Hocking Kyle M, Putumbaka Gowthami, Wise Eric S, Cheung-Flynn Joyce, Brophy Colleen M, Komalavilas Padmini
Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.
VA Tennessee Valley Healthcare System, Nashville, Tennessee, United States of America.
PLoS One. 2016 May 2;11(5):e0154460. doi: 10.1371/journal.pone.0154460. eCollection 2016.
Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation.
HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin.
These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm.
罂粟碱用于在将人隐静脉（HSV）作为旁路管道植入前的静脉移植物制备过程中预防血管痉挛。罂粟碱是磷酸二酯酶的非特异性抑制剂，可导致细胞内cGMP和cAMP均增加。我们假设罂粟碱通过降低细胞内钙浓度（[Ca2+]i）和肌球蛋白轻链磷酸化，并通过调节肌动蛋白调节蛋白磷酸化增加肌动蛋白解聚来降低张力。
将HSV在肌肉浴中平衡，先用1 mM罂粟碱预处理，然后用5 μM去甲肾上腺素处理，并同时测量张力以及[Ca2+]i水平。通过体外肌动蛋白测定法测量丝状肌动蛋白（F-肌动蛋白）水平。将组织速冻以测量肌球蛋白轻链和肌动蛋白调节蛋白磷酸化。罂粟碱预处理完全抑制了去甲肾上腺素诱导的张力产生，阻止了[Ca2+]i的增加，并导致肌球蛋白轻链磷酸化减少。罂粟碱预处理还导致热休克相关蛋白20（HSPB6）和血管舒张刺激磷蛋白（VASP）的磷酸化增加，以及丝状肌动蛋白（F-肌动蛋白）水平降低，提示肌动蛋白解聚。
这些结果表明，罂粟碱诱导的HSV张力抑制涉及[Ca2+]i介导的肌球蛋白轻链磷酸化抑制和肌动蛋白调节蛋白磷酸化介导肌动蛋白解聚。因此，罂粟碱通过调节肌球蛋白横桥形成和肌动蛋白细胞骨架动力学诱导HSV收缩的持续抑制，是预防血管痉挛的高压扩张的药理学替代方法。
