Ets Hillevi K, Seow Chun Y, Moreland Robert S
Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia PA, USA.
Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver BC, Canada.
Front Pharmacol. 2016 Dec 26;7:516. doi: 10.3389/fphar.2016.00516. eCollection 2016.
Vascular smooth muscle (VSM) is unique in its ability to maintain an intrinsic level of contractile force, known as tone. Vascular tone is believed to arise from the constitutive activity of membrane-bound L-type Ca channels (LTCC). This study used a pharmacological agonist of LTCC, Bay K8644, to elicit a sustained, sub-maximal contraction in VSM that mimics tone. Downstream signaling was investigated in order to determine what molecules are responsible for tone. Medial strips of swine carotid artery were stimulated with 100 nM Bay K8644 to induce a sustained level of force. Force and phosphorylation levels of myosin light chain (MLC), MAP kinase, MYPT1, CPI-17, and caldesmon were measured during Bay K8644 stimulation in the presence and absence of nifedipine, ML-7, U0126, bisindolylmaleimide (Bis), and H-1152. Nifedipine and ML-7 inhibited force and MLC phosphorylation in response to Bay K8644. Inhibition of Rho kinase (H-1152) but not PKC (Bis) inhibited Bay K8644 induced force. U0126 significantly increased Bay K8644-dependent force with no effect on MLC phosphorylation. Neither CPI-17 nor caldesmon phosphorylation were increased during the maintenance of sustained force. Our results suggest that force due to the influx of calcium through LTCCs is partially MLC phosphorylation-dependent but does not involve PKC or caldesmon. Interestingly, inhibition of MLC kinase (MLCK) and PKC significantly increased MAP kinase phosphorylation suggesting that MLCK and PKC may directly or indirectly inhibit MAP kinase activity during prolonged contractions induced by Bay K8544.
血管平滑肌（VSM）在维持内在收缩力水平（即张力）方面具有独特能力。血管张力被认为源于膜结合L型钙通道（LTCC）的组成性活性。本研究使用LTCC的药理学激动剂Bay K8644在VSM中引发持续的、亚最大收缩，模拟张力。研究下游信号传导以确定哪些分子负责张力。用100 nM Bay K8644刺激猪颈动脉中膜条带以诱导持续的力水平。在存在和不存在硝苯地平、ML-7、U0126、双吲哚马来酰亚胺（Bis）和H-1152的情况下，在Bay K8644刺激期间测量肌球蛋白轻链（MLC）、丝裂原活化蛋白激酶（MAP激酶）、MYPT1、CPI-17和钙调蛋白的力和磷酸化水平。硝苯地平和ML-7抑制对Bay K8644的反应中的力和MLC磷酸化。抑制 Rho激酶（H-1152）而非蛋白激酶C（Bis）抑制Bay K8644诱导的力。U0126显著增加Bay K8644依赖性力，对MLC磷酸化无影响。在持续力维持期间，CPI-17和钙调蛋白磷酸化均未增加。我们的结果表明，通过LTCCs的钙内流引起的力部分依赖于MLC磷酸化，但不涉及蛋白激酶C或钙调蛋白。有趣的是，抑制MLC激酶（MLCK）和蛋白激酶C显著增加MAP激酶磷酸化，表明在Bay K8544诱导的长时间收缩期间，MLCK和蛋白激酶C可能直接或间接抑制MAP激酶活性。
