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Cardiovascular Research 2007 73(1):190-197; doi:10.1016/j.cardiores.2006.10.020
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Copyright © 2006, European Society of Cardiology

RhoA activation and interaction with Caveolin-1 are critical for pressure-induced myogenic tone in rat mesenteric resistance arteries

Caroline Dubrocaa, Xavier Loyera, Kevin Retailleaud,e, Gervaise Loirandb, Pierre Pacaudb, Olivier Feronc, Jean-Luc Balligandc, Bernard I. Lévya, Christophe Heymesa and Daniel Henriond,e,*

aCentre de Recherche Cardiovasculaire Lariboisière, INSERM U689, Paris, France
bInstitut du Thorax, INSERM Unit 533, Nantes, France
cUnit of Pharmacology and Therapeutics UCL-FATH5349, Brussels, Belgium
dCNRS UMR 6214, Angers, France
eINSERM U771, Angers, France

* Corresponding author. Department of Integrated Neurovascular Biology, UMR CNRS 6214–INSERM 771, Faculté de Medecine, 49045 Angers, France. Tel.: +33 2 41 73 58 45; fax: +33 2 41 73 58 95. Email address: daniel.henrion{at}univ-angers.fr

Received 29 March 2006; revised 5 October 2006; accepted 25 October 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Experimental procedures
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Myogenic tone, which has a major role in the regulation of local blood flow, refers to the ability of vascular smooth muscle to adapt its contractility to changes in transmural pressure. Although Rho-kinase is involved in myogenic tone, the pathway involved remains unclear, especially concerning translocation to the plasma membrane and activation of RhoA. As caveolae have a key role in the signal transduction of membrane-bound proteins, we tested the hypothesis that RhoA might be activated by pressure and that its activation might involve caveolin-1, which has been shown to be involved in vascular functions.

Methods: Myogenic tone was studied in isolated rat mesenteric resistance arteries (118±15 µm internal diameter with a pressure of 75 mmHg) submitted to pressure steps (25, 75, and 150 mmHg). Pharmacological blockade of caveolae or RhoA–Rho-kinase pathway was assessed by confocal microscopy in pressurized arteries to analyze protein co-localization and by co-immunoprecipitation in order to confirm protein interactions. Caveolin-1-deficient mice were used to confirm the role of the protein in myogenic tone.

Results: Pressure-induced myogenic tone was significantly reduced by RhoA inactivation with TAT-C3 (90.5% inhibition at 150 mmHg) and by the Rho-kinase inhibitor Y27632 (91.8% inhibition at 150 mmHg). In arteries pressurized at 150 mmHg, RhoA was localized to the plasma membrane (localization by confocal microscopy and increased quantity of RhoA in the membrane fraction after protein extraction). Thus, translocation of RhoA to the plasma membrane was associated with pressure-induced tone. In addition, caveolae disruption with methyl-β-cyclodextrin reduced myogenic tone by 66% at 150 mmHg. Further, myogenic tone was significantly reduced to 24% of control in caveolin-1-deficient mice (active tone was 32.3±2.8 µm and 9.1±3.7 µm in +/+ and –/– mice, respectively, n=5 per group), suggesting a key role of caveolin-1 in myogenic tone. Finally, RhoA and caveolin-1 co-immunoprecipitation and co-localization significantly increased when myogenic tone developed at 150 mmHg (co-localization showed 26±13% merging at 25 mmHg versus 97±21% at 150 mmHg, n=5). Co-immunoprecipitation was prevented by TAT-C3 and by methyl β-cyclodextrin.

Conclusion: RhoA activation is critical for the development of myogenic tone in resistance arteries. This activation induced translocation of RhoA to the plasma membrane within caveolae, where the interaction of RhoA with caveolin-1 leads selectively to the activation of a Rho-kinase-dependent force development.

KEYWORDS Mechanotransduction; Contractile function; Vasoconstriction; Blood pressure; Caveolae


   1. Introduction
Top
 Abstract
 1. Introduction
2. Experimental procedures
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Myogenic tone (MT) refers to the ability of resistance arteries vascular smooth muscle to alter its state of contractility in response to changes of transmural pressure; vessels constrict in response to an increase in intravascular pressure and dilate as a result of decreased pressure [1,2]. Myogenic tone is modified in most metabolic and cardiovascular disorders although changes in myogenic tone do not affect similarly all vascular beds. The mechanisms involved in myogenic tone are not yet fully understood, mainly because of the heterogeneity of the blood vessels developing myogenic tone but also because of the size of resistance arteries; myogenic being usually absent or very low in arteries with internal diameter higher than 200 µm [1].

Myogenic tone requires membrane depolarization, opening of voltage-operated calcium channels [3–5] and Ca2+ sensitization of the contractile apparatus contributes to MT [6–8]. Rho-kinase inhibition attenuates MT through a reduction in the contractile apparatus sensitivity to calcium [8–13]. Similarly, we have shown the role of Rho-kinase in MT in rabbit facial veins [14]. Like other members of the Ras superfamily, RhoA regulates intracellular signalling pathways by cycling between active, GTP-bound and inactive, GDP-bound states. The hallmark of the Rho family is their dual subcellular localization into membrane-associated and soluble forms [15]. This reflects the presence of an inactive cytosolic pool of the proteins bound to the Rho-dissociation inhibitor (Rho-GDI) and of an active pool located to the plasma membrane [16]. The mechanisms governing the association of Rho proteins to membranes and the activation of their effector molecules remain largely unknown in a number of processes including contraction in vascular smooth muscle.

Caveolae are abundant in vascular cells and content caveolin [17,18]. They are important platforms for signal transduction [1,17,19–21]. Several studies have suggested, in cultured endothelial and smooth muscle cells, that RhoA might be linked to caveolae-enriched membrane domains, through interaction with caveolin-1 [22]. Indeed, caveolins interact with a variety of signal-transducing molecules and regulate their activity [19,23]. A short N-terminal cytoplasmic region of caveolin-1 called "scaffolding domain" is critical for these regulatory interactions. In caveolin-1 deficient mice calcium sparks frequency in cerebral arteries smooth muscle cells is decreased, suggesting a lower MT in these arteries. [24].

Thus, we hypothesized that pressure-induced MT, in resistance arteries, might involve RhoA activation through interaction with caveolae-associated proteins such as caveolin-1. This interaction with proteins contained in caveolae might have a key role in the activation of Rho-kinase and then in the calcium sensitization of the contractile apparatus needed for the development of myogenic tone.


   2. Experimental procedures
Top
 Abstract
 1. Introduction
 2. Experimental procedures
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Isolated mesenteric resistance arteries
Mesenteric resistance arteries from 12-week-old Wistar rats were isolated and cannulated at both ends in a video monitored perfusion system [25] (LSI, Burlington, VT) as previously described [26,27]. Briefly, arteries were bathed in a physiological salt solution. After stabilization (30 min) at 75 mmHg, internal diameter changes were measured when intraluminal pressure was increased from 25 to 150 mmHg. Pressure was then set at 75 mmHg, prior to a second series of pressure steps, arteries were exposed or not to TAT-C3 exoenzyme (7 µg/mL) for 2 hours, Y27632 (10 µM) for 30 min or methyl β-cyclodextrin (mβcd, 10 mM) for 2 hours. At the end of each experiment arteries were bathed in a Ca2+-free physiological salt solution containing EGTA (2 mM) plus sodium nitroprusside (10 µM) and pressure steps were repeated in order to determine the arteries passive diameter [26,27]. MT was quantified as the difference between active and passive diameter [26,27]. Third order arteries were used for the study of MT (118 internal diameter with a pressure of 75 mmHg). The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

In a separate series of experiments mesenteric arteries (second order) were isolated from mice lacking the gene encoding for caveolin-1 (gift from Pr. Balligand (Unit of Pharmacology and Therapeutics UCL-FATH5349, Brussels, Belgium). They were submitted to the protocol described above for the measurement of MT.

The procedure followed in the care and euthanasia of the study animals was in accordance with the European Community standards on the care and use of laboratory animals (authorization no. 00577).

2.2. Western blotting and immunoprecipitation experiments
Tissue extraction was performed as previously described [28,29]: arterial segments were pressurized as described above and after stabilization under a pressure of 25, 75 or 150 mmHg arterial segments were quickly frozen. Arteries (5 per group) were then pulverized in liquid nitrogen. The powders were resuspended in lysis buffer [500 mmol/L Tris-HCl pH 7.4, 20% sodium dodecyl sulfate, 100 mmol/L sodium orthovanadate, and protease inhibitors (Boehringer Mannheim)].

In a separated series of experiments, a minimum of 15 small resistance arteries were used per group in order to provide sufficient protein for reliable separation of cytosolic and particulate fractions. Frozen vascular segments were pulverized in liquid nitrogen. The powders were resuspended in ice-cold homogenization buffer of the following composition [3% chaps, 1 mol/L NaCl, 20 mmol/L Tris pH 7.4, 1 mmol/L DTT and protease inhibitors (Boehringer Mannheim)] and centrifuged at 100000 xg for 45 min at 4 °C (ultracentrifuge; Beckman). The supernatant was collected and is referred to as the cytosolic fraction. Pellets were resuspended, and the membrane proteins were extracted by incubation in 20% sodium dodecyl sulfate lysis buffer (described upper). The extract was centrifuged at 12000 xg for 10 min at 15 °C. The supernatant was collected as the membrane fraction.

For Western-blot analysis, primary antibodies against RhoA (polyclonal, Santa Cruz) or Cav-1 (monoclonal, Transduction laboratories) were used. Staining with Ponceau red or reprobing membranes with monoclonal anti-β-actin antibody were used to normalize for loading variations.

For coimmunoprecipitation experiments and in order to obtain undenaturated total caveolin to study protein–protein interaction, nOctylglucoside lysis buffer [30] was employed. Arteries lysates (75 µg, 15–20 arteries) were incubated overnight at 4 °C with mAb RhoA at a final concentration of 5 µg/mL in immunoprecipitation (IP) buffer [30]. Anti-mouse IgG-conjugated Agarose was added for 3 hours at 4 °C. After washes with IP buffer, the immunoprecipitates were separated by electrophoresis and immunoblotted with pAb Cav-1{alpha} (Santa Cruz).

2.3. Immunohistochemistry and confocal microscopy
RhoA, actin and caveolin-1 were ascertained and localized by immunohistochemistry sampled after pressurization (25 or 150 mmHg) using an arteriograph as described above. After stabilization, arterial segments were fixed in a 4% buffered formaldehyde solution as previously described [27]. Sections of arteries (7 µm thick) were incubated with primary RhoA goat antibodies (Santa Cruz, 1:200) and primary caveolin-1 rabbit antibodies (Santa Cruz, 1:100). Sections were then incubated with by the fluorescent (FITC-bound) anti-goat secondary antibody (1:200) and with fluorescent (texas-red-bound) anti-rabbit secondary antibody (1:200). Fluorescence was visualized and quantified using confocal microscopy as previously described [27]. In negative control experiments the primary antibodies were omitted.

2.4. Drugs
Y27632 was purchased from Calbiochem (La Jolla, USA); TAT-C3 exoenzyme was a kind gift of Pierre Pacaud (U533, Nantes, France); all other reagents were purchased from Sigma (St. Louis, USA).

2.5. Statistical analysis
Results are expressed as mean±SEM. Differences between means were evaluated by 1-way ANOVA or two-tailed Student's paired t-test. P values<0.05 were considered to be significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Experimental procedures
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. RhoA–Rho-kinase pathway is involved in the development of myogenic tone
Third order mesenteric resistance arteries (118±15 µm internal diameter with a pressure of 75 mmHg) developed MT in response to pressure (Figs. 1 and 2Go). Their treatment with the RhoA inhibitor, TAT-C3, or the Rho-kinase inhibitor, Y27632 strongly reduced MT at 75 and 150 mmHg (Fig. 1). At 150 mmHg, TAT-C3 and Y27632 inhibited MT by 95 and 92%, respectively.


Figure 1
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  		Fig. 1 Effect of cavaolae and RhoA–Rho-kinase alteration on myogenic tone in rat mesenteric resistance arteries. Effect of methyl β-cyclodextrin (M, 10 mM), cholesterol-saturated metyl β-cyclodextrin (M+chol), TAT-C3 (TC, 7 µg/ml), or Y27632 (Y, 10 µM) on MT in third order mesenteric resistance arteries submitted to an intraluminal pressure of 25, 75 and 150 mmHg. Data is expressed as difference between passive and active diameter induced by pressure (active tone). Mean±sem is presented (n=5 per group). *P<0.05, versus control.

 

Figure 2
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  		Fig. 2 Translocation of RhoA from the cytosol to the plasma membrane and colocalization with caveolin-1 (CAV-1) in mesenteric resistance arteries developing MT. Panel A shows an isolated artery cannulated between two glass pipettes and pressurized to 25, 75 or 150 mmHg. The internal diameter measured under different pressure levels is shown as a typical recording in panel B. Translocation of RhoA from the cytosolic fraction to plasma membrane was measured in segments of mesenteric resistance arteries under an intraluminal pressure of 25, 75 or 150 mmHg. Blots in panel C are representative of 4 experiments (15 pressurized arteries per experiment, n=4 rats). Panel D: quantification of the blots. *P<0.05, versus control (25 mmHg). In the same conditions, arteries pressurized at 25 (panel E) or 150 mmHg (panel F) were fixed and analyzed with confocal microscopy. CAV-1 and RhoA were colocalized using 2 different fluorescence intensities. Merging was quantified in the 2 conditions of pressure (n=5 arteries per condition, isolated from 5 rats, panel G). *P<0.05, 150 versus 25 mmHg.

 
3.2. Pressure-induced myogenic tone requires RhoA translocation
Fig. 2 shows an isolated mesenteric resistance artery (A) and a typical recording illustrating the effect of pressure on internal diameter (B). Panel C shows the expression of RhoA in the cytosolic and membrane fractions after extraction of the proteins in pressurized arteries. Quantification of the repartition of RhoA between the cytosolic and membrane fractions is shown in the bargraph (panel D). In the same conditions of pressure confocal microscopy was performed in order to visualize RhoA in the cytosol and at the level of the membrane (Fig. 2E and F). RhoA density was higher at the level of the plasma membrane when pressure was 150 mmHg than under a pressure of 25 mmHg (Fig. 2E and F).

3.3. Role of RhoA–Cav-1 complexes in pressure-induced myogenic tone
In order investigate the hypothesis that pressure-induced MT might involves caveolae we demonstrated that methyl β-cyclodextrin (mβcd), a caveolar structure disruptor, significantly decreased MT by 80% at 75 mmHg and by 94% at 150 mmHg (Fig. 1). In the reverse experiments (treatment of mesenteric arteries with cholesterol in addition of mβcd), MT was preserved (Fig. 1).

Confocal microscopy (Fig. 2E–G) showed an increased colocalization of RhoA with caveolin-1 at the level of the plasma membrane when pressure was 150 mmHg than under a pressure of 25 mmHg with a merging of 25±12 (arbitrary units) at 25 mmHg versus a merging of 93±17% at 150 mmHg (n=5, Fig. 2G).

Immunoprecipitation experiments demonstrated that pressure-induced MT was associated with a significant increase in the amount Cav-1–RhoA complexes (221 at 150 mmHg and 132% at 75 mmHg, as compared 25 mmHg, 100%, Fig. 3A). Total RhoA and Cav-1 protein expression was not significantly affected in the different conditions of pressure (Fig. 3B and C).


Figure 3
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  		Fig. 3 Panel A: Immunoblot showing association between RhoA and caveolin-1 (Cav-1) at 25, 75 and 150 mmHg. Mesenteric arteries lysates were immunoprecipated with anti-RhoA monoclonal antibody. Immunoprecipitates (IP) were immunoblotted (IB) for Cav-1 (n=5 rats per group, 15–20 pressurized arteries isolated per rat). Lysates of endothelial cells (EC) were used as a positive control for Cav-1 expression. Results, expressed as % of control (25 mmHg), reflect densitometric analysis. Mean±sem is presented. *P<0.05, 2-factor ANOVA, versus control (25 mmHg). Panel B and C: Western blot showing Cav-1 and RhoA expression in lysates of arteries submitted to 25, 75 and 150 mmHg (n= 4 rats per group and 15–20 segments of artery were pressurized per rat). Results, expressed as % of control (25 mmHg), reflect densitometric analysis. Mean±sem is presented. *P<0.05, 2-factor ANOVA, versus control (25 mmHg).

 
3.4. TAT-C3 and cholesterol depletion prevents RhoA–Cav-1 interaction
Both RhoA and Cav-1 protein expression were similar in arteries submitted or not to TAT-C3 exoenzyme treatment, which inactivates RhoA by ADP ribosylation. However, TAT-C3 incubation induced a marked and significant decrease in the physical association between these two proteins at 150 mmHg, as compared to control condition (Fig. 4).


Figure 4
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  		Fig. 4 Immunoblots showing association (co-immunoprecipitation) between RhoA and caveolin-1 (Cav-1) at 150 mmHg in presence or in absence (–) of mβcd or TAT-C3. Immunoprecipitates (IP) were immunoblotted (IB) for Cav-1 (n=5 rats per group, 15–20 pressurized arteries isolated per rat). Co-immunoprecipitation, representing Cav-1–RhoA interaction was quantified and is presented as a bargraph (right panel). *P<0.05, mβcd or TAT-C3 versus control (–). Total expression of Caveolin-1 (Total Cav-1), RhoA (Total RhoA) and actin was also measured in resistance arteries submitted to a pressure of 150 mmHg in presence or in absence of mβcd or TAT-C3 (n=3 per group, one representative blot is shown for each condition).

 
Although protein expression for both RhoA and Cav-1 was unchanged whatever the experimental conditions, mβcd treatment also significantly reduced pressure-induced increase in RhoA and Cav-1 co-immunoprecipitation (Fig. 4).

3.5. Myogenic tone in mice lacking caveolin-1
Second order mesenteric resistance arteries (106±8 µm internal diameter with a pressure of 75 mmHg) developed MT in response to pressure in +/+ mice whereas in –/– mice MT remained low (Fig. 5A). Indeed, MT in –/– mice represented 24% of MT in +/+ mice (calculated from the data shown in Fig. 5A at 150 mmHg for MT: active tone was 32.3±2.8 µm and 9.1±3.7 µm in +/+ and –/– mice, respectively).


Figure 5
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  		Fig. 5 Myogenic tone in mesenteric resistance arteries isolated from mice lacking the gene encoding for caveolin-1. Panel A: Myogenic tone was determined in third order mesenteric resistance arteries submitted to an intraluminal pressure of 25, 75 and 150 mmHg. Arteries-were isolated from caveolin-1 deficient mice (–/–) and their littermate control (+/+). The effect of methyl β-cyclodextrin (10 mM, panel B) and Y27632 (10 µM, panel C) on MT in third order mesenteric resistance arteries submitted to an intraluminal pressure of 25, 75 and 150 mmHg was determined in –/– and +/+ mice. Data is expressed as difference between passive and active diameter. Mean±sem is presented (n=5 per group). *P<0.05, –/– versus +/+ mice.

 
In mice mesenteric arteries treated with the mβcd MT at 75 and 150 mmHg was significantly reduced (Fig. 5B). The inhibitory effect of mβcd was significantly less important in –/– than in +/+ mice (Fig. 5B).

Similarly, the treatment of mice mesenteric arteries with the Rho-kinase inhibitor, Y27632 strongly reduced MT at 75 and 150 mmHg (Fig. 5C). The inhibitory effect of Y27632 was significantly less important in –/– than in +/+ mice (Fig. 5C).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Experimental procedures
 3. Results
 4. Discussion
Acknowledgments
 References
 
The present study demonstrates that pressure-induced myogenic tone involves the activation of RhoA through its translocation to the plasma membrane and its association to caveolin-1. Indeed, activation of RhoA through its association with caveolin-1 represents a new step in the understanding of MT.

We found that methyl β-cyclodextrin (mβcd) reduced significantly MT. This effect was due to cholesterol extraction from the plasma membrane, because cholesterol-saturated mβcd did not modify MT. This finding is consistent with previous works demonstrating that cholesterol depletion impaired contraction induced by agonists such as 5-hydroxytryptamine, vasopressin, and endothelin in the rat tail artery [31,32]. We also found that MT was strongly attenuated in caveolin-1 deficient mice. This is in agreement with a previous study showing, in cerebral arteries isolated from caveolin-1 deficient mice, that calcium sparks frequency (important for the development of MT) is decreased. [24]. Indeed, a large body of evidence suggests that cholesterol depletion affects signaling mechanisms located in caveolae including calcium sparks [31,33,34]. The possibility remains that the reduction in MT is linked, at least in part, to alter membrane fluidity and particularly liquid-ordered state normally present in either rafts or caveolae. Indeed, removal of cholesterol from the plasma membrane could increase wall stiffness and alter mechanical forces acting on resident proteins. However, it has been shown that cholesterol depletion only minimally affects myofilaments and force transmission [33].

We also found that both RhoA inactivation (TAT-C3) and Rho-kinase inhibition (Y27632) inhibited MT. This is consistent with previous studies showing that Y27632 attenuates MT due to reduction in calcium sensitivity of the contractile apparatus [8,13,14]. Rho-kinase inhibition strongly inhibits MT in the rabbit facial vein [13] and in the rat mesenteric artery [8], whereas Y27632 partly reduces MT in the rat-tail artery [14]. The use of TAT-C3 further supports the importance of this pathway in MT. The C3 enzyme selectively catalyzes the ADP-ribosylation, and consequent inactivation, of RhoA. Thus, this experiment provides an earlier event in the cascade between pressure and contraction (MT).

A main finding of the present study is that MT was associated with the translocation of RhoA from the cytosol to the plasma membrane and with its association to caveolin-1. Although several studies have demonstrated, in cultured endothelial and smooth muscle cells, the association of RhoA with caveolae-enriched membrane domains [22,23], the potential involvement of such an interaction in mediating Rho activation and a contraction, was not addressed. Our results demonstrated that 1) RhoA and caveolin-1 interact in response to pressure; 2) mβcd attenuated MT and prevented the interaction between RhoA and caveolin-1, 3) caveolin-1 deficiency strongly impaired MT and 4) TAT-C3, inhibited MT, the translocation of RhoA to the membrane and its association with caveolin-1. Thus, our results suggest that the translocation of RhoA within caveolae is necessary for the transduction of pressure into MT. This is consistent with previous studies showing that peptide blocking caveolin-1 scaffolding domain (CSD) inhibits agonist-induced RhoA translocation [23] and reduces the contractility of the ferret aorta [35].

Although caveolin-1 is generally described as a negative regulator of molecules functions [20,36–38], our study suggested that caveolin-1 may regulate in a positive manner RhoA activation. To confirm this hypothesis, we evaluated the link between RhoA activation and its association with caveolin-1 using the RhoA inhibitor TAT-C3. Treatment of mesenteric resistance arteries with TAT-C3 inhibited both MT and the formation of a complex between caveolin-1 and RhoA. Hence, the present study strongly suggests that translocation of RhoA to caveolae and that its association with caveolin-1 may facilitate the subsequent activation of Rho-kinase. This is supported by recent studies demonstrating that the sites of monoglycosylation and ADP ribosylation of RhoA, which inhibit calcium sensitization and/or RhoA translocation, are both contained within the putative caveolin-binding motif of RhoA [23,39].

As described in review articles [1,40], the rise in pressure initiating myogenic tone in resistance arteries is though to activate rapid cell architecture distension and the opening of stretch-activated cationic channels. This is followed by membrane depolarization and opening of voltage-gated Ca2+ channels [3]. In addition, other pathways are activated leading to sensitization of the contractile apparatus to calcium and cytoskeletal rearrangements [41]. This process most certainly involves integrins as recently shown [42]. Indeed, integrins are an important link between the extracellular and intracellular environments allowing transmission of inside-out and outside-in signals. Our study further supports the hypothesis that activation of integrins and focal-adhesion kinase in caveolin-1 rich domains may then participate in the Rho-kinase dependent sensitization of the contractile apparatus to calcium [7–9]. Nevertheless, the sequence is still lacking several components. We can speculate that after stimulation by pressure, integrins and focal-adhesion kinase activate tyrosine kinases. Indeed, several kinases have been shown to be involved in the development of myogenic tone [12]: PKC (6), PLC [43] and the p38-MAP kinase [14]. These kinases, in addition to the initial rise in intracellular calcium concentration [44], might play a role in the activation of RhoA as shown in contraction induced by agonists such angiotension II [45]. RhoA activation is associated with its translocation to the plasma membrane [45] and, as shown in the present study, linked to caveolin-1 in order to subsequently activate the Rho-kinase pathway. Finally, the integrin-dependent activation of kinases is certainly followed by the activation of Rho guanine exchange factors or Rho GEFs [46]. The Rho GEFs constitute a large family comprising ~70 members in humans, that activate Rho proteins by promoting the release of GDP and then facilitating the binding of GTP [47]. Although RhoA GEFs are involved in angiotensin II-dependent stimulation of RhoA in vascular smooth muscle cells (46), little is known regarding GEFs expression and activity in arteries. Thus, future experiments will have to determine the identity of GEF(s) involved in pressure-induced RhoA activation leading to myogenic tone in resistance arteries.

In summary, we demonstrated that MT required translocation of RhoA in caveolae and its activation mediated by the association of RhoA with caveolin-1. Our study provides important mechanical basis by which caveolin-1 can regulate the integration of extracellular contractile stimuli and the downstream intracellular effectors in smooth muscle. The mechanism described provides new target for investigating the role of MT in cardiovascular diseases. Indeed, MT is a key element in the control of resistance arteries tone, mainly through its interaction with other vasoactive systems; the sympathetic system being the most important one.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Experimental procedures
 3. Results
 4. Discussion
 Acknowledgments
References
 
Caroline Dubroca was supported by a grant from the French Foundation for Medical Research (Fondation pour la Recherche Médicale: FRM, Paris, France).

This work was supported in part by a grant from the French Association against Myopathies (AFM, Paris, France).


   Notes
 
Time for primary review 37 days


   References
Top
 Abstract
 1. Introduction
 2. Experimental procedures
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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