Submitted on December 16, 2006
Accepted on March 13, 2007
The Functional Consequence of RhoA knockdown by RNA Interference in Rat Cerebral Arteries
Randolph L Corteling1, Suzanne E Brett1, Hui Yin2, Xi-Long Zheng2, Michael P. Walsh3, and Donald G Welsh4*
1 Physiology & Biophysics, University of Calgary, Calgary, Canada
2 Medical Biochemistry, University of Calgary, Calgary, Canada
3 Department of Medical Biochemistry, University of Calgary, Calgary, Canada
4 Dept of Physiology & Biophysics, University of Calgary, Calgary, Canada
* To whom correspondence should be addressed. E-mail: dwelsh{at}ucalgary.ca.
Uridine triphosphate (UTP) constricts cerebral arteries by activating transduction pathways that increase cytosolic [Ca2+] and myofilament Ca2+ sensitivity. The signalling 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 interference RNA (siRNA) approach was developed to knockdown specified targets in rat cerebral arteries. siRNA directed against Gq and RhoA were 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 Gq mRNA expression by 60-70%, which correlated with a reduction in RhoA but not Gq 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 protein turnover rate is sufficiently high. They also demonstrate that RhoA's principal role in agonist-induced constriction is to facilitate the formation of F-actin, the physical structure to which phosphorylated myosin binds to elicit arterial constriction.
