Huang Yi, Smith Corey A, Chen Grace, Sharma Bharti, Miner Amy S, Barbee Robert W, Ratz Paul H
Department of Emergency Medicine and Physiology, Virginia Commonwealth University, Richmond, VA, United States.
Department of Biochemistry and Molecular Biology, School of Medicine, Virginia Commonwealth University, Richmond, VA, United States.
Front Pharmacol. 2017 Oct 18;8:756. doi: 10.3389/fphar.2017.00756. eCollection 2017.
Although recent studies reveal that activation of the metabolic and Ca sensor AMPK strongly inhibits smooth muscle contraction, there is a paucity of information about the potential linkage between pharmacological AMPK activation and vascular smooth muscle (VSM) contraction regulation. Our aim was to test the general hypothesis that the allosteric AMPK activator A-769662 causes VSM relaxation via inhibition of contractile protein activation, and to specifically determine which activation mechanism(s) is(are) affected. The ability of A-769662 to cause endothelium-independent relaxation of contractions induced by several contractile stimuli was examined in large and small musculocutaneous and visceral rabbit arteries. For comparison, the structurally dissimilar AMPK activators MET, SIM, and BBR were assessed. A-769662 displayed artery- and agonist-dependent differential inhibitory activities that depended on artery size and location. A-769662 did not increase AMPK-pT172 levels, but did increase phosphorylation of the downstream AMPK substrate, acetyl-CoA carboxylase (ACC). A-769662 did not inhibit basal phosphorylation levels of several contractile protein regulatory proteins, and did not alter the activation state of rhoA. A-769662 did not inhibit Ca- and GTPγS-induced contractions in β-escin-permeabilized muscle, suggesting that A-769662 must act by inhibiting Ca signaling. In intact artery, A-769662 immediately reduced basal intracellular free calcium ([Ca]), inhibited a stimulus-induced increase in [Ca], and inhibited a cyclopiazonic acid (CPA)-induced contraction. MET increased AMPK-pT172, and caused neither inhibition of contraction nor inhibition of [Ca]. Together, these data support the hypothesis that the differential inhibition of stimulus-induced arterial contractions by A-769662 was due to selective inhibition of a Ca mobilization pathway, possibly involving CPA-dependent Ca entry via an AMPK-independent pathway. That MET activated AMPK without causing arterial relaxation suggests that AMPK activation does not necessarily cause VSM relaxation.
尽管最近的研究表明，代谢和钙传感器AMPK的激活会强烈抑制平滑肌收缩，但关于药理学上的AMPK激活与血管平滑肌（VSM）收缩调节之间的潜在联系，目前的信息还很匮乏。我们的目的是检验一个普遍的假设，即变构AMPK激活剂A-769662通过抑制收缩蛋白激活导致VSM舒张，并具体确定哪些激活机制受到影响。在大、小肌皮动脉和内脏兔动脉中，研究了A-769662对几种收缩刺激诱导的收缩产生内皮依赖性舒张的能力。为了进行比较，还评估了结构不同的AMPK激活剂MET、SIM和BBR。A-769662表现出依赖于动脉和激动剂的差异抑制活性，这取决于动脉的大小和位置。A-769662没有增加AMPK-pT172水平，但确实增加了下游AMPK底物乙酰辅酶A羧化酶（ACC）的磷酸化。A-769662没有抑制几种收缩蛋白调节蛋白的基础磷酸化水平，也没有改变rhoA的激活状态。A-769662没有抑制β-七叶皂苷渗透肌肉中钙和GTPγS诱导的收缩，这表明A-769662必须通过抑制钙信号来发挥作用。在完整动脉中，A-769662立即降低基础细胞内游离钙（[Ca]），抑制刺激诱导的[Ca]增加，并抑制环匹阿尼酸（CPA）诱导的收缩。MET增加了AMPK-pT172，但既没有抑制收缩也没有抑制[Ca]。总之，这些数据支持了这样一个假设，即A-769662对刺激诱导的动脉收缩的差异抑制是由于对钙动员途径的选择性抑制，可能涉及通过非AMPK依赖途径的CPA依赖性钙内流。MET激活AMPK但未引起动脉舒张，这表明AMPK激活不一定会导致VSM舒张。
