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Cardiovascular Research 2006 72(3):464-472; doi:10.1016/j.cardiores.2006.08.012
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Copyright © 2006, European Society of Cardiology

Intracellular peptides of natriuretic peptide receptor-C inhibit vascular hypertrophy via Gq{alpha}/MAP kinase signaling pathways

Yuan Li, Shehla Hashim and Madhu B. Anand-Srivastava*

Department of Physiology, and Groupe de recherche sur le système nerveux autonome, (GRSNA) Faculty of Medicine, University of Montreal, Canada

* Corresponding author. Department of Physiology, Faculty of Medicine, University of Montreal, C. P. 6128, Succ. Centre-ville, Montreal, Quebec, Canada H3C 3J7. Tel.: +1 514 343 2091; fax: +1 514 343 2111. Email address: madhu.anand-srivastava{at}umontreal.ca

Received 5 June 2006; revised 19 July 2006; accepted 18 August 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: The present studies were undertaken to investigate if the activation of natriuretic peptide receptor-C (NPR-C) that has been shown to inhibit cell proliferation could also modulate the hypertrophic responses of vasoactive peptides in A10 vascular smooth muscle cells (VSMC).

Methods: For these studies A10 VSMC were incubated in the presence of angiotensin II (AngII), endothelin (ET-1) or arginine vasopressin (AVP) alone or in combination with C-ANP4–23 or NPR-C peptides for 24 h and were used for Western blotting and [3H]leucine incorporation. The different peptide fragments used were K461KYRITIERRNH472 (peptide-1) and H481RELREDSIRSH492 (peptide-3) with complete Gi activator sequences and R469RNHQEESNIGK480 (peptide-2) and I465TIERRNH472 (peptideY) with partial Gi activator sequences. The other peptide used had no structural specificity, (Q473EESNIGK480; peptide X) or was the scrambled peptide control for peptide-1 (ITIYKKRRNHRE; peptide Z).

Results: AngII, ET-1 and AVP significantly stimulated protein synthesis in these cells as determined by 3H-leucine incorporation, which was inhibited by peptides 1, 2 and 3 and not by peptides X, Y or Z in a concentration-dependent manner with an apparent Ki between 1–10 nM. In addition, C-ANP4–23 also inhibited protein synthesis stimulated by AngII, ET-1 and AVP; whereas basal protein synthesis in these cells was not inhibited by C-ANP4–23 or by the peptide fragments. Furthermore, AngII-, AVP- and ET-1-induced stimulation of protein synthesis was inhibited by PD98059 (MEK inhibitor) and wortmannin (P13K inhibitor) and this inhibition was potentiated by peptide-1. In addition, peptide-1 was also able to inhibit vasoactive peptide-induced phosphorylation of ERK1/2 and AKT and enhanced the expression of Gq{alpha} protein in these cells.

Conclusions: These data suggest that C-ANP4–23 and small peptide fragments containing 12 amino acids from different regions of cytoplasmic domain of NPR-C could modulate vasoactive peptide-stimulated protein synthesis through Gq{alpha}/MAP kinase/P13K and AKT pathways.

KEYWORDS NPR-C; Peptide-1; Vasoactive peptides; Protein synthesis; ERK1/2; Gq{alpha}; VSMC

Abbreviations: C-ANP4–23, [des(Glu18, Ser19, Glu20, Leu21, Gly22)ANP4–23-NH2] • AngII, angiotensin II • AVP, arginine vasopressin • ET-1, endothelin • MAPK, mitogen-activated protein kinase • PI3K, phosphatidylinositol 3-kinase • AKT, protein kinase B • VSMC, vascular smooth muscle cells


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Natriuretic peptides (NP) are a family of three peptide hormones termed atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) [1–3]. All of these peptides are produced in mammalian hearts including humans [4] ANP regulates a variety of physiological parameters including blood pressure, progesterone secretion, renin release, vasopressin release and endothelin release by interacting with receptors on the plasma membrane either to alter the levels of second messengers such as cAMP, cGMP [5–11] or to affect ion channels [12].

Three subtypes of natriuretic peptide receptors (NPR): (NPR)-A, NPR-B and NPR-C have been described [12]. NPR-A and NPR-B are membrane guanylyl cyclases, whereas NPR-C (clearance receptor) lacks guanylyl cyclase activity. NPR-A catalyzes the production of cGMP in response to ANP and BNP, whereas NPR-B is the target for CNP. NPR-C is coupled to adenylyl cyclase inhibition through inhibitory guanine nucleotide-regulatory protein Gi [13,14] or to activation of phospholipase C [15]. However, we have recently shown that NPR-C-mediated decrease in cAMP levels contributes to the activation of PLC signaling and suggested a cross-talk between NPR-C-mediated adenylyl cyclase and PLC signaling pathways [16].

NPR-C appears to exist as a disulfide-linked homodimer with a molecular mass of 64–66 KDa having a single transmembrane domain [17], an extracellular domain and a short 37 amino acid cytoplasmic domain or tail. We have previously demonstrated that the 37 amino acid peptide (R37A) corresponding to the cytoplasmic domain of the NPR-C inhibited adenylyl cyclase activity in rat heart particulate fractions which was completely blocked by the polyclonal rabbit antisera raised against R37A [18]. The cytoplasmic domain of NPR-C contains several Gi activator sequences [19] characterized by the presence of two basic amino acids at the amino terminal (N-terminal) and B–B–X–B or B–B–X–X–B at the carboxy terminal (C-terminal), where B and X denote basic amino acid and nonbasic amino acid respectively. We have further reported that the synthetic peptide fragments of the cytoplasmic domain of NPR-C with complete or partial Gi activator sequence inhibited adenylyl cyclase activity in heart particulate fractions and A10 VSMC, whereas the peptide fragments with no Gi activator sequences were unable to exert any inhibitory effect on adenylyl cyclase activity [19].

Hypertrophy and proliferation of vascular smooth muscle cells have been shown as an important contributor of vascular remodelling which is one of the important hall marks of vascular disease such as atherosclerosis, restenosis and hypertension. Various pathophysiological factors including AngII have been reported to cause remodelling of the vasculature via cellular hypertrophy [20,21]. Atrial natriuretic peptide (ANP) has been shown as an autocrine/paracrine modulator of cardiac hypertrophy and remodelling [22–24]. Mice with homozygous deletion of the pro-ANP deletion gene (Nppa–/–) [23] or natriuretic peptide receptor A (NPR1–/–) [24] have been shown to exhibit cardiac hypertrophy. ANP has recently been shown to inhibit cardiomyocyte hypertrophy induced by AngII and ET-1 through the induction of mitogen-activated protein kinase phosphatase-1 as well as the inhibition of MAP kinase activity [25]. In addition, a role of cGMP in antihypertrophic actions of natriuretic peptides in adult rat cardiomyocytes has also been demonstrated [22,26–28]. However, the implication of NPR-C receptor in the modulation of vascular hypertrophy induced by various factors has not been examined.

The present studies were therefore undertaken to investigate the effect of C-ANP4–23, that specifically interacts with NPR-C and small active peptide fragments of cytoplasmic domain of NPR-C on protein synthesis induced by vasoactive peptides in A10 vascular smooth muscle cells. We have shown for the first time that C-ANP4–23 and small peptide fragments containing 12 amino acids from different regions of the cytoplasmic domain of the NPR-C with complete or partial Gi activator sequence inhibit vasoactive peptide-induced protein synthesis through Gq{alpha} and MAP-kinase/P13K/AKT pathways.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
A ring-deleted analog of ANP; C-ANP4–23 was from the Peninsula Laboratories (Elmont, CA). Angiotensin II, arginine vasopressin (AVP) and endothelin (ET-1) were purchased from Sigma chemical company, St. Louis MO, USA. Peptides, #1, #2, #3, X, Y and Z were synthesized by standard solid phase techniques and were highly purified (95%–99%) by high performance liquid chromatography (Peninsula Laboratories and Chiron Technologies). Monoclonal phospho-specific-Tyr204-ERK1/2 antibody, polyclonal pAKT1/2/3 (Ser 473)-R antibody and Western blotting reagents were from Santa Cruz, California, USA).

2.1. Cell culture
The A10 cell line from rat embryonic thoracic aorta was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were plated in 75 cm2 flasks and incubated at 37 °C in 95% air and 5% CO2 humidified atmosphere in Dulbeccos's modified Eagle's medium (DMEM) (with glucose, L-glutamine, and sodium bicarbonate) containing antibiotics and 10% heat inactivated fetal calf serum (FCS) as described previously [29]. The cells were passaged upon reaching confluence with 0.5% trypsin containing 0.2% EDTA and utilized between passages 5 and 15.

2.2. Cell permeabilization
A10 VSMC were permeabilized as described previously [29]. The cells after washing once with streptolysin-0 (SLO) buffer, containing 200 mM Hepes, 50 mM NaCl, 140 mM KCl, 5 mM MgCl2 and 50 mM EGTA (pH 7.4) were incubated with SLO (0.8 U/ml) at 37 °C for 5 min. The cells were washed with DMEM without FBS. The permeabilized cells were treated with peptides in the presence or absence of hormones for 24 h and were used for Western blotting and [3H]leucine incorporation. For each experiment, non-permeabilized cells were used as control group. The cell morphology was not altered as examined under the microscope.

2.3. Cell lysis and Western blotting
Cells incubated in the absence or presence of various agents were washed twice with ice-cold PBS and lysed in 100 µl of buffer (25 mM Tris-HCl, pH 7.5, 25 mM NaCl, 1 mM Na orthovanadate, 10 mM Na fluoride, 10 mM Na pyrophosphate, 2 mM ethylene-bis(oxyethylenenitrolo)tetracetic acid, 2 mM ethylenediamine tetracetic acid, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1% Triton X-100, 0.1% sodium dodecyl sulphate (SDS), and 0.5 µg/ml leupeptin) on ice. The cell lysates were centrifuged at 12,000 g for 10 min at 4 °C. Protein concentrations were measured with the Bradford assay. Equal amounts of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membranes (Millipore, MA, USA), and incubated with the respective primary antibodies such as: monoclonal phospho-specific-Tyr204-ERK1/2 antibody (1:2000), or polyclone pAKT1/2/3 (Ser 473)-R antibody. The antigen–antibody complex was detected by a horseradish peroxidase-conjugated second antibody (1:4000), and protein bands were visualized by enhanced chemiluminescence ECL as described previously [29]. Quantitative analysis of specific bands was performed by densitometric scanning of the autoradiographs employing the enhanced laser densitometer (LKB Ultroscan XL) and quantified using the gel scan evaluation software (version 2.1) from Pharmacia (Quebec, Canada).

2.4. Methyl-[3H]leucine incorporation
Protein synthesis was evaluated by the incorporation of [3H]leucine into the cells. Subconfluent VSMC were plated in 24 well plates for 24 h and were serum deprived for 24 h to induce cell quiescence. The cells were then incubated with AngII, AVP or ET-1 10–7 M alone or in the presence of small peptide fragment of cytoplasmic domain of NPR-C (10–9–10–6M) for another 24 h with or without SLO treatment. [3H]leucine (2µCi per well) was added at the same time as that of hormones. At the end of the incubation period, the cells were harvested and the radioactivity incorporated into proteins was determined by liquid scintillation counter.

2.5. Statistical analysis
Results are expressed as mean±SEM Comparisons between groups were made with ANOVA in conjunction with the Newman–Keuls test. Results were considered significant at a value of P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Effect of C-ANP4–23 on vasoactive peptide-induced protein synthesis
A10 vascular smooth muscle cell line has been shown to demonstrate characteristics similar to those of vascular smooth muscle cells [30] has been used as model to study vascular cellular process. We have previously shown the presence of NPR-C and its modulation by vasoactive peptides such as AngII, ET-1 and AVP in these cells [31–33]. In order to investigate if AngII, AVP, ET-1 and C-ANP4–23 could modulate the protein synthesis in these cells, the effect of these peptides on protein synthesis was examined. As shown in Fig. 1, AVP, ET-1 and AngII stimulated protein synthesis in these cells by about 80%, 60%, 35% respectively as determined by [3H]leucine incorporation. However, C-ANP4–23 did not affect basal protein synthesis, but significantly inhibited AVP, ET-1 and AngII-stimulated protein synthesis. For example, AVP-induced increased protein synthesis was inhibited by about 50% by C-ANP4–23, whereas ET-1 and AngII-induced increased protein synthesis was almost abolished by C-ANP4–23.


Figure 1
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  		Fig. 1 Effect of C-ANP4–23 on vasoactive peptide-induced leucine incorporation in A-10 vascular smooth muscle cells (VSMC). A-10 VMSC were incubated in the absence (control) or presence of C-ANP4–23 (10–7 M) alone or in combination with AngII (10–7M), AVP (10–7M) or ET-1 (10–7M) for 24 h. The leucine incorporation was determined as described under «Materials and methods». The results are expressed as percentage of control taken as 100%. The values are means±SEM of three separate experiments. § P<0.05, §§ P<0.01 vs control, *P<0.05, **P<0.01 vs vasoactive peptide-induced leucine incorporation.

 
3.2. Effect of small peptide fragments of cytoplasmic domain of NPR-C on vasoactive peptide-stimulated protein synthesis
Small peptide fragments containing 12 amino acids from different regions of the cytoplasmic domain of NPR-C with partial or complete Gi activator sequences that have been shown to inhibit adenylyl cyclase activity [19] were used to investigate if these peptides could also mimic the effect of C-ANP4–23 on vasoactive peptide-induced protein synthesis. The peptide fragments used represent different parts of the cytoplasmic domain of NPR-C. These peptides consist of 12 amino acids from N-terminal, middle and C-terminal regions of the cytoplasmic domain (K461KYRITIERRNH472; peptide-1, R469RNHQEESNIGK480; peptide-2 and H481RELREDSIRSH492; peptide-3). Peptide-1 and -3 possess the required Gi activator sequences: two basic amino acids at the NH2 terminus and BBXB, BXB at the COOH terminus (where B represents a basic amino acid and X represents a nonbasic amino acid), whereas peptide-2 has two basic amino acids at the NH2 terminus but does not have the consensus sequence at the COOH terminus. On the other hand, peptides Y and X containing 8 amino acids have only the consensus sequence of the COOH terminus or lacks Gi activator sequence respectively, whereas peptide Z (12 amino acid peptide) is the scrambled peptide and serves as control for peptide-1.

As shown in Fig. 2, peptides 1, 2 and 3 (10–7M) inhibited AngII-stimulated protein synthesis in a concentration-dependent manner in SLO-treated permeabilized A10 cells with an apparent Ki between 1–5 nM (A). The maximal inhibition was about 35%. However, the scrambled peptide Z and other peptides X and Y which lack Gi activator sequences did not inhibit AngII-stimulated protein synthesis in A10 cells. In addition, none of the peptides used had any effect on vasoactive peptide-stimulated protein synthesis in non-permeabilized cells (data not shown).


Figure 2
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  		Fig. 2 Effect of different concentrations of small peptide fragments of cytoplasmic domain of NPR-C on vasoactive peptide-induced leucine incorporation in permeabilized A10 vascular smooth muscle cells (VSMC). Permeabilized A-10 VSMC were incubated in the presence of 10–7 M AngII (A), 10–7 M AVP, (B) and 10–7 M ET-1(C) alone or in combination with different peptide fragments and leucine incorporation was determined as described under "Materials and methods". The results are expressed as percentage of control taken as 100%. The values are means±SEM of three separate experiments. *P<0.05, **P<0.01 ***P<0.001 vs control.

 
Similarly, AVP as well as ET-1-stimulated protein synthesis was also inhibited by peptides 1, 2 and 3 (B, C) in a concentration-dependent manner with an apparent Ki of about 1–5 and 5–10 nM respectively. The maximal inhibitions observed were between 30–40% and 30–50% respectively. On the other hand, peptides X, Y and Z were unable to inhibit AVP- and ET-1-induced enhanced protein synthesis. Furthermore, none of the active peptides had any effect on basal protein synthesis (data not shown).

3.3. Implication of MAP kinase in vasoactive peptide-stimulated protein synthesis of A10 VSMC
Since ANP has been reported to inhibit cardiomyocyte hypertrophy through the inhibition of ERK1/2 activity [25], it was of interest to determine if the peptide fragments of cytoplasmic domain of NPR-C could also inhibit the vasoactive peptide-stimulated protein synthesis through MAP kinase pathway. For these studies, the effect of peptide-1 on vasoactive peptide-stimulated ERK1/2 phosphorylation was examined in A10 VSMC. The results shown in Fig. 3 indicate that AngII, AVP and ET-1 significantly enhanced the ERK1/2 phosphorylation by about 25%, 45% and 30% which was almost completely abolished by peptide-1. However, peptide-1 alone did not have any significant effect on ERK1/2 phosphorylation in these cells. In addition, PD98059, MEK inhibitor also abolished completely the ERK1/2 phosphorylation stimulated by all the three vasoactive peptides (data not shown). These results suggest that peptide-1 may inhibit MAP kinase activity through similar mechanism as that of PD98059. To investigate this possibility, the effect of PD98059 on vasoactive peptide-induced protein synthesis was examined in the absence or presence of peptide-1. The results shown in Fig. 4 indicate that peptide-1 inhibited AngII-(A), AVP-(B) and ET-1-(C) induced protein synthesis by about 30%, 60% and 65% respectively. PD98059 at 10 µM on the other hand, completely inhibited the stimulated protein synthesis and this inhibition was further potentiated and reached below the control levels by peptide-1. These results suggest that peptide-1-induced inhibition of protein synthesis may also involve other mechanisms in addition to MAP kinase signaling.


Figure 3
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  		Fig. 3 Effect of peptide-1 on vasoactive peptide-induced ERK1/2 phosphorylation in permeabilized A10 vascular smooth muscle cells (VSMC). Permeabilized A-10 VSMC were pretreated in the presence of 10–7 M AngII (A), 10–7 M ET-1 (B), 10–7 M AVP alone or in combination with peptide-1 (pep-1). Phosphorylated ERK1/2 protein expression was determined as described under "Materials and methods". The results are expressed as percentage of control taken as 100%. The values are means±SEM of three separate experiments. *P<0.05, **P<0.01.

 

Figure 4
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  		Fig. 4 Effect of PD98059 (PD) on vasoactive peptide-induced leucine incorporation in the absence or presence of peptide-1 in permeabilized A10 vascular smooth muscle cells (VSMC). Permeabilized A-10 VSMC were pretreated in the absence or presence of PD98059 (PD) for 30 min and were further incubated in the presence of 10–7 M AngII (A), 10–7 M AVP (B) or 10–7 M ET-1(C) alone or in combination with peptide-1 (pep-1) and the leucine incorporation was determined as described under "Materials and methods". The results are expressed as percentage of control taken as 100%. The values are means±SEM of three separate experiments. *P<0.05, **P<0.01, ***P<0.001.

 
3.4. Implication of Gq{alpha} protein in vasoactive peptide-stimulated protein synthesis
The implication of Gq{alpha} in cardiac hypertrophy has been reported [34–36]. The cardiac overexpression of wild type Gq{alpha} or constitutively active mutant form of Gq{alpha} has been shown to result in myocardial hypertrophy [34], whereas the inhibition of Gq{alpha} signaling by a peptide inhibitor of Gq{alpha} overexpressed in VSMC transgenic mice has been shown to inhibit cardiac hypertrophy [36]. Furthermore, the stimulation of Gq pathway by various agonists including AngII was also shown to induce cardiac hypertrophy [35]. Taken together it may be possible that antihypertrophic effect of peptide-1 on vasoactive peptide-stimulated protein synthesis in VSMC may also be attributed to its ability to attenuate vasoactive peptide-induced increased expression of Gq{alpha} proteins. To investigate this possibility, the effect of peptide-1 on vasoactive peptide-induced enhanced expression of Gq{alpha} protein was examined. The results shown in Fig. 5 indicate that AngII increased the expression of Gq{alpha} protein by about 70%, which was completely abolished by peptide-1. However, peptide-1 did not alter the expression of Gq{alpha} protein in control cells. Similar inhibitory effect of peptide-1 on AVP-and ET-1-induced increased expression of Gq{alpha} protein was also observed (data not shown).


Figure 5
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  		Fig. 5 Effect of peptide-1 on AngII-induced Gq{alpha} expression in permeabilized A10 vascular smooth muscle cells (VSMC). Permeabilized A-10 VSMC were pretreated in the presence of 10–7 M AngII alone or in combination with 10–7 M peptide-1 (pep-1) for 24 h. The cell lysates were used for Western blotting as described under «Materials and methods». Gq{alpha} protein expression was determinated as described under «Materials and methods». The results are expressed as percentage of control taken as 100%. The values are means±SEM of three separate experiments. **P<0.01.

 
3.5. Implication of PI3kinase/AKT on peptide1-induced attenuation of vasoactive peptide-stimulated cell proliferation
Because PI3K is an upstream regulator of ERK1/2 activation cascade and has been shown to be activated by AngII [37], it was of interest to assess if PI3K pathway is also implicated in mediating the peptide-1-induced inhibition of vasoactive peptide-stimulated protein synthesis in A10 cells. Results shown in Fig. 6 indicate that wortmannin, an inhibitor of PI3K, and peptide-1 inhibited AngII-, AVP- and ET-1-stimulated protein synthesis to various degrees. However, when the effect of both wortmannin and peptide-1 was examined together, the inhibition was potentiated further. Since AKT/PKB is a downstream signaling molecule of PI3K [38], it was of interest to investigate if peptide-1 could inhibit the activity of AKT/PKB. The results shown in Fig. 7 demonstrate that AngII, AVP and ET-1 but not peptide-1 enhanced the phosphorylation of AKT by about 40%, 35% and 30% respectively and this augmented phosphorylation was significantly inhibited by peptide-1.


Figure 6
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  		Fig. 6 Effect of wortmannin (WM) on vasoactive peptide-induced leucine incorporation in the absence or presence of peptide-1 in permeabilized A10 vascular smooth muscle cells (VSMC). Permeabilized A-10 VSMC were pretreated in the absence or presence of wortmannin for 30 min and were further incubated in the presence of 10–7 M AngII (A), 10–7 M AVP (B) or 10–7 M ET-1 (C) alone or in combination with peptide-1 (pep-1) for 24 h and the leucine incorporation was determined as described under "Materials and methods". The results are expressed as percentage of control taken as 100%. The values are means±SEM of three separate experiments. *P<0.05, **P<0.01, ***P<0.001.

 

Figure 7
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  		Fig. 7 Effect of peptide-1 on vasoactive peptide-induced AKT1/2 phosphorylation in permeabilized A10 vascular smooth muscle cells (VSMC). Permeabilized A-10 VSMC were pretreated in the presence of 10–7M AngII (A), 10–7 M ET-1 (B), 10–7 M AVP alone or in combination with peptide-1. Phosphorylated AKT1/2 protein expression was determined as described under "Materials and methods". The results are expressed as percentage of control taken as 100%. The values are means±SEM of three separate experiments. *P<0.05, **P<0.01.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Atrial natriuretic peptide (ANP) has been shown to inhibit cardiomyocyte hypertrophy through NPR-A receptor involving cGMP and MAP kinase signaling [25,27]. However, the present studies demonstrate for the first time that NPR-C agonist C-ANP4–23 and other small peptide fragments of cytoplasmic domain of NPR-C containing 12 amino acids having Gi activator sequences inhibit vasoactive-peptide-induced protein synthesis in A10 vascular smooth muscle cells without affecting the basal protein synthesis.

The small peptide fragments of the cytoplasmic domain of NPR-C containing 12 amino acids from different regions; N-terminal K461–H472 (peptide-1), middle region R469–R480 (peptide-2) and C-terminal H481–H492 (peptide 3) with consensus sequence for Gi activation at both NH2 and COOH terminus that have been reported to inhibit adenylyl cyclase [19] also inhibited protein synthesis stimulated by AngII, AVP and ET-1 in A-10 vascular smooth muscle cells. As shown previously [19], the inhibitory effect of these peptides on protein synthesis was also not due to the net positive charge present (i.e. amino acid composition), since the scrambled peptide Z with the same composition as that of peptide-1, but lacking the Gi activator sequence at the NH2 and COOH terminus did not inhibit vasoactive-induced protein synthesis in these cells. On the other hand, the absence or presence of partial COOH-terminal motif (BXB) but intact N-terminal motif (BB) in the peptide-2 and 3 respectively did not significantly change the potency of the peptide to inhibit protein synthesis suggesting that a COOH-terminal motif in the peptide may not be required to exert inhibitory effect on protein synthesis. This notion is further supported by our studies showing that C-ANP4–23 that lacks C-terminal motif (BXB) also inhibited the protein synthesis stimulated by vasoactive peptides. These results are in agreement with our previous studies showing that 12 amino acid peptide as well as C-ANP4–23 that lack COOH-terminal motif were able to exert inhibitory effect on adenylyl cyclase activity ([19]. However, the truncation of NH2-terminal motif of peptide (peptide Y) that has been shown to inactivate the peptide to inhibit adenylyl cyclase [19] was also unable to inhibit protein synthesis stimulated by AngII, AVP and ET-1 in A10 cells, suggesting that NH2-terminal motif is important for the activation of the peptide and to exert physiological functions.

Various vasoactive peptides including AngII and endothelin-1 have been shown to induce cardiomyocyte hypertrophy [36] via the interaction with their receptors coupled to phosphatidyl inositol turnover signaling through Gq{alpha} protein. The intracellular signaling pathways that are activated by these vasoactive peptides include protein kinase C and mitogen-activated protein kinase (MAPK) family, result in the induction of many specific genes and protein synthesis. Our results showing that peptide-1 attenuated the vasoactive peptide-induced enhanced ERK1/2 and AKT phosphorylation suggest that the antihypertrophic effect of peptide-1 may also be mediated through MAPK/PI3K/AKT signaling pathway. However, the fact that peptide-1 further potentiated the inhibitory effect of MEK inhibitor; PD98059 and PI3K inhibitor; wortmannin, on vasoactive peptide-induced protein synthesis suggests that the mechanisms other than MAP kinase and PI3 kinase signaling may also contribute to the antihypertrophic effect of peptide-1. In this regard, the implication of Gq{alpha} protein and associated signaling has been reported in cardiac hypertrophy [34,36]. The heterozygous transgenic overexpression of Gq{alpha} in cardiomyocytes has been shown to induce cardiac enlargement with molecular, structural and functional characteristics of pressure overload hypertrophy [39]. Our results showing the attenuation of AngII-induced enhanced expression of Gq{alpha} by peptide-1, suggest that peptide-1-induced decreased expression of Gq{alpha} may also contribute to the antihypertrophic response of peptide-1. Whether peptide-1-induced decreased expression of Gq{alpha} is responsible for the decreased MAPK activity and thereby attenuation of protein synthesis is not clear and needs to be investigated. However, the implication of MAPK signaling in Gq{alpha}-induced cardiac hypertrophy has been shown [40]. On the other hand, cardiac specific Gq{alpha} overexpression-induced hypertrophy in transgenic mice was shown to be independent of increases in ERK activity [39]. Taken together, it may be suggested that peptide-1-induced inhibition of both MAPK/PI3K/AKT signaling and decreased expression of Gq{alpha} protein may be responsible for the antihypertrophic effect of the peptide-1 (Fig. 8).


Figure 8
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  		Fig. 8 Schematic model showing potential mechanisms responsible for the antihypertrophic effect of small peptide fragment of cytoplasmic domain of NPR-C (peptide-1). Vasoactive peptides (AngII, ET-1 and AVP)-induced enhanced expression of Gq{alpha} protein as well as ERK1/2 and AKT activation contributes to the enhanced protein synthesis of VSMC. Peptide-1 exerts antihypertrophic effect through its ability to attenuate both the enhanced Gq{alpha} protein expression as well as ERK1/2 and AKT activity. Solid lines (–) denote activation; broken lines (- - - - - -) denote inhibition.

 
In conclusion, we have provided the first evidence to demonstrate that the NPR-C activation by C-ANP4–23 as well as the short cytoplasmic domain peptide consisting of 12 amino acids with Gi activator sequence irrespective of the region of NPR-C inhibit the hypertrophic responses of vasoactive peptides through Gq{alpha} protein, MAPK and PI3K/AKT signaling mechanism in A10 vascular smooth muscle cells. From these studies, it may be suggested that antihypertrophic properties of the small peptide fragments of NPR-C can be used in designing the new therapies in the treatment of cardiovascular complications.


   Acknowledgements
 
We would like to thank Christiane Laurier for her valuable secretarial help.


   Notes
 
* This study was supported by a grant from the Canadian Institutes of Health Research (MOP 13661).

Time for primary review 22 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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