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Am J Physiol Heart Circ Physiol 293: H2072-H2079, 2007. First published July 20, 2007; doi:10.1152/ajpheart.01097.2006
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Oxygen Sensing: Life and Death of a Cell

Nitric oxide attenuates endothelin-1-induced activation of ERK1/2, PKB, and Pyk2 in vascular smooth muscle cells by a cGMP-dependent pathway

Ali Bouallegue, Grace Bou Daou, and Ashok K. Srivastava

Laboratory of Cell Signaling, Montreal Diabetes Research Centre, Centre de Recherche, Centre Hospitalier de l'Université de Montréal, Technopole Angus Campus, and Department of Medicine, University of Montreal, Montreal, Quebec, Canada

Submitted 6 October 2006 ; accepted in final form 13 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Nitric oxide (NO), in addition to its vasodilator action, has also been shown to antagonize the mitogenic and hypertrophic responses of growth factors and vasoactive peptides such as endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs). However, the mechanism by which NO exerts its antimitogenic and antihypertrophic effect remains unknown. Therefore, the aim of this study was to determine whether NO generation would modify ET-1-induced signaling pathways involved in cellular growth, proliferation, and hypertrophy in A-10 VSMCs. Treatment of A-10 VSMCs with S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP), two NO donors, attenuated the ET-1-enhanced phosphorylation of several key components of growth-promoting and hypertrophic signaling pathways such as ERK1/2, PKB, and Pyk2. On the other hand, inhibition of the endogenous NO generation with NG-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, increased the ET-1-induced phosphorylation of these signaling components. Since NO mediates its effect principally through a cGMP-soluble guanylyl cyclase (sGC) pathway, we investigated the role of these molecules in NO action. 8-Bromoguanosine 3',5'-cyclic monophosphate, a nonmetabolizable and cell-permeant analog of cGMP, exhibited a effect similar to that of SNAP and SNP. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of sGC, reversed the inhibitory effect of NO on ET-1-induced responses. SNAP treatment also decreased the protein synthesis induced by ET-1. Together, these data demonstrate that NO, in a cGMP-dependent manner, attenuated ET-1-induced phosphorylation of ERK1/2, PKB, and Pyk2 and also antagonized the hypertrophic effects of ET-1. It may be suggested that NO-induced generation of cGMP contributes to the inhibition of ET-1-induced mitogenic and hypertrophic responses in VSMCs.

cell signaling; protein synthesis; protein kinase G; vasculoprotection

ENDOTHELIN-1 (ET-1) is a 21-amino acid peptide and is considered a potent vasoconstrictor (47). It also exhibits mitogenic activity in vascular smooth muscle cells (VSMCs) (5, 23, 24), suggesting a possible role for ET-1 in the pathogenesis of many diseases, such as atherosclerosis (29), hypertension (18), and restenosis after angioplasty (11).

ET-1 exerts its effects through a heteromeric G protein-coupled receptor that is linked to multiple signaling pathways that include phospholipases C and D (13), Ca2+ (32), mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), and p38mapk (7, 8, 39, 50, 51), and phosphatidylinositol 3-kinase (8, 17). Activation of receptor and nonreceptor protein tyrosine kinases (PTKs) in transducing ET-1-induced signaling responses has also been suggested (16, 26, 27, 39, 49). PTKs activated by ET-1 include epidermal growth factor (EGF) (27), c-Src (16, 26, 38), and a Ca2+-dependent PTK, Pyk2 (26, 39). Of particular interest, ET-1 mediates Pyk2 activation, which contributes to ERK1/2 (26) and JNK (27) signaling in cardiomyocytes and p38mapk (39) in mesangial cells.

Nitric oxide (NO) is a free radical that has been suggested to play an important role in cardiovascular function (38). NO mediates relaxation principally through the stimulation of soluble guanylyl cyclase (sGC), leading to enhanced production of intracellular cGMP, which in turn, activates cGMP-dependent protein kinase (PKG) (30). NO can also influence cellular events by a PKG-independent mechanism (14, 22) and is also able to react with superoxide anion to form the reactive peroxynitrite radical (25), a potent oxidant with the potential to disrupt protein structures by nitrating the tyrosine residues in protein (48). In addition to its vasodilating effect, NO has been suggested to antagonize the physiological and pathophysiological effects of several growth factors such as EGF (52), angiotensin II (ANG II) (46), as well as ET-1 (1). This is probably achieved by inhibiting one or more serine/threonine/tyrosine kinases implicated in the signaling events induced by these factors. Several studies using ANG II have shown that NO suppressed the activation of ERK1/2, p38mapk, and JNK (45) as well as Pyk2 (46) in cardiac fibroblasts. It was also recently reported that in rat neonatal pulmonary VSMCs a NO donor inhibited ET-1-induced ERK1/2 phosphorylation (3). However, to our knowledge, a possible contribution of NO to ET-1-induced activation of other signaling events has not been investigated in VSMCs. Therefore, in the present studies we have examined the effect of NO on ET-1-stimulated phosphorylation of ERK1/2, PKB, and Pyk2, the key mediators of growth-promoting, proliferative, hypertrophic survival responses. In addition, we have also examined whether NO acts via a cGMP-dependent mechanism in eliciting these responses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

ET-1 was purchased from Peninsula Laboratories (Belmont, CA), and S-nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside (SNP), 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were obtained from Calbiochem (San Diego, CA). Nitro-L-arginine methyl ester (L-NAME) was purchased from Sigma Aldrich (St. Louis, MO). Monoclonal phospho-specific-Tyr204-ERK1/2 antibody, polyclonal ERK1/2 antibody, endothelial nitric oxide synthase (eNOS) antibody, inducible nitric oxide synthase (iNOS) antibody, and horseradish peroxidase-conjugated goat anti-mouse immunoglobulin were from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-specific-Ser473-PKB and total PKB as well as phospho-specific-Tyr402-Pyk2 and total Pyk2 antibodies were procured from New England Biolabs (Beverly, MA). The enhanced chemiluminescence (ECL) detection system kit and L-[4,5-3H]leucine were from Amersham Pharmacia Biotech (Baie d'Urfé, QC, Canada). Human umbilical vein endothelial cells (HUVECs) were a gift from Dr. Eric Thorin, Montreal Cardiology Institute.

Methods

Cell culture. VSMCs derived from embryonic rat thoracic aorta A-10 cells were maintained in culture with DMEM containing 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2 as described previously (40). The cells were grown to 80–90% confluence in 60-mm plates and incubated in serum-free DMEM 20 h before treatments.

Cell lysis and immunoblotting. Cells incubated in the absence or presence of various agents were washed twice with ice-cold PBS and lysed in 200 µl of buffer [mM: 25 Tris·HCl, pH 7.5, 25 NaCl, 1 Na orthovanadate, 10 Na fluoride, 10 Na pyrophosphate, 2 benzamidine, 2 ethylenebis(oxyethylenenitrilo)tetraacetic acid, 2 ethylenediaminetetraacetic acid, and 1 phenylmethylsulfonyl fluoride, with 10 µg/ml aprotinin, 1% Triton X-100, 0.1% sodium dodecyl sulfate (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 by Bradford assay. Equal amounts of protein were subjected to either 7.5% or 10% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride membranes (Millipore), and incubated with respective primary antibodies [monoclonal phospho-specific-Tyr204-ERK1/2 antibody (1:2,000), polyclonal phospho-specific-Ser473-PKB antibody (1:4,000), phospho-specific-Tyr402-Pyk2 antibody (1:1,000), and eNOS or iNOS antibodies (1:2,000)]. The antigen-antibody complex was detected by a horseradish peroxidase-conjugated secondary antibody (1:4,000), and protein bands were visualized by ECL. The intensity of specific bands was quantified by NIH Image software as described previously (31).

Measurement of [3H]leucine incorporation. A-10 cells were treated for 20 h with ET-1 (10 nM). Protein synthesis was assessed by the addition of 2 µCi/ml of [3H]leucine (ICN Biomedicals, Costa Mesa, CA) for a period of 20 h. To assess the role of NO, cells were pretreated for 30 min with 10 or 100 µM SNAP (Calbiochem), which spontaneously generates NO. After completion of the experimental protocol A-10 cells were washed twice with cold PBS, and 1 ml of cold 5% trichloroacetic acid was added for 30 min to precipitate protein. The precipitates were subsequently washed twice with cold water and resuspended in 500 µl of 0.4 M NaOH. Aliquots were counted in a scintillation counter.

Statistics. Statistical analysis was performed by one-way repeated-measures analysis of variance (ANOVA) followed by a Fisher post hoc test. All data are reported as means ± SE. The differences between means were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Both SNAP and SNP Inhibited ET-1-Induced Phosphorylation of ERK1/2, PKB, and Pyk2 in A-10 VSMCs

To determine whether the antimitogenic and antiproliferative effects of NO are mediated by its ability to attenuate growth-promoting signaling pathway in VSMCs, we examined the effect of SNAP, which spontaneously generates NO, on ET-1-induced phosphorylation of ERK1/2, PKB, and Pyk2. As shown in Fig. 1, pretreatment of A-10 VSMCs with SNAP for 15 min dose-dependently attenuated ET-1-induced phosphorylation of all of these protein kinases. Among the kinases, PKB appeared to be more sensitive to the inhibitory effect of SNAP and exhibited almost complete attenuation of ET-1-stimulated phosphorylation at 10 µM (Fig. 1B). In contrast, ET-1-enhanced phosphorylation of ERK1/2 and Pyk2 was inhibited significantly only at 300 µM SNAP.


Figure 1
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  		Fig. 1. Dose-dependent effect of the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) on endothelin-1 (ET-1)-induced extracellular signal-regulated kinases (ERK)1/2, PKB, and Pyk2 phosphorylation in A-10 vascular smooth muscle cells (VSMCs). Serum-starved quiescent A-10 cells were pretreated without or with the indicated SNAP concentrations for 15 min, followed by 10 nM ET-1 for 5 min. Cell lysates were immunoblotted by phospho-specific-Tyr204-ERK1/2 antibodies (A), phospho-specific-Ser473-PKB antibodies (B), and phospho-specific-Tyr402-Pyk2 antibodies (C) (top). Blots were also analyzed for total ERK1/2, PKB, and Pyk2 (middle). Bottom: average data quantified by densitometric scanning of immunoblots. Values are means ± SE of at least 3 independent experiments and are expressed as % phosphorylation, where phosphorylation observed with ET-1 alone is defined as 100%. A: *P < 0.0001 vs. control; {dagger}P < 0.0001 vs. ET-1. B: *P < 0.0001 vs. control; {dagger}P < 0.0001 vs. ET-1. C: *P < 0.002 vs. control; {dagger}P < 0.0003 vs. ET-1.

 
In addition, SNP, another NO donor, also exhibited a similar effect and attenuated ET-1-induced ERK1/2, PKB, and Pyk2 phosphorylation to varying degrees (Fig. 2).


Figure 2
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  		Fig. 2. Dose-dependent effect of the NO donor sodium nitroprusside (SNP) on ET-1-induced ERK1/2, PKB, and Pyk2 phosphorylation in A-10 VSMCs. Serum-starved quiescent A-10 cells were pretreated without or with the indicated SNP concentrations for 15 min, followed by 10 nM ET-1 for 5 min. Cell lysates were immunoblotted by phospho-specific-Tyr204-ERK1/2 antibodies (A), phospho-specific-Ser473-PKB antibodies (B), and phospho-specific-Tyr402-Pyk2 antibodies (C) (top). Blots were also analyzed for total ERK1/2, PKB, and Pyk2 (middle). Bottom: average data quantified by densitometric scanning of immunoblots. Values are means ± SE of at least 3 independent experiments and are expressed as % phosphorylation, where phosphorylation observed with ET-1 alone is defined as 100%. A: *P < 0.0003 vs. control; {dagger}P < 0.0001 vs. ET-1. B: *P < 0.001 vs. control; {dagger}P < 0.0006 vs. ET-1. C: *P < 0.0003 vs. control; {dagger}P < 0.0002 vs. ET-1.

 
L-NAME Potentiated ET-1-Induced Phosphorylation of ERK1/2, PKB, and Pyk2 in A-10 VSMCs

To examine whether decreasing the endogenous NO production by inhibition of NOS activity would modify the effect of ET-1 on various signaling components, we investigated the effect of pretreatment of A-10 VSMC with L-NAME, a specific inhibitor of NOS, on ET-1-induced phosphorylation of ERK1/2, PKB, and Pyk2. As shown in Fig. 3, L-NAME treatment, at both doses used, potentiated the response of ET-1 in all three signaling components examined. L-NAME (100 µM) potentiated ET-1-induced ERK1/2 phosphorylation by fourfold (Fig. 3A), whereas only twofold potentiation in PKB and Pyk2 phosphorylation was observed under these conditions (Fig. 3, B and C).


Figure 3
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  		Fig. 3. Effect of nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, on ET-1-induced ERK1/2, PKB, and Pyk2 phosphorylation in A-10 VSMCs. Serum-starved quiescent A-10 cells were pretreated without or with the indicated L-NAME concentrations for 30 min, followed by 10 nM ET-1 for 5 min. Cell lysates were immunoblotted by phospho-specific-Tyr204-ERK1/2 antibodies (A), phospho-specific-Ser473-PKB antibodies (B), and phospho-specific-Tyr402-Pyk2 antibodies (C) (top). Blots were also analyzed for total ERK1/2, PKB, and Pyk2 (middle). Bottom: average data quantified by densitometric scanning of immunoblots. Values are means ± SE of at least 3 independent experiments and are expressed as % phosphorylation, where phosphorylation observed with ET-1 alone is defined as 100%. A: *P < 0.003 vs. control; {dagger}P < 0.005 vs. ET-1. B: *P < 0.0001 vs. control; {dagger}P < 0.0003 vs. ET-1. C: *P < 0.0001 vs. control; {dagger}P < 0.0005 vs. ET-1.

 
Although it is generally believed that VSMCs are devoid of NOS, the ability of L-NAME to potentiate ET-1-induced signaling suggested the presence of NOS in A-10 VSMCs. This possibility was evaluated by subjecting the total cellular lysates of A-10 VSMCs or HUVECs to Western blotting using specific antibodies against eNOS and iNOS. As shown in Fig. 4A, A-10 cells exhibited a significant expression of eNOS in the basal state. However, compared with HUVECs the expression level of eNOS in A-10 VSMCs was much less. Under these conditions iNOS could not be detected in A-10 VSMCs (data not shown). Furthermore, treatment of A-10 VSMCs with ET-1 or L-NAME did not alter the eNOS expression in these cells (Fig. 4B).


Figure 4
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  		Fig. 4. Expression of endothelial NOS (eNOS) in A-10 VSMCs. A: total cellular lysates from human umbilical vein endothelial cells (HUVECs; 15 µg) or A-10 VSMCs (60 µg) were subjected to SDS-PAGE, followed by immunoblotting using eNOS antibody as described in MATERIALS AND METHODS. Serum-starved quiescent A-10 cells were pretreated without or with the indicated L-NAME concentrations for 30 min, followed by 10 nM ET-1 for 5 min. B: cell lysates were immunoblotted with eNOS-specific antibody.

 
8-BrcGMP Inhibited ET-1-Induced Phosphorylation of ERK1/2, PKB, and Pyk2 in A-10 VSMCs

Since SNAP-induced production of NO would cause an elevation in cGMP, we evaluated the possibility that the effect of SNAP on ET-1-induced responses was mediated by a mechanism involving cGMP. We tested this by pretreating the cells with 8-BrcGMP, a nonmetabolizable and cell-permeant analog of cGMP. As shown in Fig. 5, treatment of cells with 8-BrcGMP decreased ET-1-induced ERK1/2, PKB, and Pyk2 phosphorylation. 8-BrcGMP (100 µM) inhibited ET-1-induced ERK1/2 and Pyk2 phosphorylation almost completely, whereas 50% inhibition of PKB phosphorylation was observed.


Figure 5
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  		Fig. 5. Effect of a stable analog of cGMP, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), on ET-1-induced ERK1/2, PKB, and Pyk2 phosphorylation in A-10 VSMCs. Serum-starved quiescent A-10 cells were pretreated without or with the indicated 8-Br-cGMP concentrations for 15 min, followed by 10 nM ET-1 for 5 min. Cell lysates were immunoblotted by phospho-specific-Tyr204-ERK1/2 antibodies (A), phospho-specific-Ser473PKB antibodies (B), and phospho-specific-Tyr402-Pyk2 antibodies (C) (top). Blots were also analyzed for total ERK1/2, PKB, and Pyk2 (middle). Bottom: average data quantified by densitometric scanning of immunoblots. Values are means ± SE of at least 3 independent experiments and are expressed as % phosphorylation, where phosphorylation observed with ET-1 alone is defined as 100%. A: *P < 0.003 vs. control; {dagger}P < 0.005 vs. ET-1. B: *P < 0.0001 vs. control; {dagger}P < 0.0003 vs. ET-1. C: *P < 0.001 vs. control; {dagger}P < 0.005 vs. ET-1.

 
ODQ Reversed Inhibitory Effect of SNAP on ET-1-Induced Phosphorylation of ERK1/2, PKB, and Pyk2 in A-10 VSMCs

Since NO stimulates cGMP production by activating a soluble form of guanylate cyclase, we wished to determine the contribution of this enzyme to SNAP-induced attenuation of ET-1 response. To validate this possibility, we used ODQ, a selective inhibitor of sGC, which prevents the generation of cGMP from GTP. For these experiments, cells were preincubated with ODQ for 15 min and then with 300 µM SNAP for 15 min and finally stimulated with 10 nM ET-1 for 5 min. As shown in Fig. 6, A–C, pretreatment with 10 µM ODQ resulted in a significant reversal in the inhibitory effect of SNAP on ET-1-stimulated ERK1/2, PKB, and Pyk2 phosphorylation. Under these conditions, however, pretreatment of cells with ODQ alone did not modify ET-1-induced ERK1/2, PKB, and Pyk2 phosphorylation (Fig. 6D).


Figure 6
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  		Fig. 6. Effect of soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) on ET-1-induced ERK1/2, PKB, and Pyk2 phosphorylation in A-10 VSMCs. A–C: serum-starved quiescent A-10 cells were pretreated without or with the indicated ODQ concentrations for 15 min before addition of 300 µM SNAP for 15 min followed by 10 nM ET-1 for 5 min. Cell lysates were immunoblotted by phospho-specific-Tyr204-ERK1/2 antibodies (A), phospho-specific-Ser473-PKB antibodies (B), and phospho-specific-Tyr402-Pyk2 antibodies (C) (top). Blots were also analyzed for total ERK1/2, PKB, and Pyk2 (middle). Bottom: average data quantified by densitometric scanning of immunoblots. Values are means ± SE of at least 3 independent experiments and are expressed as % phosphorylation, where phosphorylation observed with ET-1 alone is defined as 100%. A: *P < 0.0002 vs. control; {dagger}P < 0.007 vs. ET-1; {ddagger}P < 0.002 vs. SNAP + ET-1. B: *P < 0.0002 vs. control; {dagger}P < 0.0006 vs. ET-1; {ddagger}P < 0.003 vs. SNAP + ET-1. C: *P < 0.0005 vs. control; {dagger}P < 0.002 vs. ET-1; {ddagger} P < 0.02 vs. SNAP + ET-1. D: effect of ODQ on basal or ET-1-induced ERK1/2, PKB, and Pyk2 phosphorylation.

 
SNAP Inhibited ET-1-Stimulated [3H]Leucine Incorporation into Proteins

Activation of ERK1/2, PKB, and Pyk2 signaling has been implicated in mediating the hypertrophic response of ET-1 (42). Therefore, we next examined whether there is a correlation between the response of SNAP and ET-1-induced protein synthesis. As shown in Fig. 7, ET-1 increased [3H]leucine incorporation in total cellular proteins by ~50% over control. However, pretreatment of cells with SNAP dose-dependently decreased ET-1-induced [3H]leucine incorporation, with almost complete attenuation observed at 100 µM SNAP. SNAP alone did not significantly affect basal [3H]leucine uptake.


Figure 7
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  		Fig. 7. Effect of different concentrations of SNAP on ET-1-induced [3H]leucine incorporation into proteins. Serum-starved quiescent A-10 cells were pretreated with 10 and 100 µM SNAP for 30 min before ET-1 (10 nM) stimulation; the cells were then labeled to equilibrium with [3H]leucine for 20 h as described in MATERIALS AND METHODS. Values are means ± SE of 3 independent experiments and are expressed as % of change in [3H]leucine incorporated into total cellular proteins over basal values. *P < 0.002 vs. control; {dagger}P < 0.004 vs. ET-1.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Here we have provided evidence showing that NO generation induced by SNAP and SNP significantly attenuated ET-1-enhanced phosphorylation of ERK1/2, PKB, and Pyk2 in VSMCs. We have also demonstrated that SNAP treatment was able to antagonize ET-1-induced total protein synthesis, an index of hypertrophy, in VSMCs. Since both ERK1/2 and PKB pathways play critical roles in mediating hypertrophic and cell survival responses (8), it is reasonable to suggest that the ability of NO donors to inhibit ET-1-induced activation of these pathways is responsible for the antihypertrophic and vascular protective effect of NO. Although NO donors have been found to attenuate EGF (52)-, platelet-derived growth factor (35)-, and ANG II (44)-stimulated proliferation of VSMCs and cardiac fibroblasts, the studies reported here are the first to demonstrate an effect of NO on ET-1-induced phosphorylation of ERK1/2, PKB, and Pyk2 in A-10 VSMCs. These results are similar to those of studies in neonatal pulmonary VSMCs, in which SNP treatment was found to inhibit ET-1-induced ERK1/2 phosphorylation (3), and in rat cardiac fibroblasts, in which ANG II-induced phosphorylation of ERK1/2 and Pyk2 was blocked by SNAP (45, 46). However, our work represents the first study demonstrating that NO antagonizes ET-1-induced PKB and Pyk2 activation as well as ET-1-induced protein synthesis in VSMCs.

The demonstration that pharmacological inhibition of basal NO production with L-NAME augmented ET-1 responses on ERK1/2, PKB, and Pyk2 phosphorylation supports an inhibitory role of NO on ET-1-induced signaling events in A-10 VSMCs. A similar increase in ET-1-induced phosphorylation of ERK1/2 in L-NAME-treated pulmonary artery VSMCs has also been demonstrated (3). It is generally believed that VSMCs are devoid of NOS activity; however, recently, both iNOS and eNOS immunoreactivity as well as NOS activities have been detected in isolated VSMCs (9, 10, 34). Our results showing that A-10 VSMCs express eNOS in the basal state further support the presence of eNOS in VSMCs. Thus it is possible that L-NAME-induced inhibition of eNOS by decreasing NO bioavailability potentiates ET-1-induced signaling events in these cells.

NO is believed to exert its physiological effect through activation of sGC, a heme-containing protein (20). Binding of NO to the heme iron leads to allosteric modification of sGC, resulting in its enhanced catalytic activity to produce cGMP (21). cGMP, thus generated, elicits its downstream responses by interacting with its target proteins such as PKG (37). Additional non-sGC/cGMP-dependent mechanisms of NO action have also been suggested, which include ONOO–-catalyzed posttranslational modification of protein via nitration of tyrosine residues (6). However, our results showing that 8-BrcGMP mimicked the effect of SNAP and SNP in decreasing ET-1-induced ERK1/2, PKB, and Pyk2 phosphorylation suggest an intermediary role of cGMP in exerting this inhibitory response. Further proof for the involvement of sGC in this processes has been provided by the use of ODQ, a specific inhibitor of sGC that can block SNAP-induced elevations in cGMP levels in rat aortic VSMCs (28), A-10 VSMCs (4), endothelial cells (19), and cardiomyocytes (36). We found that ODQ treatment of A-10 VSMCs was able to significantly reverse the inhibitory effect of SNAP on ET-1-induced ERK1/2, PKB, and Pyk2 phosphorylation. A similar involvement of cGMP/PKG pathway in NO-induced inhibition of ERK1/2 phosphorylation by ET-1 in cardiomyocytes and in pulmonary artery VSMCs has also been reported (3, 12). The fact that 8-BrcGMP caused only partial inhibition of ET-1-induced PKB phosphorylation and ODQ was not able to completely reverse the SNAP-induced responses in our studies suggests a partial contribution of non-cGMP-dependent events in mediating the effect of NO donors in ET-1-induced responses. These cGMP-independent mechanisms include nitration of some upstream signaling components resulting in attenuation of their catalytic activity. The existence of a cGMP-independent mechanism in mediating the antiproliferative effects of NO has also been suggested from other studies in which ODQ, despite lowering NO-induced cGMP levels, failed to reverse the antiproliferative effect of NO donors in pulmonary microvascular smooth muscle cells (41) or in human endothelial cells (19). In these studies, however, the effect of ODQ on signaling pathways linked to proliferative responses was not investigated.

The precise mechanism by which cGMP inhibits ERK1/2 signaling remains elusive; however, the ability of PKG, the downstream effector of cGMP action, to phosphorylate c-Raf kinase on Ser43 and the resulting uncoupling between Ras-Raf might contribute to this effect (43). Since the upstream elements leading the PKB phosphorylation are different from those of ERK1/2 (8), the precise mechanism by which the cGMP/PKG system attenuates PKB phosphorylation remains undefined.

Pyk2 is a Ca2+-dependent proline-rich nonreceptor PTK that plays an essential role in ANG II-induced ERK1/2 signaling and hypertrophy in VSMCs (33) Pyk2 is activated by autophosphorylation in Tyr402 located in its catalytic domain (2). It thus may also be possible that SNAP/cGMP-induced decrease in Pyk2 phosphorylation observed in our studies contributed to the attenuating effect of SNAP on ET-1-induced signaling in A-10 VSMCs. NO generation has been shown to attenuate IGF-I- and insulin-induced elevation in H2O2 levels through a cGMP-dependent event in VSMCs (53). ET-1-induced ERK1/2 and PKB signaling is known to require activation of the NADPH-oxidase system and resultant H2O2 generation (15). Thus it is possible that a NO/cGMP-induced reduction in H2O2 generation contributes to the decrease in ET-1 response observed in our studies.

In summary, we have demonstrated that SNAP and SNP, NO donors, inhibit ET-1-stimulated increase of ERK1/2, PKB, and Pyk2 phosphorylation through a cGMP/sGC-dependent mechanism in A-10 VSMCs. We have also provided evidence showing that ET-1-stimulated protein synthesis, a hallmark of hypertrophic response, is also attenuated by the NO donor SNAP in A-10 VSMCs. Since ERK1/2, PKB, and Pyk2 play a crucial role in mediating VSMC growth and hypertrophy, it may be suggested that the ability of NO to attenuate these pathways may serve as a potential mechanism by which NO counteracts the growth-promoting and hypertrophic responses of ET-1 in VSMCs.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The work in the authors’ laboratory is supported by funding from the Heart and Stroke Foundation of Quebec and Canadian Institutes of Health Research (CIHR) Operating Grant 67037 to A. K. Srivastava. A. Bouallegue is a recipient of a CIHR Canadian Hypertension Society Doctoral Research Award.


   ACKNOWLEDGMENTS
 
We thank Ovid Da Silva, Research Support Office, Centre Hospitalier de l'Université de Montréal Research Center, for editorial assistance. We also thank Dr. Mounsif Haloui for help with protein synthesis experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. K. Srivastava, Centre de Recherche, CHUM, Angus-Campus, 2901 Rachel East, Montreal, QC, Canada H1W 4A4 (e-mail: ashok.srivastava{at}umontreal.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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