Institute of Vegetative Physiology, University of Cologne, Robert-Koch-Str. 39, 50931 Cologne, Germany.
J Muscle Res Cell Motil. 2012 Dec;33(6):471-83. doi: 10.1007/s10974-012-9306-9. Epub 2012 Jun 19.
Nitrovasodilators and agonists, via an increase in intracellular cyclic nucleotide levels, can induce smooth muscle relaxation without a concomitant decrease in phosphorylation of the regulatory light chains (RLC) of myosin. However, since cyclic nucleotide-induced relaxation is associated with a decrease in intracellular [Ca(2+)], and hence, a decreased activity of MLCK, we tested the hypothesis that the site responsible for the elevated RLC phosphorylation is not Ser19. Smooth muscle strips from gastric fundus were isometrically contracted with ET-1 which induced an increase in monophosphorylation from 9 ± 1 % under resting conditions (PSS) to 36 ± 1 % determined with 2D-PAGE. Electric field stimulation induced a rapid, largely NO-mediated relaxation with a half time of 8 s, which was associated with an initial decline in RLC phosphorylation to 18 % within 2 s and a rebound to 34 % after 30 s whereas relaxation was sustained. In contrast, phosphorylation of RLC at Ser19 probed with phosphospecific antibodies declined in parallel with force. LC/MS and western blot analysis with phosphospecific antibodies against monophosphorylated Thr18 indicate that Thr18 is significantly monophosphorylated during sustained relaxation. We therefore suggest that (i) monophosphorylation of Thr18 rather than Ser19 is responsible for the phosphorylation rebound during sustained EFS-induced relaxation of mouse gastric fundus, and (ii) that relaxation can be ascribed to dephosphorylation of Ser19, the site considered to be responsible for regulation of smooth muscle tone.
硝基血管扩张剂和激动剂通过增加细胞内环核苷酸水平，可以诱导平滑肌松弛，而不会伴随肌球蛋白调节轻链（RLC）的磷酸化减少。然而，由于环核苷酸诱导的松弛与细胞内[Ca（2+）]的减少有关，因此，肌球蛋白轻链激酶（MLCK）的活性降低，我们检验了假设，即负责 RLC 磷酸化升高的部位不是 Ser19。使用 ET-1 等张收缩胃底平滑肌条，在静息条件（PSS）下诱导单磷酸化从 9±1%增加到 36±1%，用 2D-PAGE 确定。电场刺激诱导快速、主要由 NO 介导的松弛，半衰期为 8 s，与 RLC 磷酸化的初始下降相关，在 2 s 内下降到 18%，在 30 s 后反弹到 34%，而松弛持续。相比之下，用磷酸化特异性抗体探测到的 RLC 在 Ser19 上的磷酸化与力平行下降。LC/MS 和用磷酸化特异性抗体对单磷酸化 Thr18 的 Western blot 分析表明，在持续松弛期间 Thr18 明显单磷酸化。因此，我们提出（i）在持续 EFS 诱导的小鼠胃底松弛期间，Thr18 的单磷酸化而不是 Ser19 的单磷酸化负责磷酸化反弹，以及（ii）松弛可以归因于 Ser19 的去磷酸化，该位点被认为是调节平滑肌张力的关键。
