The functional consequence of RhoA knockdown by RNA interference in rat cerebral arteries.
Uridine triphosphate (UTP) constricts cerebral arteries by activating transduction pathways that increase cytosolic [Ca(2+)] and myofilament Ca(2+) 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.
Corteling
Randolph L
RL
Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada.
Brett
Suzanne E
SE
Yin
Hao
H
Zheng
Xi-Long
XL
Walsh
Michael P
MP
Welsh
Donald G
DG
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002536:Cerebral Arteries / Q000502:physiology*; D005260:Female; D020868:Gene Silencing; D066298:In Vitro Techniques; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D014661:Vasoconstriction / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D020742:rhoA GTP-Binding Protein