Jones L G, Gause K C, Meier K E
Department of Medicine, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Life Sci. 1996;58(7):617-30. doi: 10.1016/0024-3205(95)02323-2.
The growth-promoting effects of endothelin-1 (ET-1) were examined in adult heart cells. The activity of mitogen-activated protein kinases (MAPKs) was measured in cytosolic extracts of isolated adult feline cardiac myocytes incubated with and without ET-1. Kinase activity was assessed by phosphorylation of the exogenous substrate, myelin basic protein. ET-1 stimulated the activity of MAPK up to 4-fold, with peak activation occurring between five and ten minutes after addition of ET-1. Polyclonal antisera raised against a 14-amino acid sequence of the erk-2 gene product, a MAPK isoform, identified two major bands in cytosolic extracts of the cardiac myocytes. Partial purification of kinase activities using Mono Q anion-exchange chromatography demonstrated two major peaks of myelin basic protein kinase activity. Subsequent immunoblots of the eluted fractions demonstrated that the immunoreactive bands observed in the cytosolic extracts eluted in those fractions possessing kinase activity. Overnight pretreatment of the cardiac myocytes with 100 ng/ml pertussis toxin inhibited the ET-1 stimulated increase in MAPK activity by 50 - 70%, but did not alter stimulation by 100 nM phorbol 12-myristate 13-acetate (PMA). These data suggest that stimulation of MAPK by ET-1 may be mediated by more than one pathway. MAPK has been shown to be activated in the intracellular transmission of growth factor signals. Indicative of a growth effect in this adult heart cell model, myocytes exposed to increasing concentrations of ET-1 demonstrated a dose dependent increase in [3H]-phenylalanine incorporation into cellular protein. This response was blocked by staurosporine and partially inhibited by pretreatment with pertussis toxin, again suggesting the possible involvement of multiple early signals. These data from isolated adult cardiac myocytes further support the hypothesis that ET-1 has a role in the regulation of cardiac growth.
在内皮素 -1（ET -1）对成年心脏细胞的促生长作用进行了研究。在有无ET -1的情况下，对分离的成年猫心肌细胞的胞质提取物中丝裂原活化蛋白激酶（MAPK）的活性进行了测定。通过外源性底物髓鞘碱性蛋白的磷酸化来评估激酶活性。ET -1可使MAPK活性增强达4倍，在添加ET -1后5至10分钟出现激活峰值。针对MAPK亚型erk -2基因产物的14个氨基酸序列产生的多克隆抗血清，在心肌细胞的胞质提取物中鉴定出两条主要条带。使用Mono Q阴离子交换色谱对激酶活性进行部分纯化，显示出髓鞘碱性蛋白激酶活性的两个主要峰值。随后对洗脱组分进行免疫印迹表明，在胞质提取物中观察到的免疫反应条带出现在具有激酶活性的那些组分中。用100 ng/ml百日咳毒素对心肌细胞进行过夜预处理，可使ET -1刺激的MAPK活性增加受到50 - 70%的抑制，但不改变100 nM佛波酯12 -肉豆蔻酸酯13 -乙酸酯（PMA）的刺激作用。这些数据表明，ET -1对MAPK的刺激可能由多种途径介导。MAPK已被证明在生长因子信号的细胞内传递中被激活。在这个成年心脏细胞模型中，暴露于浓度不断增加的ET -1的心肌细胞显示出[3H] -苯丙氨酸掺入细胞蛋白中的剂量依赖性增加，这表明有生长效应。这种反应被星形孢菌素阻断，并被百日咳毒素预处理部分抑制，再次表明可能涉及多个早期信号。来自分离的成年心肌细胞的这些数据进一步支持了ET -1在心脏生长调节中起作用的假说。
