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

机译:RNA干扰在大鼠脑动脉中抑制RhoA的功能后果。

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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.
机译:尿苷三磷酸(UTP)通过激活增加细胞质[Ca(2+)]和肌丝Ca(2+)敏感性的转导途径来收缩脑动脉。包含这些途径的信号传导蛋白仍不确定,最近的研究暗示了几种G蛋白的作用。为了开始弄清哪些G蛋白可以使UTP诱导的血管收缩,开发了一种小的干扰RNA(siRNA)方法来敲除大鼠脑动脉中的特定靶标。使用反向通透性将针对G(q)和RhoA的siRNA引入分离的脑动脉。在限定的器官培养期后,测定动脉的收缩功能,mRNA水平和蛋白质表达。靶向siRNA使RhoA或G(q)mRNA表达降低60-70%,这与RhoA降低但与G(q)蛋白表达降低无关。 UTP引起的收缩在RhoA耗尽的动脉中被消除,但这不是由于肌球蛋白轻链磷酸化的减少。 UTP诱导的肌动蛋白聚合在RhoA耗尽的动脉中减弱,这可以解释激动剂诱导的收缩的丧失。总而言之,这项研究表明,鉴于蛋白质的周转率足够高,siRNA方法可以有效地用于完整的动脉以诱导靶向敲低。它还表明RhoA在激动剂诱导的收缩中的主要作用是促进F-肌动蛋白的形成,F-肌动蛋白是磷酸化肌球蛋白与之结合的物理结构,可引起动脉收缩。

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