González-Montelongo MarÍa Del Carmen, Porras-González Cristina, González-Montelongo Rafaela, Revilla-González Gonzalo, Pastor MarÍa Dolores, Castellano Antonio, Ureña Juan
Instituto de Biomedicina de Sevilla (IBiS)/Hospital Universitario Virgen del Rocío/CSIC/ Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, Sevilla, Spain.
Red de Investigación Cardiovascula (RIC)/CIBERCV, Sevilla, Spain.
Cell Physiol Biochem. 2019;52(1):76-93. doi: 10.33594/000000006. Epub 2019 Feb 18.
BACKGROUND/AIMS: Protein kinase C (PKC)- and RhoA/Rho-associated kinase (ROCK) play important roles in arterial sustained contraction. Although depolarization-elicited RhoA/ROCK activation is accepted, the role of PKC in depolarized vascular smooth muscle cells (VSMCs) is a subject of controversy. Our aim was to study the role of PKC in arterial contraction and its interaction with RhoA/ROCK.
Mass spectrometry was used to identify the PKC isoenzymes. PKCα levels and RhoA activity were analyzed by western blot and G-LISA, respectively, and isometric force was measured in arterial rings.
In depolarized VSMCs RhoA and PKCα were translocated to the plasma membrane, where they colocalize and coimmunoprecipitate. Interestingly, depolarization-induced RhoA activation was downregulated by PKCα, effect reverted by PKCα inhibition. Phorbol 12,13-dibutyrate (PDBu) induced the translocation of PKCα to the plasma membrane, increased the level of RhoA in the cytosol and reduced RhoA/ROCK activity. These effects were reverted when PKC was inhibited. Pharmacological or siRNA inhibition of PKCα synergistically potentiated the vasorelaxant effect of RhoA/ROCK inhibition.
The present study provides the first evidence that RhoA activity is downregulated by PKCα in depolarized and PDBu treated freshly isolated VSMCs and arteries, with an important physiological role on arterial contractility.
背景/目的：蛋白激酶C（PKC）和RhoA/Rho相关激酶（ROCK）在动脉持续收缩中起重要作用。尽管去极化引发的RhoA/ROCK激活已被认可，但PKC在去极化的血管平滑肌细胞（VSMC）中的作用仍存在争议。我们的目的是研究PKC在动脉收缩中的作用及其与RhoA/ROCK的相互作用。
采用质谱法鉴定PKC同工酶。分别通过蛋白质印迹法和G-LISA分析PKCα水平和RhoA活性，并在动脉环中测量等长力。
在去极化的VSMC中，RhoA和PKCα转位至质膜，在那里它们共定位并共免疫沉淀。有趣的是，PKCα下调了去极化诱导的RhoA激活，PKCα抑制可逆转该效应。佛波醇12,13-二丁酸酯（PDBu）诱导PKCα转位至质膜，增加胞质溶胶中RhoA的水平并降低RhoA/ROCK活性。当PKC被抑制时，这些效应被逆转。PKCα的药理学或siRNA抑制协同增强了RhoA/ROCK抑制对血管舒张的作用。
本研究首次提供证据表明，在去极化和PDBu处理的新鲜分离的VSMC和动脉中，PKCα下调RhoA活性，这对动脉收缩性具有重要的生理作用。
