© 1995 American Heart Association, Inc.
Articles |
Regional Expression of Natriuretic Peptide Receptors During the Formation of Arterial Neointima in the Rabbit
From the Physiological Laboratory, Cambridge, England.
Correspondence to Dr J. Brown, Physiological Laboratory, Downing St, Cambridge, CB2 3EG, England.
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Abstract In vitro evidence suggests that natriuretic peptide receptors (NPR)-B and NPR-C inhibit vascular smooth muscle (VSM) proliferation. NPR-B is guanylate cyclase-coupled and selectively activated by C-type natriuretic peptide (CNP)-(1-22). NPR-C is not guanylate cyclase-coupled and, unlike NPR-B, avidly binds atrial natriuretic peptide (ANP)-(1-28) as well as CNP-(1-22). Here, we investigate these receptors during the VSM proliferation and neointimal formation found 5, 7, and 20 days after compressing the central ear artery of the rabbit. Receptors were mapped autoradiographically using [125I-Tyr0]CNP-(1-22), which binds NPR-B and NPR-C, and 125I-ANP-(1-28), which binds NPR-C and NPR-A, another guanylate cyclase-coupled receptor. Normal tunica media had NPR-B–like binding sites, and the level of these did not change significantly after compression. Consistent with this, CNP-(1-22) stimulated cGMP production equally with membranes from normal or damaged arteries and was more effective than ANP-(1-28). Neointima, which became evident 5 to 7 days after arterial damage, expressed NPR-C–like sites and no detectable NPR-B–like binding. NPR-C–like sites also appeared on the media for the first time between 5 and 7 days after compression. Immunohistochemistry for proliferating cell nuclear antigen revealed widespread mitosis in VSM at 5 days after compression, but mitosis was virtually restricted to the neointima at and beyond 7 days after compression. Thus, whereas levels of NPR-B did not change significantly after arterial injury and NPR-A was not detected, NPR-C–like receptors were unregulated as mitosis declined in the media and as a prominent neointima formed.
Key Words: natriuretic peptides • receptors • neointima
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Growth and remodeling of VSM is a key feature of diseases such as atherosclerosis and is crucial to restenosis after angioplasty.1 Several factors, including the natriuretic peptides, probably regulate these important processes.1 2 The heart secretes ANPs and BNPs into the plasma.3 Additionally, VSM itself may synthesize ANPs, and the vascular endothelium secretes two CNPs, the 22-amino acid CNP-(1-22) and the 53-amino acid CNP-(1-53), which contains the sequence of CNP-(1-22) at its carboxylic acid terminal.4 5 6 ANP-(1-28) is the major circulating form of ANP,3 and both ANP-(1-28) and CNP-(1-22) inhibit the hyperplastic responses of cultured adult VSM to serum and growth factors.2 7 8 9 Moreover, ANP-(1-28) inhibits the hypertrophic effects of angiotensin II and transforming growth factor-ß on postconfluent VSM cultured from adult rat aorta.10 The effects of BNPs and CNP-(1-53) on the growth of VSM are not known. In explaining the trophic actions of the natriuretic peptides, attention has focused on guanylate cyclase-coupled NPRs2 6 7 8 because cGMP powerfully inhibits VSM hyperplasia and hypertrophy.11 However, recent work suggests that the antiproliferative effect of natriuretic peptides on cultured VSM may not correlate with their power to stimulate cGMP production.2 9
Two groups of high-affinity receptors for the natriuretic peptides are known. The first consists of two proteins, designated NPR-A and NPR-B. These proteins are monomers of 120 and 130 kD, which have been cloned and shown to contain constitutive guanylate cyclase domains.12 NPR-A and NPR-B are conservative in their ligand selectivity. NPR-A has high affinity for ANP(1-28) but virtually none for CNP-(1-22) or for synthetic analogues of ANP-(1-28) such as des[Gln18,Ser19 ,Gly20,Leu21,Gly22]ANP-(4-23)-amide (C-ANP).12 13 14 15 16 17 18 19 20 NPR-B has high affinity for CNP-(1-22) but little for ANP-(1-28)12 13 14 15 16 17 18 ; its affinity for C-ANP has not been established. The other group of high-affinity receptors for natriuretic peptides is characterized by a disulfide-linked homodimeric protein of 60 to 70 kD units. This protein has been cloned from the cow, lacks any recognized constitutive enzymatic domain, and is designated NPR-C.12 Cloned NPR-C binds a wide range of ligands, including ANP-(1-28), CNP-(1-22), and C-ANP, all with high affinities.12 15 16 It has yet to be demonstrated that NPR-C can modulate any intracellular event when it is expressed by transfection into heterologous cells.12 Nevertheless, C-ANP competitively inhibits the cellular binding and internalization of ANP-(1-28) for lysosomal destruction in a number of tissues.12 20 As a result, cloned NPR-C has been identified as the clearance receptor of ANP-(1-28).12 Recently, however, two NPR-C–like proteins have been identified in the rat.20 These have similar apparent molecular masses, and both bind ANP-(1-28) as well as C-ANP. One of these proteins has very low affinities for CNPs and is probably the natriuretic peptide clearance receptor. The other protein has much higher affinities for CNPs and may be coupled to the inhibition of cellular levels of cAMP. Consequently, ligands such as C-ANP, previously thought to be specific for NPR-C, bind to a mixture of NPR-C–like receptor proteins.20
The mitotically quiescent VSM of adult rat aorta contains mRNA for both NPR-A and NPR-B.21 VSM subcultured from adult rat aortic media expresses mRNA for NPR-B but little or none for NPR-A.21 Consistent with this, CNP-(1-22) stimulates cGMP production and inhibits proliferation of subcultured medial VSM more powerfully than does ANP-(1-28).7 8 Exogenous CNP-(1-22) also inhibits neointimal formation in vivo.22 Such facts, together with the endothelial synthesis of CNP-(1-22),5 6 have led to the suggestion that NPR-B is selectively expressed by dividing VSM in vivo and that CNP-(1-22) acts through this receptor as a paracrine regulator of VSM hyperplasia.6 However, CNP-(1-22) has recently been found to inhibit the growth of subcultured, adult, medial VSM through NPR-C–like receptor sites rather than through the guanylate cyclase–coupled NPR-B.9 Moreover, in vitro observations of receptor expression may not translate to the in vivo situation. The population of VSM cells that proliferate from adult media in culture may be different from that involved in the proliferative response to arterial injury in vivo.23 24 25 In any case, humoral factors and interactions between different types of cell, which are absent in vitro, may modify VSM receptor expression or activity in vivo. Nothing is known of the NPRs of proliferating VSM in vivo. Consequently, we have investigated the local NPRs after a compressive injury to the central artery of the rabbit ear. We studied binding with [125I-Tyr0]CNP-(1-22) and 125I-ANP-(1-28), radioligands that respectively share the receptor selectivities of CNP-(1-22) and ANP-(1-28).15 16 17 18 19 20 26 We also examined the effects of natriuretic peptides on membrane-bound guanylate and adenylate cyclases. The results show that an NPR-C–like receptor with high affinities for both CNPs and ANPs is heavily expressed by the neointima and that this type of arterial injury does not detectably alter other classes of NPR.
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Materials
Unlabeled peptides were obtained from Peninsula Laboratories Ltd, as was [125I-Tyr0]CNP-(1-22) (specific activity, 1000 to 1400 Ci/mmol). 125I-ANP-(1-28) (specific activity, 2000 Ci/mmol) was obtained from Amersham Ltd, as were 125I standards and Hyperfilm 3H for autoradiography. Kits for radioimmunoassay of cAMP and cGMP were also from Amersham. NTB3 photographic emulsion was from Kodak Ltd. Hypnorm was obtained from Janssen Pharmaceutical Ltd. Rabma pellet rabbit food was from SDS Ltd. D-19 developer was from Kodak Ltd, and Amifix fixative was from Champion Photochemistry Ltd. Antibodies were from Dako Ltd. All other reagents were from Sigma Chemical Co.
Preparation of Arterial Lesions
All animal experiments were in accord with the guidelines for
animal care of the University of Cambridge and had Home Office
permission. Arterial lesions were prepared according to the
technique of Banai et al,27 with slight modifications.
Male New Zealand White rabbits (3 to 3.5 kg) were anesthetized
with 0.5 mL/kg IM Hypnorm (fentanyl citrate [0.315 mg/mL] and
fluanisone [10 mg/mL]). Segments of the central artery of one ear
were compressed between polyethylene disks (10x5x2 mm) applied on
either side. The disks and intervening artery were compressed for 45
minutes by Kelly clamps (Biomedical Research Instruments, Inc). Three
sites, equally spaced in the middle third of the ear, were compressed.
The other ear in each rabbit provided undamaged artery from positions
corresponding to the compressed segments in the first ear. On
recovering from the anesthetic, rabbits were allowed free access to
water and food (Rabma pellets). Rabbits were killed 5, 7, or 20 days
later by an overdose of pentobarbital (50 mg/kg IV), and the
arterial segments were used as described below.
Quantitative In Vitro
Autoradiography
We have previously described this technique in
detail.19 26 Briefly, 15-µm frozen sections of ear
tissue containing the lesioned and control arterial
segments were prepared from 6 rabbits each on the 5th and 7th days
after compression and from 7 rabbits on the 20th day after compression.
These sections were used for binding studies with
[125I-Tyr0]CNP-(1-22). Comparable sections
were obtained from a further 19 rabbits (6 on the 5th day, 6 on the 7th
day, and 7 on the 20th day after compression) for parallel studies with
125I-ANP-(1-28). Sections were preincubated at 20°C for
10 minutes in 50 mmol/L Tris-HCl (pH 7.4) containing (mmol/L) NaCl 100,
MgCl2 5, and phenanthroline 1. Sections were then immersed
in 150 mmol/L sodium chloride plus 0.5% acetic acid (pH 5.0) at 4°C
for 10 minutes. This step dissociates natriuretic peptides
from their receptors.19 20 26 The sections were
subsequently washed in three changes of preincubation buffer, each for
2 minutes at 4°C, before being incubated with either 250 pmol/L
[125I-Tyr0]CNP-(1-22) or 100 pmol/L
125I-ANP-(1-28) in fresh preincubation buffer plus 0.5%
(wt/vol) BSA (fraction V) at 20°C for 30 minutes. Competitive
inhibition of radioligand binding by unlabeled peptides was
assessed in consecutive sections by including various concentrations (1
pmol/L to 1 µmol/L) of unlabeled CNP-(1-22), ANP-(1-28), or C-ANP.
Sections from each rabbit were also incubated with
radioligand plus 1 µmol/L each CNP-(1-22) and ANP-(1-28),
radioligand plus 1 µmol/L each CNP-(1-22) and C-ANP, or
radioligand plus 1 µmol/L each ANP-(1-28) and C-ANP.
Binding of [125I-Tyr0]CNP-(1-22) in the
presence of 1 µmol/L CNP-(1-22) or binding of
125I-ANP-(1-28) in the presence of 1 µmol/L ANP-(1-28)
was considered to be nonspecific. The amino acid sequences of the
peptides used were those of the rat peptides. Preliminary studies
established that the specific binding of both radioligands
had achieved equilibrium by 30 minutes under these conditions of
incubation. Specificity of binding was assessed in the presence of
angiotensin II, vasopressin, or neuropeptide Y (all 1
µmol/L). Radioligand and ligand degradation during
incubation was measured in the incubation fluids at the end of the
incubations by using HPLC with detection by gamma counts or absorption
at 210 nm, as previously described.20 26 At the end of the
incubations, sections were washed in preincubation buffer for 5 minutes
at 4°C and rinsed with distilled water at 4°C before being
air-dried for exposure to Hyperfilm 3H with
125I standards.19 26
Autoradiograms were developed as described
previously.19 26 Sections were then fixed in formaldehyde
and stained with hematoxylin and eosin. Binding was measured
microdensitometrically, comparing autoradiograms of
sections and 125I standards.19 26 Regional
binding was assessed by measuring the dimensions of the lumen, media,
and any neointima as separated from the media by the
internal elastic lamina for each stained section. These dimensions were
then used to separate medial and any neointimal regions on
the corresponding autoradiograms. Specific gravity was
measured gravimetrically, and protein content was analyzed from
six further control arterial segments. These measurements
were used to convert values of bound ligand from femtomoles per square
millimeter to femtomoles per milligram protein.19 26
Binding of [125I-Tyr0]CNP-(1-22) was also assessed in sections from the control and damaged ear arteries of another 6 rabbits 20 days after compression. These sections were incubated with increasing concentrations (0 to 1 nmol/L) of [125I-Tyr0]CNP-(1-22), with or without 1 µmol/L CNP-(1-22).
Further sections from each rabbit were treated as above but incubated with each radioligand in the presence or absence of a single concentration (1 µmol/L) of CNP-(1-22), ANP-(1-28), or C-ANP. Once these sections were washed, they were fixed in Bouin’s solution for 30 seconds at 4°C, washed in distilled water for 1 minute at 4°C, and dried. They were then dipped in Kodak NTB3 photographic emulsion and kept in darkness at 4°C. After exposure, the emulsion was developed by immersing each slide in Kodak D-19 developer for 4 minutes and then fixative for 10 minutes at 20°C. Slides were washed in a stream of tap water for 10 minutes and counterstained with hematoxylin and eosin.
Immunohistochemistry of PCNA
Eighteen more male New Zealand White rabbits were killed, 6 at 5
days, 6 at 7 days, and 6 at 20 days after arterial
compression. The crushed and control arterial segments were
fixed in formaldehyde and embedded in paraffin. Sections (10 µm) were
cut every 100 µm along the length of the crushed segments to
determine the midpoint of each lesion. The midpoint sections were
dewaxed and rehydrated in graded ethanols. Immunohistochemistry was
performed as previously described.28
Endogenous peroxidases were quenched with 1% (vol/vol)
H2O2 in absolute methanol for 30 minutes.
Sections were then rinsed in PBS (pH 7.2) and incubated with mouse
anti-PCNA monoclonal antibody diluted 1:200 for 20 hours at 20°C.
Sections were then washed in PBS (pH 7.2) and incubated with
peroxidase-coupled rabbit anti-mouse IgG diluted 1:150
for 1 hour at 20°C. Sections were again washed in PBS (pH 7.2), and
bound secondary antibody was localized by incubating each section with
0.06% diaminobenzidine (wt/vol) in PBS (pH 7.2) containing 0.03%
H2O2 (vol/vol) for 8 minutes at 20°C.
Sections were counterstained with iron hematoxylin, dehydrated in
graded ethanols, cleared in xylene, and mounted in DPX. Areas on
sections were measured by microplanimetry, and PCNA-positive nuclei
were counted per square millimeter.
Affinity Cross-linking
Arteries from groups of three rabbits were excised 20 days after
injury, pooled, and homogenized in HBSS (mmol/L: NaCl 137,
KCl 5.4, MgSO4 0.4, Na2HPO4 0.34,
CaCl2 1.26, NaHCO3 4.17,
KH2PO4 0.44, and MgCl2 0.49) plus
10 mmol/L HEPES, 1 mmol/L phenanthroline, 1 mmol/L phenylmethylsulfonyl
fluoride, 0.1 mg/mL aprotinin, 10 µmol/L leupeptin, and 0.1
mg/mL bacitracin (pH 7.2) at 4°C. Homogenates were
centrifuged at 1200g for 10 minutes at 4°C. The
supernatants were recentrifuged at 40 000g for
60 minutes at 4°C. The resulting pellets were washed three times with
1 mL of PBS containing 1 mmol/L phenanthroline, 1 mmol/L
phenylmethylsulfonyl fluoride, 0.1 mg/mL aprotinin, 10 µmol/L
leupeptin, and 0.1 mg/mL bacitracin (pH 7.2) (solution B) and then
resuspended in this solution at 1 mg protein/mL for storage at
-80°C. Aliquots (120 µg) of membrane protein were incubated in 0.5
mL of solution B plus 0.2% gelatin and 100 pmol/L
125I-ANP-(1-28), with or without 1 µmol/L unlabeled
ANP-(1-28), CNP-(1-22), or C-ANP for 1 hour at 4°C. Membranes were
also incubated with radioligands plus 1 µmol/L
angiotensin II, vasopressin, or gastrin. Bound ligand was
cross-linked to its receptor by 1 mmol/L bis(sulfosuccinimidyl)
suberate for 40 minutes at 4°C. Cross-linking was stopped, and
membranes were dissolved in SDS with or without 50 mmol/L
dithiothreitol as described previously.20 Solubilized
samples and standard proteins (myosin, 205 kD; ß-galactosidase,
116 kD; phosphorylase B, 97.4 kD; BSA, 66 kD;
ovalbumin, 45 kD; and carbonic anhydrase, 29 kD) were
resolved by SDS-PAGE using a discontinuous buffer system on 7.5%
acrylamide separating gels. Gels were stained by Coomassie
blue, and autoradiograms were prepared on GRI-AX film
as previously described.20
cGMP Assays
Six more male New Zealand White rabbits (3 to 3.5 kg) were
killed 5 days after arterial compression, and another 6
were killed 20 days after arterial compression. Crushed and
control arterial segments were isolated by dissection.
Arterial tissue from different rabbits was treated
separately, so that a complete set of cGMP assays was performed for
each rabbit. Crushed and control arterial segments from
each rabbit were respectively pooled, finely diced, and
homogenized at 4°C in PBS (pH 7.4) by three 30-second
bursts of 27 000 rpm (Polytron), with each burst separated by 30
seconds. Each homogenate was centrifuged at
1200g 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 mmol/L
Tris-HCl (pH 7.4) and resuspended in this solution at 1 mg membrane
protein per milliliter. Aliquots of 10 µg protein of the suspension
were incubated for 20 minutes at 37°C in a final volume of 250 µL
of 50 mmol/L Tris-HCl (pH 7.6) containing 1 mmol/L
isobutylmethylxanthine, 4 mmol/L
MgCl2, 0.5 mmol/L ATP (Mg2+
salt), 1 mmol/L GTP (Mg2+ salt), 15 mmol/L creatine
phosphate, and 80 µg/mL creatine phosphokinase, plus a range of
concentrations (0 to 10 µmol/L) of either CNP-(1-22) or ANP-(1-28).
Incubations with CNP-(1-22) were paired to those with ANP-(1-28) for
each rabbit. Single incubations were performed for each concentration
of each peptide for each rabbit. Incubations were stopped by adding
0.75 mL of 50 mmol/L sodium acetate (pH 5.8) (ice cold) and boiling for
4 minutes. Samples were then centrifuged at 4000g
for 10 minutes at 4°C. cGMP was measured in supernatants by
radioimmunoassay.19 26 All samples from one rabbit were
measured in one assay. Ligand degradation during incubations of 10
µmol/L CNP-(1-22) or 10 µmol/L ANP-(1-28) was measured by HPLC,
detecting UV absorption of the natriuretic peptides at 210
nm, as previously described.20 Preliminary experiments
showed that basal and natriuretic peptide-stimulated
cGMP production by membranes from crushed or control arteries
increased linearly with time up to at least 30 minutes and with the
dose of membrane protein up to at least 20 µg.
cAMP Assay
Another 6 male New Zealand White rabbits (3 to 3.5 kg) were
killed 20 days after arterial compression, as described
above. Membranes were individually prepared from the crushed and
control arterial segments of each rabbit as described
above, and aliquots of 7.5 µg of membrane protein were incubated for
12 minutes at 37°C in a final volume of 200 µL of 50 mmol/L
Tris-HCl (pH 7.5) containing 1 mmol/L
isobutylmethylxanthine, 6.4 mmol/L
MgCl2, 0.02 mmol/L guanosine triphosphate
(Mg2+ salt), 1 mmol/L ATP (Mg2+
salt), 0.1 mg/mL myokinase, 0.2 mg/mL creatine phosphokinase, and 20
mmol/L creatine phosphate with or without 50 µmol/L forskolin and
with or without 1 µmol/L CNP-(1-22), 1 µmol/L ANP-(1-28), or 1
µmol/L C-ANP. One incubation was made for each combination of added
reagents for each rabbit, and all incubations for one rabbit were
performed as a single experiment. Incubations were stopped by adding
ice-cold trichloroacetic acid (final concentration, 6% [wt/vol])
and centrifuging at 4000g for 15 minutes at 4°C.
Supernatants were extracted with water-saturated ether, and
supernatant cAMP was then measured by radioimmunoassay.20
All measurements from one rabbit were performed in a single assay.
Peptide degradation during incubations was measured by HPLC and UV
detection as described above. Preliminary experiments showed that basal
and forskolin-stimulated cAMP production by membranes from
both crushed and control arteries increased linearly with time up to at
least 30 minutes and with the dose of membrane protein up to at least
20 µg.
Protein Assay
Protein contents were measured by a bicinchoninic acid assay
(Sigma).
Data Analysis
Apparent association constants (Ka) and
maximum binding capacities (Bmax) were estimated by
using the LIGAND program.29 Models with up to
three classes of binding sites were compared by the extra
sum-of-squares principle.29 A model with more than
one class was accepted over a simpler model if the F-test criterion
showed that the more complex model improved the goodness of fit to the
data more than expected by chance at the P>.05
level.29 Estimates of Ka are more
nearly log-normally than normally distributed about their true
mean.30 Consequently, each value of
Ka (liters per mole) is given as
-log10 (pKa) of the geometric mean
and its SEM.20 30 For reference, values of
Ka are also given in Tables 1 through 3
as their
reciprocal, the dissociation constant (Kd).
Other results are arithmetic mean±SEM. Dose-response curves were
modeled as logistics by the ALLFIT program, and their
parameters were compared by the extra
sum-of-squares principle using
ALLFIT.31 Remaining results were compared by
paired or unpaired t tests, as
appropriate.32
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Development of Neointima
The neointima developed as originally described27 for this preparation. Control arteries had no cells between the endothelium and internal elastic lamina. The neointima was still absent at many points around the arterial lumen 5 days after arterial damage but was three to nine cells thick by 7 days and had greatly narrowed the lumen by 20 days (Figs 1
and 2). PCNA immunoreactivity was used as a
marker of the S phase of mitosis.28 Control arteries
contained no PCNA-positive cells. Smooth muscle nuclei
(475±104/mm2) of sectioned media were PCNA positive 5 days
after injury, but only 24±16/mm2 were positive at 7 days.
PCNA-positive nuclei were virtually absent in media at 20 days (Fig 1).
PCNA-positive nuclei (2330±300/mm2) occurred in 7-day
neointima, and 34±10/mm2 were present in
neointima at 20 days (Fig 1).
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Autoradiographic Binding
Studies
Control Arteries
The binding of 250 pmol/L
[125I-Tyr0]CNP-(1-22) was inhibited
differently by 1 µmol/L CNP-(1-22), 1 µmol/L ANP-(1-28), or 1
µmol/L C-ANP (Fig 3). Further information suggested
that this resulted from the substantially lower affinities of
ANP-(1-28) and C-ANP compared with CNP-(1-22) for a single class of
binding sites. Thus, there were no significant differences between the
inhibitions of the binding of 250 pmol/L
[125I-Tyr0]CNP-(1-22) by 1 µmol/L
CNP-(1-22), 1 µmol/L CNP-(1-22) plus 1 µmol/L ANP-(1-28), or 1
µmol/L CNP-(1-22) plus 1 µmol/L C-ANP (not shown). This implies
that neither ANP-(1-28) nor C-ANP inhibited nonspecific
radioligand binding.19 20 26 Moreover, the
inhibitions of specific radioligand binding by increasing
concentrations of CNP-(1-22), ANP-(1-28), or C-ANP were all
consistent with a single class of sites (P>.24 for
improvements of fit by two-class versus one-class models for
each ligand) (Fig 4). This class bound CNP-(1-22),
ANP-(1-28), and C-ANP with Bmax values that did not differ
significantly from each other. It was avid for CNP-(1-22) and less so
for ANP-(1-28) (P<.01), and it bound C-ANP even less
strongly than ANP-(1-28) (P<.01) (Fig 4, Table 1
). Its low affinity for ANP-(1-28) was confirmed in
that no high-affinity binding of ANP-(1-28) contributed detectably
to the low level of specific binding of 100 pmol/L
125I-ANP-(1-28) on control media (Fig 5, Table 2). These ligand selectivities are
consistent with the presence of NPR-B in the normal tunica
media of the ear artery of the rabbit.
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Arterial Media 5 Days After Compression
As with control arteries, the binding of 250 pmol/L
[125I-Tyr0]CNP-(1-22) was inhibited
differently by 1 µmol/L CNP-(1-22), 1 µmol/L ANP-(1-28), or 1
µmol/L C-ANP (Fig 3), but there were no significant differences
between the inhibitions of the binding of 250 pmol/L
[125I-Tyr0]CNP-(1-22) by 1 µmol/L
CNP-(1-22), 1 µmol/L CNP-(1-22) plus 1 µmol/L ANP-(1-28), or 1
µmol/L CNP-(1-22) plus 1 µmol/L C-ANP (not shown). The effects of
increasing concentrations of CNP-(1-22), ANP-(1-28), and C-ANP on
specific radioligand binding were all consistent
with a single class of sites (P>.30 for improvements of fit
by two-class versus one-class models for each ligand) (Fig 4).
This class bound CNP-(1-22), ANP-(1-28), and C-ANP with
Bmax values that did not differ significantly from each
other. The binding constants for the unlabeled ligands did not differ
significantly from their constants in control arteries (Table 1). Thus,
ANP-(1-28) bound less strongly than CNP-(1-22) (P<.01), and
C-ANP bound even less well than ANP-(1-28) (P<.01).
Finally, no high-affinity binding of ANP-(1-28) was detected 5 days
after compression with 100 pmol/L 125I-ANP-(1-28) as the
radioligand (Fig 5, Table 2). Consequently,
the medial binding of natriuretic peptides 5 days after
injury does not differ significantly from that of normal arteries and
resembles NPR-B.
Arterial Media at 7 and 20 Days After
Compression
Only NPR-B–like sites were detected on normal media and on media
5 days after compression, but NPR-C–like sites were added at 7 and 20
days after injury. Thus,
[125I-Tyr0]CNP-(1-22) labeled two classes of
medial sites at both 7 and 20 days after injury. Both classes at each
time had similarly high affinities for CNP-(1-22) but differed in their
binding of ANP-(1-28) (P<.05 versus a single class at both
times) and C-ANP (P<.05 versus a single class at both
times) (Fig 4, Table 1). One class at each time matched the NPR-C–like
sites of neointima in having high affinities for ANP-(1-28)
and C-ANP. The affinities of this medial class exceeded those of the
NPR-B–like sites of, for example, 20-day control media for these
ligands (P<.01 for either ligand at both 7 and 20 days).
The other class of sites on lesioned media at both 7 and 20 days had
affinities for ANP-(1-28) and C-ANP that were significantly lower than
those of the NPR-C–like sites of 20-day neointima
(P<.05 and P<.01 for either ligand at 7 and 20
days, respectively). This class resembled the NPR-B–like sites of
control media in its affinities for ANP-(1-28) and C-ANP. The
Bmax of the medial classes of NPR-B–like binding at 5, 7,
and 20 days after compression tended to be greater than for control
media (Table 1). However, this Bmax for injured media at
any one time did not differ significantly from that of contemporary
control media. Finally, the specific binding of increasing
concentrations of [125I-Tyr0]CNP-(1-22) to
media 20 days after injury identified only one class of sites
(P>.23 for improvement of fit by two-class versus
one-class models) (Fig 6). This class had an
affinity for the radioligand
(pKa=9.01±0.63) that did not differ
significantly from the affinity of media for CNP-(1-22) (Table 1).
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The effects of increasing concentrations of ANP-(1-28), CNP-(1-22), or
C-ANP on the specific binding of 125I-ANP-(1-28) were
consistent with one class of sites at both 7 days
(P>.14 for improvements of fit by two-class versus
one-class models for each ligand) and 20 days (P>.45
for improvements of fit by two-class versus one-class models
for each ligand) (Fig 5). These sites at both 7 and 20 days after
injury bound CNP-(1-22), ANP-(1-28), and C-ANP with high affinities
(Fig 5, Table 2). At neither time did the Bmax values for
ANP-(1-28), CNP-(1-22), and C-ANP differ significantly from each other
(Table 2), nor were there significant differences in the inhibitions of
the binding of 100 pmol/L 125I-ANP-(1-28) produced by 1
µmol/L ANP-(1-28) alone, 1 µmol/L ANP-(1-28) plus 1 µmol/L
CNP-(1-22), or 1 µmol/L ANP-(1-28) plus 1 µmol/L C-ANP (not shown).
These results with 125I-ANP-(1-28) confirm that NPR-C–like
sites occur on the media at 7 and 20 days after injury.
Neointima
Specific binding of
[125I-Tyr0]CNP-(1-22) occurred throughout the
neointima but tended to be most concentrated near the
arterial lumen by 20 days (Figs 2 and 3).
Neointimal binding of 250 pmol/L
[125I-Tyr0]CNP-(1-22) at 20 days was not
inhibited differently by 1 µmol/L CNP-(1-22), 1 µmol/L CNP-(1-22)
plus 1 µmol/L ANP-(1-28), or 1 µmol/L CNP-(1-22) plus 1 µmol/L
C-ANP (not shown). Moreover, the inhibitions of specific
radioligand binding caused by increasing concentrations of
CNP-(1-22), ANP-(1-28), or C-ANP were consistent with a single
class of sites on the neointima as a whole
(P>.40 for improvements of fit by two-class versus
one-class models for each ligand) (Fig 7). This
class of sites bound CNP-(1-22), ANP-(1-28), and C-ANP with high
affinities and with Bmax values that did not differ
significantly from each other (Fig 7, Table 3). This was
confirmed by the specific binding of 125I-ANP-(1-28), which
was also almost uniformly distributed across the 20-day
neointima. Thus, there were no significant differences
between the inhibitions of the binding of 100
pmol/L125I-ANP-(1-28) caused by 1 µmol/L ANP-(1-28), 1
µmol/L ANP-(1-28) plus 1 µmol/L CNP-(1-22), or 1 µmol/L
ANP-(1-28) plus 1 µmol/L C-ANP (not shown). Moreover, the inhibitions
of the specific binding of 125I-ANP-(1-28) produced by
increasing concentrations of ANP-(1-28), CNP-(1-22), or C-ANP were
consistent with one class of sites (P>.32 for
improvements of fit by two-class versus one-class models for
each ligand), which bound these unlabeled ligands with high affinities
and Bmax values that did not differ significantly from each
other (Fig 7, Table 3). Finally, there was no significant difference
between the Bmax for ANP-(1-28) measured by its
displacement of 125I-ANP-(1-28) and that for CNP-(1-22)
measured with [125I-Tyr0]CNP-(1-22) (Table 3).
|
The specific binding of increasing concentrations of
[125I-Tyr0]CNP-(1-22) revealed a single class
of neointimal sites (P>.45 for improvement of
fit by two-class versus one-class models), with an affinity for
the radioligand of pKa=9.63±0.07
(Fig 6). This did not differ significantly from the
neointimal affinity for CNP-(1-22) (Table 3). The affinity
of 20-day neointima for CNP-(1-22)
(pKa=9.52±0.24) was similar to that of the
NPR-B–like sites of undamaged media (eg,
pKa=9.18±0.74 for undamaged arteries of the
20-day control group), but the neointima had higher
affinities for ANP-(1-28) (pKa=9.72±0.21 versus
7.51±0.30 for 20-day controls, P<.01) and C-ANP
(pKa=9.68±0.14 versus 6.71±0.50 for 20-day
controls, P<.01). Neointimal binding 20 days
after injury was therefore consistent with NPR-C rather than
NPR-B or NPR-A.
The neointima did not encircle the lumen at 5 days after injury and was still thin at 7 days. However, it was possible to measure the binding of [125I-Tyr0]CNP-(1-22) in the 2 rabbits with the thickest neointima at 7 days. In both, CNP-(1-22), ANP-(1-28), and C-ANP bound with uniformly high affinities as though to a single class of NPR-C–like sites similar to that of the neointima at 20 days (not shown).
Ligand Degradation During Binding Studies
Measurements showed that 98.1±0.3% of the initial amount of
[125I-Tyr0]CNP-(1-22) and 97.8±0.4% of the
initial amount of 125I-ANP-(1-28), added to sections from
arteries injured 20 days before, survived the binding incubations.
Similarly, 96.8±0.3% of CNP-(1-22) and 97.3±0.3% of ANP-(1-28)
survived incubation after being added initially as 1 µmol/L
solutions.
SDS-PAGE of Arterial Membrane Proteins
One protein was specifically labeled by
125I-ANP-(1-28) in membranes prepared from whole arteries
20 days after injury. The reduced protein had an
Mr of 
64 000, and its labeling was prevented
by 1 µmol/L unlabeled ANP-(1-28), CNP-(1-22), and C-ANP (Fig 8) but not 1 µmol/L angiotensin II,
vasopressin, or gastrin (not shown). An additional band, with an
apparent Mr of 130 000, was specifically
labeled by 125I-ANP-(1-28) in the absence of
dithiothreitol, and the 64-kD band became less heavily labeled (Fig 8). No proteins were detectably labeled by 125I-ANP-(1-28)
under the same conditions in membranes from control arteries. The
results affirm that injured arteries preferentially express a protein
with ligand selectivities and molecular characteristics of the
disulfide-bridged homodimeric NPR-C.
|
Cyclic Nucleotide Production
Generation of cGMP
CNP-(1-22) produced dose-dependent increments in the rate of
cGMP production by membranes prepared either from control
arteries or from arteries 20 days after injury (Fig 9).
Basal cGMP production, as estimated by ALLFIT, was
higher in the lesioned arteries (1.4±0.2 versus 1.1±0.2 pmol/mg
protein per minute, P<.05). Taking this into account, the
maximum increments in cGMP production caused by CNP-(1-22) did
not differ significantly between control and lesioned arteries (Fig 9).
Moreover, the concentration of CNP-(1-22) that achieved 50% of the
maximum increment in particulate cGMP production
(EC50) was not significantly different between control
(1.1x10-7 mol/L) and damaged (3.2x10-8
mol/L) arteries. ANP-(1-28) also caused dose-related increases in
cGMP production (Fig 9). Basal cGMP production
estimated by ALLFIT in the experiments with ANP-(1-28) was
1.0±0.2 and 1.6±0.3 pmol/mg protein per minute for control and
damaged arteries, respectively. These were not significantly different
from the corresponding basal rates in the experiments with CNP-(1-22),
but the maximum rates of cGMP production for control and
damaged arterial membranes with ANP-(1-28) (2.0±0.4 and
2.4±0.8 pmol/mg protein per minute, respectively) were significantly
lower than those with CNP-(1-22) (3.1±0.6 and 3.4±0.6 pmol/mg protein
per minute, respectively) [both P<.01 for ANP-(1-28)
versus CNP-(1-22)]. The results suggest that normal arteries and
arteries 20 days after injury express similar levels of the
CNP-stimulated cyclase-coupled NPR-B. This is consistent
with the autoradiographic binding data, which show that
injury does not significantly alter the level of arterial
NPR-B–like binding sites.
|
Generation of cAMP
Basal rates of cAMP production and the increments achieved
by 50 µmol/L forskolin were similar for membranes from control
arteries and arteries 20 days after injury. Neither basal nor
forskolin-stimulated rates were significantly affected by
CNP-(1-22), ANP-(1-28), or C-ANP, whatever the source of membranes (Fig 10).
|
Agonist Degradation During Enzymatic Studies
When 10 µmol/L CNP-(1-22) and 10 µmol/L ANP-(1-28) were
incubated with control membranes to assay cGMP production,
90.7±3.1% and 93.9±7.9%, respectively, of the peptide survived
incubation. These figures were 92.5±5.3% for CNP-(1-22) and
91.3±5.1% for ANP-(1-28) for membranes from arteries injured 20 days
earlier. Similarly, 91.1±2.0% of 1 µmol/L CNP-(1-22) and
90.1±4.6% of 1 µmol/L ANP-(1-28) survived incubations to assay cAMP
production with control membranes. These figures were
90.2±4.1% for CNP-(1-22) and 93.9±5.1% for ANP-(1-28) for membranes
from arteries injured 20 days earlier. Consequently, differential
degradation does not explain the relative agonist activities of
CNP-(1-22) and ANP-(1-28).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our results suggest that the media of the central ear artery of the rabbit normally expresses NPR-B and also develops NPR-C–like receptors between 5 and 7 days after compressive injury. NPR-C–like receptors also occur on the neointima. These findings are important because both NPR-B6 7 8 and NPR-C–like receptors2 9 are implicated in controlling VSM growth and because recent evidence suggests that neointimal VSM produces CNP-(1-22).33 CNP-(1-22) may therefore have autocrine and paracrine effects through the NPR-C–like receptors of the damaged artery, even though the normal site of synthesis for this peptide, the endothelium, may be lost over neointima.27 34
NPR-A is the only guanylate cyclase-coupled NPR identified so far in the rabbit.35 36 37 38 However, we found no NPR-A–like binding sites on the rabbit ear artery, despite the washing of tissues with acid to expose receptors occluded by endogenous ligands.19 20 26 Instead, normal media showed a single class of natriuretic peptide binding sites with high affinity for CNP-(1-22) and low affinity for ANP-(1-28), consistent with the ligand selectivity of cloned NPR-B.14 15 16 17 18 CNP-(1-22), which is the prime agonist of NPR-B and has virtually no affinity for NPR-A,12 13 14 15 16 17 18 19 was also the main agonist of guanylate cyclase in membranes prepared from normal ear arteries. Consequently, it is likely that NPR-B mediated the cGMP response of arterial membranes to low levels of CNP-(1-22). ANP-(1-28) did increase cGMP production by arterial membranes, possibly through a pool of NPR-A too small to be detected autoradiographically. However, ANP-(1-28) is a partial agonist of cloned NPR-B.16 We did not examine whether ANP-(1-28) antagonized cGMP production stimulated by CNP-(1-22), but considering the partial agonism of ANP-(1-28) at NPR-B, it would be compatible with the presence of only NPR-B that ANP-(1-28) should stimulate guanylate cyclase activity in control membranes, albeit to a significantly lower maximum than achieved with CNP-(1-22). Thus, both binding and enzymatic data suggest that the central ear artery of the rabbit normally expresses NPR-B rather than other NPRs. This contrasts with normal rabbit aorta, in which both NPR-A and NPR-C–like receptors are readily detected.35 38 39 The presence of NPR-B rather than NPR-A on the ear artery would explain why this artery, unlike the rabbit aorta, hardly relaxes in response to ANPs.40 Moreover, the rabbit ear artery is the first structure in vivo that has been shown to have both binding sites and a particulate guanylate cyclase that match the properties of cloned NPR-B.19 26 This is significant because the mapping of expressed NPR-B by its mRNA is plagued by discrepancies between the levels of mRNA and functionally expressed protein for this receptor.19 26 41
Rat aortic media normally expresses abundant NPR-A and little NPR-B,21 but NPR-A becomes undetectable and NPR-B substantially increases when rat aortic VSM adopts a synthetic phenotype and proliferates to confluence in culture.21 This has led to the suggestion that NPR-B is upregulated whenever VSM proliferates, for example, in damaged arteries.6 7 21 However, we detected no significant change in the level of NPR-B–like binding sites or in cGMP production from injured arteries compared with control arteries. Moreover, NPR-B–like sites were found only in media, whereas PCNA immunoreactivity confirmed that VSM proliferation after injury increased and then declined over a wide range in both the media and neointima.1 27 42 Consequently, measurable changes of NPR-B are not necessarily associated with changes in VSM proliferation in vivo. Species- and vessel-specific (ear artery versus aorta) differences might underlie this discrepancy from rat aortic VSM multiplying in vitro. However, we have recently shown that it is NPR-C–like receptors that become expressed in the media and neointima of the rat carotid artery after balloon angioplasty, whereas no NPR-B is detected.33 Upregulation of NPR-C–like receptors, rather than of NPR-B, may therefore be a general characteristic of the neointimal response to injury. The neointimal and medial VSM of damaged arteries in vivo is not only exposed to humoral agents but may also be influenced by surviving endothelium and cells such as platelets, macrophages, and lymphocytes,1 which are absent in culture. NPRs on VSM are homologously regulated,43 and they are also subject to differential heterologous regulation by, for example, angiotensin II.44 In addition to the circulating natriuretic peptides, VSM and macrophages can produce ANPs locally,4 45 whereas endothelium secretes CNPs5 6 and probably also ANP.46 Angiotensin II arises in the vascular wall as well as the plasma.47 Last, cytokines, in the form of transforming growth factor-ß1, have been shown to control the expression of NPRs.41 Therefore, it is unlikely that the NPRs of VSM proliferating in vitro will directly reflect those of VSM in damaged arteries. In addition, the proliferative response to arterial injury in vivo may involve a specific subpopulation of VSM that differs from that usually selected by in vitro culture.23 24 25 This could also underlie the divergent regulation of NPR-B in the VSM of damaged arteries compared with VSM in cultures from normal rat aorta.
NPR-C–like receptor sites were not found in undamaged media, which is mitotically quiescent, or in media on the 5th day after injury, when previous work has shown that the proliferation of medial VSM is at its peak.27 The proportion of VSM cells that divide in response to arterial injury may be small,42 so that NPR-C–like receptor sites might be too few to be detected if restricted to these cells. NPR-C–like sites were discovered in the media at 7 and 20 days, but it is not known whether this was because the progeny of dividing cells accumulate locally42 or because medial cells individually develop more NPR-C–like sites at these times. However, the fact that we found NPR-C–like sites throughout the neointima at 7 and 20 days, times of widely different mitotic activity, suggests that these receptor sites occur on neointimal VSM whether or not it is dividing. Evidence has recently accrued indicating that neointimal VSM in the rat derives from a particular subpopulation of phenotypically distinct VSM cells, which form only a small fraction of the adult media.23 24 25 It may be that a similar phenomenon explains the accumulation of cells expressing NPR-C–like receptors in the neointima of the rabbit, although such cells are not apparent in the normal media.
The NPR-C–like receptor sites of rabbit neointima have high affinity for CNP-(1-22) but, unlike those with high affinity for this peptide on rat glomeruli,20 are not related to the control of cAMP levels. Nevertheless, both ANP-(1-28) and CNP-(1-22) can inhibit the proliferation of rat VSM in vitro through an NPR-C–like receptor that does not modulate cAMP.2 9 This implies that the NPR-C–like sites that occur after arterial damage could limit the proliferative response of VSM to the injury. In keeping with this, we have recently demonstrated that intravenous infusions of exogenous ANP-(1-28) or CNP-(1-22) inhibit neointimal development in the present type of arterial damage.22 Consequently, endogenous natriuretic peptides may also be significant in controlling this arterial response to injury, particularly at times when other inhibitors of VSM proliferation, such as prostacyclin and nitric oxide, are diminished by endothelial damage. Such a role for NPR-C–like receptors in the control of neointimal growth would have important implications for the future design of receptor-selective agents that could modulate VSM growth therapeutically.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by the British Heart Foundation and Newton Trust. The ALLFIT program was donated by Dr P.J. Munson.
|
Footnotes |
|---|
Previously published in part in abstract form [J Physiol (Lond). 1994;475:57P].
Received March 24, 1995; accepted July 24, 1995.
|
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|---|
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]CG
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