RhoA activation and interaction with Caveolin-1 are critical for pressure-induced myogenic tone in rat mesenteric resistance arteries.
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 microm 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-beta-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 microm and 9.1+/-3.7 microm 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 beta-cyclodextrin.


CONCLUSIONS
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.
Dubroca
Caroline
C
Centre de Recherche Cardiovasculaire Lariboisière, INSERM U689, Paris, France.
Loyer
Xavier
X
Retailleau
Kevin
K
Loirand
Gervaise
G
Pacaud
Pierre
P
Feron
Olivier
O
Balligand
Jean-Luc
JL
Lévy
Bernard I
BI
Heymes
Christophe
C
Henrion
Daniel
D
D000199:Actins / Q000032:analysis; D000577:Amides / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active; D015153:Blotting, Western / Q000379:methods; D051242:Caveolin 1 / Q000032:analysis / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007150:Immunohistochemistry / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D066298:In Vitro Techniques; D008638:Mesenteric Arteries / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D014655:Vascular Resistance; D014661:Vasoconstriction / Q000187:drug effects*; D047392:beta-Cyclodextrins / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D000577:Amides; D051242:Caveolin 1; D004791:Enzyme Inhibitors; D011725:Pyridines; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C108830:Y 27632; D002784:Cholesterol; D020742:rhoA GTP-Binding Protein