Phosphorylation of Ser19 (S19-p) on the myosin regulatory light chain (MLC2) is critical for arterial contraction. It has been shown that elevated RhoA-dependent kinase (ROCK) activity or decreased MLC phosphatase (MLCP) activity leads to further phosphorylation of Thr18 (T18/S19-pp), which has been linked to vasospastic diseases. However, this phenomenon has not yet been studied in the context of pulmonary arterial hypertension (PAH). In the monocrotaline-induced PAH (PAH-MCT) rat model, we observed a significant delay in pulmonary artery (PA) relaxation following high potassium-induced contraction, which persisted even with the use of an L-type calcium channel blocker or in a calcium-free solution. Immunoblot analysis showed increased levels of both S19-p and T18/S19-pp in unstimulated PAs from PAH-MCT rats. Proteomics analysis revealed a reduction in soluble guanylate cyclase (sGC) and protein kinase G (PKG) levels, and immunoblotting confirmed decreased levels of MYPT1 (a component of MLCP) and increased ROCK in PAH-MCT. In the control PAs, the pharmacological inhibition of sGC with ODQ resulted in a prominent delay of relaxation and increased T18/S19-pp as in PAH-MCT. The delayed relaxation and the T18/S19-pp in PAH-MCT were reversed by ROCK inhibitor, Y27632, while not by membrane permeable 8-Br-cGMP. The delayed relaxation and T18/S19-diP in the ODQ-treated control PA were also reversed by Y27632. Taken together, the decreased sGC and MLCP, and increased ROCK increased T18/S19-pp, which leads to the decreased ability of PA to relax in PAH-MCT rats. PA specific inhibition of ROCK or activation of MLCP are expected to serve as potential drugs in the treatment of PAH.

肌球蛋白调节轻链 (MLC2) 上 Ser19 (S19-p) 的磷酸化对于动脉收缩至关重要。研究表明，RhoA 依赖性激酶 (ROCK) 活性升高或 MLC 磷酸酶 (MLCP) 活性降低会导致 Thr18 (T18/S19-pp) 进一步磷酸化，这与血管痉挛疾病有关。然而，这种现象尚未在肺动脉高压（PAH）的背景下进行研究。在野百合碱诱导的 PAH (PAH-MCT) 大鼠模型中，我们观察到高钾诱导的收缩后肺动脉 (PA) 舒张显着延迟，即使使用 L 型钙通道阻滞剂或使用无钙溶液。免疫印迹分析显示，PAH-MCT 大鼠未刺激的 PA 中 S19-p 和 T18/S19-pp 水平均升高。蛋白质组学分析显示，PAH-MCT 中可溶性鸟苷酸环化酶 (sGC) 和蛋白激酶 G (PKG) 水平降低，免疫印迹证实 MYPT1（MLCP 的一个组成部分）水平降低，ROCK 水平升高。在对照 PA 中，与 PAH-MCT 一样，ODQ 对 sGC 的药理学抑制导致显着的松弛延迟和 T18/S19-pp 增加。PAH-MCT 中的延迟舒张和 T18/S19-pp 可被 ROCK 抑制剂 Y27632 逆转，但不能被膜渗透性 8-Br-cGMP 逆转。ODQ 处理的对照 PA 中的延迟松弛和 T18/S19-diP 也被 Y27632 逆转。综上所述，sGC和MLCP减少，ROCK增加，T18/S19-pp增加，导致PAH-MCT大鼠PA舒张能力下降。PA特异性抑制ROCK或激活MLCP有望成为治疗PAH的潜在药物。