Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom (A.B., R.A.J.C., J.M.W.); Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing, Glenfield General Hospital, Leicester, United Kingdom (R.D.R., S.B., R.J., A.J.B.); Life Sciences Institute and Departments of Pharmacology, Biological Sciences, and Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan (H.V.W., J.J.G.T.); and Department of Biological Sciences, Purdue University, West Lafayette, Indiana (J.J.G.T.).
Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom (A.B., R.A.J.C., J.M.W.); Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing, Glenfield General Hospital, Leicester, United Kingdom (R.D.R., S.B., R.J., A.J.B.); Life Sciences Institute and Departments of Pharmacology, Biological Sciences, and Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan (H.V.W., J.J.G.T.); and Department of Biological Sciences, Purdue University, West Lafayette, Indiana (J.J.G.T.)
Mol Pharmacol. 2018 Sep;94(3):1079-1091. doi: 10.1124/mol.118.112524. Epub 2018 Jul 6.
Vasoconstrictor-driven G protein-coupled receptor (GPCR)/phospholipase C (PLC) signaling increases intracellular Ca concentration to mediate arterial contraction. To counteract vasoconstrictor-induced contraction, GPCR/PLC signaling can be desensitized by G protein-coupled receptor kinases (GRKs), with GRK2 playing a predominant role in isolated arterial smooth muscle cells. In this study, we use an array of GRK2 inhibitors to assess their effects on the desensitization of UTP and angiotensin II (AngII)-mediated arterial contractions. The effects of GRK2 inhibitors on the desensitization of UTP- or AngII-stimulated mesenteric third-order arterial contractions, and PLC activity in isolated mesenteric smooth muscle cells (MSMC), were determined using wire myography and Ca imaging, respectively. Applying a stimulation protocol to cause receptor desensitization resulted in reductions in UTP- and AngII-stimulated arterial contractions. Preincubation with the GRK2 inhibitor paroxetine almost completely prevented desensitization of UTP- and attenuated desensitization of AngII-stimulated arterial contractions. In contrast, fluoxetine was ineffective. Preincubation with alternative GRK2 inhibitors (Takeda compound 101 or CCG224063) also attenuated the desensitization of UTP-mediated arterial contractile responses. In isolated MSMC, paroxetine, Takeda compound 101, and CCG224063 also attenuated the desensitization of UTP- and AngII-stimulated increases in Ca, whereas fluoxetine did not. In human uterine smooth muscle cells, paroxetine reversed GRK2-mediated histamine H receptor desensitization, but not GRK6-mediated oxytocin receptor desensitization. Utilizing various small-molecule GRK2 inhibitors, we confirm that GRK2 plays a central role in regulating vasoconstrictor-mediated arterial tone, highlighting a potentially novel strategy for blood pressure regulation through targeting GRK2 function.
血管收缩剂驱动的 G 蛋白偶联受体 (GPCR)/磷酯酶 C (PLC) 信号转导增加细胞内 Ca2+浓度，介导动脉收缩。为了对抗血管收缩剂引起的收缩，GPCR/PLC 信号可以被 G 蛋白偶联受体激酶 (GRK) 脱敏，GRK2 在分离的动脉平滑肌细胞中起着主要作用。在这项研究中，我们使用一系列 GRK2 抑制剂来评估它们对 UTP 和血管紧张素 II (AngII) 介导的动脉收缩脱敏的影响。通过wire myography 和 Ca 成像分别测定 GRK2 抑制剂对 UTP 或 AngII 刺激的肠系膜三级动脉收缩的脱敏作用和 PLC 活性。应用刺激方案导致受体脱敏，导致 UTP 和 AngII 刺激的动脉收缩减少。预先孵育 GRK2 抑制剂帕罗西汀几乎完全阻止了 UTP 和减弱 AngII 刺激的动脉收缩的脱敏作用。相比之下，氟西汀无效。替代的 GRK2 抑制剂（武田化合物 101 或 CCG224063）的预孵育也减弱了 UTP 介导的动脉收缩反应的脱敏作用。在分离的肠系膜平滑肌细胞中，帕罗西汀、武田化合物 101 和 CCG224063 也减弱了 UTP 和 AngII 刺激的 Ca2+增加的脱敏作用，而氟西汀则没有。在人子宫平滑肌细胞中，帕罗西汀逆转了 GRK2 介导的组胺 H 受体脱敏，但没有逆转 GRK6 介导的催产素受体脱敏。利用各种小分子 GRK2 抑制剂，我们证实 GRK2 在调节血管收缩剂介导的动脉张力中起着核心作用，这突出了通过靶向 GRK2 功能调节血压的潜在新策略。
