Negative regulation of RhoA/Rho kinase by angiotensin II type 2 receptor in vascular smooth muscle cells: role in angiotensin II-induced vasodilation in stroke-prone spontaneously hypertensive rats.
OBJECTIVE
To test whether angiotensin II (Ang II) through the Ang II type 2 receptor (AT2R), downregulates RhoA/Rho kinase, which plays a role in AT1 receptor (AT1R)-mediated function.


METHODS
In vitro studies were performed in A10 vascular smooth muscle cells (VSMC) and in vivo studies in mesenteric arteries from Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive (SHRSP) rats. VSMC were stimulated with Ang II (10 mol/l), CGP42112A (10 mol/l, a selective AT2R agonist) +/- valsartan (10 mol/l, an AT1R antagonist), or the Rho kinase inhibitor fasudil (10 mol/l). AT1R and AT2R expression and myosin light chain (MLC) phosphorylation were determined by immunoblotting. RhoA activity was assessed by measuring membrane translocation. Functional significance between AT2R, RhoA/Rho kinase and vasodilation was assessed in arteries from valsartan-treated (30 mg/kg per day, 14 days) WKY and SHRSP rats. Vasodilatory responses to Ang II (10-10 mol/l) were performed in norepinephrine pre-contracted vessels +/- valsartan(10 mol/l), PD123319 (10 mol/l, an AT2R antagonist) or fasudil (10 mol/l).


RESULTS
A10 VSMC expressed AT1R and AT2R. In valsartan-treated cells, Ang II-induced RhoA translocation was reduced versus controls (42 +/- 6%, P < 0.05). Similar responses were obtained with CGP42112A (45 +/- 6%, P < 0.05). This was associated with decreased MLC activation. Fasudil abrogated Ang II- and CGP42112A-mediated effects. Ang II evoked a significant vasodilatory response only in valsartan-treated SHRSP (max dilation 40 +/- 7%). PD123319 blocked these effects. Fasudil increased AngII-induced relaxation in SHRSP vessels. AT2R expression was increased by valsartan (two- to three-fold) in SHRSP arteries. RhoA translocation was increased two-fold in untreated SHRSP (P < 0.05) and was reduced by valsartan (P < 0.05). These changes were associated with decreased MLC phosphorylation.


CONCLUSIONS
Ang II/AT2R negatively regulates vascular RhoA/Rho kinase/MLC phosphorylation. These processes may play a role in Ang II-mediated vasodilation in conditions associated with vascular AT2R upregulation, such as in SHRSP chronically treated with AT1R blockers, which may contribute to blood pressure lowering by these antihypertensive agents.
Savoia
Carmine
C
CIHR Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Quebec, Canada.
Tabet
Fatiha
F
Yao
Guoying
G
Schiffrin
Ernesto L
EL
Touyz
Rhian M
RM
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D006973:Hypertension / Q000201:enzymology* / Q000503:physiopathology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000201:enzymology*; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY; D044139:Receptor, Angiotensin, Type 2 / Q000502:physiology*; D020521:Stroke / Q000209:etiology*; D014664:Vasodilation / Q000187:drug effects*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D018994:Myosin Light Chains; D044139:Receptor, Angiotensin, Type 2; D000804:Angiotensin II; D020742:rhoA GTP-Binding Protein