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The functional consequence of RhoA knockdown by RNA interference in rat cerebral arteries

Departments of ¹Physiology and Biophysics and ²Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada

Submitted 16 December 2006 ; accepted in final form 13 March 2007

Uridine triphosphate (UTP) constricts cerebral arteries by activating transduction pathways that increase cytosolic [Ca²⁺] and myofilament Ca²⁺ sensitivity. The signaling proteins that comprise these pathways remain uncertain with recent studies implicating a role for several G proteins. To start clarifying which G proteins enable UTP-induced vasoconstriction, a small interfering RNA (siRNA) approach was developed to knock down specified targets in rat cerebral arteries. siRNA directed against G_q and RhoA was introduced into isolated cerebral arteries using reverse permeabilization. Following a defined period of organ culture, arteries were assayed for contractile function, mRNA levels, and protein expression. Targeted siRNA reduced RhoA or G_q mRNA expression by 60–70%, which correlated with a reduction in RhoA but not G_q protein expression. UTP-induced constriction was abolished in RhoA-depleted arteries, but this was not due to a reduction in myosin light chain phosphorylation. UTP-induced actin polymerization was attenuated in RhoA-depleted arteries, which would explain the loss of agonist-induced constriction. In summary, this study illustrates that siRNA approaches can be effectively used on intact arteries to induce targeted knockdown given that the protein turnover rate is sufficiently high. It also demonstrates that the principal role of RhoA in agonist-induced constriction is to facilitate the formation of F-actin, the physical structure to which phosphorylated myosin binds to elicit arterial constriction.

arterial tone; G proteins; F/G-actin; myosin light chain phosphorylation; small interfering ribonucleic acid

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