Rho/Rho kinase and PI3-kinase are parallel...

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Rho/Rho kinase and PI3-kinase are parallel pathways in the development of

spontaneous arterial tone in deoxycorticosterone acetate-salt hypertension

Erica A. Wehrwein, Carrie A. Northcott, Robert D. Loberg, and Stephanie W.

Watts.

Department of Pharmacology and Toxicology, Michigan State University, East

Lansing, MI 48824 (EAW, CAN, SWW)

Department of Physiology, Michigan State University, East Lansing, MI 48824

(EAW)

Department of Physiology, University of Michigan, Ann Arbor, MI 48109 (RDL)

JPET Fast Forward. Published on February 24, 2004 as DOI:10.1124/jpet.103.062265

Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.103.062265

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Running title: Rho and PI3-kinase in spontaneous arterial tone Correspondence, proofs and reprints should be sent to: Stephanie W. Watts, Ph.D. Department of Pharmacology and Toxicology B445 Life Science Building Michigan State University East Lansing, MI 48824-1317 Telephone: (517) 353-3900 Fax: (517) 353-8915 E-mail: [email protected] Number of text pages: 24 Number of tables: 0 Number of figures: 7 Number of references: 40 Number of words in: Abstract 235 Introduction 750 Discussion 1487 Abbreviations: 5-HT, serotonin; DMEM, Dulbecco’s Modified Eagle’s Medium; DOCA-salt, deoxycorticosterone acetate salt; EGF, epidermal growth factor; ET-1, endothelin-1; LY294002, 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; MYPT, myosin binding subunit of myosin phosphatase; PI3-kinase, phosphoinositide 3-kinase; phospho-MYPT, phosphorylated form of myosin binding subunit of myosin phosphatase; PBS, phosphate buffered saline; PSS, physiologic salt solution; ROCKI, Rho-kinase I; ROCKII, Rho-kinase II; TPR, total peripheral resistance; Y27632, [(+)-R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)] Section Assignment: Cardiovascular


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Abstract:

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 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 Rho-kinase 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 Y27632 (1 µM), a Rho-kinase inhibitor, significantly reduced spontaneous tone in the DOCA aorta, but did not affect sham aorta basal tone (DOCA:63.5 + 15.9 vs. Sham:1.2 + 0.4 Total change % phenylephrine contraction). We examined the interaction between the PI3-kinase and Rho pathways by observing the effects of LY294002 on a Rho-kinase effector, myosin phosphatase (MYPT), and Y27632 on a PI3-kinase effector, Akt, using Western blot analysis. Inhibition of PI3-kinase reduced spontaneous tone, but 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/Rho-kinase and PI3-kinase pathways in the DOCA-salt aorta, and the two pathways appear 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.


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Altered smooth muscle function and contractility are hallmarks of

hypertension. This includes enhanced agonist-stimulated contraction and

development of spontaneous arterial tone. Spontaneous tone, in which arteries

contract without exposure to exogenous stimuli, develops in arterial strips from

hypertensive rats. This phenomenon is thought to be due, in part, to altered Ca2+

sensitivity and/or handling in these tissues (Pucci et al., 1995, Thompson et al.,

1987, Webb et al., 1992). Spontaneous tone contributes to increased total

peripheral resistance and may play a role in hypertension. It is important to

understand the perturbations in vascular smooth muscle associated with

hypertension and spontaneous tone so that specifically targeted anti-

hypertensive therapy can be developed.

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 (Northcott et al., 2002, Yang et al., 2001,

Komalavilas et al., 2001, Ibitayo et al., 1998, Zheng et al., 1998) and is activated

by a variety of stimuli, including the activation of multiple types of receptors

(Vanhaesebroeck et al., 2000, Coelho et al., 2000, Cantrell et al., 2000,

Anderson et al., 1999, Rameh et al., 1999, Wymann et al., 1998). Previously,

PI3-kinase expression and activity have been shown to be upregulated in two

models of hypertension and contributes 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


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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

(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), serotonin (5-HT), and others

(Seko et al., 2002, Miao et al., 2002, Kandabashi et al., 2001, Sakurada et al.,

2001). Moreover, it has been suggested that Rho-kinase activity is elevated in

mesenteric arteries from mineralocorticoid hypertensive rats (Weber and Webb,

2000). 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


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vascular smooth muscle cells appears 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, prior to 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 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


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hypertension, there is not an associated increase in phosphorylation of Akt,

indicating that there is another downstream effector of PI3-kinase in this model.


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Methods:

Surgical and blood pressure protocol

DOCA-salt hypertension: Male Sprague Dawley rats (250-300 g; Charles River

Laboratories, Inc., Portage, MI) underwent uninephrectomy and implantation of

deoxycorticosterone acetate (DOCA; 200 mg kg-1) under isoflurane anesthesia

as described previously (Storm et al., 1990). Animals remained on the regimen

for four 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

removed. Arteries were dissected into helical strips (0.25 x 1 cm) and the

endothelial cell layer 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 (37oC), aerated (95%O2/5%CO2) physiological salt solution (PSS)

containing (mM): NaCl, 130; KCl, 4.7; KH2PO4, 1.18; MgSO4•7H2O, 1.17;

CaCl2•2H2O, 1.6; NaHCO3, 14.9; dextrose, 5.5; and CaNa2EDTA, 0.03 (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, the other attached to a force


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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 one hour. Changes in isometric force were

recorded on using PowerLab (v.3.6) and Chart (v.3.6.3) software (Mountain

View, CA) and evaluated using a Macintosh Computer. After an hour

equilibration, arteries were challenged with a maximal concentration of the α1-

adrenergic receptor agonist phenylephrine (PE, 10 µM). PE was washed out and

one of the following protocols performed. In experiments using antagonists, the

inhibitors or vehicle were added to the bath at least one hour prior to

measurements of spontaneous tone. The inhibitors used in this study, Y27632 (1

µM, Biomol, Plymouth Meeting, PA) and LY294002 (20 µM, Biomol, Plymouth

Meeting, PA), 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 (Figure 2).

Validation of phospho-MYPT activation: The contraction in response to 5-HT

(10-9- 3x10-4 M, Sigma, St. Louis, MO), a known activator of RhoA, was measured

in the presence and absence of Y27632 (1 µM), a Rho-kinase inhibitor which was

added one hour prior to the addition of agonist. The inhibitory effect of Y27632


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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 minutes 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,

Plymouth Meeting, PA) was added. Fifteen minutes after the addition of Y27632

or vehicle, Ca2+ was added back to the bath in a cumulative fashion (1x 10-6 -

3x10-3 M), with additions every 5 minutes.

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 one hour while

spontaneous tone was allowed to develop. After the development of

spontaneous tone in control tissue (1 hour), all tissues were snap-frozen and

analyzed as described below for Rho/Rho-kinase signaling elements.


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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,

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 Corning culture dish (Corning, NY) and

layered with a small amount of serum-enriched media to keep the tissues moist

[medium consisted of DMEM with D-Glucose (4500 mg/liter), L-glutamine (1%)

and HEPES buffer (25 mM; GIBCO Life Technologies, Gaithersburg, MD)

containing fetal bovine serum (40% v/v; Hyclone Laboratories, Logan UT) and

streptomycin (100 mg/ml)/penicillin (100 units/ml; GIBCO Life Technologies)].

Plates were placed in a 5 % CO2 warming incubator maintained at 37 ºC. Once

the tissues attached to the plate (~ 18 hr), additional medium was added to the

dish. After ~1 week, a sufficient number of cells had migrated from the tissue to

reach confluency in the plate. Cells were trypsinized, seeded to T75 flasks and

fed with normal serum (10 %) DMEM. 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 α-actin (Sigma Chemical, St.Louis,

MO). Cultured rat fibroblasts did not stain with this antibody.


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Vascular Smooth Muscle Cell Protein Isolation

Cells (P-100 plates) were switched to PSS (see above) and incubated for 1 hour

before the addition of agonist (final volume, 4 ml). At this same time, antagonists

or vehicle were added and equilibrated with tissues for 1 hour. Examination after

1 hour 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 minutes to stimulate the

EGF signaling cascade. After incubation, plates were placed on ice and the

incubation buffer was aspirated. Cells were washed 3 times (4 ml/wash) with

phosphate buffered saline (PBS) 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 PMSF, 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

14,000 x g for 10 minutes at 4 ºC. 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, 10% glycerol] with protease inhibitors (0.5 mM PMSF, 10 µg/ml


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aprotinin and 10 µg/ml leupeptin). Homogenates were centrifuged (11,000 g for

10 min, 4ºC) 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%, Rho-kinase I

and Rho-kinase II (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 Biotech Inc., Santa Cruz, CA), ROCKI (1:500, BD

Transduction Lab, San Diego, CA), ROCKII (1:1000, BD Transduction Lab, San

Diego, CA), MYPT (1:500, BD Transduction Lab, San Diego, CA), and phospho-

MYPT-1 Thr 850 (1 µg/mL, Upstate Cell Signaling Solutions, Lake Placid, NY)

primary antibodies. Cultured smooth muscle cells were similarly analyzed using

Akt (1:1000, Cell Signaling Solutions, Lake Placid, NY), and phospho-Akt

(1:1000, Cell Signaling Solutions, Lake Placid, NY) 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

Transduction Lab (San Diego, CA). Smooth muscle α-actin (1:400; Oncogene,

Boston, MA) was used as a marker to ensure equal loading of protein. Following

incubation in primary antibody, blots were washed and exposed to the

appropriate secondary antibody for 1 hour at 4°C with rocking. The secondary

antibody for RhoA, MYPT, ROCKI, ROCKII, and α-actin western analyses was

HRP-linked anti-mouse IgG (1:2000, Amersham Bioscience, Buckingham,

England). The secondary antibody for phospho-MYPT western analysis was


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HRP-linked anti-rabbit IgG (1:2000, Cell Signaling Solutions, Lake Placid, NY).

Finally, blots were washed and chemiluminescent detection of bands was

performed (ECL, Amersham Bioscience, Buckingham, England). All protein

immunolabeling, with the exception of α-actin, was performed on individual blots.

Measurement of α-actin was performed on all blots; this was done in a blot which

had not been stripped because α-actin is of a mass that does not interfere with

other bands. Protein quantitation is reported relative to smooth muscle α-actin.

Materials

Materials included acetylcholine hydrochloride, deoxycorticosterone acetate,

phenylephrine hydrochloride (Sigma, St. Louis, MO), 5-HT (Sigma, St. Louis,

MO), EGF (GIBCO Life Technologies, Gaithersburg, MD), LY294002 (Biomol,

Plymouth Meeting, PA), and Y27632 (Biomol, Plymouth Meeting, PA).

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 were performed on computer-scanned images of developed films

using NIH Image (v.1.61). The phospho-MYPT/MYPT ratio was calculated as

follows from Western blot data normalized to smooth muscle α-actin (arbitrary

units): phospho-MYPT density/MYPT density. This ratio was calculated to

determine changes in phosphorylation status of MYPT relative to total MYPT


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protein. When comparing two groups, an unpaired Student’s T-test was used.

In all cases, p < 0.05 was considered statistically significant.


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Results:

Rho- and PI3-kinase- mediated spontaneous tone development: contractile data

The systolic blood pressures of sham and DOCA-salt rats were 115 + 2 mm Hg

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 mg vs. 1273 + 60.6 mg, respectively).

Spontaneous tone developed in endothelium-denuded aorta isolated from

DOCA-salt but not sham rats (Figure 1, right panel and tracings 4 & 3). Y27632,

a Rho-kinase inhibitor, significantly reduced spontaneous tone in the DOCA

aorta, but did not affect sham aorta basal tone (63.5 + 15.9 % vs. 1.2 + 0.4 %

Total change) (Figure 1, right panel and tracings 2 & 1). Y27632 reduced

spontaneous tone in a concentration-dependent manner (Figure 2). The effect of

Y27632 was reversible, as 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 as

compared to sham (76.7 + 18.9 vs. 2.5 + 1.4 total change in % PE contraction)

and did so in a concentration-dependent manner (Northcott et al., 2002).

Pharmacological test of LY294002 and Y27632

LY294002 (20 µM) and Y27632 (1 µM) were tested against EGF (10 nM, 10

minutes) 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


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(10 nM) significantly increased phosphorylation of Akt, indicating that the PI3-

kinase pathway is activated by EGF stimulation (Figure 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.

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 normotensive animals. Development of tone was significantly inhibited by

Y27632 (1 µM) (Figure 4).

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- 3x10-4 M) caused

contraction of aortic strips in isolated tissue bath and 5-HT-induced contraction

was inhibited by Y27632 (1 µM) (Figure 5a). Following administration of 5-HT,

protein levels of MYPT and phospho-MYPT were analyzed using Western blot

analysis. Total MYPT protein was unchanged following 5-HT stimulation as

compared to control (Figure 5b). 5-HT stimulation increased phosphorylation of


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MYPT, indicating that the Rho pathway was activated by 5-HT (Figure 5c). To

confirm this, a Rho-kinase inhibitor, Y27632 was added to the aorta preparation

prior to 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 (Figure

5c). The phospho-MYPT/MYPT ratio was increased following 5-HT stimulation

and was reduced in the presence of Y27632 (Figure 5d).

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 if DOCA-salt hypertension resulted in alteration of protein levels

within the Rho/Rho-kinase pathway as compared to 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 (Figure 6).


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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 (Figure 7). Similarly, there was no

change in the phospho-MYPT/MYPT ratio.


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Discussion:

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, Miao et al., 2002, Kandabashi et al.,

2002). Rho-kinase inhibition causes relaxation in large and small vessels (Asano

et al., 2003). These two pathways, PI3-kinase and Rho/Rho-kinase, may be

inter-connected 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 (Chrissololis and Sobey,

2001), pressure-induced tone in rat tail artery (Schubert et al., 2002) and


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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, though these events are observed (See

figure 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 intracellular 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


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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-


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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 as 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, while 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,


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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 et al (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 altering the phosphorylation status

of MYPT.


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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 TPR. 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 as the data were collected from endothelium-

denuded tissues. We also understand that LY294002 has the ability to inhibit

casein kinase 2 (CK2) (Davies et al., 2000). Because of the lack of selective CK2

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 (PKCRK) (Davies et al., 2000). Finally, RhoA activation and

translocation from the cytosol to the membrane were not measured.


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Conclusion

The issue of Rho-PI3-kinase association is complex, as 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 appears 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.


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Acknowledgements:

We would like to thank Dr. Kanchen Chitaley (Department of Chemistry,

University of Washington) for invaluable advice on Rho/Rho-kinase pathway

antibodies and related details of Western analysis specific to Rho/Rho-kinase.


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References:

Anderson RA, Boronenkov IV, Doughman SD, Kunz J, Loijens JC (1999)

Phosphatidylinositol phosphate kinases, a multifaceted family of signaling

enzymes. J Biol Chem 274:9907-9910.

Alexander JS (2000) Rho, tyrosine kinase, Ca(2+), and junctions in endothelial

hyperpermeability. Circ Res 87(4):268-271.

Asano M, Nomura Y (2003) Comparison of inhibitory effects of Y-27632, a Rho

kinase inhibitor, in strips of small and large mesenteric arteries from

spontaneously hypertensive and normotensive Wistar-Kyoto rats. Hypertens Res

26(1):97-106.

Ayman S, Wallace P, Wayman CP, Gibson A, McFadzean I (2003) Receptor-

independent activation of Rho-kinase-mediated calcium sensitisation in smooth

muscle. Br J Pharmacol 139(8):1532-1538.

Begum N, Duddy N, Sandu O, Reinzie J, Ragolia L (2000) Regulation of myosin-

bound protein phosphatase by insulin in vascular smooth muscle cells:

evaluation of the role of Rho kinase and phosphatidylinositol-3-kinase-dependent

signaling pathways. Mol Endocrinol 14(9):1365-1376.


at ASPE

T Journals on A


Dow

nloaded from


JPET #62265

29

Cantrell DA (2000) Phosphoinositide 3-kinase signaling pathways. J Cell Sci.

114:1439-1445.

Chrissobolis S, Sobey CG (2001) Evidence that Rho-kinase activity contributes

to cerebral vascular tone in vivo and is enhanced during chronic hypertension:

comparison with protein kinase C. Circ Res 88(8):774-779.

Chong LD, Traynor-Kaplan A, Bokoch GM, Schwartz MA (1994) The small GTP-

binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in

mammalian cells. Cell 79(3):507-513.

Coelho CM, Leevers SJ (2000) Do growth and cell division rates determine cell

size in multicellular organisms? J Cell Sci 113:2927-2934.

Davies SP, Reddy H, Caivano M, Cohen P (2000) Specificity and mechanism of

action of some commonly used protein kinase inhibitors. Biochem J 351(Pt

1):95-105.

Florian JA, Watts SW (1998) Integration of mitogen-activated protein kinase

kinase activation in vascular 5-hydroxytryptamine 2A receptor signal

transduction. J Pharmacol Exp Ther 284(1)346-355.


at ASPE

T Journals on A


Dow

nloaded from


JPET #62265

30

Hosaka K, Mizuno R, Ohhashi T (2003) Rho-Rho kinase pathway is involved in

the regulation of myogenic tone and pump activity in isolated lymph vessels. Am

J Physiol Heart Circ Physiol 284(6):H2015-2025.

Ibitayo AI, Tsunoda Y, Nozu F, Owyang C, Bitar KN (1998) Src kinase and PI3-

kinase as transduction pathway in ceramide-induced contraction of colonic

smooth muscle. Am J Physiol 275:G705-G711.

Ito S, Kume H, Honjo H, Katoh H, Kodama I, Yamaki K, Hayashi H (2001)

Possible involvement of Rho kinase in Ca2+ sensitization and mobilization by

MCh in tracheal smooth muscle. Am J Physiol Lung Cell Mol Physiol

280(6):L1218-1224.

Kandabashi T, Shimokawa H, Mukai Y, Matoba T, Kunihiro I, Morikawa K, Ito M,

Takahashi S, Kaibuchi K, Takeshita A (2002) Involvement of rho-kinase in

agonists-induced contractions of arteriosclerotic human arteries. Arterioscler

Thromb Vasc Biol 22:243-248.

Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B,

Feng J, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K (1996) Regulation of

myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science

273(5272):245-248.


at ASPE

T Journals on A


Dow

nloaded from


JPET #62265

31

Komalavilas P, Mehta S, Wingard CJ, Dransfield DT, Bhalla J, Woodrum JE,

Molinaro JR, Brophy CM (2001) PI3-kinase/Akt modulates vascular smooth

muscle tone via cAMP signaling pathways. J Appl Physiol 91:1819-1827.

Kureishi Y, Kobayashi S, Amano M, Kimura K, Kanaide H, Nakano T, Kaibuchi K,

Ito M (1997) Rho-associated kinase directly induces smooth muscle contraction

through myosin light chain phosphorylation. J Biol Chem 272(19):12257-12260.

Macrez N, Mironneau C, Carricaburu V, Quignard JF, Babich A, Czupalla C,

Nurnberg B, Miranneau J (2001) Phosphoinositide 3-kinase insoforms selectively

coulple receptors to vascular L-type calcium channels. Circ Res 89:692-699.

Masumoto A, Hirooka Y, Shimokawa H, Hironaga K, Setoguchi S, Takeshita A

(2001) Possible involvement of Rho-kinase in the pathogenesis of hypertension

in humans. Hypertension 38(6):1307-1310.

Miao L, Dai Y, Zhang J (2002) Mechanism of RhoA/Rho kinase activation in

endothelin-1- induced contraction in rabbit basilar artery. Am J Physiol Heart

Circ Physiol 283(3):H983-989.

Nakamura A, Hayashi K, Ozawa Y, Fujiwara K, Okubo K, Kanda T, Wakino S,

Saruta T (2003) Vessel- and vasoconstrictor-dependent role of rho/rho-kinase in

renal microvascular tone. J Vasc Res 40(3):244-251.


at ASPE

T Journals on A


Dow

nloaded from


JPET #62265

32

Northcott CA, Poy MN, Najjar SM, Watts SW (2002) Phosphoinositide 3-kinase

mediates enhanced spontaneous and agonist-induced contraction in aorta of

deoxycorticosterone acetate-salt hypertensive rats. Circ Res 91: 360-369.

Nobe K, Paul RJ (2001) Distinct Pathways of calcium sensitization in porcine

coronary artery: Effects of Rho-related kinase and protein kinase C inhibition on

force and intracellular calcium. Circ Res 88: 1283-1290.

Nusser N, Gosmanova E, Zheng Y, Tigyi G (2002) Nerve growth factor signals

through TrkA, phosphatidylinositol 3-kinase, and Rac1 to inactivate RhoA during

the initiation of neuronal differentiation of PC12 cells. J Biol Chem

277(39):35840-35846.

Pucci ML, Tong X, Miller KM, Guan H, Nasjletti A (1995) Calcium- and protein

kinase C- dependent basal tone in the aorta of hypertensive rats. Hypertension

25(part2):752-757.

Rameh LE, Cantley, LC (1999) The role of phosphoinositide 3-kinase lipid

products in cell function. J Biol Chem 274:8347-8350.


at ASPE

T Journals on A


Dow

nloaded from


JPET #62265

33

Reif K, Nobes CD, Thomas G, Hall A, Cantrell DA (1996) Phosphatidylinositol 3-

kinase signals activate a selective subset of Rac/Rho-dependent effector

pathways. Curr Biol 6(11):1445-1455.

Sachdev P, Zeng L, Wang LH (2002) Distinct role of phosphatidylinositol 3-

kinase and Rho family GTPases in Vav3-induced cell transformation, cell motility,

and morphological changes. J Biol Chem 277(20):17638-17648.

Sakurada S, Okamoto H, Takuwa N, Sugimoto N, Takuwa Y (2001) Rho

activation in excitatory agonist-stimulated vascular smooth muscle. Am J Physiol

Cell Physiol 281(2):C571-578.

Sata M and Nagai R (2002) Phosphoinositide 3-kinase: A key regulator of

vascular tone? Circ Res 91: 273-275.

Schubert R, Kalentchuk VU, Krien U (2002) Rho kinase inhibition partly weakens

myogenic reactivity in rat small arteries by changing calcium sensitivity. Am J

Physiol Heart Circ Physiol 283(6):H2288-95.

Seko T, Ito M, Kureishi Y, Okamoto R, Moriki N, Onishi K, Isaka N, Hartshorne

DJ, Nakano T (2003) Activation of RhoA and inhibition of myosin phosphatase as

important components in hypertension in vascular smooth muscle. Circ Res

92:411-418.


at ASPE

T Journals on A


Dow

nloaded from


JPET #62265

34

Storm DS, Turla MB, Todd KM, Webb RC (1990) Calcium and contractile

responses to phorbol esters and the calcium channel agonist, BayK8644, in

arteries from hypertensive rats. Am J Hypertens 3:245S-248S.

Thompson LP, Bruner CA, Lamb FS, King CM, Webb RC (1987) Calcium influx

and vascular reactivity in systemic hypertension. Am J Cardiol 59:29A-34A.

Vanhaesebroeck B, Leevers SJ, Ahmadi K, Timms J, Katso R, Driscoll PC,

Woscholski R, Parker PJ, Waterfield MD (2001) Synthesis and function of 3-

phosphorylated inositol lipids. Annu Rev Biochem 70:535-602.

Webb RC, Schreur KD, Papadopoulos SM (1992) Oscillatory contractions in

vertebral arteries from hypertensive subjects. Clin Physiol 12:69-77.

Weber DS, Webb RC (2001) Enhanced relaxation to the rho-kinase inhibitor Y-

27632 in mesenteric arteries from mineralocorticoid hypertensive rats.

Pharmacology 63(3):129-133.

Yang ZW, Wang J, Zheng T, Altura BT, Altura BM (2001) Importance of PKC and

PI3Ks in ethanol-induced contraction of cerebral arterial smooth muscle. Am J

Physiol Heart Circ Physiol 280:H2144-H2152.


at ASPE

T Journals on A


Dow

nloaded from


JPET #62265

35

Zheng X, Renaux B, Hollenberg MD (1998) Parallel contractile signal

transduction pathways activated by receptors for thrombin and epidermal growth

factor-urogastrone in guinea pig gastric smooth muscle: blockade by inhibitors of

mitogen-activated protein kinase-kinase and phosphatidyl inositol 3'-kinase. J

Pharmacol Exp Ther 285:325-334.


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Footnotes:

a) Financial support provided by American Heart Association Established

Investigator Award 0240033N to Stephanie W. Watts and Midwest Affiliate

American Heart Association Predoctoral Grant (1102077) to Carrie A. Northcott.

b) Stephanie W. Watts, Ph.D.

Department of Pharmacology and Toxicology

B445 Life Science Building

Michigan State University

East Lansing, MI 48824-1317


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Legends for figures:

Figure 1: The Rho-kinase inhibitor, Y27632, reduced spontaneous arterial tone

in DOCA-salt aorta, but did not affect sham tone in isolated tissue bath. Left

panel shows contractile data for aortic strips from DOCA-salt and sham rats in

the presence (tracings 1,2) or absence (tracings 3,4) of Y27632 (1 µM) or

vehicle. Right panel shows contractile data normalized as percentage of PE

contraction. Y27632 significantly reduced spontaneous tone in DOCA-salt aorta

(* p<0.05 compared to DOCA vehicle, n=4), while sham tone was unaltered.

Figure 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 to DOCA vehicle,

n=4).

Figure 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 in the methods section. EGF stimulation

significantly increased pAKTSer473 ( * p<0.05 compared to control, n=6). The


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EGF-induced increase in pAKTSer473 was not inhibited by Y27632 ( * p<0.05

compared to EGF alone, n=6), but was inhibited by LY294002 ( † p<0.001, n=6).

Figure 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 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 to DOCA Y27632, n=4).

Spontaneous tone did not develop in sham tissues.

Figure 5: 5-HT-induced contraction was inhibited by Y27632. Protein levels of

MYPT were not altered following treatment with 5-HT (10-9- 3x10-4 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 (panel A), then analyzed using Western blot analysis (panels B-D). Data

were normalized to smooth muscle α-actin. Protein level of MYPT was

unchanged by stimulation with 5-HT or treatment with Y27632 (panel 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)

(panel C). Likewise, 5-HT increased the phospho-MYPT ratio (** p<0.01, n=6)

while Y27632 reduced the ratio (* p<0.05, n=6) (panel D).


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Figure 6: Protein levels of Rho pathway constituents are not altered in thoracic

aorta from DOCA-salt as compared to sham. Thoracic aorta from DOCA-salt

and sham rats was analyzed using Western blot analysis. Data were normalized

to smooth muscle α-actin. Protein level of RhoA (panel A), ROCK I (panel B),

ROCK II (panel C), MYPT (panel D), and phospho-MYPT (panel E) were

examined, and the phospho-MYPT/MYPT ratio was calculated (panel F). There

were no significant differences between sham and DOCA-salt tissues (n=11).

Figure 7: MYPT and phospho-MYPT protein levels are unchanged in aortic

strips from DOCA-salt rats following 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 (panel A) or phospho-MYPT (panel B) protein

levels, or the phospho-MYPT/MYPT ratio (panel C) (n=5). Data was normalized

to smooth muscle -actin.


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Figure 1

Y27632/Vehicle

2 min200 mg

Sham

Sham

DOCA

DOCAY27632 (1 µM)

Vehicle

Ten

sion

(m

g)

*

80

60

40

20

0

-20

-40Sham Sham DOCADOCA

Vehicle Y27632 (1 µM)

N=4

Per

cent

age

PE

( 1

0-5m

o l/ L

) C

ont r

actio

n

1)

2)

3)

4)

JPET #62265

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Fast Forward. Published on February 24, 2004 as D

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40

20

0

-20

-40

-60

-80-10 -9 -8 -7 -6 -5 -4

Log Y27632 (M)

DOCA VehicleDOCA Y27632

Sham Y27632Sham Vehicle

N=4

Per

cen t

age

PE

(1

0-5

mol

/L)

Con

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**

* * *

Figure 2JPET #62265




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pAktSer473

total Akt

EGFY27632

LY294002 -

---

--

-+ +

++

+

100

75

50

25

0

* *

Arb

i trar

y U

n it s

N=6

†

Figure 3JPET #62265




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DOCA VehicleDOCA Y27632

Sham Y27632Sham Vehicle

N=4

-7 -6 -5 -4 -3 -2

100

75

50

25

0

Log Calcium Chloride (M)

Per

cen t

age

PE

(1

0-5

mol

/L)

Con

trac

tion *

*

*

Figure 4JPET #62265




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Figure 5

MYPT

Actin

Phospho-MYPT

Actin

MYPT

Control 5-HT 5-HT+Y276320

25

50

75Control

5-HT5-HT+Y27632

N=6

phospho-MYPT

Control 5-HT 5-HT+ Y276320

50

100

150Control

5-HT*5-HT+ Y27632

**

N=6A.

B.

C.

D.

Rat thoracic aorta

-10 -9 -8 -7 -6 -5 -40

50

100

1505-HT + vehicle5-HT + Y27632 (1 uM)

N=6 ** *

log 5-HT [M]

phospho-MYPT/MYPT ratio

Control 5-HT 5-HT + Y276320

5

10

15Control5-HT5-HT + Y27632

**

*

N=6

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Figure 6

RhoA

ROCK I

ROCK II

MYPT

Phospho-MYPT

A)

B)

C)

D)

E)

E)

Rho A

Sham DOCA0

50

100

150ShamDOCA

N=11

ROCK I

Sham DOCA0

1000

2000

3000

4000ShamDOCA

N=11

ROCK II

Sham DOCA0

1000

2000

3000

4000ShamDOCA

N=11

MYPT

Sham DOCA0

1000

2000

3000

4000

5000Sham

DOCA

N=11

phospho-MYPT

Sham DOCA0

1000

2000

3000

4000Sham

DOCA

N=11


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0.75

1.00Sham

DOCA

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Figure 7

MYPT

Phospho-MYPT

Actin

Actin

A)

B)

C)

MYPT

DOCA DOCA + LY2940020

25

50

75

100DOCADOCA + LY294002

N=5

phospho-MYPT

DOCA DOCA + LY2940020

10

20

30

40

50DOCADOCA + LY294002

N=5


DOCA DOCA + LY2940020.00

0.25

0.50

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1.00DOCA

DOCA + LY294002

N=5

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Rho/Rho kinase and PI3-kinase are parallel...

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