(Received for publication, September 15, 1994; and in revised form, December 19, 1994)
From the
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-. 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.
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). ()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.
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.
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 (2.4),
MgCl
(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
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
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
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).
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 10
M rat CNP or 10
to 10
M rat ANP. Each concentration
was given at least 4 min apart or until a stable base line was
attained.
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). Average counts/min for total and
nonspecific binding were 4488 and 1323 (control), 2252 and
1762 (3G12), and 3454 and 1795 (3G12 Fab),
respectively
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.
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.
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. 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.
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.
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-
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.