Zhao Y, Rhoades R A, Packer C S
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202-5120, USA.
Am J Physiol. 1996 Nov;271(5 Pt 1):L768-74. doi: 10.1152/ajplung.1996.271.5.L768.
The signal transduction pathway of hypoxic pulmonary arterial contraction has not been elucidated. Phosphorylation of the 20-kDa myosin light chain (MLC20) is thought to be essential for vascular muscle contraction. However, there are reports that smooth muscle will contract in response to nonphysiological stimuli such as phorbol esters without the involvement of MLC20 phosphorylation. The purpose of this study was to determine if hypoxia-induced pulmonary arterial contraction is dependent on MLC20 phosphorylation. Isolated rat pulmonary and carotid (for comparative purposes) arterial strips were contracted with 80 mM KCl to establish maximum active tension in response to membrane depolarization. The strips were then stimulated with one of the following: 30 mM KCl, 1 microM phenylephrine, 0.01 microM angiotensin II, 1 microM phorbol 12-myristate 13-acetate (PMA), or hypoxia (95% N2-5% CO2). In some experiments ML-9, a myosin light chain kinase inhibitor, or calphostin C, a protein kinase C (PKC) inhibitor, was introduced into the bath before hypoxia. Isometric tension was recorded as a function of time. Muscle strips were freeze-clamped (liquid N2) at various time points during the course of responses to the various stimuli. MLC20 phosphorylation levels were measured by ureaglycerol gel electrophoresis followed by Western blot procedure. Results show that increased MLC20 phosphorylation correlates with initiation of pulmonary arterial smooth muscle contraction in response to all agonists with the exception of PMA, a known activator of PKC. The MLC20 phosphorylation levels correlate with tension development in response to hypoxia, and ML-9 abolished the hypoxic contractions. In contrast, hypoxia relaxed carotid arterial muscle, and there was a corresponding decrease in the MLC20 phosphorylation level. In conclusion, hypoxia appears to result in MLC20 phosphorylation-mediated contraction in conduit pulmonary arterial muscle and in MLC20 dephosphorylation-mediated relaxation in systemic arterial muscle.
低氧性肺动脉收缩的信号转导途径尚未阐明。20 kDa肌球蛋白轻链（MLC20）的磷酸化被认为是血管肌肉收缩所必需的。然而，有报道称平滑肌会对非生理性刺激（如佛波酯）产生收缩反应，而不涉及MLC20磷酸化。本研究的目的是确定低氧诱导的肺动脉收缩是否依赖于MLC20磷酸化。分离的大鼠肺动脉和颈动脉（用于比较）动脉条用80 mM KCl收缩，以建立对膜去极化的最大主动张力。然后用以下之一刺激这些条带：30 mM KCl、1 μM去氧肾上腺素、0.01 μM血管紧张素II、1 μM佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）或低氧（95% N2 - 5% CO2）。在一些实验中，在低氧之前将肌球蛋白轻链激酶抑制剂ML - 9或蛋白激酶C（PKC）抑制剂钙磷蛋白C引入浴槽。记录等长张力随时间的变化。在对各种刺激的反应过程中的不同时间点，将肌肉条带冷冻钳夹（液氮）。通过尿素甘油凝胶电泳，然后进行蛋白质印迹法测量MLC20磷酸化水平。结果表明，除了已知的PKC激活剂PMA外，MLC20磷酸化增加与肺动脉平滑肌对所有激动剂的收缩起始相关。MLC20磷酸化水平与对低氧的张力发展相关，并且ML - 9消除了低氧收缩。相反，低氧使颈动脉肌肉松弛，并且MLC20磷酸化水平相应降低。总之，低氧似乎导致传导性肺动脉肌肉中MLC20磷酸化介导的收缩以及全身动脉肌肉中MLC20去磷酸化介导的松弛。
