摘要目的和方法：用生长激素(GH)刺激培养的新生大鼠心肌细胞，用含MBP凝胶分离法测定细胞外信号调节酶(ERKs)活性，用Whatman Paper Filter法测定Raf-1活性，观察GH是否激活心肌细胞Raf-1-ERK级联反应。观察负显突变Ras质粒(D.N.Ras)与HA-ERK₂质粒复合转染或有关抑制剂对GH诱导ERK激活的影响。结果：GH以时间和浓度依赖性方式激活心肌细胞ERK₁和ERK₂。ERK上游调节酶Raf-1活性也升高。过度表达D.N.Ras明显抑制GH诱导的心肌细胞HA-ERK₂激活。Ras特导性抑制剂manumycin也显著阻断GH诱导的心肌细胞ERK激活。而分别用TPA和Calphostin C耗竭和抑制心肌细胞PKC，均未阻断GH对ERK的激活作用。结论：GH激活心肌细胞的Raf-1-ERK级联反应，这种激活依赖上游Ras，而不受PKC活性和含量变化的影响。

GH-induced ERK activation and upstream regulation in cardiac myocytes

YUAN Guo-Xiang, GU Ya-Ping, Issei KOMURO, Yoshio YAZAKI

Department of Pathophysiology, Nantong Medical College, Nantong(226001)

AbstractAIM and METHODS: Cultured neonatal cardiac myocytes were stimulated by growth hormone(GH), and then the activities of extracellular signal-regulated kinases(ERKs) were assayed with the method of MBP-containing gel and the activity of Raf-1 kinase was examined with the method of Whatman Paper Filter to examine whether GH activates Raf-1-ERK cascade in cardiac myocytes. Furthermore, the effects of Dominant-negative mutant Ras plasmids( D N. Ras) and HA-ERK₂ plasmids cotransfection as well as relative inhibitors on GH-induced ERK activation were observed to explore the upstream pathway leading to ERK activation stimulated by GH.RESULTS: GH activated ERK₁(42 kDa) and ERK₂(44 kDa) in cardiac myocytes in a time-and a dose-dependent manners. The activity of Raf-1, an upstream regulating enzyme of ERKs, was also increased after GH stimulation. Overexpression of D.N.Ras significantly inhibited GH-induced HA-ERK₂ activation in cardiac myocytes. Manumycin, a specific inhibitor of Ras, also strongly blocked GH-induced ERK activation in cardiac myocytes. The depletion and inhibition of PKC by long time exposure to PTA or pretreatment with calphostin C respectively had no effects on GH-induced ERK activation in cardiac myocytes.CONCLUSION: GH activated Raf-1-ERK cascade in cardiac myocytes was dependent on upstream Ras, but not affected by the changes of PKC activity and PKC quantity in cardiomyocytes.

MeSHHormones, growth; Signal transduction; Myocardizum

近年，国外有学者偿试用生长激素(growth hormone, GH)治疗扩张性心肌病和充血性心力衰竭。发现GH能增加心肌质量，缩小心腔容积，增强心肌收缩力，改善血流动力学指标，而不伴心率和耗氧量的增加^［1］。然而GH的这些有益的心脏效应机制尚不清楚。

丝裂素原激活的蛋白激酶(mitogen-activated protein kinases, MAPKs)是一个重要的细胞内信号传导酶超家族。其成员酶——细胞外信号调节酶(extracellular signal-regulated kinases, ERKs),是一个与细胞生长、增殖、分化甚至保护机制有关的重要信号分子^［2］。本研究用重组人GH(genotropin)刺激新生鼠心肌细胞，测定ERK活性变化，并采用负显质粒(dominant-negative mutant plasmide)转染试验和抑制剂研究，探讨GH诱导ERK激活的上游调控机制。

材料与方法

1.材料：重组人GH(genotropin)由Mitsui Toatsu Chemical Inc提供。［γ^-32P］ATP购于Du Pont新英格兰核公司(Boston, MA)。培养基DMEM和胎牛血清(FBS)购于GIBCO BRL公司。抗血球凝集素(hemagglutinin, HA)多克隆抗体由日本Mitsubishi生化实验室赠送。Calphostin C购于Biomol公司。12-O-tetradecanolphorbol-13-acetate(TPA)和 myelin basic protein (MBP)以及其它试剂均由Sigma化学公司提供。

2.cDNA质粒：SV₄₀起动子调控的HA-ERK₂由Karin赠送。负显突变(Asn-17)质粒(Dominant-negative mutant Ras, D.N.Ras)由Takai赠送。QIAGEN质

粒DNA制备药盒由Chatsworth提供。

3.心肌细胞培养：取1日龄新生Wistar鼠心室肌，按Simpson等的方法略加修改，进行心肌细胞原代培养^［3］。用差速贴壁分离法去除非心肌细胞。置心肌细胞于含10% FBS的DMEM液中，调细胞密度为1×10⁵／cm²培养24 h后，换成0.1% FBS的培养液继续培养48～72 h，再进行各种刺激。

4.质粒DNA转染：心肌细胞原代培养24 h后，用磷酸钙方法进行质粒DNA转染^［3］。每皿加2.5 μgHA-ERK₂ 质粒，实验组同时加入7.5 μg D.N.Ras质粒进行复合转染，14 h后抽去培养液，用磷酸盐缓冲液洗二次，再加0.1% FBS培养液，继续培养48 h，然后进行GH刺激。

5.ERK活性测定：用含MBP凝胶电泳和放射自显影法测ERK活性^［4］。用细胞溶解缓冲液(含25 mmol/L Tris-HCl pH 7.4,25 mmol/L NaCl, 1 mmol/L sodium orthovanadate, 10 mmol/L naF, 10 mmol/L焦磷酸钠，10 nmol/L Okadaic acid, 0.5 mmol/L EGTA和1 mmol/L pMSF)溶解心肌细胞。4℃ 12 000 r/min离心20 min。取上清测定蛋白含量，调节蛋白质浓度。点样于含0.5 g/L MBP的SDS-PAGE进行电泳。去除凝胶SDS后，用6 mol/L盐酸胍使酶蛋白变性，再用50 mmol/L Tris-HCl(pH 8.0,含0.04% Triton X-100和5 mmol/L 2-巯基乙醇)使酶蛋白复性。凝胶再与含92.5×10⁴ Bq［γ-³²P］ATP反应1 h，充分洗涤，干燥凝胶。放射自显影观察MBP磷酸化带。用密度仪扫描测定MBP磷酸化带的放射强度，该数据代表ERK活性。

6.HA-ERK₂活性测定：用免疫沉淀和放射自显影法测转染表达的HA-ERK₂活性^［4］。心肌细胞溶解抽提物与抗HA抗体于4℃孵育1 h。然后用蛋白A Sepharos beads沉淀免疫复合物。收集的免疫复合物洗三次，再悬浮于激酶缓冲液(含25 mmol/L Tris-HCl pH 7.4,10 mmol/L MgCl₂， 1mmol/L DTT, 40 mmol/L ATP, 7.4×10⁴ Bq［γ-³²P］ATP,2 mmol/L蛋白激酶抑制剂和0.5 mmol/L EGTA)。再与25 mmol/L MBP 25℃反应10 min,加入Laemmli缓冲液(0.002%溴酚蓝，pH 7.0的10 mmol/L磷酸盐缓冲液，10%甘油，0.4% SDS， 1% 2-巯基乙醇)终止反应。样本煮沸5 min，取上清电泳后，放射自显影观察磷酶化带。

7.Raf-1活性测定：按Yamazaki等的方法测Raf-1活性^［5］。用抗Raf-1抗体免疫沉淀Raf-1,免疫复合物与含基质10 mg syntide-2和7.4×10⁴ Bq［γ-³²P］ATP的缓冲液孵育。反应后用Whatman P-81 Paper 收集syntide-2用Cerenkov计数仪测定磷酸化的syntide放射强度。

8.统计学处理：实验结果以均数±标准差(±s)表示。统计学检验用F、t或Dunnett'st检验。

结果

一、GH激活心肌细胞ERKs:

浓度依赖关系的研究表明，GH与心肌细胞作用10 min，随着GH刺激浓度的增加，心肌细胞ERK₁(42 kDa)和ERK₂(44 kDa)活性均逐渐增加(图1)。GH浓度为500 μg/L时，两酶活性均达峰值。ERK₁活性为对照组的576.52%±34.41%,P＜0.01;ERK₂活性为对照组的388.26%±12.64%,P＜0.01。当GH浓度超过500 μg/L，ERKs活性逐渐下降。从时效关系的研究可见，用500 μg/L GH刺激心肌细胞2 min，ERKs活性开始增加，10 min时两酶活性达峰值(图2)，ERK₁活性为对照组的542.31%±38.16%,P＜0.01；ERK₂活性为对照组的358.91%±21.54%,P＜0.01。然后酶活性开始下降，30 min时恢复到基础水平。

Fig 1 GH dose-dependently activates ERK in cardiac myocytes. A representative autoradiogram of ERK bands from three independent experiments is shown. The incubation time was 10 min

图1GH浓度依赖性地激活心肌细胞ERK

Fig 2 Time course of GH-induced ERK activation in cardiac myocytes. A representative autoradiogram of ERK bands from three independent experiments is shown. The concentration of GH was 500 μg/L

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