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Cardiovascular Research 2005 68(2):268-277; doi:10.1016/j.cardiores.2005.05.031
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Copyright © 2005, European Society of Cardiology

Role of SHP-1, Kv.1.2, and cGMP in nitric oxide-induced ERK1/2 MAP kinase dephosphorylation in rat vascular smooth muscle cells

Desiree I. Palena, Souad Belmadanib, Pamela A. Lucchesia and Khalid Matrouguia,*

aDepartment of Pharmacology, LSU Health Sciences Center at New Orleans, 1901 Perdido Street, New Orleans, LA 70112, United States
bDepartment of Physiology, LSU Health Sciences Center at New Orleans, 1901 Perdido Street, New Orleans, LA 70112, United States

* Corresponding author. Tel.: +1 504 568 2837; fax: +1 504 568 2361. Email address: kmatro{at}lsuhsc.edu

Received 17 February 2005; revised 1 May 2005; accepted 20 May 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Nitric oxide (NO) elicits relaxation in vascular smooth muscle cells (VSMC) that is associated with guanylate cyclase (GC) and K+ channel activation. In this study we determined the mechanisms that lead to ERK1/2 MAP kinase dephosphorylation in response to NO.

Methods: VSMC were treated with the NO donor SNAP or sodium nitroprusside (SNP), and ERK1/2, Src homology (SH) 1 domain-containing protein tyrosine phosphatase (SHP-1), and Kv.1.2 phosphorylation were assessed by immunoprecipitation and Western blot analysis.

Results: NO decreased basal ERK1/2 phosphorylation in a dose- and time-dependent manner. NO-induced ERK1/2 dephosphorylation was detected at 1 min and sustained for 30 min. Pre-treatment with the GC inhibitor ODQ or the protein tyrosine phosphatase inhibitor I prevented ERK1/2 dephosphorylation induced by SNAP. The inhibition of protein phosphatase 1A/2A had no effect on ERK1/2 dephosphorylation induced by SNAP. Treatment with cromakalim A, a nonspecific K+ channel activator, also induced ERK1/2 dephosphorylation, while blockade of Kv.1.2 K+ channels (AM92016 hydrochloride) prevented NO-induced ERK1/2 dephosphorylation. In addition, SNAP induced SHP-1 phosphorylation, and the Kv.1.2 dephosphorylation increase and SHP-1 phosphorylation was blocked by ODQ or AM92016. The basal interaction between ERK1/2 and SHP-1 was decreased in response to SNAP stimulation. SHP-1 also interacted with Kv.1.2 under basal conditions and participates in Kv.1.2 activation. Using the mouse mesenteric resistance artery, we found that ERK1/2 MAP kinase is involved in regulation of myogenic tone.

Conclusion: Thus, our study provides the first evidence that NO controls basal ERK1/2 phosphorylation by a signaling cascade that involves a dynamic signaling complex between cGMP, Kv.1.2 and SHP-1.

KEYWORDS Nitric oxide; Tyrosine phosphatase SHP-1; Kv.1.2 potassium channel; cGMP; ERK1/2 MAP kinase; Relaxation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Nitric oxide (NO) plays an important role in the regulation of vascular tone. The mechanisms by which NO controls vascular smooth muscle cell (VSMC) relaxation have not been totally elucidated. It is well established that NO activates soluble guanylyl cyclase (GC) and K+ channels [1–4]. Activation of GC induces an increase of cyclic guanosyl monophosphate (cGMP) that activates a family of serine/threonine protein kinases known as cGMP-dependent protein kinases (PKG) [1,2]. In addition, recent study suggests that PKG activates myosin light chain (MLC) phosphatase, thereby inhibiting MLC phosphorylation and subsequent contraction [1]. On the other hand, it has also been shown that K+ channel activation plays a key role in regulating vascular relaxation [5]. In addition, voltage-gated potassium (Kv) channels represent an important dilator in the cerebral circulation [6]. Activation of these signaling pathways results in VSMC relaxation by a decrease in intracellular Ca2+ concentration and calcium desensitization. Given the heterogeneity of blood vessel phenotypes, the relative importance of these pathways in NO-induced relaxation may be different in VSMC from different arterial beds.

Many signaling cascades studies involved in the regulation of vascular tone has focused on contractile agonists such as angiotensin II, endothelin, thromboxane and phenylephrine [7]. These studies have documented the involvement of multiple signaling pathways, including Ca2+-calmodulin and the increase of phosphorylation of different proteins such as rho/rho kinase, and ERK1/2 and p38 MAP kinases [8–12]. Furthermore, a relaxing factor should decrease signaling cascades phosphorylation induced with a constrictor factor. Thus, the dynamic balance in phosphorylation/dephosphorylation of these signaling pathways involved in contraction/relaxation plays a key role in the regulation of arterial tone.

Recently it has been shown that NO donors activate VSMC protein phosphatases including the serine-threonine phosphatase PP2A, and the tyrosine phosphatases PTP-PEST and Src homology (SH) 2 domain-containing protein tyrosine phosphatase (SHP-2) [13,14]. The Src homology (SH) 1/2 domain-containing protein tyrosine phosphatase SHP-1 and -2 are particularly interesting since SH domains function not only to recruit the enzyme to tyrosine-phosphorylated molecules but also to regulate the enzymatic activity. SHP-2 activation is necessary for NO-induced VSMC motility [15] and Src homology (SH) 1 domain-containing protein tyrosine phosphatase (SHP-1) activation by estrogen has been shown to antagonize AT1 receptor-mediated ERK activation in VSMC [16].

Under in vivo condition, the arterial tone is regulated by neuro-hormones and mechanical stimuli such as pressure and flow–(shear stress). Flow-induced NO release from endothelial cells is a key factor involved in the maintenance of arterial tone. Thus, our study focuses on the mechanism how NO controls basal arterial tone using cultured VSMC with special interest on the signaling complex involving cGMP, SHP-1 and voltage-gated potassium channels Kv.1.2 in NO induced ERK1/2 dephosphorylation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Drugs
(±)-S-Nitroso-N-acetylpenicillamine (SNAP, calbiochem); sodium nitroprosside (SNP, Sigma); 1H-(1,2,4)Oxadiazolo(4,3-a]quinoxalin-1-one (ODQ, Sigma); Protein Tyrosine Phosphatase Inhibitor I (SHP-1 inhibitor, Calbiochem); protein phosphatase-1A/2A (Calbiochem); 1-(4-Methanesulfonamidophenoxy)-3-(N-methyl-3,4 dichlorophenylethylamino)-2-propanol hydrochloride: Kv.1.2 K+ channel inhibitor (AM92016, Tocris) [17]; Antibodies (ERK1/2 1:5000, Promega; SHP-1, 1:1000, Cell Signalling; phosphotyrosine 1:1000, Cell Signalling; Kv.1.2 1:1000, Cell signalling; PKG 1:1000, Stressgen). GST–ERK1/2 was kindly provided by Dr. Andrew D. Catling.

2.2 Cell culture
Vascular smooth muscle cells from the thoracic aorta of 200–250 g male Sprague–Dawley rats were isolated and maintained in DMEM supplemented with 15% fetal bovine serum and antibiotics as previously described [18,19] VSMC (passages 3–10) were grown to 75~80% confluence and then growth arrested for 48 h in serum-free DMEM. After 48 h, the serum-free DMEM was changed 1-h before the start of the experiment. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.3 Experiment protocol
VSMC were treated by different drugs for 20 min before stimulation with SNAP (nitric oxide donor): (1) no drugs, (2) ODQ (10 µM), (3) AM92016 (10 µM), (4) Cromakalim (10 µM), (5) okadaic acid (10 µM), and (6) protein tyrosine phosphatase 1 (SHP-1) inhibitor (10 µM). Another NO-donor sodium nitroprusside dihydrate (SNP) was used to strengthen our results obtained with SNAP.

2.4 Western blot analysis
Cell lysates were prepared as described [18,19] Equal amounts of protein (25 µg) were resolved by 10% SDS-PAGE and transferred to nitrocellulose. Immunoblot analysis was performed using phosphorylation state-specific antibodies against ERK1/2 MAP kinases (1:5000; Promega), SHP-1 (1:1000; Cell Signalling), Kv.1.2 (1:1000; Cell signalling) or phosphotyrosine (1:1000; Cell Signalling).

To detect the presence of PKG expression in our cells, cell lysates were prepared from different passages and immunoblot analysis was performed using antibodies against PKG (1:1000; Stressgen).

Bands were visualized by enhanced chemiluminescence (ECL; Amersham) and quantified using Fuji Image Gauge Software.

2.5 Immunoprecipitation
Cell lysates containing equal amounts of protein were incubated with anti-SHP-1, Kv.1.2, ERK1/2 MAP-kinase or phosphotyrosine overnight at 4 °C. After incubation with protein G agarose for 2 h, precipitates were washed with lysis buffer and then resuspended in SDS-PAGE sample buffer. After being denatured at 90 °C for 4 min, samples were separated by SDS-PAGE.

2.6 GST–Erk1/2 pull down assay
Cell lysates containing equal amounts of protein from control (CTR) or stimulated with either SNAP or Guanosine 3'5'-cyclic monophosphate 8-bromo-, (stable cGMP), were incubated with anti-IgG, GST or GST–ERK1/2 overnight at 4 °C. After incubation with protein-A HRP conjugate for 2 h, precipitates were washed with lysis buffer and then resuspended in SDS-PAGE sample buffer. After being denatured at 90 °C for 4 min, samples were separated by SDS-PAGE.

2.7 Functional assessment of resistance arterial tone
Mice (C57-Bl6) were obtained from Jackson Laboratories. Mesenteric resistance arteries were dissected, mounted onto two glass micropipettes in a vessel chamber and slowly pressurized to 100 mmHg by using a pressure-servo-control perfusion (Living Systems Instruments, www.livingsys.com) in order to stretch the artery and set a constant artery length [20]. Vessel diameter, (80–100 µm at 75 mm Hg), was continuously monitored by a video image analyzer as described. Cannulated arterial segments were submerged in 2 ml of physiological salt solution (pH 7.4), oxygenated with (10% O2–5% CO2 and 85% N2). The functional integrity of endothelial cell layer was assessed by testing the endothelium-dependent vasodilating effect of acetylcholine (1 µM) after precontraction with phenylephrine (1 µM). Vessels not responding by contraction and relaxation to phenylephrine and acetylcholine, respectively were discarded. Following a 45 min equilibration period diameter changes without and with ERK1/2 MAP-kinase inhibitor (U0126, 10 µM) were measured when intraluminal pressure was increased from 25–100 mm Hg. At the end of each experiment, arteries were perfused and superfused with a calcium-free physiological salt solution containing 2 mM EGTA and 100 µM sodium nitroprusside and pressure steps were repeated to determine the passive diameter of the resistance arteries. Myogenic tone was assessed as percent of passive diameter.

2.8 Statistical analysis
Results are expressed as mean ± sem. The effect of the different treatments with reference to control conditions was determined by ANOVA (with Bonferrroni post hoc analysis). Probability values p<0.05 were considered as significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Role of SNAP on basal ERK1/2 MAP-kinase
Under basal conditions, the treatment of vascular smooth muscle cell (VSMC) with SNAP (nitric oxide-donor) for 5 min causes phosphorylation decrease, concentration-dependent manner (0.01–100 µM), of ERK1/2 MAP-kinase (Fig. 1A). The dephosphorylation of ERK1/2 MAP-kinase was also obtained using SNP (another nitric oxide-donor) (Fig. 1A, right panel). SNAP at one concentration (10 µM) decreased ERK1/2 MAP-kinase phosphorylation in a time dependent manner that was detected at 1 min and sustained for 30 min (Fig. 2).


Figure 1
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  		Fig. 1 ERK1/2 MAP-kinase dephosphorylation in a dose dependent manner in response to nitric oxide donor. (A) Immunoblot showing ERK1/2 MAP-kinase dephosphorylation in response to nitric oxide-donors SNAP (left panel) and SNP (right panel). VSMC were treated with SNAP dose–response manner (0.01–10–100 µM) for 5 min. One single dose of SNP was used to confirm results obtained with SNAP. Cell lysates were blotted with anti-phosphorylated ERK1/2 MAP-kinase, and then membrane was stripped and reprobed with rabbit anti-total ERK1/2 MAP-kinase antibody. (B) Cumulative data showing the effect of nitric oxide-donor dose–response manner on basal ERK1/2 MAP-kinase phosphorylation. Each experiment is a representative of 4 experiments. P<0.05 *statistically significant CTR vs. SNAP.

 

Figure 2
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  		Fig. 2 ERK1/2 MAP-kinase dephosphorylation in response to nitric oxide donor (SNAP) in a time dependent manner. VSMC were treated with SNAP (10 µM) time-dependent manner (1–5–10–30 min). Cell lysates were blotted with anti-phosphorylated ERK1/2 MAP-kinase or with rabbit anti-total ERK1/2 MAP-kinase antibody. Immunoblot shows ERK1/2 MAP-kinase dephosphorylation in response to one single dose of nitric oxide-donors SNAP (upper panel) in a time dependent manner. Cumulative data shows ERK1/2 MAP-kinase dephosphorylation in response to one single dose of nitric oxide-donors SNAP (upper panel) in a time dependent manner (down panel). Quantified densitometry data expressed as fold decreases relative to control. Each experiment is a representative of 4 experiments. P<0.05 *statistically significant CTR vs. SNAP.

 
3.2 Role of signaling cascades on ERK1/2 MAP-kinase dephosphorylation
It is well known that nitric oxide activates guanylate cyclase (GC) leading to cGMP increase. Thus, the pretreatment of VSMC with GC inhibitor (ODQ, 10 µM) prevented ERK1/2 MAP-kinase dephosphorylation in VSMC exposed to SNAP (Fig. 3). Similar data were found using Guanosine 3'5'-cyclic monophosphate 8-bromo (stable cGMP) (Fig. 3A-1). Since previous studies have reported passage-dependent loss of protein kinase G (PKG) expression in VSMC, we determined the presence of this cGMP effector in our cultured VSMC using the same antibody as the previous group [21]. Equivalent PKG expression was detected in VSMC at passages 2–9 (Fig. 3A), indicating that the GC–cGMP–PKG pathway is intact in our cultured VSMC model.


Figure 3
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  		Fig. 3 ERK1/2 MAP-kinase dephosphorylation in response to nitric oxide donor (SNAP) under guanylate cyclase inhibition with ODQ (10 µM). (A) Western blot analysis showing the presence of PKG in VSMC at different passages (P). (A-1) Western blot analysis showing a decrease of basal ERK1/2 phosphorylation under Guanosine 3'5'-cyclic monophosphate 8-bromo (cGMP) stimulation. (B) Immunoblot showing the effect of cGMP on ERK1/2 MAP-kinase dephosphorylation induced by nitric oxide-donor (SNAP). VSMC were treated with SNAP (10 µM) for 5 min with or without ODQ. Cell lysates were blotted with anti-phosphorylated ERK1/2 MAP-kinase or with rabbit anti-total ERK1/2 MAP-kinase antibody. (C) Cumulative data on the role of cGMP on ERK1/2 MAP-kinase dephosphorylation induced by nitric oxide-donor (SNAP). Quantified densitometry data expressed as fold decreases relative to control. Each experiment is a representative of 4 experiments. P<0.05 *statistically significant CTR vs. SNAP.

 
On the other hand, SNAP has also been shown to activate potassium channel to induce artery relaxation. Moreover, cromakalim (a non-specific potassium channel activator) induced also ERK1/2 MAP-kinase dephosphorylation (Fig. 4). The inhibition of Kv.1.2 potassium channel prevented the ERK1/2 MAP-kinase dephosphorylation induced by SNAP (Fig. 4).


Figure 4
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  		Fig. 4 ERK1/2 MAP-kinase dephosphorylation in response to nitric oxide donor (SNAP) under a non specific potassium channels activator (cromakalim: crom, 10 µM) or a specific inhibitor of Kv.1.2 potassium channel (AM92, 10 µM). VSMC were treated with SNAP (10 µM) for 5 min ± Crom or AM92. Cell lysates were blotted with anti-phosphorylated ERK1/2 MAP-kinase or with rabbit anti-total ERK1/2 MAP-kinase antibody. Immunoblot (upper panel) and cumulative data (down panel) show the involvement of potassium channels especially Kv.1.2 on ERK1/2 MAP-kinase dephosphorylation under stimulation of VSMC with SNAP. Quantified densitometry data expressed as fold decreases relative to control. Each experiment is a representative of 4 experiments. P<0.05 *statistically significant CTR vs. SNAP.

 
The ERK1/2 MAP-kinase dephosphorylation induced by SNAP should involve protein phosphatase activation. Thus, ERK1/2 MAP-kinase dephosphorylation induced by SNAP still occurs under protein phosphatase 1A and 1B inhibition (okadaic acid, 10 µM) (Fig. 5). On the other hand, inhibition of SHP-1 protein tyrosine prevented the ERK1/2 MAP-kinase dephosphorylation induced by SNAP (Fig. 6). To support our data, we have shown that SNAP increased SHP-1 phosphorylation (Fig. 7A, B), which was completely inhibited under guanylyl cyclase and Kv.1.2 channel inhibition (Fig. 7C).


Figure 5
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  		Fig. 5 ERK1/2 MAP-kinase dephosphorylation in response to nitric oxide donor (SNAP) with or without okadaic acid. VSMC were treated with SNAP (10 µM) for 5 min with or without okadaic acid. Cell lysates were blotted with anti-phosphorylated ERK1/2 MAP-kinase or with rabbit anti-total ERK1/2 MAP-kinase antibody. Immunoblot (upper panel) and cumulative data (down panel) show the involvement of okadaic acid on ERK1/2 MAP-kinase dephosphorylation with nitric oxide-donor (SNAP). Quantified densitometry data expressed as fold decreases relative to control. Each experiment is a representative of 4 experiments. P<0.05 *statistically significant CTR vs. SNAP.

 

Figure 6
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  		Fig. 6 ERK1/2 MAP-kinase dephosphorylation in response to nitric oxide donor (SNAP) with or without protein phosphatase 1 (SHP-1) inhibitor (10 µM). VSMC were treated with SNAP (10 µM) for 5 min with or without protein tyrosine phosphatase inhibitor I. Cell lysates were blotted with anti-phosphorylated ERK1/2 MAP-kinase or with rabbit anti-total ERK1/2 MAP-kinase antibody. Immunoblot (upper panel) and cumulative data (down panel) show the involvement of SHP-1 on ERK1/2 MAP-kinase dephosphorylation with nitric oxide-donor (SNAP). Quantified densitometry data expressed as fold decreases relative to control. Each experiment is a representative of 4 experiments. P<0.05 *statistically significant CTR vs. SNAP.

 

Figure 7
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  		Fig. 7 Relationship between nitric oxide donor (SNAP), SHP-1 and Kv.1.2 potassium channel. VSMC were treated with SNAP (10 µM) for 5 min. (A) Cell lysates were immunoprecipitated with mice anti-SHP-1 and blotted with anti-phosphotyrosine antibody. The same membrane was stripped and re-probed with mouse anti-total SHP-1 antibody. (B) Quantified densitometry data expressed as fold decreases relative to control. P<0.05 *statistically significant CTR vs. SNAP. (C) VSMC were treated with SNAP (10 µM) for 5 min with or without ODQ and AM92. Cell lysates were immunoprecipitated with mice anti-SHP-1 and blotted with anti-phosphotyrosine antibody. (D) VSMC were treated with SNAP (10 µM) for 5 min. Cell lysates were immunoprecipitated with mice anti-Kv.1.2 potassium channel and blotted with anti-phosphotyrosine antibody. The membrane was stripped and reprobed with mouse anti-total Kv.1.2 potassium channel antibody. (F) VSMC were treated with SNAP (10 µM) for 5 min with or without ODQ and AM92. Cell lysates were immunoprecipitated with mice anti-Kv.1.2 potassium channel and blotted with anti-phosphotyrosine antibody. Each experiment is a representative of 4 experiments.

 
It has been shown that SNAP activates K+ channels leading to hyperpolarization and vasodilation. In our study, the application of SNAP activates Kv.1.2 K+ channel (by dephosphorylation) (Fig. 7D) and prevented under guanylate cyclase and SHP-1 inhibition (Fig. 7F).

3.3 Interaction between SHP-1 and EKR1/2 MAP-kinase (immunoprecipitation and GST–ERK1/2 pull down assay)
In order to understand the mechanism of the ERK1/2 dephosphorylation induced by SNAP, we next determined whether this kinase interacts with SHP-1. Using immunoprecipitation and Western blot analysis we found in non-stimulated VSMC that ERK1/2 MAP-kinase interacts with SHP-1 (Fig. 8A, B). Similar data were obtained using GST–ERK1/2 pull down assay (Fig. 8C). Upon stimulation with SNAP, SHP-1 was phosphorylated and EKR1/2 MAP-kinase was dephosphorylated, resulting in dissociation of the two molecules (Fig. 8D).


Figure 8
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  		Fig. 8 ERK1/2 MAP-kinase and SHP-1 interaction. VSMC were treated with SNAP (10 µM) for 5 min. (A) Cell lysates were immunoprecipitated with mouse anti-SHP-1 antibody and blotted with rabbit anti-total ERK1/2 MAP-kinase antibody. The membrane was stripped and reprobed with mouse anti-total SHP-1 antibody. (B) Cell lysates were immunoprecipitated with rabbit anti-total ERK1/2 MAP-Kinase antibody and blotted with mouse anti-total SHP-1 antibody. The membrane was stripped and reprobed with rabbit anti-total ERK1/2 MAP-Kinase antibody. (C) GST–ERK1/2 pull down assay; Cell lysates from control (CTR) or stimulated with SNAP or Guanosine 3'5'-cyclic monophosphate 8-bromo (cGMP) were incubated with GST, GST–ERK1/2 or IgG for overnight and then blotted with SHP-1 antibody. The membrane was stripped and reprobed with rabbit anti-total ERK1/2 MAP-kinase antibody. (D) Cell lysates were immunoprecipitated with rabbit anti-phosphorylated ERK1/2 MAP-kinase antibody and blotted with rabbit anti-phosphorylated ERK1/2 MAP-kinase antibody. The membrane was stripped and reprobed with mouse anti-total SHP-1 antibody. Each experiment is a representative of 4 experiments.

 
3.4 Role of ERK1/2 MAP-kinase in arterial tone regulation
In isolated resistance artery, stepwise increases in pressure induced myogenic tone (MT) development (Fig. 9A, B). MT (which is a contraction induced in response to pressure changes) significantly decreased when resistance artery was treated with the ERK1/2 MAP-kinase inhibitor U0126 (10 µM) (Fig. 9A, B) (59.9 ± 3.4 vs. 87.13 ± 0.89% of passive diameter at 75 mm Hg under control and U0126 treatment, respectively, p<0.05).


Figure 9
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  		Fig. 9 ERK1/2 MAP-kinase and arterial tone. (A) Pressure–diameter relationship under control conditions (CTR), in the presence of ERK1/2 MAP-kinase inhibitor (U0126, 10 µM) and passive diameter (PD) determined in response to increases in pressure levels in mesenteric resistance arteries. (B) Myogenic tone response normalized to passive diameter (PD) at stepwise increases in intraluminal pressure in mice mesenteric resistance arteries under control conditions (CTR) and in the presence of ERK1/2 MAP-kinase inhibitor (U0126, 10 µM). n = 5 per group. *P<0.05; 2-factor ANOVA, statistically significant CTR vs. U0126; #P<0.05 statistically significant CTR vs. PD.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Nitric oxide is of critical importance in the regulation of arterial tone. Our study provides the first evidence that nitric oxide induced ERK1/2 MAP-kinase dephosphorylation in a concentration-dependent manner in VSMC isolated from large arteries (rat thoracic aorta). This dephosphorylation involved a complex interaction between guanylate cyclase, Kv.1.2 channels and the SHP-1 tyrosine phosphatase.

Nitric oxide plays an important role in pathological and physiological regulation of conduit and resistance arterial tone. Under in vivo conditions, both large and small arteries are submitted continuous exposure to neuro-hormones, pressure and flow–(shear stress)–induced nitric oxide release from endothelial cells. The in vivo basal tone of these arteries is dependent upon many factors, although nitric oxide release from endothelial cell plays a predominant role. The mechanisms by which nitric oxide controls VSMC relaxation have not been totally elucidated. It is well known that nitric oxide can activate soluble GC as well as K+ channels [1,5]. GC activation increases cyclic guanosyl monophosphate (cGMP), which, in turn, activates a family of cGMP-dependent serine/threonine protein kinases (PKG). PKG is a major effector for cGMP in VSMCs that leads to artery relaxation [1,2]. In addition, it has been shown that K+ channels also play a key role in regulating vascular tone [5]. It is likely that no single pathway acts exclusively or independently in any one type of smooth muscle cells; therefore the relative importance of the different pathways inducing relaxation is likely to be different in VSMC from different artery beds.

K+ channel activity is a major regulator of VSMC membrane potential and therefore vascular tone regulation. Numerous studies have shown that nitric oxide activates K+ channels with subsequent artery relaxation. Studies have indicated that Kv.1.2 activity is dependent on the dephosphorylation status of the channel and that both tyrosine and serine/threonine phosphorylation are essential in regulating the ion channel current [22]. In support of this notion, it has been found that K+ current is dramatically decreased in response to the increased tyrosine phosphorylation of the Kv.1.2 channel. Taken together, these results suggest that the status of tyrosine phosphorylation can be effectively employed to regulate Kv.1.2 activity. In a previous study, we found that A7r5 VSMC treated with 100 pM AVP increased Kv.1.2 channel tyrosine phosphorylation through the non-receptor tyrosine kinase, PYK2 [23]. On the other hand, Vanhoutte's laboratory showed that AVP induced artery relaxation in an endothelium-dependent manner [24]. Therefore, while Kv.1.2 channel activity in isolated embryonic VSMC led to Ca2+ oscillations, Kv.1.2 could also be involved in relaxation of intact arteries and therefore could participate in regulation of vascular tone.

In this study, we showed that nitric oxide dephosphorylated Kv.1.2 through cGMP and SHP-1, suggesting that tyrosine dephosphorylation may be linked to increased activity. ERK1/2 MAP-kinase dephosphorylation occurred secondary to Kv.1.2 activation, since Kv.1.2 inhibition blocked ERK1/2 dephosphorylation by nitric oxide. ERK1/2 dephosphorylation involved cGMP and SHP-1 tyrosine phosphatase. Taken these data together it is more likely that these signaling interact with each other directly or through mediators. The interaction is a dynamic since Kv.1.2 and SHP-1 interact with each other and their activity could be controlled by each other (Fig. 10). In addition, GC and Kv.1.2 pathways could line in series in the signaling cascades since the pre-blockade of GC and Kv.1.2 showed restoration of phosphorylation level of ERK1/2 MAP-kinase under SNAP. Hassid's laboratory has also shown that nitric oxide increases protein tyrosine phosphatase activity in VSMC [25,26]. Thus, our data show that nitric oxide increased SHP-1 phosphorylation in VSMC and prevented guanylate cyclase-dependent Kv.1.2 phosphorylation. On the other hand okadaic acid, at concentrations that inhibit PP2A/B, had no effect on ERK1/2 MAP-kinase dephosphorylation.


Figure 10
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  		Fig. 10 Proposed mechanism by which nitric oxide induces VSMC basal ERK1/2 MAP-kinase dephosphorylation.

 
The mechanism by which nitric oxide decreases ERK1/2 MAP-kinase phosphorylation appears to be very complex and complicated. Using immunoprecipitation and Western blot analysis of lysates from non-stimulated VSMC, ERK1/2 MAP-kinase appeared to interact with SHP-1. Alternatively we used GST–ERK1/2 pull down assay and found similar interaction between ERK1/2 and SHP-1 under basal conditions, which almost disappear under stimulation with either nitric oxide donor or a stable cGMP (Fig. 8). It is unclear whether this interaction leads to a direct ERK1/2 inactivation since ERK1/2 activation requires phosphorylation of both Ser/Thr and Tyr residues. Using immunoprecipitation and–GST-pull down assay, does not mean that the interaction between ERK1/2 MAP-kinase and SHP-1 is direct but could also involve other intermediate molecules. It is also possible that ERK1/2 dephosphorylation by nitric oxide involves interaction with molecules other than SHP-1, cGMP and Kv.1.2. Further studies are needed to clarify the molecules involved.

Upon stimulation with nitric oxide, SHP-1 was phosphorylated and EKR1/2 MAP-kinase was dephosphorylated, resulting in a dissociation of the two molecules from the complex. These finding may have potential implications in cardiovascular diseases such as hypertension. For example, it is well known that flow-induced dilation involves nitric oxide release from endothelial cells, which is significantly altered in different arterial beds in hypertensive rats [27,28]. However, Qiu et al. have shown a significant decrease in flow-induced dilation and at the same time an increase in cGMP production from endothelial mesenteric arteries in spontaneously hypertensive rats [29]. Our data may suggest that the response to nitric oxide, in addition its production, could be altered in hypertension thereby affecting the level of ERK1/2 MAP-kinase phosphorylation. This possibility will be addressed in future studies.

We used small artery, which develops a spontaneously tone (contraction called myogenic tone) under intraluminal pressure in order to show that ERK1/2 MAP-kinase is involved in arterial tone regulation. Thus, under ERK1/2 MAP-kinase inhibition, MT was significantly decreased. These data are in agreement to our previous and other studies showing the relationship in resistance artery between pressure and ERK1/2 activation [10,30,31]. These data provide evidence that ERK1/2 MAP-kinase play key role in arterial tone regulation.

In conclusion, the present study provides novel insight into the mechanism by which acute nitric oxide stimulation may control arterial tone through ERK1/2 MAP-kinase dephosphorylation. This dephosphorylation involves a complex of signaling cascades and interaction between cGMP, Kv.1.2 and SHP-1. These results may identify new therapeutic targets for the control of vascular function and therefore important implications in cardiovascular physiology and disease.


   Acknowledgements
 
This work was supported by the American Heart Association, National 0430278N (KM) and South East Affiliate 0365217B (KM) and NIH RO1HL56046 (PAL). The authors thank Dr. Andrew D. Catling for generously providing (GST and GST–ERK1/2) needed to performed experiments.


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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