Vascular Biology Research Centre, Molecular and Clinical Research Institute, St. George's, University of London, Cranmer Terrace, London SW17 0RE, UK.
Leeds Institute of Cardiovascular and Metabolic Medicine, Faculty of Medicine and Health, University of Leeds, Leeds LS2 9JT, UK.
Vascul Pharmacol. 2020 Sep;132:106776. doi: 10.1016/j.vph.2020.106776. Epub 2020 Jul 21.
Phosphatidylinositol 4,5-bisphosphate (PIP) acts as substrate and unmodified ligand for Gq-protein-coupled receptor signalling in vascular smooth muscle cells (VSMCs) that is central for initiating contractility. The present work investigated how PIP might perform these two potentially conflicting roles by studying the effect of myristoylated alanine-rich C kinase substrate (MARCKS), a PIP-binding protein, on vascular contractility in rat and mouse mesenteric arteries. Using wire myography, MANS peptide (MANS), a MARCKS inhibitor, produced robust contractions with a pharmacological profile suggesting a predominantly role for L-type (CaV1.2) voltage-gated Ca channels (VGCC). Knockdown of MARCKS using morpholino oligonucleotides reduced contractions induced by MANS and stimulation of α-adrenoceptors and thromboxane receptors with methoxamine (MO) and U46619 respectively. Immunocytochemistry and proximity ligation assays demonstrated that MARCKS and CaV1.2 proteins co-localise at the plasma membrane in unstimulated tissue, and that MANS and MO reduced these interactions and induced translocation of MARCKS from the plasma membrane to the cytosol. Dot-blots revealed greater PIP binding to MARCKS than CaV1.2 in unstimulated tissue, with this binding profile reversed following stimulation by MANS and MO. MANS evoked an increase in peak amplitude and shifted the activation curve to more negative membrane potentials of whole-cell voltage-gated Ca currents, which were prevented by depleting PIP levels with wortmannin. This present study indicates for the first time that MARCKS is important regulating vascular contractility and suggests that disinhibition of MARCKS by MANS or vasoconstrictors may induce contraction through releasing PIP into the local environment where it increases voltage-gated Ca channel activity.
磷脂酰肌醇 4,5-二磷酸（PIP）作为底物和未修饰的配体，参与血管平滑肌细胞（VSMCs）中的 Gq 蛋白偶联受体信号转导，这对于启动收缩性至关重要。本研究通过研究肌酰化丙氨酸丰富的 C 激酶底物（MARCKS），一种 PIP 结合蛋白，对大鼠和小鼠肠系膜动脉血管收缩性的影响，来研究 PIP 如何发挥这两个潜在冲突的作用。使用线描记法，MARCKS 抑制剂 MANS 肽（MANS）产生了强大的收缩作用，其药理学特征表明其主要作用是 L 型（CaV1.2）电压门控钙通道（VGCC）。使用 MORFOLINO 寡核苷酸（MO）对 MARCKS 进行敲低，可减少 MANS 诱导的收缩以及刺激α-肾上腺素能受体和血栓素受体分别用甲氧胺（MO）和 U46619 引起的收缩。免疫细胞化学和接近性连接测定表明，MARCKS 和 CaV1.2 蛋白在未刺激的组织中在质膜上共定位，MANS 和 MO 减少了这些相互作用，并诱导 MARCKS 从质膜向细胞质易位。斑点印迹显示，在未刺激的组织中，MARCKS 与 PIP 的结合比 CaV1.2 更多，而这种结合模式在受到 MANS 和 MO 刺激后发生逆转。MANS 引起全细胞膜电压门控钙电流的峰值幅度增加，并将激活曲线移至更负的膜电位，这可以通过用渥曼青霉素耗尽 PIP 水平来预防。本研究首次表明 MARCKS 对血管收缩性很重要，并表明 MANS 或血管收缩剂对 MARCKS 的抑制作用可能通过将 PIP 释放到局部环境中，从而增加电压门控钙通道的活性来引起收缩。
