siRNA knock down of casein kinase 2 increases force and cross-bridge cycling rates in vascular smooth muscle.
Contraction of smooth muscle involves myosin light chain (MLC) kinase catalyzed phosphorylation of the regulatory MLC, activation of myosin, and the development of force. However, this cannot account for all aspects of a smooth muscle contraction, suggesting that other regulatory mechanisms exist. One potentially important technique to study alternative sites of contractile regulation is the use of small interfering RNA (siRNA). The goal of this study was to determine whether siRNA technology can decrease the levels of a specific protein and allow for the determination of how that protein affects contractile regulation. To achieve this goal, we tested the hypothesis that casein kinase 2 (CK2) is part of the complex regulatory scheme present in vascular smooth muscle. Using intact strips of swine carotid artery, we determined that siRNA against CK2 produced a tissue that resulted in a approximately 60% knockdown after 4 days in organ culture. Intact strips of vascular tissue depleted of CK2 produced greater levels of force and exhibited an increased sensitivity to all stimuli tested. This was accompanied by an increase in cross-bridge cycling rates but not by a change in MLC phosphorylation levels. alpha-Toxin-permeabilized vascular tissue depleted of CK2 also showed an increased sensitivity to calcium compared with control tissues. Our results demonstrate that siRNA is a viable technique with which to study regulatory pathways in intact smooth muscle tissue. Our results also demonstrate that CK2 plays an important role in the mechanism(s) responsible for the development of force and cross-bridge cycling by a MLC phosphorylation-independent pathway.
Smolock
Elaine M
EM
Dept. of Pharmacology and Physiology, 245 N. 15th St., MS 488, Philadelphia, PA 19102, USA.
Wang
Tanchun
T
Nolt
Jocelyn K
JK
Moreland
Robert S
RS
D000185:Actinin / Q000378:metabolism; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000502:physiology; D002339:Carotid Arteries / Q000187:drug effects / Q000502:physiology; D047390:Casein Kinase II / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D015536:Down-Regulation; D066298:In Vitro Techniques; D009119:Muscle Contraction; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics*; D013552:Swine; D010738:Type C Phospholipases / Q000494:pharmacology
D002148:Calmodulin-Binding Proteins; D018994:Myosin Light Chains; D034741:RNA, Small Interfering; D000185:Actinin; D047390:Casein Kinase II; D010738:Type C Phospholipases