Small cytoplasmic domain peptides of natriuretic peptide receptor-C ...

Page 1 Articles of 43 in PresS. Am J Physiol Heart Circ Physiol (August 18, 2006). doi:10.1152/ajpheart.00327.2006

Small cytoplasmic domain peptides of natriuretic peptide receptor-C attenuate cell proliferation through Gi protein /MAPkinase /PI3kinase/AKT pathways*

Shehla Hashim, Yuan Li and Madhu B. Anand-Srivastava** Department of Physiology, and Groupe de recherche sur le système nerveux autonome (GRSNA), Faculty of Medicine, University of Montreal, Montreal, Canada

Correspondance should be sent Dr. Madhu B. Anand-Srivastava, Ph.D. Professor Department of Physiology Faculty of Medicine University of Montreal C.P. 6128, Succ. Centre-ville Montreal, Quebec, Canada H3C 3J7 Tel : (514) 343-2091 Fax: (514) 343-2111 E-mail: [email protected]

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Copyright Information Copyright © 2006 by the American Physiological Society.

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ABSTRACT The present studies were undertaken to investigate the effect of C-ANP4-23, and several peptide fragments containing 12 amino acids from different regions of cytoplasmic domain of NPR-C on cell proliferation in the absence or presence of angiotensin II (Ang II), endothelin (ET-1) and arginine-vasopressin (AVP) in A10 vascular smooth muscle cells (VSMC). The peptide fragments used have either complete Gi activator sequences K461-H472 (peptide-1) and H481-H493 (peptide 3) or partial Gi activator sequences R469-K480 (peptide 2) and I465-H472 (peptide Y) with truncated carboxyl or amino terminus respectively. The other peptide used had no structural specificity Q473-K480 (peptide X) or was the scrambled peptide control for peptide-1 (peptide Z). Ang II, ET-1 and AVP significantly stimulated DNA synthesis in these cells as determined by 3H-thymidine incorporation that was inhibited by peptides 1, 2 and 3 and not by peptides X, Y and Z in a concentration-dependent manner, with an apparent Ki between 1-10 nM. In addition, C-ANP4-23, that interacts with NPR-C, also inhibited DNA synthesis stimulated by vasoactive peptides; however, the inhibition elicited by C-ANP4-23 was not additive with the inhibition elicited by peptide-1. On the other hand, basal DNA synthesis in these cells was not inhibited by C-ANP4-23 or by the peptide fragments. Furthermore, vasoactive peptide-induced stimulation of DNA synthesis was inhibited by PD98059 and wortmannin, and this inhibition was potentiated by peptide-1. In addition, peptide-1 also inhibited vasoactive peptide-induced phosphorylation of ERK1/2 and AKT and enhanced expression of Gi

proteins. These data

suggest that C-ANP4-23 and small peptide fragments containing 12 amino acids irrespective of the region of cytoplasmic domain of NPR-C inhibit the proliferative responses of vasoactive peptides through Gi protein and MAP kinase/PI3K/AKT pathways. Key words: NPR-C, vasoactive peptides, DNA synthesis, ERK1/2, peptide-1, VSMC.

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INTRODUCTION 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) (13,43,44). All of these peptides are produced in mammalian hearts including humans (29, 49). 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 (1,2,4,5, 9,10), cGMP (20, 46, 47) or to affect ion channels (3). The other members of the natriuretic peptide family act as antagonists to vasopressin, endothelin and the renin-angiotensin-aldosterone system (13, 38). CNP has also been shown to be a vasodilator produced by endothelial cells (44,45). Compared with ANP, BNP has an additional six amino acid sequence at its amino terminal end (13, 43) whereas CNP lacks the carboxy terminal extension (44). Molecular cloning techniques revealed three subtypes of natriuretic peptide receptors (NPR):

(NPR)-A (17, 31), NPR-B (16, 42) and NPR-C (8,19).

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 NPRB is the target for CNP.

NPR-C is coupled to adenylyl cyclase inhibition through inhibitory

guanine nucleotide-regulatory protein Gi (6, 8) or to activation of phospholipase C (22). 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 (32). NPR-C is a disulfide-linked homodimer of 64-66 KDa having a single transmembrane domain (19, 26, 40), an extracellular domain of ~ 440 amino acids and a short 37-amino acid

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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 (7). The cytoplasmic domain of NPR-C contains several Giactivator sequences (34) 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 carboxy terminal (C-terminal), where B and X denote basic amino acid and non-basic 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 (34). The present studies were undertaken to investigate if small active peptide fragments of 12 amino acids of cytoplasmic domain of NPR-C that have been shown to inhibit adenylyl cyclase could also modulate the proliferative effect of vasoactive peptides such as Ang II, ET-1 and AVP in A10 vascular smooth muscle cells. For these studies, the effect of small peptides of NPR-C on cell proliferation determined by [3H] thymidine incorporation was examined in the absence or presence of Ang II, ET-1 and AVP in A 10 VSMC. We have shown for the first time that 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 cell proliferation through GiM/MAP kinase/P13K/AKT pathways.

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EXPERIMENTAL Materials: A ring-deleted analog of ANP; c-ANP4-23 was from Peninsula Laboratories (Belmont, 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. Methods: Cell culture: The A10 cell line from rat embryonic thoracic aorta was obtained from American Type Culture Collection (Manassas, VA, USA). The cells were plated in 7.5 cm2 flasks and incubated at 370C 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 (36). The cells were passaged upon reaching confluence with 0.5% trypsin containing 0.2% EDTA and utilized between passages 5 and 15. Cell permeabilization: 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 37oC for 5 minutes. The cells were washed with DMEM without FBS. The permeabilized cells were treated with peptides in the presence or absence of hormones for 24 hrs. For each experiment non-permeabilized cells were used as control group.

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Cell lysis and Western blotting: Cells incubated in the absence or presence of various agents were washed twice with icecold PBS and lysed in 100 Ql 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 ethylenebis(oxyethylenenitrolo)tetraacetic acid, 2 mM ethylenediamine tetracetic acid, 1 mM phenylmethylsulfonyl fluoride, 10 Qg/ml aprotinin, 1% Triton X-100, 0.1% sodium dodecyl sulphate (SDS), and 0.5 Qg/ml leupeptin) on ice. The cell lysates were centrifuged at 12,000 g for 10 min at 4oC. Protein concentrations were measured with the Bradford assay.

Equal

amounts of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDSPAGE), transferred to PVDF membranes (Millipore, MA, USA), and incubated with respective primary antibodies: monoclonal phospho-specific-Tyr204-ERK1/2 antibody (1:2000) or polyclonal pAKT1/2/3 (ser473)R antibody (1:1000).

The antigen-antibody complex was

detected by a horseradish peroxidase-conjugated second antibody (1:400), and protein bands were visualized by enhanced chemiluminescence ECL as described previously (35). 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). Methyl-[3H]thymidine Incorporation: DNA synthesis was evaluated by incorporation of [3H]thymidine into cells. Subconfluent A10 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 Ang II, AVP or ET-1 (10-7M) alone or in the presence of small fragments of cytoplasmic domain of NPR-C (10-9-10-6 M) for 24 hours with or without SLO treatment. [3H] thymidine (1 QCi) was added and further

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incubated for 4 h before harvesting the cells. The cells were rinsed twice with ice-cold phosphate buffered saline (PBS) and incubated with 5% trichloroacetic acid (TCA) for 1 h at 4oC. After washing twice with ice cold water, the cells were incubated with 0.4 N sodium hydroxide solution for 30 minutes at room temperature and the radioactivity was determined by liquid scintillation counter. The cell viability was checked by trypan blue exclusion technique. Pertussis toxin (PT) treatment: The serum-deprived cells were pretreated with or without PT (500 ng/ml) for ½ h at 37oC. The cells were further incubated in the absence or presence of Ang II, AVP, or ET-1 (107

M ) for 24 h and used for thymidine incorporation .

Statistical analysis: Results are expressed as mean ± S.E.M. 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. RESULTS Effect of C-ANP4-23 on vasoactive peptides-induced cell proliferation A10 vascular smooth muscle cell line has been shown to demonstrate characteristics similar to those of vascular smooth muscle cells (25) and 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 Ang II, ET-1 and AVP in these cells (11,12, 35). In order to investigate if Ang II, AVP, ET-1 and C-ANP4-23 could modulate cell proliferation in these cells, the effect of these peptides on DNA synthesis was examined. As shown in Fig. 1, AVP, ET-1 and Ang II stimulated DNA synthesis ìn these cells by about 70%, 50%, 25% respectively as determined by [3H]thymidine incorporation. However, C-ANP4-23, a ring deleted peptide that

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interacts specifically with NPR-C did not affect basal DNA synthesis, but significantly inhibited AVP, ET-1- and Ang II-stimulated DNA synthesis. For example AVP-induced increased DNA synthesis was inhibited by about 65% by C-ANP4-23 whereas ET-1 and Ang II-induced increased DNA synthesis was almost abolished by C-ANP4-23. Effect of small peptide fragments of cytoplasmic domain of NPR-C on vasoactive peptide stimulated DNA 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 (34) were used to investigate if these peptides could also mimic the effect of C-ANP4-23 on vasoactive peptide-induced DNA synthesis. The peptide fragments used represent different parts of the cytoplasmic domain as shown in Fig. 2. These consist of 12 amino acids (peptides 1, 2, 3 and Z) or 8 amino acids (peptides X and Y). 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, peptide Y has only the consensus sequence of the COOH terminus, and peptide X lacks Gi activator sequence, whereas peptide Z is the scrambled peptide and serves as control for peptide-1. As shown in Fig. 3, the peptides 1, 2 and 3 (10-7M) inhibited Ang II-stimulated DNA synthesis (A) in a concentration-dependent manner in SLO-treated permeabilized VSMC. The maximal inhibition observed was about 60%, however, peptide-1 was more potent than peptides 2 and 3 and inhibited DNA synthesis with an apparent Ki of 5 nM, whereas a Ki of about 10 nM

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was observed for peptides 2 and 3. On the other hand, the scrambled peptide Z and peptides X and Y with no Gi activator sequences were unable to inhibit Ang II-stimulated DNA synthesis in A10 VSMC. In addition, none of the peptides used had any effect on vasoactive peptidestimulated cell proliferation in non permeabilized cells (data not shown). Similarly, AVP-stimulated DNA synthesis was also inhibited in a concentrationdependent manner by peptides 1, 2 and 3. All the three peptides were almost equipotent and inhibited DNA synthesis with an apparent Ki of about 1 nM , however, peptide-1 inhibited the DNA synthesis by about 45%, whereas about 30 % inhibition was observed by peptide-2 and 3 (B). On the other hand, the scrambled peptide Z and inactive peptides (X and Y) without Gi activator sequences did not inhibit AVP-induced DNA synthesis in A10 VSMC. In addition, peptides 1, 2 and 3 but not X, Y and Z were also able to inhibit ET-1 stimulated DNA synthesis in a concentration dependent manner (C). The maximal inhibition of DNA synthesis by peptide1 was about 60% and by peptides 2 and 3 was about 30% with an optimal Ki between 5-10 nM. However, the peptide fragments did not have any effect on basal DNA synthesis (data not shown). In order to investigate if NPR-C ligand C-ANP4-23 as well as NPR-C peptide-1 act via the same pathway, the effect of both C-ANP4-23 (10-7M) and peptide-1 (10-7M) on AVP-induced enhanced DNA synthesis was examined together. The results shown in Fig. 4 indicate that CANP4-23 as well as peptide-1 inhibited AVP-stimulated DNA synthesis by about 30%, however, no additive effect was observed when both the ligands were added together. Implication of MAP Kinase in vasoactive peptide-stimulated cell proliferation Since C-ANP4-23 has been shown to inhibit cell proliferation through the MAP kinase pathway in astrocytes (37), it was of interest to determine if the inhibition of the vasoactive

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peptide-stimulated vascular smooth muscle cell proliferation by the peptide fragments of cytoplasmic domain of NPR-C is also mediated through MAP kinase pathway. For these studies, the effect of peptide-1 on vasoactive peptides-stimulated

ERK1/2 phosphorylation was

examined in A10 VSMC. The results shown in Fig. 5 indicate that Ang II, AVP and ET-1 significantly enhanced the ERK1/2 phosphorylation by about 40, 40 and 50% ( n=6 ), which was 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 DNA synthesis was examined in the absence or presence of peptide1. The results shown in Fig. 6 indicate that peptide-1 inhibited Ang II-(A), AVP-(B) and ET-1(C) induced DNA synthesis by about 65, 50 and 80% respectively. PD98059 at 10 QM on the other hand, completely inhibited the stimulated DNA 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 cell proliferation may also involve some other mechanisms in addition to MAP kinase pathway. Implication of Gi protein in vasoactive peptide-stimulated cell proliferation Gi

proteins have been shown to be implicated in cell proliferation (33). We have

recently shown that the enhanced cell proliferation of VSMC from spontaneously hypertensive rats (SHR) was attributed to the enhanced levels of Gi

proteins, because the treatment of

VSMC from SHR with pertussis toxin (PT) restored the enhanced cell proliferation to control levels (30). To investigate if the Gi protein is also implicated in vasoactive-induced enhanced

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cell proliferation, the effect of PT treatment on Ang II-, AVP- and ET-1-induced DNA synthesis was examined and the results are shown in Fig. 7. As reported before, Ang II, AVP, ET-1 increased DNA synthesis to various degrees and this increase was significantly attenuated by PT. Thus, taken together, it may be possible that the additive antiproliferative effect of peptide-1 and MAP kinase inhibitor; PD98059 on vasoactive peptide-stimulated proliferation of VSMC (Fig.6) may be attributed to its ability to attenuate vasoactive peptide-induced increased expression of Gi

proteins.

To investigate this possibility, the effect of peptide-1 on the

expression of Gi proteins was examined and the results are shown in Fig .8. As reported earlier (11), ET-1 increased the expression of Gi -2 and Gi -3 protein by about 45% and 30%, which was completely abolished by peptide-1. However, peptide-1 did not alter the expression of Gi proteins in control cells. Similar effect of peptide-1 on AVP and Ang II-induced increased expression of Gi

protein was observed (data not shown). It may be possible that peptide-1-

induced decreased expression of Gi protein results in decreased ERK1/2 phosphorylation and thereby decreased cell proliferation. To investigate the relationship between the Gi

protein and

MAP Kinase activation, we tested the effect of PT on Ang II-induced increased ERK1/2 phosphorylation. The results shown in Fig. 9 indicate that PT treatment was ineffective in altering Ang II-induced increased phosphorylation of ERK1/2, suggesting that Gi

protein is

not implicated in MAP kinase activation. However, ERK1/2 phosphorylation and not AKT phosphorylation was inhibited by PD98059, whereas wortmannin; an inhibitor of PI3K was able to inhibit pAKT and not pERK!/2 in these cells. Implication of PI3kinase/AKT on peptide1-induced attenuation of vasoactive peptide stimulated cell proliferation

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Because PI3K is an upstream regulator of ERK1/2 activation cascade and has been shown to be activated by Ang II (39), it was of interest to assess if PI3K pathway is also implicated in mediating the peptide-1-induced inhibition of vasoactive peptide-stimulated cell proliferation of A10 cells. Results shown in Fig. 10 indicate that wortmannin, and peptide-1 inhibited Ang IIAVP- and ET-1-stimulated cell proliferation 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 signalling molecule of PI3K (18), it was of interest to investigate if peptide-1 could inhibit the activity of AKT/PKB. The results shown in Fig.11 demonstrate that Ang II, AVP and ET-1 but not peptide-1 enhanced the phosphorylation of AKT1/2 by about 80% ,40% and 30% respectively. Peptide-1, on the other hand completely abolished the AVP- and ET-1-induced augmented phosphorylation of AKT1/2 , whereas about 85% attenuation of AngII- induced-enhanced AKT1/2 phosphorylation was observed.

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DISCUSSION Natriuretic peptides have been shown to inhibit the proliferation of several cell types including VSMC (14, 23, 28). Prins et al. (37) have shown that C-ANP4-23, inhibits mitogenic factors-stimulated cell proliferation of astrocytes via MAP kinase pathway. Recently, Lelièvre et al. (27) have reported proliferative and antiproliferative action of ANP in neuroblastoma cells; lower concentration of ANP and CNP stimulate cell proliferation in a cGMP-dependent mechanism, and higher concentration of ANP and CNP and low concentration of C-ANP4-23 exert antiproliferative action mediated through MAP kinase pathway. However, the present studies demonstrate for the first time that the small peptide fragments of cytoplasmic domain containing 12 amino acids of NPR-C having complete or partial Gi activator sequences inhibit vasoactive-peptide-induced cell proliferation in A10 vascular smooth muscle cells, however these peptides did not have any effect on basal cell proliferation. The antiproliferative effect of peptide-1 may not be attributed to apotosis, because the cell viability checked by trypan blue exclusion indicate that more than 90-95% cells were viable and secondly, peptide-1 did not inhibit DNA synthesis in control cells. 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 H481-493 (peptide 3) with consensus sequence for Gi activation at both NH2 and COOH terminus that have been reported to inhibit adenylyl cyclase activity (34), inhibited cell proliferation stimulated by Ang II, AVP and ET-1 in A-10 vascular smooth muscle cells. As shown previously (34), the inhibitory effect of these peptides on cell proliferation 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

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NH2 and COOH terminus did not inhibit vasoactive-induced cell proliferation in these cells. On the other hand, the absence or presence of partial COOH-terminal motif (BXB) but intact Nterminal motif (BB) in the peptide 2 and 3 respectively did not significantly change the potency of the peptide to inhibit cell proliferation suggesting that a COOH-terminal motif in the peptide may not

be required to exert inhibitory effect on cell proliferation.

These results are in

agreement with the studies of Kanwal et al. (24), who have shown that 15 amino acid peptide fragment of NPR-C (Arg1-Gln15) lacking COOH-terminal motif attenuated dopamine efflux in pheochromocytoma cells (PC12). However, the truncation of NH2-terminal motif of peptide (peptide Y) that has been shown to inactivate the peptide to inhibit adenylyl cyclase (34) was also unable to inhibit cell proliferation stimulated by Ang II, 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. Our results showing that C-ANP4-23, like small peptide fragments also inhibited vasoactive-induced cell proliferation in A10 VSMC without affecting basal levels, are in accordance with the studies of other investigators (14) who were also unable to show any effect of C-ANP4-23 on basal cell proliferation. In addition, our results showing that inhibitory effect of C-ANP4-23 and peptide-1 on AVP-induced DNA synthesis was not additive further suggests that both NPR-C ligand and NPR-C peptide-1 act via the same pathway. A role of cAMP in cell proliferation has been reported (21, 41), however, cAMP that has been shown to be decreased by C-ANP4-23 as well as by small peptide fragment in A10 VSMC (34) does not appear to contribute to the antiproliferative effect of the peptides, because decreased cAMP levels have been reported to exert hyperproliferation of the cells (21, 41). This notion is supported by the studies of other investigators who have also shown that C-ANP4-23 inhibits cell proliferation by cAMP-independent mechanism (37). The implication of MAP

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kinase and P13 kinase in cell proliferation has been reported (48). Our results showing that the peptide-1 attenuated the vasoactive peptide-induced enhanced Erk1/2 and AKT phosphorylation suggest that the antiproliferative effect of peptide-1 may also be mediated through MAPK/P13K/AKT signalling pathway. However, the fact that peptide-1 further potentiated the inhibitory effect of MEK inhibitor; PD98059 and P13K inhibitor; wortmannin on vasoative peptide-induced cell proliferation suggest that mechanisms other than MAP kinase and P13 kinase sigaling may also contribute to the antiproliferative effect of peptide-1. In this regard, we have shown that treatment of A10 cells with peptide-1 resulted in the decreased expression of Gi

protein stimulated by vasoactive peptides, which may also be responsible for the

antiproliferative effect of peptide-1. This notion is substantiated by our studies showing that PT treatment that inactivates Gi proteins attenuated vasoactive peptide-evoked enhanced cell proliferation in A10 cells. The implication of Gi protein in the cell proliferation has also been demonstrated by other investigators (30, 33). On the other hand, decreased expression of Gi

the peptide-1-induced

protein may not be responsible for the decreased Erk1/2

phosphorylation, because PT treatment was ineffective in decreasing Ang II-induced enhanced Erk1/2 phosphorylation in A10 VSMC. The implication of MAP kinase signaling in Ang IIinduced enhanced expression of Gi protein in A10 VSMC has been reported (15). However, the results showing that PT treatment inhibits Ang II-induced Gi protein, DNA synthesis but not Erk1/2 phosphorylation suggests that MAP kinase-induced increased DNA synthesis may not be mediated through Gi proteins. Taken together, it may be suggested that peptide-1 induced inhibition of both MAPK/PI3K/AKT signalling and decreased expression of Gi protein may be responsible for the antiproliferative effect of the peptide-1 (Fig.12).

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In conclusion, we have provided the first evidence to demonstrate that the short cytoplasmic domain peptide consisting of 12 amino acids with

Gi activator sequence

irrespective of the region of NPR-C inhibit the proliferative responses of vasoactive peptides through Gi

protein, MAPK and PI3K/AKT signaling mechanism in A10 vascular smooth

muscle cells. From these studies, it may be suggested that antimitogenic properties of the small peptide fragments of NPR-C can be used in designing the new therapies in the treatment of cardiovascular complications.

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FOOTNOTES *

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

**

To whom correspondence should be addressed. ABBREVIATIONS

C-ANP4-23, [des(Glu18,Ser19,Glu20, Leu21, Gly22)ANP4-23-NH2]; Ang II, angiotensin II; AVP, arginine vasopressin; ET-1, endothelin, MAPK, mitogen-activated protein kinase; MEK, mitogen extracellular signal regulated kinase kinase; ERK1/2,extracellular signal regulated kinase; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B, VSMC, vascular smooth muscle cells.

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ACKNOWLEDGEMENTS

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

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Figure legends Figure 1.

Effect of C-ANP4-23 on vasoactive peptide-induced thymidine 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 ( 10-7 M), AVP( 10-7 M) or ET-1 (10-7 M) for 24 h. The thymidine incorporation was determined as described under the "Experimental". The result are expressed as percentage of control taken as 100%. The values are means ± SEM of three separate experiments. § p