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CARDIOVASCULAR

Rho/Rho Kinase and Phosphoinositide 3-Kinase Are Parallel Pathways in the Development of Spontaneous Arterial Tone in Deoxycorticosterone Acetate-Salt Hypertension

Departments of Pharmacology and Toxicology (E.A.W., C.A.N., S.W.W.) and Physiology (E.A.W.), Michigan State University, East Lansing, Michigan; and Department of Physiology (R.D.L.), University of Michigan, Ann Arbor, Michigan

Received for publication October 30, 2003
Accepted February 24, 2004.

	Abstract

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Hypertension is characterized by abnormal vascular contractility and function. Arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive rats develop spontaneous tone that is not observed in arteries from normotensive rats. Inhibition of phosphoinositide 3-kinase (PI3-kinase) by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) reduces spontaneous tone development. The Rho/Rho-kinase pathway has been suggested to play a role in hypertension and may be dependent on PI3-kinase activity. We hypothesized that Rhokinase is involved in spontaneous tone development and that Rho/Rho-kinase is a downstream effector of PI3-kinase. Using endothelium-denuded aortic strips in isolated tissue bath, we demonstrated that (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) (Y27632) (1 µM), a Rho-kinase inhibitor, significantly reduced spontaneous tone in the DOCA aorta but that it did not affect sham aorta basal tone (DOCA 63.5 ± 15.9 versus sham 1.2 ± 0.4 total change in percentage of phenylephrine contraction). We examined the interaction between the PI3-kinase and Rho pathways by observing the effects of LY294002 on a Rhokinase effector, myosin phosphatase (MYPT), and Y27632 on a PI3-kinase effector, Akt, using Western blot analysis. Inhibition of PI3-kinase reduced spontaneous tone, but it had no effect on the phosphorylation status of MYPT, indicating that PI3-kinase is not a downstream effector of Rho/Rho-kinase. These data indicate that there is little interaction between the Rho/Rhokinase and PI3-kinase pathways in the DOCA-salt aorta, and the two pathways seem to operate in parallel in supporting spontaneous arterial tone. These data reflect spontaneous tone only and do not rule out the possibility of interaction between these pathways in agonist-stimulated tone.

Two intracellular signaling pathways associated with altered contractility have been receiving attention recently, namely, the phosphoinositide 3-kinase (PI3-kinase) and Rho/Rho-kinase pathways. PI3-kinase has been linked to altered smooth muscle contractility (Ibitayo et al., 1998; Zheng et al., 1998; Komalavilas et al., 2001; Yang et al., 2001; Northcott et al., 2002) and is activated by a variety of stimuli, including the activation of multiple types of receptors (Anderson et al., 1999; Rameh and Cantley, 1999; Cantrell, 2000; Coelho and Leevers, 2000; Vanhaesebroeck et al., 2001). Previously, PI3-kinase expression and activity have been shown to be upregulated in two models of hypertension and contribute to enhanced contractility (Northcott et al., 2002). It is curious that arteries from hypertensive rats show a decrease in phosphorylation of Akt, an effector molecule of PI3-kinase, even though the activity of PI3-kinase is increased (Northcott et al., 2002). It is thus of significant interest to understand the events upstream and downstream of PI3-kinase in the development of spontaneous tone (Sata and Nagai, 2002).

Rho/Rho-kinase is a logical candidate to consider when discussing altered contractility, calcium sensitization, and spontaneous tone due to its prominent role in altering the phosphorylation status of myosin. Activation of RhoA ultimately leads to phosphorylation and inactivation of the myosin binding subunit (MYPT) of myosin phosphatase. This occurs through the action of Rho-kinase I/II (ROCKI/ROCKII) (Kimura et al., 1996). MYPT removes a phosphate from myosin to stop actin/myosin cycling; however, when inactivated by phosphorylation (due to RhoA activation and consequent activity of Rho-kinase), myosin remains in a phosphorylated state, thus maintaining contraction.

Agonist-induced activation of RhoA in normotensive models is associated with contraction stimulated by norepinephrine, phenylephrine, endothelin-1 (ET-1), angiotensin II, epidermal growth factor (EGF), 5-hydroxytryptamine (serotonin) (5-HT), and others (Sakurada et al., 2001; Kandabashi et al., 2002; Miao et al., 2002; Seko et al., 2003). Moreover, it has been suggested that Rho-kinase activity is elevated in mesenteric arteries from mineralocorticoid hypertensive rats (Weber and Webb, 2001). RhoA is activated in vasospasm (Miao et al., 2002), and in several models of hypertension, including deoxycorticosterone acetate (DOCA)-salt, renal hypertensive, stroke-prone spontaneously hypertensive, and N-nitro-L-arginine methyl ester-treated rats (Seko et al., 2003). Thus, RhoA activation in vascular smooth muscle cells seems to an important event leading to altered contractility in hypertensive vessels.

It has been suggested that activation of RhoA is associated with activation of PI3-kinase. Miao et al. (2002) demonstrated that PI3-kinase is involved in the activation of RhoA in response to ET-1 in rabbit basilar artery. In their study, PI3-kinase was repressed by an inhibitor, LY294002, before ET-1 stimulation, and this inhibition resulted in decreased RhoA activation, suggesting that RhoA is a downstream effector of PI3-kinase. Conversely, PI3-kinase did not induce the Rho signaling pathways in other systems. Reif et al. (1996) analyzed activation of Rho-related signaling associated with upstream activation of PI3-kinase and concluded that PI3-kinase stimulation was not sufficient to activate Rho-mediated responses, such as stress fiber and focal adhesions formation.

Given the above-mentioned findings, we must consider that the interaction of Rho/Rho-kinase and PI3-kinase may depend on cell type and conditions. It has not been demonstrated conclusively whether Rho/Rho-kinase is upstream or downstream of PI3-kinase, or a separate parallel pathway in development of spontaneous tone in aorta from DOCA-salt rats. Therefore, the purpose of this study was to determine the interaction, if any, between Rho/Rho-kinase and PI3-kinase in aorta from DOCA-salt rats. It is hypothesized that Rho/Rho-kinase functions downstream of PI3-kinase and, as such, serves as an alternative effector to the well known PI3-kinase effector Akt. This hypothesis is aimed at addressing the apparent discrepancy noted by Northcott et al. (2002). Specifically, that although the activity of PI3-kinase is increased in DOCA-salt hypertension, there is not an associated increase in phosphorylation of Akt, indicating that there is another downstream effector of PI3-kinase in this model.

	Materials and Methods

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Surgical and Blood Pressure Protocol
DOCA-Salt Hypertension. Male Sprague-Dawley rats (250–300 g; Charles River Breeding Laboratories, Portage, MI) underwent uninephrectomy and implantation of DOCA (200 mg kg^-1) under isoflurane anesthesia as described previously (Storm et al., 1990). Animals remained on the regimen for 4 weeks. Systolic blood pressures were measured using standard tail cuff methods. This study was reviewed and approved by University Laboratory Animal Resources at Michigan State University.

Isolated Tissue Bath Protocol
Rats were euthanized (80 mg kg^-1 pentobarbital i.p.), and the thoracic aorta was removed. Arteries were dissected into helical strips (0.25 x 1 cm), and the endothelial cell layer was removed by rubbing the luminal side of the vessel with a moistened cotton swab. Tissues were pair-mounted (sham/DOCA) in isolated tissue baths for measurement of isometric force. Muscle baths were filled with warmed (37°C), aerated (95% O₂,5%CO₂) physiological salt solution (PSS) containing 130 mM NaCl, 4.7 mM KCl, 1.18 mM KH₂PO₄, 1.17 mM MgSO₄·7H₂O, 1.6 mM CaCl₂·2H₂O, 14.9 mM NaHCO₃, 5.5 mM dextrose, and 0.03 mM CaNa₂EDTA (pH 7.2). This slightly acidic buffer was used to reduce oscillatory tone (small, phasic contractions, not spontaneous tone) in the preparation. Oscillatory tone masks development of spontaneous tone and makes it difficult to quantify. One end of the preparation was attached to a glass rod, and the other was attached to a force transducer (FT03; Grass Instruments, Quincy, MA), and the strip was placed under optimum resting tension (1500 mg for all tissues, determined previously) and allowed to equilibrate for 1 h. Changes in isometric force were recorded using PowerLab (version 3.6) and Chart (version 3.6.3) software (ADIn-struments, Mountain View, CA) and evaluated using a Macintosh computer. After an hour of equilibration, arteries were challenged with a maximal concentration of the ₁-adrenergic receptor agonist phenylephrine (PE; 10 µM). PE was washed out, and one of the following protocols was performed. In experiments using antagonists, the inhibitors or vehicle was added to the bath at least 1 h before measurements of spontaneous tone. The inhibitors used in this study, Y27632 (1 µM; BIOMOL Research Laboratories, Plymouth Meeting, PA) and LY294002 (20 µM; BIOMOL Research Laboratories), have been shown to be relatively selective at the stated concentrations (Davies et al., 2000). Spontaneous tone was measured such that a positive number represents an increase above basal tone as a percentage of PE maximal contraction, and a negative number indicates a decrease below basal tone as a percentage of PE maximal contraction. Data were normalized to a maximal PE contraction.

Validation of Phospho-MYPT Activation. The contraction in response to 5-HT (10^-9–3 x 10^-4 M; Sigma-Aldrich, St. Louis, MO), a known activator of RhoA, was measured in the presence and absence of Y27632 (1 µM), a Rho-kinase inhibitor that was added 1 h before the addition of agonist. The inhibitory effect of Y27632 on 5-HT-induced cumulative contraction was observed. The developed tension was normalized and expressed as a percentage of the maximal PE response.

Calcium Study. For Ca²⁺ experiments, arteries were first incubated in normal Ca²⁺ (1.6 mM) and challenged with PE (10^-5 M). Once the contraction stabilized, PE was washed out, and tissues were incubated for 30 min in Ca²⁺-free PSS supplemented with 1 mM EGTA. The buffer was changed every 10 min to allow for equilibration to the new buffer. Tissues were then switched to a Ca²⁺-free EDTA (0.03 mM) PSS, equilibrated for 15 min, and Y27632 (1 µM; BIOMOL Research Laboratories) was added. Fifteen minutes after the addition of Y27632 or vehicle, Ca²⁺ was added back to the bath in a cumulative manner (1 x 10^-6–3 x 10^-3 M), with additions every 5 min.

Role of Rho-Kinase and PI3-Kinase in Spontaneous Tone. Aortic strips from DOCA-salt and sham rats were pair-mounted (sham/DOCA) in isolated tissue baths for measurement of isometric force. LY294002 (20 µM) or vehicle was added to the bath and allowed to incubate for at least 1 h while spontaneous tone was allowed to develop. After the development of spontaneous tone in control tissue (1 h), all tissues were snap-frozen and analyzed as described below for Rho/Rho-kinase signaling elements.

Aortic Vascular Smooth Muscle Cell Culture
Vascular smooth muscle cells were derived from the aorta of male Sprague-Dawley rats. Aorta were excised in an aseptic manner, cleaned of fat and connective tissue, and cut into helical strips. The endothelium was removed by gently rubbing the luminal face of these strips with a moistened cotton swab. The strips were cut into small squares (2 x 2 mm). These pieces of tissue were placed lumen side down in a P-60 culture dish (Corning Glassworks, Corning, NY) and layered with a small amount of serum-enriched media to keep the tissues moist [medium consisted of Dulbecco's modified Eagle's medium with D-glucose (4500 mg/l), L-glutamine (1%), and HEPES buffer (25 mM; Invitrogen, Carlsbad, CA) containing fetal bovine serum (40% v/v; Hyclone Laboratories, Logan, UT) and streptomycin (100 mg/ml)/penicillin (100 units/ml; Invitrogen)]. Plates were placed in a 5% CO₂ warming incubator maintained at 37°C. Once the tissues attached to the plate (18 h), additional medium was added to the dish. After 1 week, a sufficient number of cells had migrated from the tissue to reach confluence in the plate. Cells were trypsinized, seeded to T75 flasks and fed with normal serum (10%) Dulbecco's modified Eagle's medium. Cells were plated onto P-100 plates and used when confluent between passages 2 and 9. With each new isolation, cells were positively stained for smooth muscle -actin (Sigma-Aldrich). Cultured rat fibroblasts did not stain with this antibody.

Vascular Smooth Muscle Cell Protein Isolation. Cells (P-100 plates) were switched to PSS (see above) and incubated for 1 h before the addition of agonist (final volume, 4 ml). At this same time, antagonists or vehicle was added and equilibrated with tissues for 1 h. Examination after 1 h indicated that treatment with agonist or vehicle did not destroy cells or cause the cells to lift off the plate. Each dish was incubated with one agonist concentration. EGF (10 nM) or vehicle was added for 10 min to stimulate the EGF signaling cascade. After incubation, plates were placed on ice and the incubation buffer was aspirated. Cells were washed three times (4 ml/wash) with phosphate-buffered saline containing sodium orthovanadate as a tyrosine phosphatase inhibitor (10 mM sodium phosphate, 150 mM NaCl, and 1 mM sodium orthovanadate, pH 7.0). Five hundred microliters of supplemented lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EGTA, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/µl aprotinin, 10 µg/µl leupeptin, and 1 mM sodium orthovanadate) was added to each dish, and cells were lysed with a rubber policeman. Lysate was transferred into 1.5-ml centrifuge tubes and centrifuged at 14,000g for 10 min at 4°C. The supernatant was aspirated from the pellet of cellular debris, and Western blot analysis was performed.

Western Blot Protocol
Protein Isolation. Aorta were cleaned, quick frozen, pulverized in liquid nitrogen-cooled mortar and pestle, and solubilized in lysis buffer (0.5 M Tris-HCl, pH 6.8, 10% SDS, and 10% glycerol) with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Homogenates were centrifuged (11,000g for 10 min, 4°C), and supernatant total protein was measured.

Western Blotting. Equivalent amounts of aortic protein from sham and DOCA-salt rats were separated on SDS-polyacrylamide gels (RhoA-15%, ROCKI and ROCK II-5%, MYPT and phospho-MYPT-10%) and transferred to Immobilon-P membrane for standard Western analyses using RhoA (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), ROCKI (1:500; BD Biosciences, San Diego, CA), ROCKII (1:1000; BD Biosciences), MYPT (1:500; BD Biosciences), and phospho-MYPT-1 Thr 850 (1 µg/ml; Upstate Biotechnology, Lake Placid, NY) primary antibodies. Cultured smooth muscle cells were similarly analyzed using Akt (1:1000; Upstate Biotechnology) and phospho-Akt (1:1000; Upstate Biotechnology) primary antibodies. Incubation of primary antibodies was performed at 4°C overnight, rocking. Positive controls for ROCKI, ROCK II, and MYPT were purchased from BD Biosciences. Smooth muscle -actin (1:400; Oncogene, Boston, MA) was used as a marker to ensure equal loading of protein. After incubation in primary antibody, blots were washed and exposed to the appropriate secondary antibody for 1 hat 4°C with rocking. The secondary antibody for RhoA, MYPT, ROCKI, ROCKII, and -actin Western analyses was horseradish peroxidase-linked anti-mouse IgG (1:2000; Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK). The secondary antibody for phospho-MYPT Western analysis was horseradish peroxidase-linked anti-rabbit IgG (1:2000; Upstate Biotechnology). Finally, blots were washed and chemiluminescent detection of bands was performed (Amersham Biosciences UK, Ltd.). All protein immunolabeling, with the exception of -actin, was performed on individual blots. Measurement of -actin was performed on all blots; this was done in a blot that had not been stripped because -actin is of a mass that does not interfere with other bands. Protein quantitation is reported relative to smooth muscle -actin.

Materials
Materials included acetylcholine hydrochloride, deoxycorticosterone acetate, phenylephrine hydrochloride, 5-HT (all from Sigma-Aldrich), EGF (Invitrogen), and LY294002 and Y27632 (both from BIOMOL Research Laboratories).

Data Analysis
Data are presented as means ± standard error of the mean for the number of animals indicated. Contraction is reported as tension (milligrams) or as a percentage of response to maximum contraction to PE. Quantitation of Western blot analyses was performed on computer-scanned images of developed films using NIH Image (version 1.61). The phospho-MYPT/MYPT ratio was calculated as follows from Western blot data normalized to smooth muscle -actin (arbitrary units): phospho-MYPT density/MYPT density. This ratio was calculated to determine changes in phosphorylation status of MYPT relative to total MYPT protein. When comparing two groups, an unpaired Student's t test was used. In all cases, p 0.05 was considered statistically significant.

	Results

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Rho- and PI3-Kinase-Mediated Spontaneous Tone Development: Contractile Data. The systolic blood pressures of sham and DOCA-salt rats were 115 ± 2 and 178 ± 5 mm Hg, respectively. Data were normalized as percentage of PE initial response (10^-5 M), which was not statistically different in aorta from sham versus DOCA-salt animals (1190 ± 44.1 versus 1273 ± 60.6 mg, respectively). Spontaneous tone developed in endothelium-denuded aorta isolated from DOCA-salt but not sham rats (Fig. 1, right, tracings 4 and 3). Y27632, a Rho-kinase inhibitor, significantly reduced spontaneous tone in the DOCA aorta, but it did not affect sham aorta basal tone (63.5 ± 15.9 versus 1.2 ± 0.4% total change) (Fig. 1, right, tracings 2 and 1). Y27632 reduced spontaneous tone in a concentration-dependent manner (Fig. 2). The effect of Y27632 was reversible, because spontaneous tone was restored upon washing out of Y27632. Similarly, previous experiments have shown that LY294002 significantly inhibited spontaneous tone development in aorta from DOCA-salt rats compared with sham (76.7 ± 18.9 versus 2.5 ± 1.4 total change in percentage of PE contraction) and did so in a concentration-dependent manner (Northcott et al., 2002).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Rho-kinase inhibitor Y27632 reduced spontaneous arterial tone in DOCA-salt aorta, but it did not affect sham tone in isolated tissue bath. Left, contractile data for aortic strips from DOCA-salt and sham rats in the presence (tracings 1 and 2) or absence (tracings 3 and 4) of Y27632 (1 µM) or vehicle. Right, contractile data normalized as percentage of PE contraction. Y27632 significantly reduced spontaneous tone in DOCA-salt aorta (*, p < 0.05 compared with DOCA vehicle; n = 4), whereas sham tone was unaltered.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Blockade of Rho-kinase with Y27632 inhibited spontaneous arterial tone in a concentration-dependent manner in isolated tissue bath. Data are normalized as a percentage of PE contraction. Spontaneous tone developed in aortic strips from DOCA-salt but not sham animals. Aortic strips from sham rats were not significantly affected by Y27632 (1 µM). However, Y27632 significantly inhibited spontaneous arterial tone in aortic strips from DOCA-salt rats and did so in a concentration-dependent manner (*, p < 0.05 compared with DOCA vehicle; n = 4).

Pharmacological Test of LY294002 and Y27632. LY294002 (20 µM) and Y27632 (1 µM) were tested against EGF (10 nM; 10 min) in rat aortic smooth muscle cells cultured from a normal rat to demonstrate pharmacological selectivity by examining the effects of these compounds on the phosphorylation of Akt, a PI3-kinase effector molecule. EGF (10 nM) significantly increased phosphorylation of Akt, indicating that the PI3-kinase pathway is activated by EGF stimulation (Fig. 3). The selective PI3-kinase inhibitor LY294002 (20 µM) abolished phosphorylation of Akt in the presence of EGF. By contrast, Y27632 (1 µM), a selective Rho-kinase inhibitor, did not alter the phosphorylation status of Akt during EGF stimulation.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Blockade of PI3-kinase with LY294002 inhibited EGF-induced phosphorylation of Akt (pAKTSer473); however, EGF-induced phosphorylation of Akt was unchanged by inhibition of Rho-kinase using Y27632. Cultured aortic smooth muscle from normal rat was treated with EGF (10 nM) in the presence or absence of LY294002 (20 µM) or Y27632 (1 µM). pAKTSer473 was analyzed using Western blot analysis as described under Materials and Methods. EGF stimulation significantly increased pAKTSer473 (*, p < 0.05 compared with control; n = 6). The EGF-induced increase in pAKTSer473 was not inhibited by Y27632 (*, p < 0.05 compared with EGF alone; n = 6) but was inhibited by LY294002 (, p < 0.001; n = 6).

Rho-Kinase and Calcium. Calcium is required for the development of spontaneous tone in DOCA aorta. Calcium was removed from the bath and added back cumulatively. As calcium was added, spontaneous tone developed in aorta from hypertensive animals but not from normotensive animals. Development of tone was significantly inhibited by Y27632 (1 µM) (Fig. 4).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Calcium was required for Rho-kinase-mediated spontaneous arterial tone in aortic strips from DOCA-salt aorta. Calcium was removed from the isolated tissue bath and then added back in a cumulative manner while spontaneous arterial tone was allowed to develop. As calcium was added, spontaneous tone developed in aortic strips from DOCA-salt rat but not in sham tissue. Development of tone in the DOCA-salt preparations was inhibited by Rho-kinase blockade using Y27632 (1 µM) (*, p < 0.05 compared with DOCA Y27632; n = 4). Spontaneous tone did not develop in sham tissues.

Validation of Association of Phospho-MYPT with Contraction. Aorta isolated from normal Sprague-Dawley rats was used to validate the role of the Rho/Rho kinase pathway in mediating contractions, to confirm that we could accurately measure phosphorylation of MYPT, and to test the ability of Y27632 to block Rho-kinase activity. Stimulation by 5-HT (10^-9–3 x 10^-4 M) caused contraction of aortic strips in isolated tissue bath and 5-HT-induced contraction was inhibited by Y27632 (1 µM) (Fig. 5A). After administration of 5-HT, protein levels of MYPT and phospho-MYPT were analyzed using Western blot analysis. Total MYPT protein was unchanged after 5-HT stimulation compared with control (Fig. 5B). 5-HT stimulation increased phosphorylation of MYPT, indicating that the Rho pathway was activated by 5-HT (Fig. 5C). To confirm this, a Rho-kinase inhibitor, Y27632, was added to the aorta preparation before the addition of 5-HT. 5-HT-induced contraction was inhibited by Y27632, and this was associated with a decrease in the levels of phospho-MYPT (Fig. 5C). The phospho-MYPT/MYPT ratio was increased after 5-HT stimulation and was reduced in the presence of Y27632 (Fig. 5D).

View larger version (33K): [in this window] [in a new window]   Fig. 5. 5-HT-induced contraction was inhibited by Y27632. Protein levels of MYPT were not altered following treatment with 5-HT (10-9–3 x 10-4 M) and Y27632 (1 µM), but the phosphorylation status of MYPT was significantly increased. Aortic strips from sham rats were studied in isolated tissue bath and stimulated by 5-HT in the presence or absence of the Rho-kinase inhibitor Y27632 (A) and then analyzed using Western blot analysis (B–D). Data were normalized to smooth muscle -actin. Protein level of MYPT was unchanged by stimulation with 5-HT or treatment with Y27632 (B). 5-HT stimulation significantly increased the levels of phospho-MYPT (*, p < 0.05; n = 6), which was reduced by Rho-kinase blockade using Y27632 (**, p < 0.01; n = 6) (C). Likewise, 5-HT increased the phospho-MYPT ratio (**, p < 0.01; n = 6), whereas Y27632 reduced the ratio (*, p < 0.05; n = 6) (D).

Expression of Rho/Rho-Kinase Pathway-Related Proteins in DOCA-Salt Hypertension. We analyzed the components of the Rho pathway in aorta from DOCA-salt rats to determine whether DOCA-salt hypertension resulted in alteration of protein levels within the Rho/Rho-kinase pathway compared with sham. A similar analysis was published by Seko et al. (2003). Our study was performed to confirm the results from the aforementioned study and to examine fully the Rho pathway in our model. The expression levels of RhoA, ROCKI, ROCKII, MYPT, and phospho-MYPT were analyzed in the unstimulated thoracic aorta from sham and DOCA-salt animals. The phospho-MYPT/MYPT ratio was also calculated. Protein expression levels for RhoA, ROCKI, ROCKII, MYPT, phospho-MYPT, and the phospho-MYPT/MYPT ratio were unchanged (Fig. 6).

View larger version (32K): [in this window] [in a new window]   Fig. 6. Protein levels of Rho pathway constituents are not altered in thoracic aorta from DOCA-salt compared with sham. Thoracic aorta from DOCA-salt and sham rats was analyzed using Western blot analysis. Data were normalized to smooth muscle -actin. Protein level of RhoA (A), ROCKI (B), ROCKII (C), MYPT (D), and phospho-MYPT (E) were examined, and the phospho-MYPT/MYPT ratio was calculated (F). There were no significant differences between sham and DOCA-salt tissues (n = 11).

Rho- and PI3-Kinase-Mediated Spontaneous Tone Development: Biochemical Data. The changes in protein expression and phosphorylation status of MYPT that occur upon inhibition of spontaneous tone with LY294002 were examined in DOCA-salt aorta. In the presence of LY294002 (20 µM), we confirmed that development of spontaneous tone in DOCA-salt aorta was inhibited (Northcott et al., 2002); however, protein levels of the Rho effector MYPT were unchanged and levels of phospho-MYPT were unchanged (Fig. 7). Similarly, there was no change in the phospho-MYPT/MYPT ratio.

View larger version (28K): [in this window] [in a new window]   Fig. 7. MYPT and phospho-MYPT protein levels are unchanged in aortic strips from DOCA-salt rats after inhibition of spontaneous tone development with LY294002 (20 µM), a PI3-kinase inhibitor. Aortic strips in isolated tissue bath were allowed to develop spontaneous arterial tone in the presence or absence or LY294002 then analyzed using Western blot analysis. There were no significant differences in MYPT (A) or phospho-MYPT (B) protein levels, or the phospho-MYPT/MYPT ratio (C) (n = 5). Data were normalized to smooth muscle actin.

	Discussion

Top Abstract Materials and Methods Results Discussion Conclusions References

 
PI3-kinase plays a role in numerous cellular processes, including growth, apoptosis, and vascular contraction. The latter is evident in studies using a PI3-kinase inhibitor, LY294002, to inhibit development of spontaneous tone in vitro (Northcott et al., 2002). Likewise, increased RhoA activation in aorta has been demonstrated in four models of hypertension, including the DOCA-salt model, indicating that the Rho pathway could play an important role in hypertension (Seko et al., 2003). Rho-kinase inhibitors have been used in vivo to demonstrate the involvement of Rho-kinase in hypertension (Masumoto et al., 2001). Similarly, Rho-kinase inhibition by Y27632 or fausidil reduces vascular contraction in vitro to ET-1 and 5-HT (Weber and Webb, 2001; Kandabashi et al., 2002; Miao et al., 2002). Rho-kinase inhibition causes relaxation in large and small vessels (Asano and Nomura, 2003). These two pathways, PI3-kinase and Rho/Rho-kinase, may be interconnected and the nature of this interaction in spontaneous tone in aorta from DOCA-salt rats was the goal of the present study. This study is designed as an initial determination of interaction between Rho and PI3-kinase in arterial spontaneous tone.

Rho-Kinase in Spontaneous Tone Development. Rho/Rho-kinase has been implicated in altered contractility and tone, including cerebral vascular tone during hypertension (Chrissobolis and Sobey, 2001), pressure-induced tone in rat tail artery (Schubert et al., 2002), and lymphatic vessels (Hosaka et al., 2003). However, the role of Rho/Rho-kinase in arterial spontaneous tone is unclear.

Our experiments showed that the Rho/Rho-kinase pathway functions independently from the PI3-kinase pathway in the development of spontaneous tone in aortic strips isolated from DOCA-salt rats. Contractile experiments using Y27632, a Rho-kinase inhibitor, demonstrate that Rho-kinase inhibition is capable of reducing spontaneous tone development in DOCA-salt aorta. Notably, we have not measured the mechanical activity as presented as phasic, small oscillations in aorta from DOCA rats, although these events are observed (Fig. 1). These are distinct from the gradual increase in tone that we have defined as spontaneous tone. We have not quantified these events and view the oscillations as a different event than spontaneous tone.

Calcium in Rho-Mediated Spontaneous Tone Development. Rho-kinase modulates contraction in smooth muscle independently from calcium-calmodulin dependent myosin light chain kinase, indicating that Rho/Rho-kinase can modulate contraction independent of calcium (Kureishi et al., 1997). Nobe and Paul (2001) show that, although in a transient phase of contraction Y27632 reduced force and intra-cellular calcium, treatment during the sustained phase of contraction indicates that inhibition of MYPT can alter force without changing calcium. In contrast, Rho/Rho-kinase is involved with voltage-dependent calcium channels in regulating tone of the renal afferent arteriole (Nakamura et al., 2003) and may have a role in mediating intracellular calcium signaling (Ayman et al., 2003). Also, Rho/Rho-kinase contributes to calcium mobilization induced by agonist stimulation in cultured cells (Chong et al., 1994). Rho inhibition by Y27632 has the ability to relax NE contraction and to inhibit calcium signaling from voltage- or store-operated channels in mesenteric arteries and aorta (Ghisdal et al., 2003). Y27632 also inhibits calcium mobilization induced by methacholine stimulation (Ito et al., 2001). The present study used a calcium-free buffer to demonstrate that calcium is partially required for the development of spontaneous tone in aorta from DOCA-salt rats, a tone that is Rho-kinase dependent. These results support the idea that the actions of Rho/Rho-kinase in spontaneous tone are partially dependent on calcium.

Interaction of PI3-Kinase and Rho-Kinase in Spontaneous Tone. An important question is whether Y27632 has the capability of interfering directly with PI3-kinase, thereby decreasing spontaneous tone. We observed that Y27632 likely does not have the capabilities of interfering in the PI3-kinase pathways between the level of receptor and Akt. This can be stated only for the EGF-exposed cultured aortic smooth muscle cells. In the same experiment, the PI3-kinase inhibitor LY294002 inhibits EGF-induced Akt phosphorylation. These experiments are meant to demonstrate the selectivity of the pharmacological tools used and were not performed in intact tissue. This control experiment suggests that Y27632 does not likely have the ability to inhibit PI3-kinase directly.

To test whether Rho/Rho-kinase was downstream of PI3-kinase, aorta from DOCA-salt rats was tested in isolated tissue bath in the presence of a PI3-kinase inhibitor, LY294002. If Rho/Rho-kinase were a downstream effector of PI3-kinase, then blockade of PI3-kinase would inhibit the phosphorylation of the Rho pathway effector. LY294002, although it reduced spontaneous tone, did not alter protein levels or phosphorylation status of the Rho pathway effector MYPT. This indicates that Rho-kinase is not acting directly downstream of PI3-kinase and is contradictory to previously published data.

Miao et al. (2002) demonstrated that RhoA activation was associated with PI3-kinase in normal rabbit basilar artery such that LY294002 inhibited RhoA activation, indicating that PI3-kinase was upstream of Rho/Rho-kinase. Likewise, in vascular muscle inhibition of PI3-kinase interferes with insulin-mediated dephosphorylation of MYPT, further suggesting that Rho and PI3-kinase are associated (Begum et al., 2000). In this case, it was suggested that insulin inhibited the action of Rho-kinase, thus allowing for the action of MYPT to cleave a phosphate from myosin and limit contraction. However, PI3-kinase is involved in this pathway as an intermediary between insulin receptor activation and Rho-kinase activation because blockade of PI3-kinase inhibited the activation of Rho-kinase, i.e., PI3-kinase acts upstream of Rho activation (Begum et al., 2000).

Conversely, a distinct role of PI3-kinase and Rho/Rho-kinase has been shown in NIH 3T3 cells, such that the Rho pathway is required for focus formation, morphological alterations, and cell motility, whereas PI3-kinase is differentially required for cell transformation and cell motility but not for morphological alterations (Sachdev et al., 2002). This demonstrates a situation where Rho/Rho-kinase and PI3-kinase function as unique pathways. In addition, activation of TrkA receptor on PC12 cells stimulates PI3-kinase, which inactivates RhoA during neurite outgrowth (Nusser et al., 2002).

Our data demonstrate that there is little or no interaction between these pathways in the development of spontaneous tone in aorta from DOCA-salt rats. Therefore, we believe that the interaction between Rho and PI3-kinase may be tissue/disease-specific. Tissue heterogeneity in the Rho response was also suggested by Alexander (2000).

Role of PI3-Kinase Inhibition in Relaxation of Spontaneous Tone. PI3-kinase inhibition is capable of reducing spontaneous tone, yet does not affect the phosphorylation of MYPT. The mechanism by which PI3-kinase inhibition reduces spontaneous tone development is currently unknown. Previous work suggests that PI3-kinase activity is increased in aorta from DOCA-salt rats and contributes to enhanced contractility by interacting with calcium channels (Northcott et al., 2002). Macrez et al. (2001) demonstrates that intracellular infusion of the catalytic subunit of PI3-kinase, the p110 subunit, can activate barium current flow in smooth muscle cells. These findings suggest that the p110 subunits may interact directly with the calcium channel to stimulate activity; it is currently unclear whether phosphorylation of the channel in necessary. It follows then that PI3-kinase inhibition could relieve the activation of calcium channels to reduce tone rather than alter the phosphorylation status of MYPT.

Limitations. Several limitations of these studies should be acknowledged. Our studies are basic and more complex interactions may be occurring in the cell relating to Rho and PI3-kinase. Next, in these studies we used the thoracic aorta because is it well characterized, and due to its larger size, it is easier to use for protein isolation. This is not a resistance artery and thus we cannot state that our observations apply to resistance arteries, which are responsible for total peripheral resistance. Our data relate to spontaneous tone only, and there may be interactions between Rho/Rho-kinase and PI3-kinase in agonist stimulated tone that would not be evident from the present study. In addition, the role of the endothelium in the observed response in unknown, because the data were collected from endothelium-denuded tissues.

We also understand that LY294002 has the ability to inhibit casein kinase 2 (Davies et al., 2000). Because of the lack of selective casein kinase 2 inhibitors, it was not possible to control for this limitation. Also, we have studied the interaction of Rho/Rho-kinase and PI3-kinase only after the development of hypertension, making it difficult to draw any conclusions when these changes in the vasculature occur. Furthermore, we have used only Y27632 as a Rho-kinase inhibitor. We realize that Y27632 has the ability to inhibit protein kinase C-related protein kinase (Davies et al., 2000). Finally, RhoA activation and translocation from the cytosol to the membrane were not measured.

	Conclusions

Top Abstract Materials and Methods Results Discussion Conclusions References

 
The issue of Rho-PI3-kinase association is complex, because various tissues under different conditions reveal seemingly contradictory results. Our data in aorta from DOCA-salt rats show that Rho/Rho-kinase and PI3-kinase are involved in the development of spontaneous tone; however, Rho/Rho-kinase does not work directly upstream or downstream of PI3-kinase. This indicates that the Rho/Rho-kinase and PI3-kinase pathways are separate, parallel pathways in the development of spontaneous tone in aorta from DOCA-salt rats. Although both pathways are activated in DOCA-salt hypertension, it seems that each is activated through unique mechanisms, not due to an interconnected pathway. However, it is possible that these parallel pathways converge further downstream to activate a common effector such as a calcium channel.

	Acknowledgements

 
We thank Dr. Kanchen Chitaley (Department of Chemistry, University of Washington, Seattle, WA) for invaluable advice on Rho/Rho-kinase pathway antibodies and related details of Western analysis specific to Rho/Rho-kinase.

	Footnotes

 
This study was supported by American Heart Association Established Investigator Award 0240033N to S.W.W. and Midwest Affiliate American Heart Association Predoctoral Grant (1102077) to C.A.N.

DOI: 10.1124/jpet.103.062265.

ABBREVIATIONS: PI3-kinase, phosphoinositide 3-kinase; MYPT, myosin binding subunit of myosin phosphatase; phospho-MYPT, phosphorylated form of myosin binding subunit of myosin phosphatase; ET-1, endothelin-1; ROCKI, Rho-kinase I; ROCKII, Rho-kinase II; EGF, epidermal growth factor; 5-HT, 5-hydroxytryptamine (serotonin); DOCA-salt, deoxycorticosterone acetate salt; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PSS, physiologic salt solution; PE, phenylephrine; Y27632, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl).

Address correspondence to: Dr. Stephanie W. Watts, Department of Pharmacology and Toxicology, B445 Life Science Bldg., Michigan State University, East Lansing, MI 48824-1317. E-mail: wattss{at}msu.edu

	References

Top Abstract Materials and Methods Results Discussion Conclusions References

 

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