©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Natriuretic Peptide Receptor-B (Guanylyl Cyclase-B) Mediates C-type Natriuretic Peptide Relaxation of Precontracted Rat Aorta (*)

(Received for publication, September 15, 1994; and in revised form, December 19, 1994)

James G. Drewett (1)(§) Brian M. Fendly (2) David L. Garbers (3) David G. Lowe (2)(¶)

From the  (1)Department of Pharmacology and Toxicology and the Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, (2)Genentech, Inc., South San Francisco, California 94080, and the (3)Howard Hughes Medical Institute and Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The most potent known agonist for the natriuretic peptide receptor-B (NPR-B)/guanylyl cyclase-B is C-type natriuretic peptide (CNP). A homologous ligand-receptor system consists of atrial natriuretic peptide (ANP) and NPR-A/guanylyl cyclase-A. A third member of this family is NPR-C, a non-guanylyl cyclase receptor. Monoclonal antibodies were raised against NPR-B by immunizing mice with a purified receptor-IgG fusion protein consisting of the extracellular domain of NPR-B and the Fc portion of human IgG-(1). One monoclonal antibody, 3G12, did not recognize NPR-A or NPR-C and bound to human and rat NPR-B. CNP binding to NPR-B and stimulation of cGMP synthesis were inhibited by 3G12. With cells isolated from either the media or adventitia layers of rat thoracic aorta, 3G12 did not interfere with ANP-stimulated cGMP synthesis, but it inhibited CNP-stimulated cGMP levels in cells from both layers. CNP (IC = 10 nM) and ANP (IC = 1 nM) caused relaxation of phenylephrine-contracted rat aortic rings. 3G12 caused a marked increase in the IC for CNP, from 10 nM to 140 nM, but failed to affect ANP-mediated relaxation. Therefore, our results for the first time demonstrate that CNP relaxes vascular smooth muscle by virtue of its binding to NPR-B.


INTRODUCTION

de Bold et al.,(1981) discovered that atrial extracts contain a potent natriuretic and diuretic activity which was subsequently associated with a factor termed atrial natriuretic peptide (ANP). (^1)It turned out to be the first of three related endogenous peptides identified. ANP and a second natriuretic peptide, BNP, are found predominantly in the heart and are released into the systemic circulation under various conditions (Brenner et al., 1990). At first ANP was thought of as only an endocrine hormone acting on targets distal to the heart, perhaps most importantly the kidney (Maack and Kleinhert, 1986). However, both ANP and BNP have been subsequently found in various other tissues including the adrenal medulla, brain, intestine, kidney, lung, and stomach (Nemer and Gutkowska, 1989; Drewett and Garbers, 1994) suggesting they could also function locally. The third natriuretic peptide CNP (Sudoh et al., 1990) initially was found only within the central nervous system and adrenal medulla (Tawaragi et al., 1990), but low levels of CNP have been detected in rat small intestine, colon, and kidney (Komatsu et al., 1991; Dean et al., 1994), and high levels have been found in seminal vesicle fluid and tracheal mucosa (Chrisman et al., 1993).

The three natriuretic peptides appear to act by binding either to membrane-associated members of the guanylyl cyclase-coupled receptor family or to a truncated, guanylyl cyclase-uncoupled binding site, the natriuretic peptide clearance receptor (NPR-C) (Anand-Srivastava and Trachte, 1994; Drewett and Garbers, 1994; Maack, 1992). At low concentrations (i.e. submicromolar) ANP and BNP bind preferentially to the natriuretic peptide receptor-A (NPR-A; also called guanylyl cyclase-A) and CNP to the natriuretic peptide receptor-B (NPR-B; also called guanylyl cyclase-B) (Koller et al., 1991, Bennett et al., 1991; Suga et al., 1992b).

Results within the last 2 years suggest that CNP is produced by endothelial cells in culture (Suga et al., 1992a). In addition, low amounts of immunoreactive CNP are present in human plasma (6 pg/ml) (Stingo et al., 1992). Previous work has demonstrated that CNP relaxes several isolated vascular preparations from the rat (Furuya et al., 1990), suggesting that CNP produced by endothelial cells could be involved in the regulation of vascular tone. Furuya et al.,(1990) found that ANP was about 16-fold more potent than CNP at relaxing the rat aorta in vitro which is similar to the results reported in the present study. A subsequent study in dog reported that CNP was more effective at relaxing isolated veins in comparison to arteries (Wei et al., 1993).

In the present study we report the identification of an antagonistic mAb, 3G12, against the extracellular domain of NPR-B. This antibody blocks the ability of CNP to activate guanylyl cyclase in 293 cells over-expressing either rat or human NPR-B, and it also blocks the ability of CNP to increase intracellular accumulation of cGMP in rat aortic adventitia and vascular smooth muscle preparations. Furthermore, this antibody antagonizes the ability of CNP but not ANP to relax phenylephrine-contracted rat aorta in vitro, demonstrating for the first time that NPR-B mediates the vasorelaxant effect of CNP.


MATERIALS AND METHODS

Cell Lines

Human embryonic kidney 293 cell lines expressing recombinant natriuretic peptide receptors were derived by co-transfection of expression constructs and the marker plasmid pNeoDHFR (Lowe and Goeddel, 1987). The hNPR-B (Chang et al., 1989) and rNPR-B (Schulz et al., 1989) cDNA expression vectors previously described were used for stable expression in 293 cells. Stable expression of the hNPR-A cDNA (Lowe et al., 1989) is described by Lowe and Fendly(1992), and expression of the hNPR-C6 cDNA (Lowe et al., 1990) is described in Cunningham et al.,(1994). The 293 BDeltaKC cell line expresses NPR-B with a cytoplasmic domain truncation (Koller et al., 1991).

Monoclonal and Polyclonal Antibodies

10 BALB/c mice were immunized with 4 µg of hB-IgG (Bennett et al., 1991) complexed with 40 µg of mouse anti-human IgG (Rockland, Gilbertsville, PA or Jackson Immunoresearch Labs, Inc. West Grove, PA) on days 0, 7, and 21. 5 µg of hB-IgG complexed with 5 µg of mouse anti-human IgG was used for immunization on days 35 and 80. Serum samples from hyperimmunized mice were tested by flow cytometry on 293BDeltaKC cells using 2-h incubations on ice and an F(ab)`2 preparation of goat anti-mouse IgG-fluorescene isothiocyanate (Boehringer Mannheim). The lymph node cells (inguinal and popliteal) from five mice were fused on day 84 with the mouse myeloma line X63.Ag8.653, as described previously (Lucas et al., 1990). Supernatants from parental wells were combined into pools of 12 and screened by flow cytometry on 293BDeltaKC cells as described above. Ultimately, six parental lineages (2A8, 3G12, 4D2, 4E6, 6D8, and 6E5) were chosen for limiting dilution cloning, ascites production, purification, and further characterization. Antibody purification from ascites was achieved by affinity chromatography on protein A (Bennett et al., 1991), and Fab preparation by papain cleavage was performed using an ImmunoPure Fab purification kit (Pierce).

NPR-B Signal Transduction

A stable 293 cell line expressing hNPR-B was maintained in glycine free F-12/Dulbecco's minimal essential medium (50/50, v/v) with 10% dialyzed fetal calf serum, 10 mM HEPES, 400 ng/ml of G418 (Geneticin, Life Technologies, Inc), pH 7.2, in a humidified incubator at 37 °C with 7% CO(2), 93% air. For signal transduction experiments cells were passaged using 136.8 mM NaCl, 2.6 mM KCl, 7.9 mM Na(2)HPO(4), 1.4 mM KH(2)PO(4), pH 7.2 (PBS) with 0.5 mM EDTA, and plated into 12-well tissue culture plates at 2 times 10^5 cells/well. For concentration-response experiments, log phase cultures of 293 cell lines were passaged to 12-well plates at a density of 2.5 times 10^5 cells/well. Cultures were approximately 50% confluent after 48 h and were used for stimulation of cGMP production. Peptide treatments were performed as described (Lowe and Fendly, 1992). Dried peptide aliquots were resuspended in 0.1% (v/v) acetic acid to a stock concentration of 50 µM and diluted to 1 µM in F-12/Dulbecco's minimal essential medium (F12/DMEM) (50/50, v/v) with 25 mM HEPES, 0.1% (w/v) bovine serum albumin, 0.1 mM isobutylmethylxanthine at pH 7.2 (stimulation medium). Serial dilutions were prepared in stimulation medium and prewarmed to 37 °C before use. Culture medium was aspirated prior to adding peptides in 0.5 ml of stimulation medium for 10 min at 37 °C. Reactions were stopped by adding of 0.5 ml of ice-cold 12% trichloroacetic acid to a final volume of 1 ml. For measurement of cGMP by radioimmunoassay (Biomedical Technologies, Inc.), samples were processed as described by Koller et al.(1991). For mAb inhibition, samples were diluted to a concentration of 1 µg/ml in stimulation medium and cooled to 4 °C. Cells in tissue culture plates were chilled to 4 °C, growth media were aspirated, and cold stimulation media with or without (control) antibodies were added and further incubated for 2 h at 4 °C. At the end of the incubation, one set of cultures was treated by aspirating the stimulation media and adding 1 ml of ice-cold 6% trichloroacetic acid. This corresponds to the T = 0 condition (Fig. 1). The remainder of the cells were divided into two treatment groups that did or did not have CNP added to a final concentration of 3 nM. These cultures were placed on a 37 °C aluminum plate for rapid warming. At the end of a 10-min incubation, the stimulation media were aspirated, 1 ml of 6% trichloroacetic acid was added, and samples were processed for cGMP radioimmunoassay as described above. For the mAb 3G12 concentration-response inhibition experiment, cells were incubated with serial dilutions of antibody as described above, prior to shifting to 37 °C and adding CNP to a final concentration of 3 nM. Cells were processed as described above for cGMP radioimmunossay.


Figure 1: Monoclonal antibody inhibition of NPR-B signal transduction. 293 cells expressing hNPR-B were incubated with antibody at 4 °C prior to shifting to 37 °C for 0 or 10 min (A, 0` and 10`). CNP was added to a concentration of 3 nM to one set of cultures prior to incubation at 37 °C for 10 min (B, 10` + 3 nM CNP). The mAb legend is shown at the top of the figure; control indicates no antibody addition. Results are the mean of duplicate determinations.



Inhibition of CNP Binding

The 293 hNPR-B cells were removed from plastic culture dishes with PBS, 0.5 mM EDTA, collected by centrifugation, and resuspended in PBS, 0.5% (w/v) bovine serum albumin, 0.02% (w/v) sodium azide at 4 °C. Four aliquots of 2 times 10^5 cells were used for each incubation with 10 µg/ml of either goat anti-mouse IgG as a control, mAb 3G12, or mAb 3G12 Fab for 2 h at 4 °C. Binding reactions were then initiated by adding I-Y-CNP (Koller et al., 1991) to a final concentration of 50 pM in a volume of 1 ml, either without or with (two reactions each) the addition of 0.5 µM CNP, to measure total and nonspecific binding, respectively. Samples were incubated at 4 °C overnight then harvested by vacuum filtration through a Whatman GF-B membrane pretreated with 1% (v/v) polyethyleneimine.

Fluorescence-activated Cell Sorting

Cells were removed from monolayer culture with PBS, 0.5 mM EDTA, collected by centrifugation, and resuspended in ice-cold PBS, 2.0% (v/v) calf serum. 2 times 10^5 cells in 100 µl were incubated on ice with neat cell culture supernatant for screening clones or with antibodies at a concentration of 10 µg/ml of IgG for the receptor specificity assay. Cells were washed by centrifugation three times in PBS, 2.0% (v/v) serum, and incubated with goat anti-mouse H+L (Fab`)2 phycoerytherin conjugate for 1 h. Cells were washed as above and analyzed by flow cytometry.

Collagenase Treatment of Rat Aorta

Male Sprague-Dawley Rats (200-300 g) were given a lethal intraperitoneal injection of sodium pentobarbitol (50 mg/kg). Thoracic aortae were removed and immediately placed in 1 times PBS. In each experiment with either rat CNP or ANP treatments, seven aortae were rinsed several times with 1 times PBS and the fascia removed gently with forceps. The aortae were placed in 10-cm culture dishes containing DMEM buffered with 25 mM HEPES at pH 7.4 (HEPES-DMEM) containing 300 µg/ml collagenase (Type 4, Worthington), 300 µg/ml soybean trypsin inhibitor (ICN), and 300 µg/ml bovine serum albumin (Sigma) at 37 °C and allowed to incubate for 10 min. The collagenase activity was inhibited by the addition of E-64 (Calbiochem) to a final concentration of 10 µM (Bond, 1989), and 1,10-o-phenanthroline (Sigma) to a final concentration of 0.1 mM (Salveson and Nagase, 1989).

The aortae were rinsed twice with 1 x PBS and the adventitia gently removed with microforceps. The muscle and adventitia were cut up into small pieces and placed into separate 10-ml aliquots of HEPES buffer pH 7.4, consisting of the following in mM concentrations shown in parentheses: HEPES (10), NaCl (148), KCl (5), CaCl(2) (2.4), MgCl(2) (2.1), and D-glucose (5.6). Both tissues were subjected to two brief low speed Polytron homogenizations (15 s on ice with 1 min between), then centrifuged at 1000 times g, and the pellets resuspended in 7 ml of cold HEPES buffer. 1 ml of each preparation was placed in two sets of six 12 times 75-mm polystyrene tubes at 37 °C. In each group for the CNP treatments, there were six treatments of one/tube: control + antibody vehicle, control + 2A8, control + 3G12, 30 nM CNP, 30 nM CNP + 2A8, and 30 nM CNP + 3G12. 30 min prior to the initiation of the assay the appropriate cells were treated with antibody (20 µg/ml) or antibody vehicle. 20 min later each tube was treated with isobutyl-methyl xanthine (final concentration 0.25 mM). After 10 min, either CNP or CNP-vehicle (1 times PBS) were given. In the experiments with ANP treatments, ANP was substituted for CNP. Cyclic GMP was allowed to accumulate over 5 min prior to termination of the reaction with an equal volume of 1 N perchloric acid. The tubes were stored at -20 °C until column purification of cGMP and radioimmunoassay (Hansborough and Garbers, 1981).

Vasorelaxation Studies

Thoracic aorta obtained from male Sprague-Dawley rats were cleaned of their fascia in a similar manner as that previously described. Two aortic rings were obtained from the anterior end of the thoracic aorta. Each ring was placed in a 5-ml tissue bath in oxygenated (95% O(2); 5% CO(2)) Krebs-bicarbonate buffer at 37 °C (Drewett et al., 1989), hanging between two wires, one attached to a stabilizing rod, and the other attached to a Grass FT03C force transducer. 1 g of resting tension was placed on each ring. Vascular responses were recorded on a Grass 7D Polygraph. To inhibit nonspecific protein binding to glass, all tissue baths were treated with 2% dichloro-dimethyl-silane in toluene for 2 min followed by five rinses with methanol. The baths were allowed to dry and rinsed several times with deionized water prior to the initiation of these experiments.

Each ring was allowed to equilibrate for 2 h at room temperature, prior to the initiation of KCl-induced contractions. The rings were given fresh buffer every 15-20 min over this preincubation period. Following preincubation, the rings were contracted with 40 mM KCl over 4 min followed by rinsing and a 2-min rest period. Once maximal responses to KCl were attained the rings were rinsed. One ring was treated with 2A8 (20 µg/ml) and the other with 3G12 (20 µg/ml). The rings were allowed to incubate in the presence of each antibody for 15 min prior to contraction with 100 nML-phenylephrine (Sigma). After 15 min the relaxant assays were initiated in both rings. Following treatment with the natriuretic peptide-vehicle (PBS), the rings were treated in a cumulative manner with 10 to 10M rat CNP or 10 to 10M rat ANP. Each concentration was given at least 4 min apart or until a stable base line was attained.


RESULTS

Monoclonal Antibodies against NPR-B

The goal was to develop receptor-specific antibodies to the native extracellular domain of NPR-B. Such antibodies might function as specific agonists or antagonists of the receptor. Using purified chimeric human B-IgG fusion protein (Bennett et al., 1991), mice were immunized using a complex of B-IgG and mouse anti-human Fc. A total of six clones, 2A8, 3G12, 4D2, 4E6, 6D8, and 6E5, that secrete mAbs recognizing the extracelluar domain of native hNPR-B were identified by flow cytometry. Evaluation of mAb agonist or antagonist activity was carried out by preincubating 293 hNPR-B cells with each mAb followed by stimulation of cGMP production using a submaximal concentration (3 nM) of CNP. One mAb, 2A8, potentiated CNP signal transduction (Fig. 1B) and did not recognize rNPR-B^3. This mAb was therefore used as a control in experiments on rNPR-B. Of the remaining five mAbs, none showed appreciable agonist activity alone (Fig. 1A), and each inhibited CNP-stimulated signaling of hNPR-B (Fig. 1B). The mAb 3G12 appeared to be the most effective CNP antagonist and was chosen to study NPR-B function.

Inhibition of CNP Binding

The inhibitory activity of 3G12 on hNPR-B could be explained by various mechanisms including inhibition of CNP binding. There is also the possibility that mAb bivalency is required for the inhibition of NPR-B. Preincubation of 293 hNPR-B cells in suspension with 3G12 or 3G12-Fab before addition of I-Y^0-CNP resulted in a reduction in the amount of specific binding (Fig. 2). At a protein concentration of 10 µg/ml, the monovalent Fab was less effective, 51% inhibition, than the intact bivalent mAb, 86% inhibition, possibly due to an avidity effect. This experiment is consistent with the hypothesis that 3G12 inhibits NPR-B signal transduction by antagonizing CNP binding.


Figure 2: mAb 3G12 inhibition of CNP Binding. 293 cells expressing hNPR-B were preincubated with goat anti-mouse H+L (Fab`)2 (control), 3G12, or 3G12 Fab prior to measurement of CNP specific binding. Total and background binding were each measured in duplicate. Results are expressed as the fraction of maximum specific binding in the presence of control antibody (B/B(max)). Average counts/min for total and nonspecific binding were 4488 and 1323 (control), 2252 and 1762 (3G12), and 3454 and 1795 (3G12 Fab), respectively



Receptor Subtype Specificity

Natriuretic peptide receptor specificity of 3G12 was assessed by flow cytometry of 293 cell lines expressing either hNPR-B, hNPR-A, or hNPR-C (Fig. 3). Peak fluorescence intensity only showed a positive shift for mAb 3G12 on hNPR-B. The expression of hNPR-A and hNPR-C was confirmed using rabbit polyclonal antisera (^2)raised against the respective extracellular domain-IgG fusion proteins. These antisera do not show cross-reactivity to other natriuretic peptide receptors. From this flow cytometry experiment, we conclude that mAb 3G12 is specific for NPR-B.


Figure 3: Receptor specificity of 3G12. Flow cytometry histograms are shown for 293 cells expressing human NPR-B, NPR-A, and NPR-C. For NPR-B the control panel is with an inactive antibody. Expression of NPR-A and NPR-C is demonstrated by sorting each with antibodies raised to IgG fusion proteins (Anti-AIgG and Anti-CIgG). Data are presented as relative cell number versus the logarithm of the phycoerythrin fluorescence intensity.



Inhibition of Rat NPR-B

The antagonistic activity of 3G12 suggested that this mAb could serve as a valuable reagent to determine the role of NPR-B in cellular signaling in various tissues. Although raised against the human receptor, initial experiments using flow cytometry demonstrated that 3G12 also reacted with rNPR-B^3. We therefore characterized in detail the concentration responses of CNP and 3G12 on a stable 293 rNPR-B cell line (Fig. 4) as a prelude to experiments with rat tissue. CNP had an ED of 2.2 nM for stimulation of cGMP production, whereas ANP and rBNP were very weak agonists (Fig. 4A). In the presence of 3 nM CNP, we measured concentration-dependent inhibition of cGMP production by 3G12 (Fig. 4B). Half-maximal inhibition was at approximately 0.2 µg/ml of 3G12, a value very similar to that obtained with the human receptor.^2 However, despite a clear concentration response relationship for inhibition, there was not complete inhibition, with approximately 10 pmol cGMP/10^5 cells. In contrast, unstimulated levels are typically less than 0.1 (see Fig. 1A). Lack of complete inhibition could be due to the mAb/receptor binding not going to equilibrium after 2 h at 4 °C. Alternatively, 3G12 may be a weak antagonist of NPR-B signal transduction.


Figure 4: CNP and 3G12 concentration responses on rNPR-B. The concentration-response of 293 rNPR-B cells is shown in A for CNP, rBNP, and ANP treatments. Inhibition of 3 nM CNP stimulated rNPR-B signal transduction with dilutions of 3G12 is shown in B.



Receptor Inhibition in Dissociated Rat Aorta

In an attempt to determine the distribution of NPR-B within rat aorta, collagenase treatment of segments of thoracic aorta was employed to facilitate separation of the muscle from the adventitia. Separation of the two layers was assessed by histology following tissue fixation in 10% buffered formalin (Fig. 5). By this treatment most of the adventitia was removed from the aortic vascular smooth muscle. Collagenase treatment identical to that used on rat aortae had no effect on the ability of ANP or CNP to increase cGMP levels in 293 cells over-expressing either rNPR-A and rNPR-B, and also did not alter the inhibitory effect of 3G12 on rNPR-B^3.


Figure 5: Histology of collagenase-treated aorta. Untreated aorta (panel A) is shown with the tunica media (M) and tunica adventitia (A). After treatment with collagenase (panel B), only tunica media (M) is present. Tissue samples were fixed in formalin, stained with hematoxylin and eosin, and photographed by bright field microscopy.



In both aorta adventitia and muscle preparations, the presence of NPR-B and NPR-A was indicated by the ability of 30 nM CNP or 30 nM ANP to induce significant increases in cGMP (Fig. 6). The control mAb 2A8 had no effect on the ability of CNP to increase cGMP levels.^2 MAb 3G12 blocked the ability of 30 nM CNP to increase cGMP concentrations in both the muscle (Fig. 6A) and adventitia (Fig. 6B). This result confirms the presence of the NPR-B in both aortic adventitia and vascular smooth muscle. Neither antibody had any effect on ANP-induced increases in cGMP concentrations in either the muscle or adventitia preparations (Fig. 6). The inability of the 3G12 antibody to block ANP effects is consistent with its selectivity for the NPR-B and suggests the presence of NPR-A or other natriuretic peptide receptors.


Figure 6: Stimulation of cGMP production in aortic media and adventitia. Preparations of rat cells from the smooth muscle media layer (A) and the fibroblast adventitia layer (B) were preincubated with control mAb 2A8 or NPR-B CNP antagonist 3G12 prior to stimulation with buffer (control), ANP, or CNP. Results are presented as mean ± standard error from five experiments. Antibody concentrations were 20 µg/ml.



3G12 Antagonism of Aortic Ring Relaxation

Because the presence of NPR-B within the aortic vascular smooth muscle was confirmed, experiments were designed to determine whether 3G12 could block the vasorelaxant effect of CNP in intact aortic rings. Preliminary experiments verified that neither 2A8 nor 3G12 had any effect on basal tension.^2 Tracings showing the effect of CNP on a pair of phenylephrine-contracted aortic rings from one rat are shown in Fig. 7. The lower portion of Fig. 7shows the relaxation to CNP in the presence of the control mAb 2A8. The upper tracing (Fig. 7) shows the effect of 3G12 to attenuate the ability of CNP to relax the phenylephrine-contracted aortic ring. The summary data for six of these experiments on paired rings is shown in Fig. 8. Data are presented as a percentage of maximal relaxation attained in the presence of peptide. The effect of CNP appears to be directly upon the smooth muscle, since either removal of the endothelium or treatment with 100 µM methylene blue, a compound thought to act as an NO-electron acceptor and inhibitor of NO synthase (Mayer et al., 1993), does not affect the response.^2 In the presence of either 2A8 (20 µg/ml) or 3G12 (20 µg/ml), CNP results in a concentration-dependent vasorelaxant effect on the precontracted aortae (Fig. 8A), based upon the results of linear regression analysis of mean contraction (% of control) versus log[CNP] (2A8: R = 0.965, p < 0.05; 3G12: R = 0.971, p < 0.05). The IC for CNP relaxation of phenylephrine-induced contractions in the presence of control mAb 2A8 is 10.7 ± 1.6 nM (Fig. 8A). Treatment with 3G12 results in an increase in the IC to 140 ± 26 nM. In contrast to the competitive inhibition for CNP relaxation, 3G12 has no effect on the relaxant activity of ANP in similar experiments (Fig. 8B), with an IC of approximately 1 nM in the presence of either 3G12 or 2A8.


Figure 7: Typical relaxation tracing for phenylephrine-contracted aorta. Samples were incubated with antagonistic mAb 3G12 (top) or control mAb (2A8) bottom prior to contraction with phenylephrine (PE, 100 nM). Relaxation was effected by increasing concentrations of CNP after treatment with vehicle control (VEH). Antibody concentrations were 20 µg/ml.




Figure 8: Concentration response for relaxation of aortic rings. These data summarize the results of multiple experiments performed as described in the legend for fig. 7. Results are presented as the mean ± standard error from six experiments.




DISCUSSION

The present study reports the identification of a monoclonal antibody (3G12) which binds NPR-B but does not bind to the other known natriuretic peptide receptors NPR-A and NPR-C. This antibody blocks the ability of CNP to elevate cGMP concentrations in cell lines overexpressing NPR-B and in aortic adventitia and smooth muscle. The presence of NPR-B in the adventitia is consistent with the observations of high levels of NPR-B expression in fibroblasts (Chrisman et al., 1993) and the effect of CNP on vascular remodeling following vessel injury (Furuya et al., 1993). The NPR-B-specific antibody 3G12 also antagonizes the effect of CNP on phenylephrine-precontracted rat aorta demonstrating the importance of this receptor in mediating smooth muscle relaxation.

For ANP, there is strong evidence that cGMP is the second messenger mediating increased glomerular filtration (Huang et al.,1986) and inhibition of renal tubular sodium reabsorption (Light et al., 1989; Stanton, 1991). Previous studies have suggested a likely role for guanylyl cyclase in mediating vascular smooth muscle relaxation based upon correlations of increases in intracellular cGMP concentrations with concomitant vasorelaxation (Waldman and Murad, 1987). However, there are not yet any specific pharmacological or biochemical agents developed that antagonize the increase in intracellular cGMP levels and the relaxant effect of guanylyl cyclase activators such as ANP, CNP, sodium nitroprusside, and morpholino-sydnonimine. Determining the mechanism of action of the natriuretic peptides has been hampered due to the lack of receptor antagonists which differentiate between NPR-A, NPR-B, and NPR-C. Several peptide analogs which block the action of ANP on NPR-A may still bind to NPR-C or exhibit other nonspecific effects (von Geldern et al., 1990; Delporte et al., 1992). Morishita and co-workers(1991) have reported the identification of a non-peptide, microbiol polysaccharide ANP receptor antagonist, HS-142-1. It has been used in several studies (Morishita et al., 1991; Sano et al., 1992; Ohyama et al., 1992; Imura et al., 1992; Stevens et al., 1994). This agent has been represented as specific for the ANP receptor (Morishita et al., 1991). However, a more careful examination of the literature revealed that it not only blocked the NPR-A but also the NPR-B. In rabbit aortic vascular smooth muscle cells, HS-142-1 blocked the generation of cGMP in response to ANP, and in intact rabbit aortic rings it also antagonized the vasorelaxant effects of ANP (Imura et al., 1992).

From their results Imura et al.(1992) conclude that guanylyl cyclase and the second messenger cGMP mediate the relaxant effects of the natriuretic peptides. This conclusion is supported by data indicating that activators of soluble and membrane guanylyl cyclase result in vasorelaxation and that membrane permeable analogs of cGMP mimic the vasorelaxant effect (Waldman and Murad, 1987; Lincoln and Cornwell, 1993). However, Imura et al.(1992) in their HS-142-1 studies do not point out other possibilities such as another portion of the guanylyl cyclase receptor may signal (i.e. the protein kinase-like domain), or interact with some other downstream effector(s) independent of the catalytic region. These possibilities are not eliminated in the present study either, but what is shown is that an NPR-B receptor-specific antibody can block a known action of CNP in vascular tissue.

Until this study it was unclear whether the NPR-B was present within aortic vascular smooth muscle. Previous studies showed the presence of NPR-B and NPR-A mRNA in whole aorta, together with ANP and CNP pharmacology consistent with these receptors (Suga et al., 1992c). Previously employed standard isolation procedures leave the adventitia intact and remove only the loose connective tissue on the outer surface of the vessel. Using histological examination we found that collagenase treatment could provide nearly complete removal of the adventitia layer, allowing for functional localization of NPR-B.

The results of the present study demonstrate that CNP has effects which are separable from those of ANP through specific activation of NPR-B. A recent study in human patients with congestive heart failure has shown that CNP can act to reduce peripheral forearm resistance in a manner distinct from ANP (Nakamura et al., 1994). Our results combined with those of Nakamura et al.(1994) suggest that CNP may be a physiological regulator of vascular tone and support the possibility that CNP acts as an endothelial cell-derived relaxing factor (Suga et al., 1992a) targeting NPR-B in vascular smooth muscle. If CNP acts as an endothelial cell-derived relaxing factor, it would seem necessary for some agent to result in its release to cause vasorelaxation. TGF-beta has been suggested as a potential stimulator of CNP synthesis, although this would likely be over a longer time period (i.e. requiring protein synthesis) (Suga et al., 1992a), compared to that observed with acetylcholine-mediated increases in the putative nitric oxide-containing endothelial cell-derived relaxing factor (Furchgott and Zawadzki, 1980). Some have suggested that bradykinin releases a non-nitric oxide, non-prostaglandin factor to mediate relaxation of canine arteries (Richard et al., 1990). Perhaps this novel factor is CNP.

In conclusion, we have developed a novel antibody which is able to block the NPR-B receptor. Unlike several of the other agents previously employed to antagonize the natriuretic peptide receptor family, this tool specifically blocks NPR-B. This is the first report showing that NPR-B mediates the vasorelaxant effect of CNP in rat aortic rings and provides strong evidence that activation of guanylyl cyclase is intimately associated with vascular smooth muscle relaxation.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
A portion of this work was performed while this author was an Associate with the Howard Hughes Medical Institute and Department of Pharmacology at the University of Texas Southwestern Medical Center, Dallas, Texas.

To whom correspondence should be addressed: David G. Lowe, Cardiovascular Research Dept., Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, CA. Tel.: 415-225-2738, Fax: 415-225-6327; lowe{at}gene.com.

(^1)
The abbreviations used are: ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; CNP, C-type natriuretic peptide; NPR, natriuretic peptide receptor; NPR-A, natriuretic peptide receptor-A; hNPR-A, human NPR-A; rNPR-A, rat NPR-A; NPR-B, natriuretic peptide receptor-B; hNPR-B, human NPR-B; rNPR-B, rat NPR-B; NPR-C, natriuretic peptide receptor-C; hNPR-C, human NPR-C; cGMP, cyclic 3`,5` guanosine monophosphate; mAb, monoclonal antibody; PBS, phosphate-buffered saline.

(^2)
J. G. Drewett, B. M. Fendly, D. L. Garbers, and D. G. Lowe, unpublished observations.


ACKNOWLEDGEMENTS

We greatly appreciate the technical assistance of Brian Bennett, Mike Mensing, Jim Porter, Jill Schoenfeld, Cecelia Green, and Deborah Miller; Dr. Nancy Gillett for histology, Allison Bruce for artwork; and Dr. Ted D. Chrisman for helpful discussions.


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