| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
From the Department of Physiology and Ophthalmology, Medical School Institute for Medical Sciences, Jeonbug National University, Jeonju, Korea.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 (Kd) 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.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 tissue10 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 body14 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 Viader20 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 2x
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, 100
µg/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 (Kd) and their maximal binding capacities (Bmax) 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
|
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 Glembotski13 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.
|
Footnotes |
|---|
Submitted for publication December 13, 1999; revised March 1, 2000; accepted March 8, 2000.
Commercial relationships policy: N.
Corresponding author: Suhn Hee Kim, 2-20 Keum-Am-Dong-San, Department of Physiology, Jeonbug National University Medical School, Jeonju 560-181, Korea. shkim{at}moak.chonbuk.ac.kr
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  J.-F. Tolsa, Y. Gao, F. C. Sander, A.-C. Souici, A. Moessinger, and J. U. Raj Differential responses of newborn pulmonary arteries and veins to atrial and C-type natriuretic peptides Am J Physiol Heart Circ Physiol, January 1, 2002; 282(1): H273 - H280. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) 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).
