Higher proportions of type C than of types A and B natriuretic peptide receptors exist in the rat ciliary body

Vision Research 38 (1998) 3833 – 3841

Higher proportions of type C than of types A and B natriuretic peptide receptors exist in the rat ciliary body F. Jose´ Moya, Jose´ A. De Juan, Ainhoa Ripodas, Rosa Bernal, Arturo Fernandez-Cruz, Raquel Fernandez-Durango * Dapartmento Medicina Interna III, Hospital Clı´nico San Carlos, Ciudad Uni6ersitaria, 28040 Madrid, Spain Received 6 November 1997; received in revised form 4 February 1998

Abstract We investigate the interaction of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) with their receptors (NPRA, NPRB and NPRC), as well as the proportion and localization of those receptors in the rat ciliary body. Binding assays and affinity cross-linking experiments demonstrated the presence of the NPRC receptor type. However, the three natriuretic peptides stimulate the guanylate cyclase activity in the ciliary body membranes suggesting the presence of the NPRA and NPRB receptor type. Microautoradiographic data show that the NPRs are localized in the whole ciliary body. Our results indicated that NPRC is the most prominent receptor type in this tissue. © 1998 Elsevier Science Ltd. All rights reserved. Keywords: Natriuretic peptides; Receptor; Rat; Ciliary body

1. Introduction The natriuretic peptide family is composed of at least three ligands: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). ANP and BNP regulate body fluid homeostasis and blood pressure [1,2]. CNP acts mainly as a vasodilator with little natriuretic activity, and is found principally in the central nervous system and endothelial cells [3,4]. The natriuretic peptides can stimulate the production of cGMP through the activation of specific receptors: the type A natriuretic peptide receptor (NPRA) and the type B natriuretic peptide receptor (NPRB). These receptors are membrane-bound insoluble guanylyl cyclases with molecular sizes of 120 – 140 kDa [5]. The NPRA receptor selectively responds to ANP and, to a lesser extent, to BNP [6], whereas NPRB is selectively activated by CNP and, in a lesser degree, by ANP and BNP [7]. The third natriuretic peptide receptor, NPRC, which is composed of two identical subunits with the * Corresponding author. Present address: Unidad de Investigacio´n Experimental, Hospital Clı´nico San Carlos, C/ Martı´n Lagos s/n, 28040 Madrid, Spain. Fax: +34 1 5445432 0042-6989/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved. PII: S0042-6989(98)00105-9

molecular size of 60–70 kDa held together by disulfide bonds, is known to have its major role in the clearance of natriuretic peptides from blood circulation [8]. The NPRC receptor has no intrinsic ability to generate cGMP although it mediates changes in cAMP [9,10] and phosphoinositides [11]. It binds the three natriuretic peptides with approximately equal affinity, as well as ring-deleted and truncated linear peptides [4,7,12]. The five amino acid, ring-deleted ANP analog,(des (Gln18Ser19Gly20Leu21Gly22) rANF-(3-23)NH2), named C-ANP, has been reported to be specific for the NPRC receptor [8,13], even though it interacts with NPRA and NPRB receptors at high concentrations (\ 1 mM) [14]. This peptide has been used to distinguish and quantify NPRC receptors [4,14]. In the eye, it has been described that ANF increases cGMP production in the rabbit ciliary processes [15,16], and that BNP increases the concentration of cGMP in the aqueous humour of the rabbit eye [17]. These results predict the existence of natriuretic peptide receptors in the eye. Indeed, Bianchi et al. [18] identified ANP binding sites in rabbit and rat ciliary processes by binding assay and autoradiography. Binding sites for 125 I-ANF have also been found in rat retina [19]. Re-

3834

F.J. Moya et al. / Vision Research 38 (1998) 3833–3841

cently, specific high affinity binding sites for 125I[Tyr0]-CNP have been identified in human trabecular meshwork cells and ciliary muscle cells [20]. These data, together with the observations that the natriuretic peptides can decrease the intraocular pressure (IOP) [15–17,21,22] and alter ciliary blood flow [23], suggest that the peptides may act on specific receptors to control IOP. In addition, Ferna´ndez-Durango et al. [24] reported that messenger RNAs encoding the natriuretic peptides and their receptors are expressed in the eye. Thus, the eye appears to be able to synthesize all the components of the natriuretic peptide system necessary to modulate IOP independently of changes in the plasma concentration of these peptides. The interaction of the three ligands, ANP, BNP, CNP with their three types of receptors to produce biological effects in the ciliary body is unknown. Furthermore, the proportion of the three natriuretic peptide receptors and their localization in the ciliary body remain to be elucidated. For these reasons, we studied in the rat ciliary body: (1) the characterization of ANP, BNP and CNP specific binding sites and their proportions by binding assay and affinity cross-linking; (2) the existence of particulate guanylate cyclase activity and its activation by ANP, BNP and CNP; and (3) the localization of the specific binding sites by microautoradiography.

2. Materials and methods

2.1. Materials Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), CANP and 125I-[Tyr0]-CNP (1627 Ci/mmol) were obtained from Peninsula Laboratories (Merseyside, UK). 125 I-ANP (2000 Ci/mmol), 125I-BNP (2000 Ci/mmol) and the photographic emulsion (LM1) for microautoradiography were obtained from Amersham International (Buckinghamshire, UK). Dissuccinimidyl suberate (DSS) was purchased from Pierce Chemical Co.(Rockford, IL). Standard proteins for electrophoresis (SDS-PAGE standards LMW) were from BIO-RAD (Richmond CA). Whatman GF/C filters were from Whatman International (Maidstone, UK). Aprotinin, phenylmethylsulphonylfluoride (PMSF), EDTA, pepstatin, triethanolamine, 3-isobutil-1methylxanthine, theophyline, Cl2Mn, GTP, creatinine phosphokinase, phosphocreatinine, poly-L-lysine, paraformaldehide and glutaraldehide were obtained from Sigma (St.Louis, MO). Developer liquid was purchased from Kodak, and fixative from Ilford (Cheshire, UK).

2.2. Preparation of ciliary body membranes Adult male Wistar rats (250–350 g) (n = 100) were used in all experiments. The animals were kept in a temperature-controlled room on a 12 h light/dark cycle and fed ad libitum. Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). The eyes of the rats were removed immediately after decapitation. The ciliary bodies were dissected free of retina and vitreous humour. Ciliary body membranes were prepared as previously described [19]. Briefly, ciliary bodies were homogenized on ice in 5 mM Tris–HCl buffer pH 7.4, containing 0.32 M sucrose, 0.5 mM PMSF, 0.2 mM pepstatin, and 0.8 mM aprotinin. The homogenates were centrifuged at 30000×g for 30 min in the same buffer. The pellets were resuspended in 50 mM Tris–HCl buffer pH 7.4 at a protein concentration of 0.75–1 mg/ml. The protein concentration was estimated by the method of Lowry et al. [25]. Tissue preparations were stored in aliquots at −70°C and used within 1 month.

2.3. ANP, BNP, and CNP binding assays Ciliary body membranes (15–25 mg of protein) were incubated for 60 min at 25°C in 300 ml (final volume) of 50 mM Tris–HCl buffer pH 7.4 containing 0.5 mM PMSF, 0.2 mM pepstatin, 0.1% BSA, and 0.03% bacitracin. In the competitive experiments, the binding assay was performed with 5–8 pM of 125I-ANP, 125I-BNP or 125I-[Tyr0]-CNP and varying concentrations of unlabeled ANP, BNP, CNP or C-ANP (10 − 13 to 10 − 7 M). Increasing concentrations of labelled ANP, BNP or CNP (0.38–80 pM) were used in the saturation experiments. The specific binding of 125I-ANP, 125I-BNP and 125I-[Tyr0]-CNP was calculated by subtracting non-specific binding (125I-ANP bound in the presence of 1 mM ANP, 125I-BNP bound in the presence of 1 mM BNP and 125I-[Tyr0]-CNP bound in the presence of 1 mM CNP) from total 125I-ANP, 125I-BNP or 125I-[Tyr0]-CNP binding (in absence of unlabelled ANP, BNP or CNP). The binding reaction was terminated by diluting the reaction mixture with 3 ml of 50 mM Tris–HCl buffer pH 7.4, followed by rapid vacuum filtration through Whatman GF/C filters previously soaked for 1h in 0.3% vol/vol polyethylenimine solution. Each filter was washed twice with 3 ml of 50 mM Tris–HCl buffer, dried, and removed for counting on a LKB gamma-counter with 75% efficiency. Several unrelated peptides, including somatostatin, endothelin, tyroid stimulating hormone (TSH), growth hormone (GH) or insulin, at the concentration of 1 mM, were tested for 125I-ANP, 125 I-BNP or 125I-[Tyr0]-CNP binding inhibition under the same conditions as in the competitive experiments.

F.J. Moya et al. / Vision Research 38 (1998) 3833–3841

2.4. Affinity cross-linking studies

3835

This study was made as previously described in rat retina [19]. The labelled peptides (125I-ANP, 125I-BNP or 125 I-[Tyr0]-CNP), 35 pM, were incubated with ciliary body membranes (45 mg of protein) in a total volume of 300 ml of 10 mM Hepes buffer, pH 7.4, containing 0.5 mM PMSF, 0.2 mM pepstatin, 0.1% BSA, and 0.03% bacitracin, for 60 min at 25°C. The reaction was terminated by centrifugation at 30000× g for 15 min at 4°C. The pellets were resuspended in 10 mM Hepes buffer, pH 7.4, and labelled peptides were cross-linked on receptors with 0.5 mM DSS. The reaction was quenched 20 min later with 2 M ammonium acetate. The specificity of the binding was determinated by the addition of 0.1 mM ANP, BNP, CNP or C-ANP to the incubation mixture. Samples were denatured under reducing (2% b-mercaptoethanol) and non-reducing conditions prior to polyacrylamide gel electrophoresis.

decapitation, were immersed in 2-methylbutane on liquid nitrogen. Sections (12 mm) were cut in a cryostat at − 28°C, thaw-mounted onto poly-L-lysine treated slides, and dried for 4 h in a desiccator under vacuum at 4°C. The sections were then preincubated in 50 mM Tris– HCl buffer pH 7.4 containing 100 mM NaCl, 5 mM ClMg2, 0.5 mM PMSF, 2 mM pepstatin, 10.000 U.I.C. aprotinin, and 0.5% BSA for 15 min. at 25°C. Sections were then incubated with 40 pM 125I-ANP or 125I-[Tyr0]CNP in the same buffer for 60 min at room temperature. Specific binding was determined by radioligand displacement with 0.1 mM ANP, BNP, CNP or C-ANP. After incubation, the slides containing the sections were washed three times with fresh buffer for 4 min, then rinsed in fresh bidistillated water and fixed in 4% paraformaldehyde and 1% glutaraldehyde in 100 mM phosphate buffer for 4 h. The sections were covered with LM1 Amersham emulsion, stored in the dark at 4°C for 10 days and developed with Kodak D19 developer.

2.5. Sodiumdodecylsulfate (SDS) -polyacrylamide gel electrophoresis

2.8. Data analysis

Gel electrophoresis was performed according to the method of Lammeli [26], using 8% SDS-polyacrylamide gels. Samples were run for 1 h 45 min at 125 V. After electrophoresis, proteins were visualised by Coomassie blue staining; the gels were then dried and exposed to a X-OMAT RP6 Kodak film with intensifying screen at − 70°C.

2.6. Guanylate cyclase acti6ity Natriuretic peptides-activated guanylate cyclase activity was measured as the rate of conversion of GTP to cyclic GMP as previously described by Inagami et al. [27]. For this assay, reaction tubes were prepared to contain (in 0.1 ml final volume): Tris – HCl 50 mM, pH 7.4; 50 mM triethanolamine, 2 mM 3-isobutil-1methylxanthine, 10 mM theophyline, 3 mM Cl2Mn, 1 mM GTP, 75 U/ml creatinine phosphokinase, 72 mM phosphocreatinine, 0.1 mM natriuretic peptides and ciliary body membranes (6 mg of protein). The reaction was started by the addition of GTP. The tubes were incubated for 30 min. at 37°C, and the reaction was stopped by addition of 2 ml 30 mM EDTA at 80°C, followed by boiling for 3 min. The cyclic GMP formed was subsequently measured by specific radioimmunoassay (Amersham RPA 525) using a non acetylation protocol. The minimal detectable limit of cGMP by this assay was 50 fmol/tube.

2.7. Microautoradiography of 125 I-[Tyr 0] -CNP binding

125

I-ANP and

The eyes of the rats, removed immediately after

The binding data for the determination of the density and affinity of binding sites were evaluated by computer-assisted non-linear regression analysis with the LIGAND program [28], after preliminary treatment of data with the EBDA program [29] The inhibition constant (ki) was calculated according to the method of Cheng and Prusoff [30]. The values are presented as means9SEM. The curves were plotted using the SigmaPlot Scientific Graphing System (version 4.10) computer program by Jandel Corporation

3. Results

3.1. Saturation studies Specific 125I-ANP and 125I-BNP binding to rat ciliary body membranes was saturable in a concentration-dependent manner. Scatchard analysis of saturation binding data of 125I-ANP and 125I-BNP indicated the presence of a single class of high-affinity binding sites. KD and Bmax values evaluated from 125 I-ANP saturation binding data by non-linear regression analysis were 12.3 92.9 pM and 198 9 46 fmol/mg of protein (n=3), respectively (Fig. 1), and the Hill coefficient was 0.94 9 0.03 (n = 4). KD and Bmax values calculated from 125I-BNP saturation binding data by the same method were 27912 pM and 238 9 54 fmol/mg of protein (n= 4), respectively (Fig. 1), and the Hill coefficient was 1.01690.01 (n= 4).

3836

F.J. Moya et al. / Vision Research 38 (1998) 3833–3841

3.2. Competition studies Competitive binding experiments were performed using 5–8 pM 125I-ANP, 125I-BNP or 125I-[Tyr0]-CNP as labeled hormones. Unlabeled ANP, BNP, CNP and C-ANP competed for labeled hormones specific binding sites in a dose-dependent manner (Fig. 2). Analysis of the competition curves showed that ANP, BNP and CNP competed with 125I-ANP, 125I-BNP and 125I[Tyr0]-CNP with similar affinity (Tables 1 and 2). On the other hand, C-ANP displaced 100% of 125I-ANP and 125I-[Tyr0]-CNP binding. These results suggest the presence of NPRC receptors, without confirming the presence of guanylate cyclase-coupled receptors (NPRA or NPRB).

3.3. Cross-linking 125 I-ANP, 125I-BNP and 125I-[Tyr0]-CNP (35 pM) were used to identify the apparent molecular weight of the natriuretic receptors in rat ciliary body membranes. Affinity labeling of these membranes by cross-linking with 125I-ANP (Fig. 3(A)), 125I-BNP (Fig. 3(B)) and 125 I-[Tyr0]-CNP (Fig. 3(B)), using disuccinimidyl suberate (DSS), indicated the presence of two specific bands with molecular weights of :140 and 66 kDa (Fig. 3(A), lane 1; Fig. 3(B), lanes 1 and 5). The labeling of these binding proteins was specifically inhibited by the presence of excess nonradioactive ANP (Fig. 3(A), lane

2;Fig. 3(B), lanes 2 and 6), BNP (Fig. 3, lanes 3) or CNP lanes (Fig. 3(A), lane 5; Fig. 3(B), lane 7) (0.1 mM) indicating that the labeling of the two bands was specific. In the presence of 0.1 mM C-ANP (Fig. 3(A), lane 4; Fig. 3(B), lanes 4 and 8) the labeling was also specifically inhibited. The : 140 kDa radiolabeled protein migrated in the gel to the 66 kDa position when similar experiments were performed with 125I-ANP and 125I-[Tyr0]-CNP under reducing conditions (2% b-mercaptoethanol) (Fig. 3(C)). These results suggest the unique presence of NPRC receptors.

3.4. Microautoradiography Specific binding of 125I-ANP and 125I-[Tyr0]-CNP was observed in the rat ciliary body. The silver grains are localized in pigmented and non-pigmented epithelium and in vascular stroma. These results are shown in Fig. 4(A) (total binding of 125I-ANP) and Fig. 5(A) (total binding of 125I-[Tyr0]-CNP). These figures represent dark-field photomicrographs. Their correspondent bright-field photomicrographs of rat ciliary body stained with toluidine blue are shown in Fig. 4(B) and Fig. 5(B), respectively. The unlabeled peptides ANP, BNP, CNP and C-ANP completely displaced the 125IANP (Fig. 4(C, D, E and F)) as well as the 125I-[Tyr0]CNP (Fig. 5(C, D, E and F)).

3.5. Guanylate cyclase acti6ity In the rat ciliary body membranes, the baseline values of guanylate cyclase activity were 64.69 1.7 pmol/ mg protein per min. In the presence of 10 − 7 M ANP, 10 − 7 M BNP (specific for NPRA receptor) or 10 − 7 M CNP (specific for NPRB receptor) the values were 82.89 8.9 pmol/mg protein per min, 80.397.1 pmol/ mg protein/min and 88.79 9.5 pmol/mg protein per min, respectively, showing a statistically significant increase (PB 0.05, n= 4) in cGMP production over baseline (Fig. 6).

4. Discussion

Fig. 1. (A) Saturacion of specific binding of 125I-ANP () and 125 I-BNP (“) to rat ciliary body membranes. (B) Scatchard transformation of the specific binding of 125I-ANP () and 125I-BNP (“) from A. Data points are means of duplicate measurements in a representative experiment.

This study demonstrates the existence of high-affinity binding sites for ANP, BNP and CNP in the rat ciliary body, and that those peptides stimulate guanylyl cyclase activity in this tissue. Scatchard analysis derived from saturation binding data revealed one high-affinity binding site for ANP and BNP. The KD obtained using 125I-ANP and 125IBNP were 12.39 2.9 pM and 27912, respectively. The Bmax values obtained for 125I-ANP was 198 946 fmol/ mg protein, and 2389 54 fmol/mg protein for 125IBNP. These results suggest a single class of receptors,

F.J. Moya et al. / Vision Research 38 (1998) 3833–3841

3837

Fig. 2. Competitive inhibition of 125I-ANP (A), 125I-BNP (B) and 125I-[Tyr0]-CNP (C) binding to rat ciliary body membranes by unlabeled ANP (), BNP (“), CNP () and C-ANP (). The specific binding of labeled peptides in absence of competitor was normalized to 100% (Bo). The results (B/Bo) are expressed as the percentage of labeled peptides binding in the presence of competitor. Data points are means 9 SEM of four independent experiments performed in duplicated.

multiple receptors with similar affinities, or an abundance of one receptor over another. The KD values that we obtained were similar to those obtained in rabbit ciliary process [18] and hog ciliary body [31]. Analysis of the competition binding experiments showed that the rat ciliary body has a single class of high-affinity binding sites for ANP, BNP and CNP, and that unlabeled ANP, BNP, CNP and C-ANP displaced the total binding of 125I-ANP, 125I-BNP and 125 I-[Tyr0]-CNP to the ciliary body membranes. These data also suggest the unique presence of the NPRC receptor or a great abundance of NPRC receptor over NPRA and NPRB receptors. As Ferna´ndez-Durango et al. [24] have reported that the messenger RNAs encoding the three natriuretic peptide receptors are expressed in rat and rabbit ciliary bodies, our results could be

explained as indicating that the majority of natriuretic receptors in the rat ciliary body belongs to the NPRC type. Affinity cross-linking of 125I-ANP, 125I-BNP and 125I[Tyr0]-CNP to rat ciliary body membranes resulted in two specific bands with approximate molecular masses of : 140 and 66 kDa. Both bands were totally displaced by unlabeled ANP, BNP, CNP and C-ANP. However, under reducing conditions, we only observed a single band of :66 kDa, which corresponds to the molecular weight of the non-guanylate cyclase linked receptor (NPRC receptor). These results, in agreement with the data obtained using the binding assay, indicate that either there are fewer of the guanylate cyclase-coupled receptors (NPRA and NPRB), or the efficiency of cross-linking to these receptors is diminished. Since we

F.J. Moya et al. / Vision Research 38 (1998) 3833–3841

3838

Table 1 Ki (pM) values for ANP, BNP, CNP and C-ANP in competing for 125 I-ANP, 125I-BNP and 125I-[Tyr0]-CNP binding to rat ciliary body membranes Labeled peptides

125

I-ANP

125

I-BNP

125

I-[Tyr0]-CNP

Unlabeled ligands

Ki pM

ANP BNP CNP C-ANP

289 6 299 5 24 9 5 24609 500

ANP BNP C-ANP

909 3 59 9 12 11779 373

ANP BNP CNP C-ANP

209 4 80 9 19 23 96 11459 215

The data presented are the mean9S.E.M. of four determinations performed in duplicate. The Ki was calculated according to the formula of [30].

have previously cross-linked both receptor types simultaneously using the same methodology in rat retina [19], these data again indicate that there is a low number of guanylyl cyclase-coupled receptors relative to NPRC receptors. Similar data have been reported with ANP on hog ciliary body [31]. On the other hand, Leitman et al. [32] suggest that three molecular forms of ANP binding sites may exist (1) a non reducible 130 kDa site (2) a 130 kDa site that is probably comprised of two identical 66 kDa subunits joined by disulphide bond and (3) a non-reducible 66 kDa monomer. The band that we observed of :140 kDa is probably comprised of two identical 66 KDa subunits joined by a disulphide bond. The non-reducible 130 kDa band, associated with guanylate cyclase-coupled receptors, is absent in our experiments. Thus, our cross-linking experiments indicate the unique presence of NPRC receptors. In order to investigate the presence of the guanylate cyclase-linked receptor types (NPRA and NPRB) in rat ciliary body membranes, we have examined the effects of ANP, BNP and CNP on guanylate cyclase activity in those membranes. Our baseline values of guanylate Table 2 Receptor density (Bmax) and dissociation constant (KD) values for ANP, BNP and CNP in competing for 125I-ANP, 125I-BNP and 125 I-[Tyr0]-CNP binding to rat ciliary body membranes 125

125

125

289 5 1459 8

4598 225 948

30 97 1239 10

I-ANP

KD (pM) Bmax (fmol/mg of protein)

I-BNP

I-[Tyr0]-CNP

The data presented are the mean9 S.E.M. of four determinations performed in duplicate. Data were analyzed using the LIGAND program [28].

Fig. 3. (A) and (B), Autoradiogram of 125I-ANP ((A), lanes 1 to 5), 125 I-BNP ((B), lanes 1 to 4) and 125I-[Tyr0]-CNP (B. lanes 5 to 8) cross-linked to natriuretic peptide receptors on rat ciliary body membranes. Membranes were incubated with labeled peptides in absence ((A), lane 1; (B) lanes 1 and 5) or presence of 0.1 mM ANP ((A), lane 2; (B) lanes 2 and 7), 0.1 mM BNP ((A) and (B), lane 3), 0.1 mM C-ANP ((A), lane 4; (B). lanes 4 and 8), and 0.1 mM CNP ((A), lane 5; (C), lane 6). (C): Autoradiogram of 125I-ANP ((C). lanes 1 to 4) and 125I-[Tyr0]-CNP ((C), lanes 5 to 8), under reducing conditions ((C), lanes 3, 4, 7 and 8) in absence ((C), lanes 1, 3, 5 and 7) or presence of 0.1 mM ANP ((C), lanes 2 and 4) or 0.1 mM CNP ((C), lanes 6 and 8).

cyclase activity were similar to those obtained by Nathanson [15] in rabbit ciliary bodies. The three natriuretic peptides increase the guanylate cyclase activity, CNP being slightly more potent that ANP and BNP. Since the NPRA is selectively activated by ANP and BNP [6], and the NPRB selectively responds to CNP [7], our results suggest the presence of the functional receptor types, NPRA and NPRB, in the membrane of the rat ciliary body. The ciliary body productions of cGMP induced by ANP, BNP and CNP were similar and is consistent with the similar KD values obtained in the binding assay when 125I-ANP, 125I-BNP and 125I[Tyr0]-CNP were used as radioligand. In agreement with our results, Nathanson [15] and Mittag et al. [16] reported that ANP activates guanylate cyclase in rabbit ciliary body. The presence of NPRB receptors has also been demonstrated in human fetal non-pigmented epithelial cells (NPE) [33]. Our microautoradiography data showed specific binding of 125I-ANP and 125I-[Tyr0]-CNP in the rat ciliary body. Silver grains are uniformly distributed in the ciliary body, and specific binding is totally displaced in presence of unlabeled natriuretic peptides (10 − 7M

F.J. Moya et al. / Vision Research 38 (1998) 3833–3841

3839

Fig. 4. Dark-field photomicrographs showing the specific 125I-ANP binding sites in rat ciliary body. Cryostat sections were incubated with I-ANP in absence (A) or in presence of 0.1 mM unlabeled ANP (C), BNP (D), CNP (E) or C-ANP (F). Bright-field photomicrograph stained with toluidine blue at similar level (B). 125

ANP, 10 − 7M BNP, 10 − 7M CNP and 10 − 7M CANP). In contrast with our results, Bianchi et al. [18], demonstrated by ‘in vivo’ microautoradiography that the competitive binding sites for 125I-ANP were localized mostly on the pigmented epithelium of the rat ciliary body. However, the presence of 8% and 7% of silver grains on non-pigmented epithelium and vascular stroma, respectively, 2 min after 125I-ANP injection, led these authors to suggest that binding sites for 125I-ANP may also be present in these tissues. This idea was sustained by the fact that silver grains seem to be progressively internalized at later time intervals after injection of 125I-ANP [18]. In fact, Tsukara et al. [34], 30 min after the injection of 125IANP observed binding sites on the whole ciliary body of mice. From a physiological point of view, the presence of guanylate cyclase-linked receptors in the ciliary body suggests an active role for natriuretic peptides in the regulation of the aqueous humour secretion, and thus of the IOP. In fact, ANP [15,22,35] and BNP [17] decrease IOP in various species. In agreement with this idea, Carre´ and Civan [36] presented evidence

that the cGMP-mediated transmitters ANP and NO both modulate ion transport across the rabbit ciliary epithelium. Interestingly Ferna´ndez-Durango et al. [37] reported than in rabbits with experimental glaucoma the NPRs are down-regulated in the ciliary body and that immunoreactive ANP levels in aqueous humour rise as IOP increases [38]. On the other hand, the great abundance of NPRC receptors that we found in rat ciliary body was also observed in specialized endothelial cells composing the blood-brain barrier [39], suggesting that a high rate of natriuretic peptides clearance will prevent inappropriate fluctuations of the levels of the natriuretic peptides in these tissues which are specifically sensitive to changes in fluid and electrolyte homeostasis. In conclusion, in the rat ciliary body the natriuretic peptides ANP, BNP and CNP stimulate the guanylyl cyclase activity, probably through the interaction with NPRA and NPRB receptors. These peptides also bind to NPRC receptors which are the most prominent natriuretic receptor types in this tissue. Microautoradiographic data reveal that NPRs are localized throughout the whole ciliary body.

3840

F.J. Moya et al. / Vision Research 38 (1998) 3833–3841

Fig. 5. Dark-field photomicrographs showing the specific 125I-[Tyr0]-CNP binding sites in rat ciliary body. Cryostat sections were incubed with 125 I-[Tyr0]-CNP in absence (A) or in presence of 0.1 mM unlabeled ANP (C), BNP (D), CNP (E) or C-ANP (F). Bright-field photomicrograph stained with toluidine blue at similar level (B).

Acknowledgements This work was supported by grant PB92-0737 from DGICYT.

Fig. 6. Effect of 0.1 mM ANP, BNP and CNP on guanylate cyclase activity. Values are means 9 SEM of four independent experiment performed in duplicated (* PB 0.05. Two-tailed Student’s t-test).

References [1] de Bold AJ, Flynn T. Cardionatrin I. A novel heart peptide with potent diuretic and natriuretic properties. Life Sci 1983;33:297– 303. [2] McGregor A, Richards M, Espiner E, Yandle T, Ikram H. Brain natriuretic peptide administered to man; actions and metabolism. J Clin Endocrinol Metab 1990;70:1103 – 7. [3] Sudoh T, Minamino N, Kangawa K, Matsuo H. C-Type natriuretic peptide (CNP): a new member of natriuretic family identified in porcine brain. Biochem Biophys Res Commun 1990;168:863 – 70. [4] Suga SI, Nakao K, Hosada K, et al. Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide. Endocrinology 1992;130:229 – 39. [5] Koller KJ, Goeddel DV. Molecular biology of the natriuretic peptides and their receptors. Circulation 1992;86:1081 –8. [6] Schulz S, Singh S, Bellet RA, et al. The primary structure of a plasma membrane guanylate cyclase demonstrates diversity within this new receptor family. Cell 1989;58:1155 – 62. [7] Koller KJ, Lowe DG, Bennet GL, et al. Selective activation of the B natriuretic peptide receptor by C-type natriuretic peptide (CNP). Science 1991;252:120 – 3. [8] Maack T, Suzuk M, Almeida FA, et al. Physiological role of silent receptors of atrial natriuretic factor. Science 1987;238:675– 8.

F.J. Moya et al. / Vision Research 38 (1998) 3833–3841 [9] Anand-Srivastava MB, Cantin M. Atrial natriuretic factor receptors are negatively coupled to adenylate cyclase in cultured atrial and ventricular cardiocytes. Biochem Biophys Res Commun 1986;138:427 – 36. [10] Anand-Srivastava MB, Cantin M, Genest J. Inhibition of pituitary adenylate cyclase by atrial natriuretic factor. Life Sci 1985;36:1873 – 9. [11] Berl T, Mansour J, Teitlebaum I. ANP stimulates phospholipase C in cultured RMICT cells: roles of protein kinases and G protein. Am J Physiol 1991;260:F590–5. [12] Maack T. Receptors of atrial natriuretic factor. Annu Rev Physiol 1992;54:11 – 27. [13] Bovy PR, O’Neal JM, Olins GM, Patton DR. Identification of structural requirements for analogues of atrial natriuretic peptide (ANP) to discriminate between ANP receptor subtypes. J Med Chem 1989;32:869–74. [14] Konrad EM, Thibault G, Schiffrin EL, Cantin M. Atrial natriuretic factor receptor subtypes in the rat central nervous system. Hypertension 1991;17:1144–51. [15] Nathanson JA. Atriopeptin-activated guanylate cyclase in the anterior segment. Identification, localization, and effects of atriopeptins on IOP. Invest Ophthalmol Vis Sci 1987;28:1357 – 64. [16] Mittag TW, Tormay A, Ortega M, Severin C. Atrial natriuretic peptide (ANP), guanylate cyclase, and intraocular pressure in the rabbit eye. Curr Eye Res 1987;6:1189–96. [17] Takashima Y, Taniguchi T, Yoshida M, Haque MSR, Yoshimura N, Honda Y. Ocular hypotensive mechanism of intravitreally injected brain natriuretic peptide in rabbit. Invest Ophthalmol Vis Sci 1996;37:2671–7. [18] Bianchi C, Anand-Srivastava MB, De Lean A, et al. Localization and characterization of specific receptors for atrial natriuretic factor in the ciliary processes of the eye. Curr Eye Res 1986;5:283 – 93. [19] Ferna´ndez-Durango R, Sanchez D, Gutkowska J, Carrier F, Ferna´ndez-Cruz A. Identification and characterization of atrial natriuretic factor receptors in the rat retina. Life Sci 1989;44:1837 – 46. [20] Pang IH, Shade DL, Matsumoto S, Steely HT, DeSantis L. Presence of functional type B natriuretic peptide receptor in human ocular cells. Invest Ophthalmol Vis Sci 1996;37:1724 – 31. [21] Diestelhorst M, Krieglstein GK. The intraocular pressure response of human atrial natriuretic factor in glaucoma. Int Ophthalmol 1989;13:99–101. [22] Samuelsson A, Nilsson S, Maepea O, Bill A. Effects of atrial natriuretic factor (ANF) on intraocular pressure and aqueous humor flow in the cynomolgus monkey. Exp Eye Res 1991;53:253 – 60. [23] Funk R, Rohen JW. Reactions of efferent venous segments in the ciliary process vasculature of albino rabbits. Exp Eye Res 1988;46:95 – 104.

.

3841

[24] Ferna´ndez-Durango R, Nunez DJR, Brown MJ. Messenger RNAs encoding the natriuretic peptides and their receptors are expressed in the eye. Exp Eye Res 1995;61:723 – 9. [25] Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265 – 75. [26] Lammeli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680– 5. [27] Inagami T, Takayanagi R, Snajdar RM. Copurification of atrial natriuretic factor receptors and guanylyl cyclase from adrenal cortex. Methods Enzymol 1991;195:404 – 13. [28] Munson PJ, Rodbard D. LIGAND: A versatile computerized approach for the characterization of ligand binding systems. Anal Biochem 1980;107:220 – 39. [29] Macpherson GA. Analysis of radioligand binding experiments: A collection of computer programs for the IBM PC. J Pharmacol Methods 1985;14:213 – 24. [30] Cheng Y, Prusoff W. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 1973;22:3099 – 108. [31] Shinjo M, Kim S, Miyazaki H, Usuki Y, Murakami K. Atrial natriuretic peptide binding sites on hog ciliary bodies and choroid. Biomed Res 1988;9:21 – 6. [32] Leitman DC, Andresen JW, Catalano RM, Waldman SA, Tuan J, Murad F. Atrial natriuretic peptide binding, cross– linking, and stimulation of cyclic GMP accumulation and particulate cyclase activity in cultured cells. J Biol Chem 1988;263:3720– 8. [33] Crook RB, Chang AT. Differential regulation of natriuretic peptide receptors on ciliary body epithelial cells. Biochem J 1977;324:49 – 55. [34] Tsukara S, Sasaki T, Yamabayashi S, Furuta M, Ushiyama M, Yamamoto T. Effect of alpha-human atrial natriuretic peptides on intraocular pressure in normal albino rabbits. Ophthalmol Basel 1988;197:104 – 9. [35] Wolfensberger TJ, Singer DRJ, Freegard T, Markandu ND, Buckley MG, MacGregor GA. Evidence for new role of natriuretic peptides: control of intraocular pressure. Br J Ophthalmol 1994;78:446 – 8. [36] Carre´ DA, Civan MM. cGMP modulates transport across the ciliary epithelium. J Membr Biol 1995;146:293 – 305. [37] Tarentino, A.L., Gomez, C.M. and Plummer, T.H. (1985). Biochemistry 24: 4665 – 4671 [38] Ferna´ndez-Durango R, Trivin˜o A, Ramirez JM, et al. Immunoreactive atrial natriuretic factor in aqueous humor: its concentration is increased with high intraocular pressure in rabbit eyes. Vis Res 1990;30:1305 – 10. [39] Whitson PA, Huls MH, Sams CF. Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells. J Cell Physiol 1991;146:43 – 51.