Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Tufts Medical Center, Boston, Massachusetts, United States of America.
PLoS One. 2018 Apr 12;13(4):e0195780. doi: 10.1371/journal.pone.0195780. eCollection 2018.
Contraction of human pulmonary artery smooth muscle cells (HPASMC) isolated from pulmonary arterial hypertensive (PAH) and normal (non-PAH) subject lungs was determined and measured with real-time electrical impedance. Treatment of HPASMC with vasoactive peptides, endothelin-1 (ET-1) and bradykinin (BK) but not angiotensin II, induced a temporal decrease in the electrical impedance profile mirroring constrictive morphological change of the cells which typically was more robust in PAH as opposed to non-PAH cells. Inhibition with LIMKi3 and a cofilin targeted motif mimicking cell permeable peptide (MMCPP) had no effect on ET-1 induced HPASMC contraction indicating a negligible role for these actin regulatory proteins. On the other hand, a MMCPP blocking the activity of caldesmon reduced ET-1 promoted contraction pointing to a regulatory role of this protein and its activation pathway in HPASMC contraction. Inhibition of this MEK/ERK/p90RSK pathway, which is an upstream regulator of caldesmon phosphorylation, reduced ET-1 induced cell contraction. While the regulation of ET-1 induced cell contraction was found to be similar in PAH and non-PAH cells, a key difference was the response to pharmacological inhibitors and to siRNA knockdown of Rho kinases (ROCK1/ROCK2). The PAH cells required much higher concentrations of inhibitors to abrogate ET-1 induced contractions and their contraction was not affected by siRNA against either ROCK1 or ROCK2. Lastly, blocking of L-type and T-type Ca2+ channels had no effect on ET-1 or BK induced contraction. However, inhibiting the activity of the sarcoplasmic reticulum Ca2+ ATPase blunted ET-1 and BK induced HPASMC contraction in both PAH and non-PAH derived HPASMC. In summary, our findings here together with previous communications illustrate similarities and differences in the regulation PAH and non-PAH smooth muscle cell contraction relating to calcium translocation, RhoA/ROCK signaling and the activity of caldesmon. These findings may provide useful tools in achieving the regulation of the vascular hypercontractility taking place in PAH.
从肺动脉高压（PAH）和正常（非 PAH）患者肺部分离的人肺动脉平滑肌细胞（HPASMC）的收缩通过实时电阻抗来确定和测量。血管活性肽内皮素-1（ET-1）和缓激肽（BK）而非血管紧张素 II 处理 HPASMC 会导致电阻抗谱的暂时下降，反映出细胞的收缩性形态变化，这种变化在 PAH 细胞中比在非 PAH 细胞中更为明显。LIMKi3 抑制和模拟细胞渗透性肽（MMCPP）的原肌球蛋白靶向基序对 ET-1 诱导的 HPASMC 收缩没有影响，表明这些肌动蛋白调节蛋白的作用可以忽略不计。另一方面，一种阻断钙调蛋白活性的 MMCPP 减少了 ET-1 促进的收缩，表明该蛋白及其激活途径在 HPASMC 收缩中起调节作用。该 MEK/ERK/p90RSK 途径是钙调蛋白磷酸化的上游调节剂，其抑制减少了 ET-1 诱导的细胞收缩。虽然发现 ET-1 诱导的细胞收缩在 PAH 和非 PAH 细胞中相似，但一个关键区别是对药理学抑制剂和 Rho 激酶（ROCK1/ROCK2）的 siRNA 敲低的反应。PAH 细胞需要更高浓度的抑制剂来消除 ET-1 诱导的收缩，而其收缩不受针对 ROCK1 或 ROCK2 的 siRNA 的影响。最后，阻断 L 型和 T 型 Ca2+通道对 ET-1 或 BK 诱导的收缩没有影响。然而，抑制肌浆网 Ca2+ATP 酶的活性减弱了 ET-1 和 BK 诱导的 PAH 和非 PAH 来源的 HPASMC 的收缩。总之，我们这里的研究结果以及以前的通讯一起说明了 PAH 和非 PAH 平滑肌细胞收缩的调节之间的异同，涉及钙转运、RhoA/ROCK 信号转导和钙调蛋白的活性。这些发现可能为调节 PAH 中发生的血管高收缩性提供有用的工具。
