Lazou A, Bogoyevitch M A, Clerk A, Fuller S J, Marshall C J, Sugden P H
Department of Cardiac Medicine, National Heart and Lung Institute, London, UK.
Circ Res. 1994 Nov;75(5):932-41. doi: 10.1161/01.res.75.5.932.
The regulation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) was studied in freshly isolated adult rat heart preparations. In contrast to the situation in ventricular myocytes cultured from neonatal rat hearts, stimulation of MAPK activity by 1 mumol/L phorbol 12-myristate 13-acetate (PMA) was not consistently detectable in crude extracts. After fast protein liquid chromatography, MAPK isoforms p42MAPK and p44MAPK and two peaks of MEK were shown to be activated > 10-fold in perfused hearts or ventricular myocytes exposed to 1 mumol/L PMA for 5 minutes. The identities of MAPK or MEK were confirmed by immunoblotting and, for MAPK, by the "in-gel" myelin basic protein phosphorylation assay. In retrogradely perfused hearts, high coronary perfusion pressure (120 mm Hg for 5 minutes), norepinephrine (50 mumol/L for 5 minutes), or isoproterenol (50 mumol/L for 5 minutes) stimulated MAPK and MEK approximately 2- to 5-fold. In isolated myocytes, endothelin 1 (100 nmol/L for 5 minutes) also stimulated MAPK, but stimulation by norepinephrine or isoproterenol was difficult to detect. Immunoblotting showed that the relative abundances of MAPK and MEK protein in ventricles declined to < 20% of their postpartal abundances after 50 days. This may explain the difficulties encountered in assaying the activity of MAPK in crude extracts from adult hearts. We conclude that potentially hypertrophic agonists and interventions stimulate the MAPK cascade in adult rats and suggest that the MAPK cascade may be an important intracellular signaling pathway in this response.
在新鲜分离的成年大鼠心脏标本中研究了丝裂原活化蛋白激酶（MAPK）和MAPK激酶（MEK）的调节。与新生大鼠心脏培养的心室肌细胞的情况相反，在粗提物中，1μmol/L佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）对MAPK活性的刺激作用并不始终能检测到。经过快速蛋白质液相色谱分析，MAPK亚型p42MAPK和p44MAPK以及两个MEK峰在灌注心脏或暴露于1μmol/L PMA 5分钟的心室肌细胞中被激活超过10倍。通过免疫印迹法确认了MAPK或MEK的身份，对于MAPK，则通过“凝胶内”髓鞘碱性蛋白磷酸化试验进行确认。在逆行灌注的心脏中，高冠状动脉灌注压（120 mmHg，持续5分钟）、去甲肾上腺素（50μmol/L，持续5分钟）或异丙肾上腺素（50μmol/L，持续5分钟）可使MAPK和MEK刺激约2至5倍。在分离的心肌细胞中，内皮素1（100 nmol/L，持续5分钟）也可刺激MAPK，但去甲肾上腺素或异丙肾上腺素的刺激作用难以检测到。免疫印迹显示，50天后心室中MAPK和MEK蛋白的相对丰度下降至产后丰度的<20%。这可能解释了在检测成年心脏粗提物中MAPK活性时遇到的困难。我们得出结论，潜在的肥大激动剂和干预措施可刺激成年大鼠的MAPK级联反应，并表明MAPK级联反应可能是这种反应中重要的细胞内信号通路。
