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Cardiovascular Research 1997 35(1):158-167; doi:10.1016/S0008-6363(97)00086-2
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Mechanisms of natriuretic-peptide-induced growth inhibition of vascular smooth muscle cells

Howard G Hutchinson1, Pedro T Trindade2, Dolores B Cunanan, Can-Fang Wu3 and Richard E Pratt*

Falk Cardiovascular Research Center, Division of Cardiovascular Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA

* Corresponding author. Current address: Laboratory of Genetic Physiology, Brigham and Women's Hospital, Thorn-12, 75 Francis Street, Boston, MA 02115, USA. Tel.: +1 (617) 732-8799; fax: +1 (617) 975-0995; e-mail: rpratt@bustoff.bwh.harvard.edu

Received 28 August 1996; accepted 5 March 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: While natriuretic peptides can inhibit growth of vascular smooth muscle cells (VSMC), controversy exists as to whether this effect is mediated via the guanylate cyclase-coupled receptors, NPR-A and NPR-B, or the clearance receptor, NPR-C. The original aim of this study was to examine the mechanism by which the NPR-C receptor regulates growth. Methods: Rat VSMC were characterized with regard to natriuretic peptide receptor expression by RT/PCR and radioligand binding studies. The effect on growth following addition of the peptides and the ligands for NPR-C was measured by [3H]thymidine incorporation. Cyclic guanosine monophosphate (cGMP) levels were determined by radioimmunoassay and mitogen activating protein kinase activity was based on the phosphorylation of myelin basic protein. Results: In rat VSMC, passages 4–12, both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) dose-dependently inhibited serum and PDGF-induced VSMC growth. In contrast, NPR-C specific ligands alone had no effect on cell growth but enhanced growth inhibition when co-administered with ANP and CNP. ANP and CNP also decreased PDGF-BB-stimulated MAP kinase activity. Once again, NPR-C specific ligands alone had no effect but enhanced the effects of ANP. Furthermore, a cGMP specific phosphodiesterase inhibitor dose-dependently inhibited VSMC growth and markedly enhanced natriuretic-peptide-induced inhibition at low peptide concentrations. To examine a potential mechanism for the controversy concerning the NPR-C, we investigated the autocrine expression of ANP and CNP by VSMC and found that mRNA encoding both peptides could be detected by RT/PCR. Conclusion: Our findings indicate that the guanylyl-cyclase-linked receptors mediate the antiproliferative actions of the natriuretic peptides on vascular smooth muscle cell growth. Moreover, we hypothesize that the apparent inhibition of growth by NPR-C specific ligands reported by others may be due to stabilization of natriuretic peptides produced by the cultured VSMC and subsequent action of these peptides at guanylyl-cyclase-linked receptors.

KEYWORDS ANP; Growth; Radioimmune assay; cGMP; MAP kinase; Receptors; Vascular smooth muscle cell proliferation; Rat, vascular smooth muscle cells


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
A common thread in several forms of vascular disease is the abnormal growth of vascular smooth muscle cells (VSMC). The growth of VSMC is controlled by a balance of growth inhibitors and growth promoters and, in the normal adult vessel, this balance results in a very low rate of growth of the smooth muscle. However, following vascular injury by either mechanical or chemical means, this balance shifts such that proliferation of the smooth muscle cells occurs. The identity of the factors which regulate this growth is the subject of intense investigation and to date is not fully understood. What is becoming increasingly apparent is that vasoactive substances seem to play a major role.

One example of a vasoactive agent which may influence vascular growth is the family of natriuretic peptides, whose initial member, ANP, was isolated from the atria [1–3]. In addition to direct natriuretic and diuretic properties, these peptides also regulate sodium and water homeostasis indirectly via changes in glomerular filtration rate (GFR), inhibition of renin release and of aldosterone secretion [4, 5]. Moreover, ANP has been shown to be a direct smooth muscle relaxant, and it is becoming apparent that these factors may also be potent regulators of growth. We and others have shown that ANP antagonizes the growth-promoting effects of angiotensin II, serum, and purified growth factors on vascular smooth muscle and endothelial cells in vitro [6–11].

ANP was the first member of a family of natriuretic peptides isolated. It is highly abundant in the atria of the heart where its mRNA constitutes ~2% of the total mRNA [12]. ANP mRNA, albeit present at much lower levels, was also found in multiple tissues including hypothalamus, pituitary, aortic arch, lung, adrenal and kidney [13–17]. The second distinct form—brain natriuretic peptide (BNP)—was originally isolated from the brain, but has since been found to be more abundant in the atria [18]. BNP is structurally similar to ANP, containing a highly homologous 17-member ring which is the hallmark of the natriuretic peptides. However, BNP differs from ANP in its carboxyl and amino terminal tails. A third peptide in this family, CNP, was again isolated from brain [19, 20]but is also expressed at high levels by the endothelium [21]. This peptide contains the 17-member ring and is similar to ANP except that it lacks the C terminal extension.

The actions of ANP and the other natriuretic peptides are mediated by a family of membrane-bound receptors [22–24]. To date, three such receptors have been cloned and characterized. NPR-A (previously termed GC-A) is a protein of ~130 kDa consisting of an extracellular ligand binding domain, a single transmembrane domain and an intracellular domain consisting of a kinase domain and a guanylyl cyclase domain. The cyclase domain generates cyclic GMP upon ligand binding to the extracellular domain while the kinase region apparently is involved in the regulation of cyclase activity [22]. NPR-B has a low degree of homology to NPR-A, exhibiting similar distribution of domains, but preserving only 44% homology in the extracellular region, and 74% in the intracellular region. In contrast to these two receptors, the NPR-C or clearance receptor contains the extracellular and transmembrane domains but lacks the intracellular enzymatic activities. These receptors differ in ligand specificity. NPR-A binds ANP >BNP > >CNP while NPR-B binds CNP > >ANP≥BNP. NPR-C apparently shows less discrimination between the peptides binding ANP >CNP≥BNP [25].

Previous data suggested that NPR-C functions as a clearance receptor, responsible for internalization of ANP and the other peptides resulting in degradation [26]. However, it was reported, but not widely confirmed, that this receptor may be G-protein-linked, resulting in calcium channel activation, IP3 formation and/or adenylate cyclase inhibition [27–29]. Interestingly, Cahill and Hassid [10, 11]as well as Levin and co-workers [30, 31]presented several lines of evidence that the NPR-C receptor isoform mediates the antiproliferative effects of the natriuretic peptides. These results are based on the observation that NPR-C specific ligands, as well as ANP and CNP, are able to induce the antiproliferative actions. However, at this point it is unclear how such an action is mediated.

Thus, the original aim of this current study was to examine the mechanism by which NPR-C regulates growth. However, to our surprise, we were unable to confirm NPR-C-mediated effects on the growth of VSMC. Our data are entirely consistent with the hypothesis that the guanylyl-cyclase-linked receptors and these receptors alone mediate the antiproliferative actions of the peptides. Moreover, based on our data, we hypothesize that the apparent inhibition of growth induced by NPR-C specific ligands may be due to the stabilization of natriuretic peptides produced in an autocrine fashion by the cultured smooth muscle cells.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
Atrial natriuretic peptide (ANP99–126), C-type natriuretic peptide (CNP), the ring-deleted ANP analog des[Gln18,Ser19,Gly20,Leu21,Gly22]ANP[4–23]NH2 (C-ANP-4–23]) and the linearized ANP analog des[Cys105,Cys121]rANP104–126 were obtained from Peninsula Laboratories Inc. (Belmont, CA). Peptide purities were >99% as determined by HPLC. Sodium nitroprusside (SNP) was obtained from Sigma Laboratories. The phosphodiesterase V inhibitor, zaprinast (M&B 22,948) was purchased from Biomol (Plymouth Meeting, PA). [methyl-3H]Thymidine (20 Ci/mmol) was obtained from Amersham (Arlington Heights, IL). ANP and CNP RIA kits were from Peninsula Laboratories (Belmont, CA), the cGMP assay kit was obtained from NEN/Dupont (Boston, MA) and [125I]rANP99–126, [125I]CNP and [125I]des[Cys105,Cys121]rANP104–126 were prepared by Dr. Robert Speth (Washington State University, Tacoma, WA). Oligonucleotides were synthesized at Stanford University using a Model 394 Applied Biosystems DNA synthesizer (Foster City, CA). All other reagents were of the highest purity available and were obtained from either Sigma or Fisher unless noted.

2.2 Cell culture
This method conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Cultured aortic VSMC were derived from explants [32]of the thoracic aortae of male Sprague-Dawley rats (200–300 g) and were grown in a 1:1 mixture of DMEM and Ham's F12 medium (Gibco, Grand Island, NY) with 10% Fetal Calf Serum (FCS), penicillin (100 U/ml), streptomycin (100 µg/ml) and 25 mM Hepes, pH 7.4 (DMEM-F12). The cells were grown in a humidified atmosphere of 5% CO2/95% air at 37°C and were passaged at 85% confluence using 0.125% trypsin/EDTA. Cells from passages 4–12 were utilized for all experiments. These cells had the typical hill-and-valley appearance of VSMC and stained positive for {alpha}-actin.

2.3 Measurement of DNA synthesis
Estimates of DNA synthesis were made using [3H]thymidine incorporation. Cultured aortic VSMC were seeded in 24-well culture plates (Corning, Corning, NY) at a density of 2.0x104 cells/cm2 and grown in medium plus 10% FCS until 2 days post-confluence (~5–7 days). Quiescence was induced by washing cells once in a defined serum-free (DSF) medium (6) containing insulin (5x10–7 mol/l), transferrin (5 µg/ml) and ascorbate (0.2 mmol/l) and followed by 2 days growth in the same medium. The cells were stimulated using 2.5% FCS or PDGF-BB supplemented medium in the presence or absence of experimental agents. Cell toxicity as assessed by trypan blue exclusion was not observed under these conditions. Twelve hours post-stimulation, 5 µCi of [methyl-3H]thymidine was added. At 36 h post-stimulation, the cells were washed with ice-cold 10% TCA and then incubated for 30 min in 10% TCA. The cells were subsequently washed with 95% ethanol, air-dried and solubilized in 0.25% NaOH for 1 h at room temperature. The radioactivity of an aliquot of the extract was measured by scintillation counting.

2.4 Radioimmunometric assays
2.4.1 Measurement of cGMP production
To determine the effects of the natriuretic peptides on cGMP production, cells were seeded onto 24-well culture plates as described above. After induction of quiescence, the cells were incubated at 37°C for 10 min in 1 ml of DSF containing 0.5 mmol/l isobutylmethylxanthine (IBMX). ANP, CNP, C-ANP, des[Cys105,Cys121]rANP104–126, ANP in combination with C-ANP, and CNP in combination with C-ANP were added at various concentrations and the cells incubated for an additional 10 min. After the incubation, the medium was rapidly removed and 6% ice-cold TCA was added to the cells and the cells scraped from the plates. The TCA was then removed from the samples by extracting 3 times with water-saturated ether. cGMP concentration was determined by radioimmunoassay as previously described [33].

2.4.2 Measurement of ANP and CNP in conditioned media
To determine the potential expression of the natriuretic peptides in the VSMC, cells were made quiescent in flasks as described above. Conditioned media was collected after 24 h and assayed for the presence of ANP or CNP using radioimmunometric assays from Peninsula Laboratories, Inc. (Belmont, CA). The sensitivity of these assays was 11 and 15 pM for ANP and CNP, respectively. The levels of ANP and CNP in the conditioned media were below the lower limits of the assay.

2.4.3 Radioligand binding assays
Binding assays were performed in 24-well culture plates. Cell monolayers were washed twice with DSF and incubated in a binding buffer which consisted of DSF supplemented with 0.2% essentially fatty-acid-free bovine serum albumin, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 5 µg/ml aprotinin. To assess competition for [125I]rANP99–126, [125I]CNP and [125I]des[Cys105,Cys121]rANP104–126 binding, radioactive peptide (0.1–5.0 nmol/l) in the presence or absence of non-radioactive peptide (1.0 µmol/l) was added to the binding buffer for 2 h at 4°C. Incubation for longer periods of time did not substantially increase the level of binding (data not shown). Following incubation, the cells were washed rapidly 4 times with ice-cold PBS and solubilized in 1 mol/l NaOH for 15 min at 37°C. Aliquots were removed and counted on a Wallac LKB 1274 gamma counter (Turku, Finland). Parallel aliquots were examined for protein content using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Nonspecific binding was determined in the presence of 1 µmol/l non-radioactive peptide.

2.5 Quantitation of receptor mRNA
2.5.1 RNA extraction
RNA was isolated from cultured aortic VSMC using the guanidinium thiocyanate-phenol/CHCl3/isoamyl alcohol method of Chomczynski et al. [34]. RNA was isolated from excised thoracic aortae of male Sprague-Dawley rats (500 g) following stripping of the adventitia and endothelium. Sections of the stripped aortae were examined microscopically to confirm that the adventitia and endothelium were adequately excised. Samples were frozen in liquid nitrogen and stored at –80°C until used for further experiments.

2.5.2 Oligonucleotides for PCR amplification
The primers used for PCR were as follows: NPR-A primers (from the rat sequence (22, 35)), forward: 5' AAG AGC CTG ATA ATC CTG AGT ACT 3', reverse: 5' TTG CAG GCT GGG TCC TCA TTG GCA 3' (product 451 bp); NPR-B receptor primers (from the rat sequence [24, 35]), forward: 5' AAC GGG CGC ATT GTG TAT ATC TGC GGC 3', reverse: 5' TTA TCA CAG GAT GGG TCG TCC AAG TCA 3' (product 692 bp); NPR-C receptor primers (from the bovine sequence [36]): forward: 5' AGA CAA ACA CGA CTT TGA AGC TAA 3', reverse: 5' CGC ATT TCA AAA CGA CCT TCT TTT 3' (product 428 bp); ANP primers (from the rat sequence [35, 37]), forward: 5' TAC AGT GCG GTG TCC AAC ACA GAT CTG ATG GAT TTC AAG 3', reverse: 5' GCA ATG CGA CCA AGC TGT GTG ACA CAC CGC 3' (product 451 bp); CNP primers (from the rat sequence [38]), forward: 5' TGC TCG CGC TAC TCT CAC T 3', reverse: 5' CGC TGC ACT AAC ATC CCA GAC CGC 3' (product 356 bp); β-actin primers (from the rat sequence [39]), forward: 5' ATC ATG AAG TGT GAC GTT GAC 3', reverse: 5' AAC CAA CTG CGG TCG CCT TCA 3' (product 508 bp).

2.5.3 Reverse transcription and polymerase chain reaction
Reverse transcription (RT) of RNA was performed in a mixture containing 5 mmol/l MgCl2, PCR buffer (50 mmol/l KCl, 10 mmol/l Tris-HCl), 1 mmol/l each dATP, dTTP, dCTP, dGTP, 2.5 µmol/l random hexamers, 1 U RNase Inhibitor and 2.5 U Moloney Murine Leukemia Virus (MMLV) reverse transcriptase. 1 µg of RNA was added to the reaction mixture and the volume brought to 20 µl with nuclease-free dH2O. The mixture was overlaid with mineral oil and incubated at 42°C for 1 h. Afterwards, the mixture was heated to 99°C for 10 min, then placed on ice.

The polymerase chain reaction (PCR) was performed in a solution containing 2 mmol/l MgCl2, PCR buffer (50 mmol/l KCl, 10 mmol/l Tris-HCl), 0.2 mmol/l each dATP, dTTP, dCTP, dGTP and 0.5 µmol/l of forward and reverse primers. The reactants were overlaid with mineral oil. 1 µl of AmpliTaq DNA Polymerase (Gibco, BRL, Gaithersburg, MD) was added to the reaction tubes during a 3 min incubation at 80°C (‘hot start’ method). PCR was performed for 25–40 cycles at 94°Cx1 min, 58°Cx2 min, and 72°Cx3 min and finally, 1 cycle of 72°Cx15 min. 15 µl of each PCR product was separated by electrophoresis through a 1.5% agarose gel using TBE buffer (45 mM Tris borate, 1 mM EDTA). The bands were visualized following ethidium bromide staining. Visual and densitometric evaluation of the bands revealed exponential amplification over the range of 25 to 40 cycles. As an internal control, RT/PCR of the housekeeping gene ß-actin was performed for each sample. Also, RNAse A treatment of RNA samples prior to RT/PCR abolished the resultant signals confirming that the signals were of RNA origin.

2.5.4 Measurement of MAP kinase activity
Post-confluent rat aortic SMC were incubated in DSF for 48 h to induce quiescence. Cells were then stimulated with 10–7 mol/l ANP, 40 ng/ml PDGF-BB, and 40 ng/ml PDGF-BB plus a series of natriuretic peptides (10–7 mol/l each). Following a 10 min incubation, MAP kinase activity was assessed by Western blot analysis of protein phosphotyrosine [40]. Briefly, the cells were quickly washed 3 times with ice-cold phosphate-buffered saline (PBS; 10 mmol/l sodium phosphate, pH 7.4, 137 mmol/l NaCl, 1.2 mmol/l MgSO4) containing 1 mmol/l sodium vanadate and lysed in 10 mmol/l Tris-HCl, pH 7.6, 5 mmol/l EDTA, 1 mmol/l ethyleneglycol-bis-β-aminoethyl ether -N,N'-tetraacetic acid (EGTA), 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 100 mmol/l NaCl, 1% sodium deoxycholate, 1 mmol/l phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mmol/l sodium vanadate. The proteins (150 µg per lane) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in 7.5% polyacrylamide and transferred to a polyvinylidene fluoride membrane. The membrane was incubated with monoclonal anti-phosphotyrosine antibody (4G10: UBI, Lake Placid, NY) for 3 h at room temperature, washed, and incubated for 1 h with goat anti-mouse IgG conjugate to horseradish peroxidase (Bio-Rad, Richmond, CA). The position of MAP kinase was determined by comparison with a parallel Western blot which was developed with an anti-rat MAP kinase R1 antibody (UBI, cat #06-183). Proteins were visualized by the Amersham ECL (enhanced chemiluminescence; Arlington Heights, IL) detection system.

A second method was used to quantitate MAP kinase activity based on its ability to phosphorylate myelin basic protein (MBP) as described previously [41]with modification. Cells were lysed in 25 mmol/l Tris/HCl, pH 7.5, 25 mmol/l NaCl, 0.5 mmol/l EGTA, 10 mmol/l NaF, 20 mmol/l β-glycerophosphate, 1 mmol/l Na3VO4 and 1 mmol/l PMSF. Cell lysates (100 µg) were incubated with 5 µg of anti-rat MAP kinase R2 antibody (UBI, cat #06-182) and 40 µl suspension of protein G-Sepharose. The immunoprecipitates were incubated with 24 µl of reaction buffer (25 mmol/l Tris/HCl, pH 7.5, 10 mmol/l MgCl2, 1 mmol/l DTT, 2 µmol/l protein kinase inhibitor (Sigma), 0.5 mmol/l EGTA, 1 mg/ml MBP and 40 µmol/l [{gamma}-32P]ATP) for 10 min at room temperature. The samples were separated on 14% SDS/PAGE and radiolabeled MBP, detected by autoradiography, was eluted from the gel and quantitated by scintillation counter.

2.6 Data analysis
Binding data were analyzed by Scatchard analysis. Statistical comparisons were performed by the use of unpaired, two-tailed Student's t-test, or analysis of variance, as appropriate. Parametric analysis of the raw data was employed.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Previous reports have shown that culturing aortic VSMC results in up-regulation of NPR-B and NPR-C and down-regulation of NPR-A [42]. For the purposes of this study, all experiments were performed on rat VSMC grown for 2 days post-confluence with serum and then 2 additional days in a defined serum-free (DSF) medium to induce quiescence. We characterized our cell line for natriuretic peptide receptor expression using both RT/PCR analysis and radioligand binding studies. RT/PCR analysis, a semi-quantitative analytic tool, revealed the presence of mRNA for all three receptor isoforms in our cell line (Fig. 1, inset). This result was followed by receptor binding studies to quantify further the receptor subtype expression (Fig. 1). Scatchard analysis of the competitive inhibition of [125I]des[Cys105,Cys121]rANP104–126 binding to confluent VSMC by unlabeled des[Cys105,Cys121]rANP104–126, a specific NPR-C antagonist, revealed that the density of NPR-C receptors (Bmax) was 1.70x105 receptors/cell. Competitive inhibition of [125I]rANP by ANP and [125I]CNP by CNP was used to estimate the density of total ANP and CNP binding sites, respectively. Based on the relative affinities for the different receptor isoforms, we would anticipate that the radiolabeled ANP would bind to the NPR-A and NPR-C while radiolabeled CNP would bind to the NPR-B and NPR-C [23, 25]. The total binding for ANP was 1.83x105 sites/cell. Since ANP binds both NPR-A and NPR-C, by subtraction, the number of NPR-A receptors was calculated to be 0.13x105 receptors/cell. Similarly, the total biding for CNP was 2.07x105 sites/cell, yielding 0.37x105 receptors/cell for the NPR-B receptor. These data indicated that the predominant receptor isoform in our cell line was NPR-C > >NPR-B > NPR-A.


Figure 1
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  		Fig. 1 Natriuretic peptide receptor levels in rat VSMC. Cultured VSMC were grown until 2 days post-confluence in 10% FCS and then switched to a defined serum free media for 2 days to induce quiescence. Shown are the competitive binding of [125I]ANP by unlabeled ANP (open box), [125I]CNP by unlabeled CNP (open triangle), and [125I]des[Cys105,Cys121]rANP104–126 by unlabeled des[Cys105,Cys121]rANP104–126 (closed box). Scatchard analysis showed that the predominant receptor was NPR-C; however, NPR-A and NPR-B were also present with greater expression of NPR-B than NPR-A. VSMC from several passages were analyzed and in each case the results were comparable. Insert: RT/PCR analysis revealed the presence of the mRNA encoding NPR-A, NPR-B and NPR-C in cultured VSMC. This is representative of several independent experiments.

 
Consistent with these results, in a separate series of experiments, des[Cys105,Cys121]rANP104–126 competed for approximately 93 and 84% of the total [125I]rANP and [125I]CNP binding sites, indicating the presence of other receptors. Finally, [125I]des[Cys105,Cys121]rANP104–126 displacement by ANP, CNP, C-ANP and des[Cys105,Cys121]rANP104–126 was assessed in order to establish the relative binding affinities for NPR-C. We found that the binding affinities were similar (~0.35 pmol/l).

We next examined the ability of the different peptides to affect the growth of smooth muscle cells. Following the induction of quiescence, cells were simulated with 2.5% FCS or PDGF-BB (20 ng/ml) in the presence of various natriuretic peptides or peptide analogs. These levels of serum were chosen since, in preliminary experiments, they gave a near-maximum increase in DNA synthesis. Following stimulation, the rank order of receptor subtype expression was similar to that observed in the quiescent cells. Fig. 2 shows a characteristic response of rat VSMCs to ANP, CNP and the C-receptor specific ANP analogs, C-ANP and des[Cys105,Cys121]rANP104–126, following stimulation with 2.5% fetal calf serum. Both ANP and CNP dose-dependently inhibited serum-induced increases in DNA synthesis. Consistent with the greater expression of NPR-B, at decreasing peptide concentrations, greater inhibition was maintained with CNP than with ANP. However, no significant effect on growth was observed with C-ANP nor did we observe a growth-inhibitory effect for des[Cys105,Cys121]rANP104–126. The ability of ANP and CNP to inhibit growth occurred independent of whether the peptides were administered at the time of cell stimulation from a quiescent state or at a time when the cells were actively cycling (data not shown).


Figure 2
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  		Fig. 2 Effect of natriuretic peptides on VSMC growth. Confluent, quiescent VSMC were grown as in Fig. 1 and then treated with 2.5% FCS plus ANP (open box), CNP (open triangle), C-ANP (open circle) or des[Cys105,Cys121]rANP104–126 (closed box) and [3H]thymidine incorporation was measured and compared to 2.5% FCS alone. Results were calculated as cpm per well and expressed as the percent of inhibition of serum-induced DNA synthesis (n = 5, c.p.m. in defined serum-free wells, 12 136±891; c.p.m. in 2.5% FCS-stimulated wells, 48 113±2225). A dose-dependent growth-inhibitory response was consistently found with ANP and CNP with CNP having the greater effect at lower concentrations. Maximum growth inhibition from several experiments ranged between 60 to 80%. Similar inhibition of PDGF-BB (20–40 ng/ml)-induced DNA synthesis was observed (data not shown). Note that neither of the NPR-C specific ligands affected growth in this cell culture.

 
In order to further clarify the potential role of NPR-C in the growth-inhibitory response to natriuretic peptides, we examined the effect of increasing concentrations of the NPR-C antagonist, C-ANP, when added to various concentrations of ANP or CNP. As shown in Fig. 3, C-ANP did not block the growth-inhibitory effects of ANP or CNP. In fact, to the contrary, C-ANP potentiated the effects of low ANP or CNP concentrations. Similar results were observed using des[Cys105,Cys121]rANP104–126; however, C-ANP and des[Cys105,Cys121]rANP104–126 added together at various concentrations produced no effect.


Figure 3
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  		Fig. 3 C-ANP enhances the growth-inhibitory effects of the natriuretic peptides. Confluent, quiescent VSMC were grown as in Figs. 1 and 2Go and then treated with 2.5% FCS plus vehicle, 0.01 µmol/l ANP or 0.01 µmol/l CNP alone (filled bars) or in combination with 1 µmol/l C-ANP (striped bars). DNA synthesis was measured as [3H]thymidine incorporation and the results were expressed relative to the rates of DNA synthesis in the untreated confluent quiescent culture (set equal to 1 (n = 5;, c.p.m. in defined serum-free wells, 10 432±763; c.p.m. in 2.5% FCS-stimulated wells, 31 818±2156). Note that, alone, C-ANP had no effect on DNA synthesis rates but when added to ANP and CNP would markedly enhance the growth-inhibitory effects of the ligands. A similar potentiation in growth inhibition was found with des[Cys105,Cys121]rANP104–126 (data not shown). This experiment was repeated three times with similar results.

 
Further insight into the mechanisms of natriuretic-peptide-mediated growth inhibition can be obtained by studying events downstream of receptor activation. Mitogen-activated protein (MAP) kinase can be activated by various growth factors and may play an important role in cell growth [43]. Since platelet-derived growth factor-BB (PDGF-BB) has been reported to promote cell growth by activating MAP kinase, we examined ANP, CNP, C-receptor specific ligands, sodium nitroprusside (SNP) and 8-Br-cGMP for potential effects on the MAP kinase cascade. Initial experiments (data not shown) were performed by Western blot analysis using an anti-phosphotyrosine antibody [40]. The position of MAP kinase was determined with a parallel Western blot developed with an anti-MAP kinase R1 antibody. In these experiments, ANP and CNP reduced phosphorylated MAP kinase levels. In contrast, C-ANP and des[Cys105,Cys121]rANP104–126 had no effect. To confirm the above results, MAP kinase was selectively precipitated using an anti-MAP kinase R2 antibody and quantitated based on its ability to phosphorylate myelin basic protein [41]. Fig. 4 shows that ANP, CNP, SNP and 8-Br-cGMP decreased MAP kinase activity. Consistent with their growth-inhibitory potential, CNP was a more potent inhibitor than ANP. C-ANP, which had no effect on SMC growth, had no effect on MAP kinase activity but, as above, potentiated the effects of ANP and CNP. The fact that SNP and 8-Br-cGMP also inhibited PDGF-BB-induced increases in MAP kinase suggested that cGMP could be acting to inhibit MAP kinase activity and ultimately inhibit cellular growth.


Figure 4
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  		Fig. 4 Effect of natriuretic peptides, SNP and 8-Br-cGMP on MAP kinase activity. Confluent, quiescent VSMC, grown as in Figs. 1–3GoGo were treated with vehicle, PDGF-BB (40 ng/ml), or PDGF-BB plus 0.1 µmol/l of the indicated peptides, 10 µmol/l SNP or 100 mmol/l 8-Br-cGMP (n = 3). Cells were harvested 10 min later and MAP kinase activity assessed by selective immunoprecipitation and quantitation of its ability to phosphorylate myelin basic protein. Approximately a 3-fold increase in MAP kinase activity was observed following exposure to PDGF-BB (c.p.m. in defined serum-free wells, 40 332; c.p.m. in PDGF-BB-stimulated wells, 124 362). The results are expressed as percent inhibition of PDGF-BB stimulation. ANP and CNP each inhibited MAP kinase activity. CNP had the greater effect, which is consistent with the greater number of NPR-B receptors and its greater growth-inhibitory potential. NPR-C receptor ligands which had no effect on cell growth had no effect on MAP kinase activity. Simultaneous exposure to ANP and C-ANP further inhibited MAP kinase activity similar to the additive effect on growth inhibition. SNP and 8-Br-cGMP also decreased MAP kinase activity, suggesting that decreases in MAP kinase activity could be related to increases in cGMP.

 
Taken together, the above results suggest that the anti-growth effects of the natriuretic peptides are mediated by the guanylyl-cyclase-linked receptors. In support of this conclusion, growth inhibition by ANP and CNP correlated with the ability of these peptides to induce cGMP production (Fig. 5a). Moreover, consistent with their ability to enhance growth inhibition, C-ANP and des[Cys105,Cys121]rANP104–126 potentiated the effects of ANP and CNP on cGMP production (Fig. 5b). To examine further the effects of cGMP on VSMC growth, we utilized a cGMP specific phosphodiesterase inhibitor, zaprinast (M&B 22,948). This compound is a highly specific inhibitor of phosphodiesterase V, the major cGMP phosphodiesterase in smooth muscle. Treatment of confluent, quiescent smooth muscle cells with zaprinast (0.01 µmol/l to 100 µmol/l) in the presence of 2.5% FCS resulted in dose-dependent growth inhibition (Fig. 6a). Of note, low-dose zaprinast (0.01 µmol/l), which alone had no effect on DNA synthesis in the VSMC, markedly potentiated the growth-inhibitory effects of ANP and CNP especially at low peptide concentrations (10–8 mol/l, Fig. 6b). This potentiation was not observed with addition of C-ANP or des[Cys105,Cys121]rANP104–126. Since the co-administration of zaprinast with either ANP or CNP augments cGMP levels, this result again suggests the involvement of cGMP in the inhibition of VSMC growth.


Figure 5
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  		Fig. 5 Effects of natriuretic peptides on intracellular cGMP levels. Upper panel: Confluent, quiescent VSMC, grown as in Figs. 1–4GoGoGo, were pretreated with IBMX for 10 min. Vehicle or increasing doses of ANP (open box), CNP (open triangle), C-ANP (open circle) or des[Cys105,Cys121]rANP104–126 (closed box) were added for 10 min. Cells were harvested and the lysates were assayed for cGMP (n = 3). Lower panel: As above, cells were pretreated with IBMX followed by vehicle or 0.01 µmol/l ANP or 0.01 µmol/l CNP alone or with 1 µmol/l C-ANP. Ten minutes later, cells were harvested and the lysates assayed for cGMP (n = 3). Note that ANP and CNP dose dependently increased cGMP levels while C-ANP and des[Cys105,Cys121]rANP104–126 alone had little effect (upper panel). However, in combination with ANP or CNP, C-ANP potentiated the increase in cGMP (lower panel).

 

Figure 6
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  		Fig. 6 Effects of zaprinast on growth of VSMC. Upper panel: Confluent, quiescent VSMC, grown as in Figs. 1–5GoGoGoGo, were stimulated with 2.5% FCS alone or with increasing concentrations of zaprinast, a specific cGMP phosphodiesterase inhibitor. DNA synthesis was measured and expressed relative to the rates observed in the confluent, quiescent culture (n = 6, c.p.m. in defined serum-free wells, 10 735±501; c.p.m. in 2.5% FCS-stimulated wells, 33 493±1633). Lower panel: As above, confluent, quiescent VSMC were stimulated with 2.5% FCS alone or in combination with ANP, CNP or C-ANP (all 0.01 µmol/l). Zaprinast (closed bars) or vehicle (hatched bars) was also added. DNA synthesis was measured and expressed relative to the rates observed in the non-treated confluent, quiescent cultures. Zaprinast dose-dependently inhibited serum stimulated DNA synthesis in rat VSMC and at a concentration which by itself had no effect, Zaprinast (0.01 µmol/l) also potentiated the ability of ANP and CNP to inhibit DNA synthesis (lower panel). This concentration of zaprinast had no effect in combination with up to 1 µmol/l C-ANP or des(Cys,Cys)rANP on cGMP levels.

 
How then do we explain previously published results demonstrating a growth-inhibitory effect of the NPR-C specific ligands, des[Cys105,Cys121]rANP104–126 and C-ANP? A clue is provided in the data above (Figs. 3 and 5Gob) which demonstrated that C-ANP potentiated the growth-inhibitory effects and the cGMP-generating effects of ANP and CNP. If smooth muscle cells were capable of low-level expression of ANP and/or CNP, administration of C-ANP may stabilize these ligands, allowing the autocrine action of these ligands at the NPR-A or NPR-B receptor, respectively. To examine if VSMC are capable of expressing either ANP or CNP, RT-PCR analysis was performed on RNA isolated from confluent, quiescent cells. The results (Fig. 7) demonstrate the presence of low levels of both ANP and CNP transcripts. Therefore, it is possible that smooth muscle cells might produce these peptides that could act in a autocrine and/or paracrine manner. We next assayed ANP and CNP in conditioned media from our cells. However, the levels of the ANP and CNP were below the sensitivity of the assay (11 and 15 pM, respectively, data not shown). This is consistent with the fact that in our cell line, we do not observe an anti-growth effect of the C-receptor ligands, suggesting that the expression of the endogenous ligands must be very low. However, these results raise the possibility that in other cell cultures the endogenous production of the natriuretic peptides may influence the growth of the cells.


Figure 7
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  		Fig. 7 Cultured VSMC express ANP and CNP. mRNA was isolated from confluent, quiescent VSMC, grown as in Figs. 1–6GoGoGoGoGo. RT/PCR analysis using primers specific for ANP (upper panel), CNP (middle panel) and B-actin (lower panel) mRNA was performed. PCR products analyzed by agarose gel electrophoresis were visualized by ethidium bromide staining. Note that mRNA transcripts encoding both ANP and CNP were detected. Neither ANP nor CNP transcripts were observed in the control lanes where RNA was omitted.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Several lines of evidence from this study suggest that growth inhibition of VSMC by natriuretic peptides is mediated by NPR-A and NPR-B. First, we were unable to observe a growth-inhibitory effect with the NPR-C ligands, C-ANP or des[Cys105,Cys121]rANP104–126. In fact, these ligands enhanced growth inhibition when co-administered with ANP and CNP to VSMC. Second, only ANP and CNP were able to inhibit growth-factor-induced increases in MAP kinase activity. The latter effect was mimicked with SNP and 8-Br-cGMP but not NPR-C ligands, suggesting a potential effect of cGMP production by natriuretic peptides on growth. And third, we observed that the levels of growth inhibition in response to ANP and CNP correlated with the ability of these peptides to induce cGMP.

Evidence that the guanylyl cyclase receptors mediate the antiproliferative effects of ANP are compelling. First, agents which increase intracellular cGMP in cultured cells (8-bromo-cGMP, nitrovasodilators) inhibit growth of VSMC [44], suggesting cGMP can inhibit growth. Secondly, HS-142-1, a non-peptide antagonist which exhibits relatively high binding to NPR-A and NPR-B but low binding to NPR-C, blocks the antimitogenic effects of CNP [45]. Finally, as we have shown in this report, in cells which express NPR-B and NPR-C, CNP, the ligand specific for the NPR-B receptor, induces a larger increase in cGMP and a more potent inhibition of cell growth compared to ANP. This should be viewed in light of the fact that ANP and CNP had a similar affinity for NPR-C in our cell line.

We were initially intrigued by the possibility that NPR-C may mediate a biological effect other than the clearance of the natriuretic peptides. Cahill and Hassid [10, 11]reported that the compound des[Cys105,Cys121]rANP104–126, which specifically interacts with NPR-C, inhibited growth of vascular SMC in a manner similar to that observed with ANP and CNP but without increasing cGMP. They observed that the NPR-C antagonist, C-ANP, resulted in the attenuation of the growth inhibition induced by ANP or des[Cys105,Cys121]rANP104–126. Moreover, in cells of various passages, the ability of ANP to inhibit growth correlated better with the expression of NPR-C than with NPR-A. Similarly, others have reported that C-ANP was an NPR-C agonist and could inhibit growth of other cell cultures such as astroglial cells and cardiac fibroblasts [30, 31, 46].

The possibility that ANP could signal through a guanylyl-cyclase-linked receptor and a separate, structurally distinct receptor (or at least a structurally distinct intracellular domain) would be fairly unique. True, {alpha}-adrenergic agonists may signal through the {alpha}1-adrenergic receptor, which couples to Gq and stimulates phospholipase C, or through the {alpha}2-adrenergic receptor, which couples to Gi and inhibits adenylate cyclase [47]. However, these signaling mechanisms are mediated by receptors that are highly homologous and couple via the same general mechanism (i.e., the heterotrimeric GTP binding proteins). Coupling through NPR-C would require a different mechanism than that observed for NPR-A and NPR-B since NPR-C has a much shorter intracellular tail and would presumably require accessory proteins in order to signal. Using our cell cultures, we were not successful in observing changes in cAMP or calcium in response to NPR-C ligands.

Several potential explanations exist to explain the differing results. Different isolates of smooth muscle cells may have differing phenotypes and, as a result, the receptors may mediate different responses. However, we have performed these experiments on multiple cell isolates using both enzymatically dispersed cultures as well as explant cultures and have found similar results. Another explanation for differing growth-inhibitory effects could be differing levels of autocrine production of ANP and/or CNP by different cultures of VSMC. In our cultures, the endogenous expression of the ligands is low. However, it is conceivable that under certain circumstances, the endogenous expression may be greater. Under these conditions, exposure to the NPR-C ligands may stabilize the endogenous peptides, resulting in an inhibition of growth which is, in fact, being mediated by the guanylyl-cyclase-linked receptors. In support of a potential local autocrine natriuretic system, careful examination of Fig. 5a,b reveals that addition of C-ANP or des[Cys105,Cys121]rANP104–126 to rat VSMC resulted in an increase in cGMP levels above those seen in untreated cells. In addition, Cahill and Hassid [10, 11]reported that administration of IBMX alone (i.e., no exogenously added natriuretic peptides) resulted in an increase in cGMP above basal levels and an inhibition of growth. Several examples exist for the regulation of growth by autocrine factors. For example, it is becoming evident that endogenously produced angiotensin II regulates myocyte growth in an autocrine manner [48, 49].

However, this hypothesis does not explain all the differing results since Cahill and Hassid report that des[Cys105,Cys121]rANP104–126 but not C-ANP exerts antiproliferative actions and that C-ANP will block the antiproliferative actions of ANP or des[Cys105,Cys121]rANP104–126. Some of the conflicting data may be the methods employed to assess growth inhibition. For example, we have observed that certain isolates of VSMC will undergo apoptosis in response to natriuretic peptides [50]. However, the isolates used in the current study do not undergo apoptosis under these conditions, neither do they become necrotic. Therefore, some of the discrepancies in the literature regarding the growth-inhibitory effects of the natriuretic peptides as well as their mechanism of action may be due to heterogeneity of the apoptotic response of the cells to the natriuretic peptides. Heterogeneity in response to an apoptotic stimulus may be a common cell culture phenomenon [51, 52].

In summary, our results lend further support to the concept that growth inhibition is mediated via NPR-A and NPR-B. We were unable to reproduce the results of others in our cell line and, in fact, observed a contradictory response. Des[Cys105,Cys121]rANP104–126 produced no growth inhibition and C-ANP either had no effect on growth inhibition by ANP and CNP or potentiated their growth-inhibitory effects. In addition, our cGMP growth-inhibition experiments with SNP, ANP and CNP suggest that a cGMP-dependent pathway may mediate the growth-inhibitory response.

Time for primary review 21 days.


   Acknowledgements
 
This work was supported by NIH grants HL42663, NIH training grant HL07708 and a fellowship from the Fonds National Suisse pour la Recherche Scientifique (PTT). HGH was a fellow of the AHA Bugher Foundation for Molecular Biology. This work was presented in part at the 68th Scientific Session of the American Heart Association, 1995, Anaheim, California and the 30th Annual Scientific Meeting of the European Society for Clinical Investigation, 1996, Interlaken, Switzerland.


   Notes
 
1 Current address: Cardiovascular and Licensing, Zeneca Pharmaceuticals, 1800 Concord Pike, PO Box 15437, Wilmington, DE 19850, USA. Back

2 Current address: Centre de Cardiologie, Hôpital Cantonal Universitaire de Genève, 24, rue Micheli-du-Crest, 1211 Geneva 4, Switzerland. Back

3 Current address: Cardiovascular Research Department, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080, USA. Back


   References
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 1 Introduction
 2 Methods
 3 Results
 4 Discussion
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