Calponin is required for agonist-induced signal transduction - evidence from an antisense approach in ferret smooth muscle
Calponin is required for agonist-induced signal transduction - evidence from an antisense approach in ferret smooth muscle
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DOI：
10.1111/j.1469-7793.2001.00567.x
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发表时间：
2001-12-01
影响因子：
5.5
通讯作者：
Morgan, KG
中科院分区：
文献类型：
--
作者：
Je, HD;Gangopadhyay, SS;Morgan, KG
1. 本研究旨在确定钙钙蛋白（CaP）是否参与血管平滑肌收缩的调节，如果参与，则探讨其机制。通过PCR同源克隆，确定了雪貂碱性（h1） CaP的cDNA序列，合成了硫代反义核苷酸和随机寡核苷酸，并通过化学加载法导入到雪貂主动脉条带中。用CaP反义寡核苷酸处理雪貂主动脉导致CaP蛋白水平下降到随机序列加载肌肉的54%，但钙素（CaD）、肌动蛋白、desmin或细胞外调节蛋白激酶（ERK）的蛋白水平没有变化。与随机序列加载的对照相比，反义处理的肌肉对苯肾上腺素或佛波酯的收缩反应明显减少，基础固有张力和对51毫米KCl的收缩反应都没有受到反义处理的显著影响。在苯肾上腺素收缩期间，磷酸化erk水平升高，肌球蛋白轻链（LC20）磷酸化也升高。苯肾上腺素诱导的ERK磷酸化和CaD磷酸化在ERK位点被CaP反义显著降低。肌球蛋白轻链磷酸化的增加不受影响。这些数据表明，CaP在收缩调节中起着重要作用，并表明在强直活动的平滑肌中，CaP可能作为一种信号蛋白促进erk依赖性信号传导，但不是肌丝水平上肌动球蛋白相互作用的直接调节剂。
1. The present study was undertaken to determine whether calponin (CaP) participates in the regulation of vascular smooth muscle contraction and, if so, to investigate the mechanism.2. By PCR homology cloning, the cDNA sequence of ferret basic (h1) CaP was determined and phosphorothioate antisense and random oligonucleotides were synthesized and introduced into strips of ferret aorta by a chemical loading procedure.3. Treatment of ferret aorta with CaP antisense oligonucleotides resulted in a decrease in protein levels of CaP to 54% of that in random sequence-loaded muscles, but no change in the protein levels of caldesmon (CaD), actin, desmin or extracellular regulated protein kinase (ERK).4. Contraction in response to phenylephrine or a phorbol ester was significantly decreased in antisense-treated muscles compared to random sequence-loaded controls, Neither basal intrinsic tone nor the contraction in response to 51 mM KCl was significantly affected by antisense treatment.5. During phenylephrine contractions, phospho-ERK levels increased, as did myosin light chain (LC20) phosphorylation. Phenylephrine-induced ERK phosphorylation and CaD phosphorylation at an ERK site were significantly decreased by CaP antisense. Increases in myosin light chain phosphorylation were unaffected.6. The data indicate that CaP plays a significant role in the regulation of contraction and suggest that in a tonically active smooth muscle CaP may function as a signalling protein to facilitate ERK-dependent signalling, but not as a direct regulator of actomyosin interactions at the myofilament level.