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CARDIOVASCULAR

The Role of Rho Kinase and Extracellular Regulated Kinase-Mitogen-Activated Protein Kinase in ₂-Adrenoceptor-Mediated Vasoconstriction in the Porcine Palmar Lateral Vein

Institute of Cell Signalling and Department of Obstetrics and Gynaecology, University of Nottingham, Medical School, Queen's Medical Centre, Nottingham, United Kingdom

Received May 7, 2004; accepted June 28, 2004.

	Abstract

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₂-Adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon activation of the extracellular signal-regulated kinase-mitogen-activated protein (ERK-MAP) kinase signal transduction pathway. Recent studies have shown that ₂-adrenoceptor-mediated vasoconstriction in the rat aorta is also dependent upon activation of Rho kinase. The aim of this study was to determine whether Rho kinase and ERK-MAP kinase are part of the same signaling pathway. The Rho kinase inhibitor Y27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride) (10 µM) almost completely inhibited the contractile response to the ₂-adrenoceptor agonist UK14304 (5-bromo-6-[2-imidazolin-2-ylamine]-quinoxaline bitartrate) in segments of porcine palmar lateral vein [maximum response 2.9 ± 2.3% of 60 mM KCl response (mean ± S.E.M.) in the presence of Y27632, compared with 64.9 ± 7.1% in control tissues, n = 4]. However, Y27632 had no effect on ₂-adrenoceptor-mediated ERK activation, as measured by Western blotting. ₂-Adrenoceptor-mediated vasoconstriction was associated with an increase in phosphorylation of the myosin phosphatase-targeting subunit (MYPT) at Thr696 (the Rho kinase phosphorylation site). This phosphorylation was inhibited by 10 µM Y27632. In contrast, inhibition of ERK activation with the MAP kinase kinase inhibitor PD98059 (2-amino-3-methoxyflavone) (50 µM) had no effect on MYPT phosphorylation. Both Y27632 and PD98059 inhibited myosin light chain phosphorylation. These data indicate that ₂-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon both Rho kinase and ERK activation, although these are separate pathways. Rho kinase causes vasoconstriction through inhibition of myosin phosphatase and an increase in myosin light chain phosphorylation, whereas ERK causes vasoconstriction through a myosin phosphatase-independent pathway.

Recently, Carter et al. (2002) showed that ₂-adrenoceptor-mediated vasoconstriction in the rat aorta involves activation of Rho kinase. Like the porcine palmar lateral vein, ₂-adrenoceptor-mediated vasoconstriction in the rat aorta also involves activation of ERK (Carter and Kanagy, 2002). Therefore, it is possible that there is some form of cross talk between the Rho kinase signal transduction pathway and the ERK signal transduction pathway or even that they are part of the same pathway. Angiotensin II-induced contraction of pressurized mesenteric arteries involves activation of ERK, which is inhibited by the Rho kinase inhibitor Y27632, suggesting that Rho kinase is upstream of ERK activation (Matrougi et al., 2001). Furthermore, lysophosphatidic acid-induced ERK activation has been reported to be dependent upon Rho kinase activity in vascular smooth muscle (Rhoades et al., 2001), and Rho kinase activity is also required for sustained ERK activity in fibroblasts (Roovers and Assoian, 2003).

Rho kinase is activated by the small GTPase Rho (Matsui et al., 1996). Activation of Rho kinase can cause vasoconstriction through either direct phosphorylation of myosin light chains (although this is not thought to be a physiologically significant mechanism in smooth muscle; Amano et al., 1996; Somlyo and Somlyo, 2003) or inhibition of myosin phosphatase (Kimura et al., 1996). Rho kinase has been implicated in calcium sensitization of the contractile proteins, allowing calcium-independent smooth muscle contraction (Somlyo and Somlyo, 2003).

The porcine palmar lateral vein contains a relatively high density of functional ₂-adrenoceptors (Blaylock and Wilson, 1995; Wright et al., 1995). Although many arteries do respond to ₂-adrenoceptor activation, prior pharmacological manipulation is often required to observe these responses (Roberts et al., 1999; Bhattacharya and Roberts, 2003). On the other hand, ₂-adrenoceptor stimulation in the porcine palmar lateral vein produces a large contractile response without the need for prior pharmacological manipulation, thus making it a good model for studying ₂-adrenoceptor-mediated vasoconstriction.

The aim of this study was to determine whether Rho kinase and ERK-MAP kinase are part of the same signal transduction pathway mediating ₂-adrenoceptor-induced vasoconstriction in the porcine palmar lateral vein.

	Materials and Methods

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Isometric Tension Recordings. Porcine trotters were obtained from a local abattoir and transported to the laboratory on ice. Palmar lateral veins were dissected out and placed in Krebs-Henseleit buffer containing 2% Ficoll, which had been pregassed with 95% O₂/5% CO₂, and stored overnight at 4°C. The following day, veins were carefully cleaned of fat and connective tissue, dissected into 5-mm ring segments, and suspended in a 5-ml isolated tissue bath containing Krebs-Henseleit buffer maintained at 37°C and constantly gassed with 95% O₂/5% CO₂. The lower support was fixed, and the upper support was connected to a force transducer (Lectromed, Letchworth, UK) linked to a PowerLab data acquisition system (AD Instruments Ltd., Hastings, UK) via an amplifier. After a 20-min equilibration period, tension was applied to the tissue, which was allowed to relax to a final resting tension of between 0.5 and 1.0 g wt. Before each experiment, the tissues were contracted with 60 mM KCl, until the final two responses differed by less than 10%.

Effect of Rho Kinase Inhibitor on UK14304 Responses. Tissues were incubated for 1 h with the Rho kinase inhibitor Y27632 (10 µM; Uehata et al., 1997). Cumulative concentration response curves to the ₂-adrenoceptor agonist UK14304 (1 nM to 10 µM) were then performed. Alternatively, tissues were contracted with 10 µM UK14304 and responses allowed to reach a plateau before the cumulative addition of Y27632 (1-10 µM). Similarly, in a second set of tissues contracted with 10 µM UK14304, the MEK inhibitor PD98059 was added in a noncumulative fashion (10, 30, and 100 µM).

ERK Immunoblotting. Segments of porcine palmar lateral vein were set up in tissue baths as above, in the absence or presence of 10 µM Y27632. Tissues were then exposed to a maximum concentration of UK14304 (10 µM). Control tissues were not exposed to any compound (basal conditions). When the contractions to UK14304 reached a plateau (3-4 min after addition of the agonist), the segments were quickly removed from the tissue baths and immediately frozen on dry ice. Frozen segments were then homogenized in ice-cold buffer [80 mM sodium -glycerophosphate, 20 mM imidazole, pH 7.0, 1 mM dithiothreitol, 1 mM sodium fluoride, 500 µM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 µM trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane (E-64), 10 µg/ml aprotonin, 1 µM leupeptin, and 500 µM EDTA). After removal of a sample for a protein assay, the homogenate was diluted 1:1 in 2x Laemmli sample buffer and heated at 95°C for 5 min. Equal amounts of protein from each sample were separated on 10% SDS-PAGE gels and then transferred onto nitrocellulose membranes by Western blotting. After incubating in blocking solution (5% powdered milk in Tris-buffered saline containing 0.1% Tween 20), nitrocellulose blots were incubated overnight at 4°C with antibodies against the double phosphorylated (activated) forms of both isoforms of ERK (ERK 1 and 2) or total ERK (Cell Signaling Technology Inc., Beverly, MA). After washing in Tris-buffered saline containing 0.1% Tween 20, the blots were incubated with the appropriate, hydrogen peroxidase-conjugated secondary antibody. Proteins were visualized using the ECL system (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK). Bands were analyzed by densitometry using the Bio-Rad molecular analyst software package (Bio-Rad, Hercules, CA).

Protein Kinase C-Related Kinase (PRK) Immunoblotting. Samples were prepared as above and separated on a 7% SDS-PAGE. After Western blotting, immunoblots were then probed with antibodies against either phosphorylated PRK (Thr778 PRK-1/Thr816 PRK-2) or total PRK2 (both Cell Signaling Technology Inc.) overnight at 4°C. Bands were then incubated with the appropriate, hydrogen peroxidase-conjugated secondary antibody. Proteins were visualized using the ECL system (Amersham Biosciences UK, Ltd.). Bands were analyzed by densitometry as above.

Myosin Phosphatase-Targeting Subunit (MYPT) Immunoblotting. Samples were prepared as above and separated on a 7% SDS-PAGE. After Western blotting, immunoblots were probed with antibodies against either the MYPT of myosin phosphatase (Upstate, Milton Keynes, UK) or MYPT phosphorylated at Thr696 (Upstate) overnight at 4°C. Bands were then incubated with the appropriate, hydrogen peroxidase-conjugated secondary antibody. Proteins were visualized using the ECL system (Amersham Biosciences UK, Ltd.). Bands were analyzed by densitometry as above.

Myosin Light Chain Immunoblotting. Samples were prepared as above and separated on a 15% SDS-PAGE. After Western blotting immunoblots were probed with antibodies against either total myosin light chain (Sigma Chemical, Poole, Dorset, UK) or myosin light chain 2 phosphorylated at Thr18/Ser19 (Cell Signaling Technology Inc.). Bands were then incubated with the appropriate, hydrogen peroxidase-conjugated secondary antibody. Proteins were visualized using the ECL system (Amersham Biosciences UK, Ltd.). Bands were analyzed by densitometry as above.

Drugs. UK14304 was obtained from Tocris Cookson Inc. (Bristol, UK); PD98059 was obtained from Calbiochem (San Diego, CA), and Y27632 was obtained from Tocris Cookson. All other compounds were obtained from Sigma Chemical.

Data Analysis. Contractile responses were expressed as a percentage of the response to 60 mM KCl, and results expressed as mean ± S.E.M. Bands obtained by immunoblotting were analyzed by densitometry. Statistical evaluations were carried out using a two-tailed Student's paired or unpaired t test for normally distributed data.

	Results

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Effect of Rho Kinase Inhibitor Y27632 on UK14304 Responses. Preincubation with the Rho kinase inhibitor Y27632 (10 µM) almost completely inhibited the contractile response to the ₂-adrenoceptor agonist UK14304 in the porcine palmar lateral vein (Fig. 1). The maximum response to UK14304 alone was 64.9 ± 7.1% of the response to 60 mM KCl (mean ± S.E.M., n = 4). In the presence of Y27632, the maximum response was reduced to 2.9 ± 2.7%.

View larger version (10K): [in this window] [in a new window]   Fig. 1. Concentration response curves showing contractile responses to UK14304 in segments of porcine palmar lateral vein. Responses are expressed as a percentage of the response to 60 mM KCl and are means ± S.E.M. of four separate experiments. ***, p < 0.001 versus control, two-tailed, Student's unpaired t test.

Y27632 also inhibited the maintained UK14304 response. In tissues contracted with 10 µM UK14304, 1 µM Y27632 caused a 78.0 ± 6.6% relaxation (n = 6). This increased to 99.1 ± 1.1% when the concentration of Y27632 was increased to 10 µM.

Effect of Rho Kinase Inhibitor Y27632 on ERK Activation. In segments of porcine palmar lateral vein, contraction with 10 µM UK14304 was associated with an increase in the level of ERK1 and 2 phosphorylation (Fig. 2). Because the level of ERK1 was often at the limit of analysis, we have concentrated on the effects on ERK2 phosphorylation. There was no change in levels of total ERK, indicating an increase in ERK activation. Preincubation with 10 µM Y27632 had no significant effect on the level of ERK2 phosphorylation (Fig. 2).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Effect of Y27632 (10 µM) on UK14304-stimulated ERK2 phosphorylation in porcine palmar lateral vein segments. A, representative immunoblot showing phosphorylated ERK levels in segments of porcine palmar lateral vein stimulated with 10 µM UK14304 in the absence (UK) or presence of 10 µM Y27632 (Y2). Control tissues (C) were not exposed to any compounds (basal conditions). B, bar chart showing levels of phosphorylated and total ERK2 (mean ± S.E.M. of eight separate experiments) in tissues contracted with 10 µM UK14304 in the absence (UK) or presence of 10 µM Y27632 (Y2), expressed as a percentage of control levels. Control tissues (C) were not exposed to any compounds (basal conditions). *, p < 0.05 versus control, Student's two-tailed paired t test (direct comparison of densitometric values).

Effect of UK14304 on PRK Phosphorylation. Y27632 can inhibit PRK as well as Rho kinase (Davies et al., 2000). Therefore, we measured the level of PRK phosphorylation at Thr778 or Thr816 for PRK1 and PRK2, respectively, as an indication of activation state. In tissues contracted with 10 µM UK14304, there was no increase in either PRK1 phosphorylation (102.2 ± 11.6% of control, n = 10) or PRK2 phosphorylation (115.4 ± 16.9% of control, n = 10) compared with controls.

Effect of Y27632 or PD98059 on MYPT1 Phosphorylation. A double band was observed in immunoblots probed with an antibody against MYPT1 (see Figs. 3 and 4). This double band represents different isoforms of MYPT1 as identified by others in vascular tissue (Payne et al., 2004). In segments of porcine palmar lateral vein, contraction with 10 µM UK14304 was associated with an increase in the level of MYPT1 phosphorylation at Thr696 (Figs. 3 and 4). There was no change in levels of total MYPT1. Preincubation with 10 µM Y27632 caused a significant reduction in the level of MYPT1 phosphorylation (Fig. 3). On the other hand, preincubation with the MEK inhibitor 50 µM PD98059 to inhibit ERK activation had no significant effect on MYPT1 phosphorylation (Fig. 4).

View larger version (30K): [in this window] [in a new window]   Fig. 3. A, representative immunoblot of porcine palmar lateral vein proteins separated by SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with a primary antibody against phosphorylated MYPT1 (Thr696). Segments of porcine palmar lateral vein were set up in a tissue bath and contracted with 10 µM UK14304 in the absence (UK) or presence of 10 µM Y27632 (Y2). Nonstimulated segments kept under basal conditions were also obtained (C). B, bar chart showing changes in MYPT1 phosphorylation expressed as a percentage of control levels. Results are mean ± S.E.M. of six experiments. **, significant difference from UK, p < 0.01, Student's unpaired t test.

View larger version (24K): [in this window] [in a new window]   Fig. 4. A, representative immunoblot of porcine palmar lateral vein proteins separated by SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with a primary antibody against phosphorylated MYPT1 (Thr696). Segments of porcine palmar lateral vein were set up in a tissue bath and contracted with 10 µM UK14304 in the absence (UK) or presence of 50 µM PD98059 (PD). Nonstimulated segments kept under basal conditions were also obtained (C). B, bar chart showing changes in MYPT1 phosphorylation expressed as a percentage of control levels. Results are mean ± S.E.M. of eight experiments. *, significant difference from control, p < 0.05, paired Student's t test (direct comparison of densitometric values).

Effect of Y27632 or PD98059 on MLC Phosphorylation. In segments of porcine palmar lateral vein, contraction with 10 µM UK14304 was associated with an increase in the level of MLC2 phosphorylation at Thr18/Ser19 (Figs. 5 and 6). There was no change in levels of total MLC. Preincubation with 10 µM Y27632 caused a significant reduction in the level of MLC phosphorylation (Fig. 5). Similarly, preincubation with 50 µM PD98059 also caused a significant reduction of MLC phosphorylation (Fig. 6). The degree of inhibition with Y27632 (105.8 ± 12.0%, n = 8) correlated with the degree of inhibition of contraction in these tissues (95.5 ± 1.0%, n = 8). Likewise, the degree of inhibition with PD98059 (66.2 ± 15.1%, n = 9) also correlated with the degree of inhibition of contraction in these tissues (61.6 ± 4.4%, n = 9).

View larger version (23K): [in this window] [in a new window]   Fig. 5. A, representative immunoblot of porcine palmar lateral vein proteins separated by SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with a primary antibody against phosphorylated MLC (Thr18/Ser19). Segments of porcine palmar lateral vein were set up in a tissue bath and contracted with 10 µM UK14304 in the absence (UK) or presence of 10 µM Y27632 (Y2). Nonstimulated segments kept under basal conditions were also obtained (C). B, bar chart showing changes in MLC phosphorylation expressed as a percentage of control levels. Results are mean ± S.E.M. of eight experiments. **, indicates significant difference from UK, p < 0.01 Student's unpaired t test.

View larger version (24K): [in this window] [in a new window]   Fig. 6. A, representative immunoblot of porcine palmar lateral vein proteins separated by SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with a primary antibody against phosphorylated MLC (Thr18/Ser19). Segments of porcine palmar lateral vein were set up in a tissue bath and contracted with 10 µM UK14304 in the absence (UK) or presence of 50 µM PD98059 (PD). Nonstimulated segments kept under basal conditions were also obtained (C). B, bar chart showing changes in MLC phosphorylation expressed as a percentage of control levels. Results are mean ± S.E.M. of 10 experiments. *, significant difference from UK, p < 0.05, Student's unpaired t test.

Effect of PD98059 on Maintained UK14304 Response. PD98059 inhibited the maintained UK14304 response. In tissues contracted with 10 µM UK14304, 10 µM PD98059 caused a 56.8 ± 11.4% relaxation, 30 µM caused a 73.2 ± 4.5% relaxation, and 100 µM caused a 82.0 ± 3.5% relaxation (n = 6). These relaxations were associated with 29.2 ± 11.7%, 52.6 ± 6.4%, and 59.9 ± 8.2% inhibition of ERK activation. On the other hand, there was no apparent effect on myosin light chain phosphorylation at any of these concentrations of PD98059.

	Discussion

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Previous studies have demonstrated that ₂-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon activation of ERK (Roberts, 2001, 2003). The mechanism by which ERK causes vasoconstriction is unknown. A recent study has indicated that endothelin-1-stimulated ERK activation in the rat thoracic aorta leads to vasoconstriction through a myosin light chain phosphorylation-independent pathway, although the nature of that pathway was not identified (Kwon et al., 2003). However, this does not appear to be the case for ₂-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein. This present study demonstrates that ₂-adrenoceptor-mediated vasoconstriction is associated with an increase in myosin light chain phosphorylation, and this increase is inhibited by preincubation with the MEK inhibitor PD98059. PD98059 inhibits ₂-adrenoceptor-mediated vasoconstriction and ERK activation in the porcine palmar lateral vein (Roberts, 2001). Taken together, these data would seem to indicate that ₂-adrenoceptor vasoconstriction in the porcine palmar lateral vein occurs through an ERK-dependent phosphorylation of myosin light chains. Similar effects are seen in the porcine carotid artery in which endothelin-1-stimulated vasoconstriction is also inhibited by PD98059, and this inhibition is also associated with inhibition of myosin light chain phosphorylation (D'Angelo and Adam, 2002). PD98059 also inhibits phenylephrine-stimulated contraction and myosin light chain phosphorylation in ovine uterine artery (Xiao et al., 2004). Interestingly, both of these studies call into question the role of ERK-mediated caldesmon phosphorylation in mediating vasoconstriction. Rather, both groups speculate that ERK causes activation of myosin light chain kinase, leading to the phosphorylation of myosin light chains. The fact that preincubation with PD98059 prevents ₂-adrenoceptor-mediated myosin light chain phosphorylation could be explained by this theory. However, PD98059-induced relaxation of ₂-adrenoceptor-induced contractions did not seem to be associated with a reduction in myosin light chain phosphorylation but was associated with a reduction in ERK activation. This suggests that the mechanism by which PD98059 inhibits the initiation of contraction by UK14304 may be different from the mechanism by which PD98059 causes relaxation of UK14304-contracted tissues, although an inhibition of ERK activation is associated with both mechanisms.

Role of Rho Kinase. ₂-Adrenoceptor-mediated vasoconstriction in the rat aorta is associated with an increased activation of RhoA and Rho kinase and is inhibited by the Rho kinase inhibitor Y27632 (Carter et al., 2002). This contraction is also sensitive to inhibition of ERK activation (Carter and Kanagy, 2002), suggesting that ERK and Rho kinase may be part of the same pathway. Because the ₂-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon ERK activation (Roberts, 2001), we were interested in determining whether, like the rat aorta, this contraction is also sensitive to inhibition of Rho kinase. Interestingly, preincubation of tissues with the Rho kinase inhibitor Y27632 almost completely inhibited the ₂-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein, indicating that Rho kinase plays a major role in the ₂-adrenoceptor-mediated response in this tissue. On the other hand, Y27632 had no effect on the increase in ERK activation associated with the ₂-adrenoceptor-mediated vasoconstriction. This indicates that, unlike ERK activation in vascular smooth muscle in response to activation of other receptors (Matrougi et al., 2001; Rhoades et al., 2001), Rho kinase is not upstream of ERK activation.

Y27632 can also inhibit other kinases with a similar potency as against Rho kinase (Davies et al., 2000; Eto et al., 2001). Of these, PRK and PKC could be involved in vasoconstriction. PRK can phosphorylate the myosin phosphatase inhibitory protein CPI-17 in vitro (Hamaguchi et al., 2000) and could therefore also regulate vasoconstriction through inhibition of myosin phosphatase activity. PRK is activated by phosphatidylinositol-dependent kinase (PDK) and Rho (Flynn et al., 2000). PDK, in turn, is activated by phosphatidylinositol 3,4,5 trisphosphate, the formation of which is catalyzed by phosphatidylinositol 3-kinase. We have previously shown that ₂-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon activation of phosphatidylinositol 3-kinase; therefore, it is possible that the action of Y27632 is to inhibit PRK activity rather than Rho kinase. PRK is phosphorylated by PDK (Thr778 for PRK1 and Thr816 for PRK2) (Balendran et al., 2000; Flynn et al., 2000), causing activation. Therefore, we determined whether there was an increase in PRK1 and 2 phosphorylation in ₂-adrenoceptor-contracted palmar lateral vein segments. The results showed that there was no increase in phosphorylation of either PRK1 or PRK2, suggesting that PRK activation is not involved in ₂-adrenoceptor-mediated vasoconstriction, and that this is not the site of action of Y27632. Furthermore, there is no evidence that PRK phosphorylates MYPT1 at Thr696, the Rho kinase phosphorylation site. Because ₂-adrenoceptor-mediated vasoconstriction causes phosphorylation of MYPT1 at this site, and this is inhibited by Y27632, it is unlikely that this action of Y27632 is mediated through inhibition of PRK.

Y27632 has also been reported to inhibit PKC (Eto et al., 2001). PKC has been identified in porcine coronary arteries (Kandabashi et al., 2003), although attempts to determine whether PKC is present in the porcine palmar lateral vein by Western blotting, using the same antibodies, proved inconclusive (data not shown). On the other hand, like PRK, PKC causes inhibition of myosin phosphatase through phosphorylation of CPI-17 rather than phosphorylation of MYPT. As stated above, because Y27632 inhibits ₂-adrenoceptor-mediated phosphorylation of MYPT at the Rho kinase phosphorylation site, it is unlikely that this action of Y27632 is mediated through inhibition of PKC.

Role of Myosin Phosphatase. Rho kinase phosphorylates myosin phosphatase, thereby inhibiting this enzyme (Kimura et al., 1996). Myosin phosphatase dephosphorylates myosin light chains. Therefore, by inhibiting myosin phosphatase, Rho kinase increases the level of myosin light chain phosphorylation, without activating myosin light chain kinase (Nagumo et al., 2000; Somlyo and Somlyo, 2003). Myosin phosphatase is phosphorylated by Rho kinase on MYPT1 at Thr695 in the chicken gizzard (Feng et al., 1999), which is equivalent to Thr696 in human MYPT1 (Hartshorne, 1998). ₂-Adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein was associated with an increase in MYPT1 phosphorylation at this site, and this was inhibited by Y27632, suggesting that ₂-adrenoceptor-mediated vasoconstriction is dependent upon Rho kinase-induced myosin phosphatase inhibition. Myosin light chain phosphorylation was also inhibited by Y27632, indicating that the result of inhibition of myosin phosphatase is an increase in MLC phosphorylation.

Interestingly, inhibition of ERK activation by the MEK inhibitor PD98059 had no effect on MYPT1 phosphorylation at the Rho kinase phosphorylation site. This suggests that either inhibition of myosin phosphatase plays no role in ERK-mediated vasoconstriction or that inhibition of myosin phosphatase by ERK occurs through phosphorylation of a different site. These data also indicate that Rho kinase is not downstream of ERK because inhibition of Rho kinase inhibits MYPT1 phosphorylation, but inhibition of ERK activation does not. Taken together, these data indicate that, although Rho kinase and ERK are both mediators of ₂-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein, they are part of independent signal transduction pathways. However, there must be some degree of cooperation between these signaling pathways because inhibition of either Rho kinase or ERK activation causes large inhibitions of contraction. In other words, inhibition of one of the pathways prevents vasoconstriction mediated through the other. Similar cooperation between these signaling pathways has been observed in urokinase-type plasminogen activator-induced cell migration (Jo et al., 2002).

In conclusion, these data demonstrate that ₂-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is induced through activation of separate Rho kinase and ERK signal transduction pathways. The Rho kinase pathway causes a vasoconstriction through inhibition of myosin phosphatase and an increase in myosin light chain phosphorylation. On the other hand, the ERK pathway causes vasoconstriction through a pathway independent of inhibition of myosin phosphatase. However, further work is required to clarify the role of myosin light chain phosphorylation in the ERK-mediated pathway.

	Acknowledgements

 
We thank G. Woods and Sons Ltd. (Clipstone, Nottinghamshire, UK) for the supply of porcine tissues.

	Footnotes

 
This study was supported by The Wellcome Trust.

doi:10.1124/jpet.104.071100.

ABBREVIATIONS: ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; MEK, MAP kinase kinase; Y27632, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride; UK14304, 5-bromo-6-[2-imidazolin-2-ylamine]-quinoxaline bitartrate; MLC, myosin light chain; PAGE, polyacrylamide gel electrophoresis; PRK, protein kinase C-related kinase; MYPT, myosin phosphatase-targeting subunit; PDK, phosphatidylinositol-dependent kinase.

Address correspondence to: Dr. Richard Roberts, Institute of Cell Signalling and Department of Obstetrics and Gynaecology, University of Nottingham, Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK. E-mail: richard.roberts{at}nottingham.ac.uk

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