding 375 μl cold 50 mM sodium acetate (pH 5.8) and boiling for 5 minutes. Samples were then centrifuged at 10,000g for 5 minutes at 4°C. 
The amount of cGMP generated in the supernatant was measured by RIA. 25 In brief, standards or samples were introduced in a final volume of 100 μl of 50 mM sodium acetate buffer (pH 4.8), and 100 μl each of diluted cGMP antiserum (Calbiochem–Novabiochem, San Diego, CA) and iodinated cGMP (10,000 cpm/100 μl, Specific activity, 2200 Ci/mmol, Du Pont-New England Nuclear, Wilmington, DE) was added. After incubation at 4°C for 24 hours, the bound form was separated from the free form by charcoal suspension. The measurement of cGMP generated was performed on the day of experiments, and all samples in an experiment were analyzed in a single assay. Nonspecific binding was less than 2.5%. The 50% intercept was 0.39 ± 0.03 picomoles per tube (n = 15). The intra- and interassay coefficients of variation were 6.7% (n = 12) and 8.6% (n = 9), respectively. 
In Vitro Autoradiographic Binding of 125I-[Tyr0]-CNP(1-22)
Serial 20-μm sections were cut on a cryostat at −20°C, thaw-mounted onto gelatin-chrom-alum–coated slides and dried in a desiccator at 4°C overnight before incubation. The incubation conditions of 125I-[Tyr0]-CNP(1-22) were as previously reported. 25 Briefly, the sections were washed with 150 mM NaCl-0.5% acetic acid (pH 5.0) at room temperature for 10 minutes to remove the endogenous CNP and then preincubated with 30 mM phosphate buffer (pH 7.2) containing 120 mM NaCl and 1 mM phenanthrolene at room temperature for 10 minutes. The sections were incubated with 125I-[Tyr0]-CNP(1-22) in fresh preincubation buffer containing 40 μg/ml bacitracin, 100μ g/ml phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 0.5% BSA at room temperature. The adjacent section was incubated in 125I-[Tyr0]-CNP(1-22) plus 1 μM unlabeled CNP(1-22). After incubation, the sections were washed with fresh preincubation buffer at 4°C for 5 minutes, rinsed three times in cold distilled water at 4°C, and quickly dried under a stream of cold air. 
Autoradiographic images were generated by the exposure of the slides with dried 125I-[Tyr0]-CNP(1-22)–labeled eye sections to Hyperfilm-3H (Amersham) in x-ray cassettes together with 20-μm-thick 125I-labeled polymer standard strips (Amersham) at room temperature for 7 days. Autoradiograms were developed (D-19 developer; Kodak) for 3 minutes and fixed in a rapid fixer (Kodak) for 5 minutes at room temperature. 
Autoradiographic images were viewed with a macroscope (Wild M 420; Leica, Deerfield, IL) and captured using a video camera (Sony, Tokyo, Japan) with a charge-coupled device iris and an AC adaptor (Hamamatsu; Bridgewater, NJ) connected to a computer (Power Macintosh 8100/80AV; Apple, Cupertino, CA). Regional bindings of 125I-[Tyr0]-CNP(1-22) in the cornea were analyzed for mean gray-scale value by image analysis software (PRISM ver. 3.6-1; Improved Vision, Coventry, UK). The number of ligand binding sites of different affinities, their apparent dissociation constants (K d) and their maximal binding capacities (B max) on particular structures were derived separately in each individual by Scatchard analysis using an iterative model-fitting computer program (Ligand; Elsevier-BIOSOFT, Cambridge, UK). 
Statistical Analysis
Statistical significance of differences was tested using analysis of variance and Dunnett test and the results expressed as mean ± SEM. 
Results
Presence of NPs
The presence of irANP in the bovine cornea was demonstrated by a specific and sensitive RIA with HPLC. The serial dilutions of corneal extracts displaced 125I-APIII in a manner parallel to the synthetic standard (Fig. 1A ). The concentration of ANP in the cornea was 3.58 ± 0.5 pg/mg of tissue wet weight (n = 6), and the total content of ANP was 1.66 ± 0.23 ng per eye. Figure 1B shows the molecular profile of irANP in corneal extracts by reversed-phase HPLC. Two major immunoreactive peaks were observed: the elution time of major peak corresponded to that of the synthetic APIII. The synthesis of ANP in the cornea was confirmed by RT-PCR. The agarose gel analysis after electrophoresis of RT-PCR products is shown in Figure 1C . A band of DNA is present in both atrial and corneal lanes corresponding to the predicted size (364 bp) according to the base pair ladders. 
The serial dilution of corneal extracts also displaced 125I-[Tyr0]-CNP(1-22) in a manner parallel to the synthetic standard (Fig. 2A ). The concentration of CNP in the cornea was 0.073 ± 0.004 pg/mg of tissue wet weight (n = 4), and the total content of CNP in the eye was 45.9 ± 3.11 pg. The molecular profile of CNP in corneal extracts by reversed-phase HPLC showed a major immunoreactive peak corresponding to synthetic CNP(1-53) (Fig. 2B) . The first round PCR with CNP primers of large size produced a product of the expected size of 571-bp in both pituitary gland and cornea, but other nonspecific products were also amplified from the cornea (data not shown). When the other set of CNP primers was used, the nonspecific bands disappeared. This band was confirmed as CNP by Southern blot analysis (Fig. 2C)
Particulate GC Activation by NPs
To determine the presence of NPR-A and NPR-B in the cornea, the activation of particulate GC by NPs in the corneal membrane was measured. The basal rate of cGMP production by particulate GC activation was 3.21 ± 0.36 picomoles/mg protein/min (n= 6). By the addition of ANP, BNP, or CNP (10−6 M) in the corneal membrane, the rates of cGMP production were 4.30 ± 0.26, 5.00 ± 0.36, or 8.19 ± 0.19 picomoles/mg protein/min, respectively. An increase in cGMP production by CNP, selective ligand to NPR-B, was significantly higher than those by other NPs (Fig. 3) . This means that NPR-B may be the predominant NPR in the cornea. 
Autoradiographic Localization of 125I-[Tyr0]- CNP(1-22)–Binding Sites
Specific 125I-[Tyr0]-CNP(1-22)–binding sites were demonstrated in the cornea by in vitro autoradiography (Fig. 4) . The specifically reversible and strong binding of 125I-[Tyr0]-CNP(1-22) was observed in the corneal endothelium (Fig. 4A) . In the presence of 1 μM unlabeled CNP(1-22), the binding of 125I-[Tyr0]-CNP(1-22) to the corneal endothelium was completely displaced (Fig. 4B) . However, the displacement of 125I-[Tyr0]-CNP(1-22) binding to the retina, ciliary body, or iris by 1 μM unlabeled CNP(1-22) was not observed. Unrelated peptides including angiotensin II or arginine vasopressin (10 μM) did not displace the binding of 125I-[Tyr0]-CNP(1-22) (data not shown). 
Analysis of the competitive inhibition of the binding of 125I-[Tyr0]-CNP(1-22) to the corneal endothelium by increasing the concentrations of unlabeled CNP(1-22) was consistent with a single high-affinity binding site for 125I-[Tyr0]-CNP(1-22) (Fig. 5) . The mean K d and B max values of these sites were 3.06 ± 0.73 nM and 3.40 ± 0.63 femtomole/mm2, respectively. 
Detection of NPR-A and NPR-B mRNAs by RT-PCR
Figure 6 shows the agarose gel analysis after electrophoresis of RT-PCR products. The positive control for NPR-A was renal medulla and that for NPR-B was pituitary gland. A band of DNA was present in both the positive control and corneal lanes corresponding to the predicted sizes (451 bp for NPR-A and 692 bp for NPR-B) according to the base pair ladders. 
Discussion
This study demonstrates for the first time the presence of mRNAs for NPs and NPRs in bovine cornea and the localization of NPR-B in the corneal endothelium. 
There are many reports about the presence of ANP in the eye. ANP immunoreactivity has been found in the anterior uvea (31 ng/g tissue), retina (8 ng/g tissue), 13 and aqueous humor (3 pg/ml). 21 ANP mRNA was detected in the choroid and ciliary bodies but not in the retina by RT-PCR. 15 However, there has been no systematic study to determine the presence of the NP system in the cornea. In the present study, the concentration of ANP in the bovine cornea was 3.58 ± 0.5 pg/mg tissue, and the total content of ANP was 1.66 ± 0.23 ng. In the molecular profile of corneal irANP, two peaks were found; the major one corresponded to the circulating form of ANP, and the other one was located between APIII and proANP. Stone and Glembotski 13 found that major forms of irANP in the uvea and retina have approximate molecular weights of 2400 and 1750 Da, respectively, similar in size to the hypothalamic form but clearly distinguishable from the larger cardiac form. We do not know why proANP was not detectable in the cornea. One possibility is the processing of the proANP to the low-molecular-weight form and other fragments during extraction, even though the extraction of ANP was performed in the presence of several protease inhibitors at 4°C after rapid freezing of cornea. However, we found evidence of the synthesis of ANP in the cornea, which was confirmed by the detection of ANP mRNA. 
CNP, which is located in vascular endothelium and brain, also is found in other tissues. There is no report about the presence and synthesis of CNP in the eye. Recently, Takashima et al. 27 have reported that CNP injected intravitreally causes ocular hypotension. Therefore, CNP may have an important paracrine function similar to ANP. The total content in the cornea was 45.9 ± 3.1 pg, which was 35 times less than the ANP content. We confirmed the synthesis of CNP in the cornea by Southern blot analysis and detection of CNP(1-53). The synthesis of CNP itself in the cornea suggests that it has a paracrine or autocrine function. Therefore, we searched for the presence of biological receptors of NPs in the cornea. 
A high density of ANP receptor has been observed on the retina, ciliary body, and cornea. Bianchi et al. 16 have reported that the receptor on the epithelium of ciliary body is negatively coupled to adenylyl cyclase and positively coupled to GC. Pang et al. 19 have observed ANP receptor in the cultured human trabecular meshwork cells and ciliary muscle cells, in which NPR-B is the primary functional NPR. NPR-A is also found in the retina. 17 We found both NPR-A and -B in the cornea. In the corneal membrane, particulate GC activity was markedly induced by the addition of CNP and was more prominent than the activity induced by ANP and BNP. The binding site of CNP was observed in the corneal endothelium. With RT-PCR, the mRNAs for NPR-A and -B were also detected. These results, showing that the major type of biologic receptors in the corneal endothelium is NPR-B, suggest that CNP may be an important hormonal system, even though the level of CNP is low compared with that of ANP. However, Walkenbach et al. 22 have reported that ANP receptor in the corneal endothelium is the clearance type and that cGMP is not generated by ANP in corneal endothelial cell culture. The discrepancy in the subtype of NPR in the cornea may be due to the changes in NPR subtypes in the culture system. 
The presence of NPs and their receptors in the eye led us to investigate their paracrine actions. In experimental glaucoma, the concentration of ANP in the aqueous humor is increased, 21 and the ANP binding sites of ciliary processes are downregulated. 14 The physiological role for ANP has been suggested to decrease intraocular pressure in rabbits. 14 20 21 Recently, Fernandez–Durango et al. 28 have found that CNP is the most potent agent in decreasing intraocular pressure stimulating GC activity in the membrane of rabbit ciliary processes. At present, the physiological significance of ANP and CNP systems in the cornea is unknown. Walkenbach et al. 22 have found no ANP effect (1 nM-1 μM) on corneal deturgescence and cGMP production in cultured corneal endothelial cells. From these results, they suggest that corneal endothelial NPR-C may sequester ANP and its metabolites to provide a more constant supply of ANP to the trabecular meshwork. 
However, we found the localization of CNP binding sites in the endothelial cells of bovine cornea. The endothelial cells on the inner surface of the cornea are the major cell type responsible for the maintenance of corneal transparency and thickness. The transparency of the cornea is due to its uniform structure, avascularity, and deturgescence. Deturgescence is maintained through passive movement of water out of the cornea by active ion transport. Although the humoral regulation of this process remains poorly understood, CNP may influence water transport in the endothelial cells. Another role of CNP may be to inhibit the proliferation of corneal endothelial cells. In pathologic conditions in which NPR-B is downregulated, corneal transparency may be disturbed. More studies are needed to define the physiological role of NP systems in the cornea. 
Our data suggest that the ANP and CNP systems coexist in the bovine cornea and the NPR-B is the predominant receptor subtype. 
 
Figure 1.
 
Presence of ANP in bovine cornea. (A) A representative dilution curves of tissue extracts. The serial dilutions of corneal extracts (○) displaced 125I-AP III in a manner parallel to the synthetic standard (•). (B) Reversed-phase HPLC profiles of ANP in tissue extracts. Arrows: Retention time of AP III and pro-ANP, respectively. (C) Detection of ANP mRNA by RT-PCR. Lane 1: atrium as a positive control of ANP mRNA; lane 2: cornea; lane MM: DNA molecular size marker (174 RF DNA, HaeIII cut).
Figure 1.
 
Presence of ANP in bovine cornea. (A) A representative dilution curves of tissue extracts. The serial dilutions of corneal extracts (○) displaced 125I-AP III in a manner parallel to the synthetic standard (•). (B) Reversed-phase HPLC profiles of ANP in tissue extracts. Arrows: Retention time of AP III and pro-ANP, respectively. (C) Detection of ANP mRNA by RT-PCR. Lane 1: atrium as a positive control of ANP mRNA; lane 2: cornea; lane MM: DNA molecular size marker (174 RF DNA, HaeIII cut).
Figure 2.
 
Presence of CNP in bovine cornea. (A) Representative dilution curves of tissue extracts. The serial dilutions of corneal extracts (○) displaced 25I-[Tyr0]-CNP(1-22) in a manner parallel to the synthetic standard (•). (B) Reversed-phase HPLC profiles of CNP in tissue extracts. Arrows: Retention time of CNP(1-22) and CNP(1-53), respectively. (C) Detection of CNP mRNA by RT-PCR (a) and Southern blot analysis (b). Lane1: cornea; lane 2: pituitary gland as a positive control of CNP mRNA; lane MM: DNA molecular size marker (174 RF DNA, HaeIII cut).
Figure 2.
 
Presence of CNP in bovine cornea. (A) Representative dilution curves of tissue extracts. The serial dilutions of corneal extracts (○) displaced 25I-[Tyr0]-CNP(1-22) in a manner parallel to the synthetic standard (•). (B) Reversed-phase HPLC profiles of CNP in tissue extracts. Arrows: Retention time of CNP(1-22) and CNP(1-53), respectively. (C) Detection of CNP mRNA by RT-PCR (a) and Southern blot analysis (b). Lane1: cornea; lane 2: pituitary gland as a positive control of CNP mRNA; lane MM: DNA molecular size marker (174 RF DNA, HaeIII cut).
Figure 3.
 
cGMP production by the activation of guanylyl cyclase stimulated by ANP, BNP, and CNP in corneal cell membrane (n = 6). More cGMP was produced by CNP than ANP (P < 0.01) and BNP (P < 0.01).
Figure 3.
 
cGMP production by the activation of guanylyl cyclase stimulated by ANP, BNP, and CNP in corneal cell membrane (n = 6). More cGMP was produced by CNP than ANP (P < 0.01) and BNP (P < 0.01).
Figure 4.
 
Dark-field photomicrograph of autoradiograms of bovine eye sections incubated in the presence of 250 pM 125I-[Tyr0]-CNP(1-22) (A) and the adjacent section incubated in 250 pM 125I-[Tyr0]-CNP(1-22) plus 1 μM unlabeled CNP(1-22) (B). Specific 125I-[Tyr0]-CNP(1-22) binding sites were localized in the endothelial cell layers of cornea. CE, corneal endothelium; I, iris; CB, ciliary body; R, retina.
Figure 4.
 
Dark-field photomicrograph of autoradiograms of bovine eye sections incubated in the presence of 250 pM 125I-[Tyr0]-CNP(1-22) (A) and the adjacent section incubated in 250 pM 125I-[Tyr0]-CNP(1-22) plus 1 μM unlabeled CNP(1-22) (B). Specific 125I-[Tyr0]-CNP(1-22) binding sites were localized in the endothelial cell layers of cornea. CE, corneal endothelium; I, iris; CB, ciliary body; R, retina.
Figure 5.
 
Competitive inhibition curves of specific 125I-[Tyr0]-CNP(1-22) binding to frozen sections of the bovine cornea. Mean values from five eyes were plotted for the competition of binding of 250 pM 125I-[Tyr0]-CNP(1-22) to the cornea by increasing concentrations of unlabeled CNP(1-22). Inset: Representative Scatchard plot obtained from one eye of these bovines.
Figure 5.
 
Competitive inhibition curves of specific 125I-[Tyr0]-CNP(1-22) binding to frozen sections of the bovine cornea. Mean values from five eyes were plotted for the competition of binding of 250 pM 125I-[Tyr0]-CNP(1-22) to the cornea by increasing concentrations of unlabeled CNP(1-22). Inset: Representative Scatchard plot obtained from one eye of these bovines.
Figure 6.
 
Detection of NPR mRNAs by RT-PCR in the cornea (lanes 2, 4), medulla (lane 1), and pituitary gland (lane 3) of the rat as positive control. Lane MM: DNA molecular size marker (174 RF DNA, HaeIII cut).
Figure 6.
 
Detection of NPR mRNAs by RT-PCR in the cornea (lanes 2, 4), medulla (lane 1), and pituitary gland (lane 3) of the rat as positive control. Lane MM: DNA molecular size marker (174 RF DNA, HaeIII cut).
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Figure 1.
 
Presence of ANP in bovine cornea. (A) A representative dilution curves of tissue extracts. The serial dilutions of corneal extracts (○) displaced 125I-AP III in a manner parallel to the synthetic standard (•). (B) Reversed-phase HPLC profiles of ANP in tissue extracts. Arrows: Retention time of AP III and pro-ANP, respectively. (C) Detection of ANP mRNA by RT-PCR. Lane 1: atrium as a positive control of ANP mRNA; lane 2: cornea; lane MM: DNA molecular size marker (174 RF DNA, HaeIII cut).
Figure 1.
 
Presence of ANP in bovine cornea. (A) A representative dilution curves of tissue extracts. The serial dilutions of corneal extracts (○) displaced 125I-AP III in a manner parallel to the synthetic standard (•). (B) Reversed-phase HPLC profiles of ANP in tissue extracts. Arrows: Retention time of AP III and pro-ANP, respectively. (C) Detection of ANP mRNA by RT-PCR. Lane 1: atrium as a positive control of ANP mRNA; lane 2: cornea; lane MM: DNA molecular size marker (174 RF DNA, HaeIII cut).
Figure 2.
 
Presence of CNP in bovine cornea. (A) Representative dilution curves of tissue extracts. The serial dilutions of corneal extracts (○) displaced 25I-[Tyr0]-CNP(1-22) in a manner parallel to the synthetic standard (•). (B) Reversed-phase HPLC profiles of CNP in tissue extracts. Arrows: Retention time of CNP(1-22) and CNP(1-53), respectively. (C) Detection of CNP mRNA by RT-PCR (a) and Southern blot analysis (b). Lane1: cornea; lane 2: pituitary gland as a positive control of CNP mRNA; lane MM: DNA molecular size marker (174 RF DNA, HaeIII cut).
Figure 2.
 
Presence of CNP in bovine cornea. (A) Representative dilution curves of tissue extracts. The serial dilutions of corneal extracts (○) displaced 25I-[Tyr0]-CNP(1-22) in a manner parallel to the synthetic standard (•). (B) Reversed-phase HPLC profiles of CNP in tissue extracts. Arrows: Retention time of CNP(1-22) and CNP(1-53), respectively. (C) Detection of CNP mRNA by RT-PCR (a) and Southern blot analysis (b). Lane1: cornea; lane 2: pituitary gland as a positive control of CNP mRNA; lane MM: DNA molecular size marker (174 RF DNA, HaeIII cut).
Figure 3.
 
cGMP production by the activation of guanylyl cyclase stimulated by ANP, BNP, and CNP in corneal cell membrane (n = 6). More cGMP was produced by CNP than ANP (P < 0.01) and BNP (P < 0.01).
Figure 3.
 
cGMP production by the activation of guanylyl cyclase stimulated by ANP, BNP, and CNP in corneal cell membrane (n = 6). More cGMP was produced by CNP than ANP (P < 0.01) and BNP (P < 0.01).
Figure 4.
 
Dark-field photomicrograph of autoradiograms of bovine eye sections incubated in the presence of 250 pM 125I-[Tyr0]-CNP(1-22) (A) and the adjacent section incubated in 250 pM 125I-[Tyr0]-CNP(1-22) plus 1 μM unlabeled CNP(1-22) (B). Specific 125I-[Tyr0]-CNP(1-22) binding sites were localized in the endothelial cell layers of cornea. CE, corneal endothelium; I, iris; CB, ciliary body; R, retina.
Figure 4.
 
Dark-field photomicrograph of autoradiograms of bovine eye sections incubated in the presence of 250 pM 125I-[Tyr0]-CNP(1-22) (A) and the adjacent section incubated in 250 pM 125I-[Tyr0]-CNP(1-22) plus 1 μM unlabeled CNP(1-22) (B). Specific 125I-[Tyr0]-CNP(1-22) binding sites were localized in the endothelial cell layers of cornea. CE, corneal endothelium; I, iris; CB, ciliary body; R, retina.
Figure 5.
 
Competitive inhibition curves of specific 125I-[Tyr0]-CNP(1-22) binding to frozen sections of the bovine cornea. Mean values from five eyes were plotted for the competition of binding of 250 pM 125I-[Tyr0]-CNP(1-22) to the cornea by increasing concentrations of unlabeled CNP(1-22). Inset: Representative Scatchard plot obtained from one eye of these bovines.
Figure 5.
 
Competitive inhibition curves of specific 125I-[Tyr0]-CNP(1-22) binding to frozen sections of the bovine cornea. Mean values from five eyes were plotted for the competition of binding of 250 pM 125I-[Tyr0]-CNP(1-22) to the cornea by increasing concentrations of unlabeled CNP(1-22). Inset: Representative Scatchard plot obtained from one eye of these bovines.
Figure 6.
 
Detection of NPR mRNAs by RT-PCR in the cornea (lanes 2, 4), medulla (lane 1), and pituitary gland (lane 3) of the rat as positive control. Lane MM: DNA molecular size marker (174 RF DNA, HaeIII cut).
Figure 6.
 
Detection of NPR mRNAs by RT-PCR in the cornea (lanes 2, 4), medulla (lane 1), and pituitary gland (lane 3) of the rat as positive control. Lane MM: DNA molecular size marker (174 RF DNA, HaeIII cut).
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