生长激素诱导心肌细胞外信号调节酶激活及其上游调控
作者：袁国祥 顾亚平 小室一成 矢崎义雄
单位：袁国祥 顾亚平 南通医学院病理生理学教研室 (南通226001)；小室一成 矢崎义雄 日本东京大学医学部第3内科 (东京113-8655)
关键词：激素类；生长；信号传递；心肌
中国病理生理杂志990106 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。
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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)
Abstract AIM 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.
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MeSH Hormones, 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。
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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
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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。
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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。
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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's t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。
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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1 GH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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图2 GH诱导心肌细胞ERK激活的时间过程
Fig 3 Time course of Raf-1 activation by GH in cardiac myocytes. The activity of Raf-1 is shown as percent increase of 32P-syntide-2 radioactivity compared with unstimulated controls(100%).±s,P<0.05， vs control图3 GH激活心肌细胞Raf-1的时间过程
二、GH激活心肌细胞Raf-1:
GH引起心肌细胞Raf-1活性变化的时间过程见图3。GH刺激后2 min，心肌细胞Raf-1活性迅速升高，8 min时达峰值。
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三、GH诱导的心肌细胞ERK激活依赖Ras:
质粒转染试验显示，单独HA-ERK2质粒转染的心肌细胞经GH刺激，引起显著的HA-ERK2激活，是对照组酶活性的437.86%±41.62%(P<0.01)。而D.N.Ras与HA-ERK2质粒复合转染的心肌细胞，经GH刺激，未引起明显的HA-ERK2激活，与对照组复合转染的细胞相比无显著差异，而与GH刺激的单独HA-ERK2转染的细胞相比，有显著差异(P<0.05)(图4A)。表明过度表示的D.N.Ras阻断了GH对HA-ERK2的激活作用。
用Ras抑制剂manumycin与心肌细胞孵育30 min后，进行GH刺激。发现三种浓度的manumycin均阻断GH对心肌细胞ERK的激活作用(图4B)。以上结果均表明心肌细胞GH信号传递途径中，Ras起着关键作用。
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Fig 4 Ras is critical for GH-induced ERK activation in cardiac myocytes.
A. D.N.Ras suppresses HA-ERK2 activation by GH in cardiomyocytes. B. Manumycin inhibites GH-induced ERK activation in cardiomyocytes.Cardiac myocytes were pretreated with manumycin at indicated concentration for 30 min and then stimulated with 500 μg/L GH for 10 min. A representative autoradiogram of ERK bands is showm
P<0.05,vs control; P<0.05, vs sample with only HA-ERK transfection followed by GH stimulation
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图4 GH激活心肌细胞ERK需要Ras
四、GH激活心肌细胞ERK不依赖PKC：
用Calphostin C(10-6mol/L)与心肌细胞孵育60 min以抑制细胞PKC，以及用PKC激动剂PTA(10-7mol/L)与心肌细胞孵育24 h以耗竭细胞PKC。结果发现，单独两种预处理本身对细胞ERK无激活作用；而两种预处理后进行GH刺激，心肌细胞ERK活性明显升高。提示心肌细胞PKC的抑制或下调不影响GH对ERK的激活作用(图5)。
讨 论
MAPKs超家族是细胞内重要的信号分子。已发现该家族的成员酶有：ERK，JNK(c-jun NH2-terminal kinase)和P38 MAPK。迄今关于MAPK通路的激活机制及生理病理意义仍未完全清楚。初步发现ERK途径与细胞生长、增殖、分化和转化有关；而JNK和P38MAPK途径可能与应激、损伤和细胞凋亡有关[6]。已发现GH激活某些细胞株如3T3-F442A前脂肪细胞、成纤维细胞等的ERK[7]。用MEK抑制剂PD 98059阻断ERK激活，能加重H2O2诱导的心肌细胞凋亡[3，8]。心肌缺血预处理诱导的保护作用也与ERK的激活有关。这些研究表明ERK对细胞的生存起着关键作用。本研究发现GH能激活培养的新生鼠心肌细胞ERK1和ERK2，可能是GH改善充血性心力衰竭病人心功能的重要机制之一。
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Fig 5 GH-induced ERK activation in cardiac myocytes is independent of PKC. Cardiac myocytes were incubated with 10-7mol/L TPA for 24 h or with 10-6mol/L calphostin C for 60 min followed by stimulation with 500 μg/L GH for 10 min
图5 GH激活心肌细胞ERK不依赖PKC
双蛋白激酶MEK(mitogen-activated protein kinase kinase)磷酸化ERK的苏氨酸和酪氨酸残基使ERK活化。而MEK可被几种蛋白激酶(mitogen-activated protein kinase kinase kinases, MAPKKKs)磷酸化丝氨酸残基而激活，形成MAPKKK→MEK→ERK瀑布激活链。对一些细胞株的研究表明Raf-1在丝裂素原信号链中可能起着MAPKKK的作用。本研究发现GH刺激后，心肌细胞Raf-1活性迅速增高，提示Raf-1在GH的心脏效应中可能起了重要作用。
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Raf-1-ERK级联反应的上游调控机制因细胞类型和刺激物的不同而有高度差异性。据报道AⅡ激活心肌细胞ERK通过PKC，而不依赖Ras[4]；相反，AⅡ激活心脏成纤维细胞ERK以及H2O2激活心肌细胞ERK均依赖上游Ras和Src家族酪氨酸激酶[3，9]。Winston等发现GH激活肾胚胎细胞ERK需要Ras[10]。而GH诱导的前脂肪细胞ERK激活与PKC密切相关。本研究经质粒转染试验发现D.N.Ras的过度表达明显抑制了GH对HA-ERK2的激活作用。Ras抑制剂manumycin也显著阻断GH的ERK激活作用。两个实验均表明GH诱导的心肌细胞ERK激活对上游Ras有较强的依赖性。而心肌细胞PKC的下调或抑制，对GH的ERK激活作用无影响。虽然本研究不能排除GH对PKC的激活作用，但可以认为GH诱导心肌细胞Raf-1-ERK途径激活不依赖PKC，而Ras可能起了关键的作用，关于心肌细胞GH受体信号与Ras激活因子之间的偶联过程尚需进一步的研究。
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*国家教委留学回国人员科研基金和日本文部省心血管研究基金资助
参考文献
1 Volterrani M, Desenzani P, Lorusso R, et al. Haemodynamic effects of intravenous growth hormone in congestive heart failure. Lancet, 1997, 349:1067.
2 Cano E, Mahadevan LC. Parallel signal processing among mammalian MAPKs. Trends Biochem Sci, 1995, 20:117.
3 Aikawa R, Komuro I, Yamazaki T, et al. Oxidative stress activates extracellular signal regulated kinase through Src and Ras in cultured cardiac myocytes of neonatal rats. J Clin Invest, 1997, 100:1813.
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4 Zou Y, Komuro I, Yamazaki T, et al. Protein kinase C, but not tyrosine kinase or Ras, plays a critical role in angiotensin Ⅱ-induced protein kinases in cardiac myocytes. J Biol Chem, 1996, 271:33592.
5 Yamazaki T, Komuro I, Kudoh S, et al. Endothelin-1 is involved in mechanical stress induced cardiomyocyte hypertrophy. J Biol Chem, 1996, 271:3221.
6 Xia Z, Dickens M, Raingeaud J, et al. Opposing effects of ERK and JNK-p38MAPK kinases on apoptosis. Science, 1995, 270:1326.
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7 Anderson NG. Growth hormone activates mitogen-activated protein kinase and S6 kinase and promotes intracellular tyrosine phosphorylation in 3T3-F442A preadipocytes. Biochem J, 1992, 284:649.
8 Gu Y, Komuro I, Yamazaki T, et al. Growth hormone protects cardiac myocytes against H2O2-induced apoptosis through extracellular signal-regulated kinase. Jpn Circ J, 1998, 62(suppl):18.
9 Zou Y, Komuro I, Yamazaki T, et al. Cell type-specific angiotensin Ⅱ-evoked signal transduction pathways: critical roles of Gβy subunit, Src family, and Ras in cardiac fibroblasts. Circ Res, 1998, 82:328.
10 Winston LA, Hunter T. JAK2, Ras, and Raf are required for activation of extracellular signal-regulated kinase/mitogen-activated protein kinase by growth hormone. J Biol Chem, 1995, 270:30837.
收稿日期：1998年5月18日
修稿日期：1998年8月28日
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单位：袁国祥 顾亚平 南通医学院病理生理学教研室 (南通226001)；小室一成 矢崎义雄 日本东京大学医学部第3内科 (东京113-8655)
关键词：激素类；生长；信号传递；心肌
中国病理生理杂志990106 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。
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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)
Abstract AIM 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.
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MeSH Hormones, 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。
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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
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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。
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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。
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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's t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。
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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1 GH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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图2 GH诱导心肌细胞ERK激活的时间过程
Fig 3 Time course of Raf-1 activation by GH in cardiac myocytes. The activity of Raf-1 is shown as percent increase of 32P-syntide-2 radioactivity compared with unstimulated controls(100%).
±s,P<0.05， vs control图3 GH激活心肌细胞Raf-1的时间过程二、GH激活心肌细胞Raf-1:
GH引起心肌细胞Raf-1活性变化的时间过程见图3。GH刺激后2 min，心肌细胞Raf-1活性迅速升高，8 min时达峰值。
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三、GH诱导的心肌细胞ERK激活依赖Ras:
质粒转染试验显示，单独HA-ERK2质粒转染的心肌细胞经GH刺激，引起显著的HA-ERK2激活，是对照组酶活性的437.86%±41.62%(P<0.01)。而D.N.Ras与HA-ERK2质粒复合转染的心肌细胞，经GH刺激，未引起明显的HA-ERK2激活，与对照组复合转染的细胞相比无显著差异，而与GH刺激的单独HA-ERK2转染的细胞相比，有显著差异(P<0.05)(图4A)。表明过度表示的D.N.Ras阻断了GH对HA-ERK2的激活作用。
用Ras抑制剂manumycin与心肌细胞孵育30 min后，进行GH刺激。发现三种浓度的manumycin均阻断GH对心肌细胞ERK的激活作用(图4B)。以上结果均表明心肌细胞GH信号传递途径中，Ras起着关键作用。
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Fig 4 Ras is critical for GH-induced ERK activation in cardiac myocytes.
A. D.N.Ras suppresses HA-ERK2 activation by GH in cardiomyocytes. B. Manumycin inhibites GH-induced ERK activation in cardiomyocytes.Cardiac myocytes were pretreated with manumycin at indicated concentration for 30 min and then stimulated with 500 μg/L GH for 10 min. A representative autoradiogram of ERK bands is showm
P<0.05,vs control; P<0.05, vs sample with only HA-ERK transfection followed by GH stimulation
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图4 GH激活心肌细胞ERK需要Ras
四、GH激活心肌细胞ERK不依赖PKC：
用Calphostin C(10-6mol/L)与心肌细胞孵育60 min以抑制细胞PKC，以及用PKC激动剂PTA(10-7mol/L)与心肌细胞孵育24 h以耗竭细胞PKC。结果发现，单独两种预处理本身对细胞ERK无激活作用；而两种预处理后进行GH刺激，心肌细胞ERK活性明显升高。提示心肌细胞PKC的抑制或下调不影响GH对ERK的激活作用(图5)。
讨 论
MAPKs超家族是细胞内重要的信号分子。已发现该家族的成员酶有：ERK，JNK(c-jun NH2-terminal kinase)和P38 MAPK。迄今关于MAPK通路的激活机制及生理病理意义仍未完全清楚。初步发现ERK途径与细胞生长、增殖、分化和转化有关；而JNK和P38MAPK途径可能与应激、损伤和细胞凋亡有关[6]。已发现GH激活某些细胞株如3T3-F442A前脂肪细胞、成纤维细胞等的ERK[7]。用MEK抑制剂PD 98059阻断ERK激活，能加重H2O2诱导的心肌细胞凋亡[3，8]。心肌缺血预处理诱导的保护作用也与ERK的激活有关。这些研究表明ERK对细胞的生存起着关键作用。本研究发现GH能激活培养的新生鼠心肌细胞ERK1和ERK2，可能是GH改善充血性心力衰竭病人心功能的重要机制之一。
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Fig 5 GH-induced ERK activation in cardiac myocytes is independent of PKC. Cardiac myocytes were incubated with 10-7mol/L TPA for 24 h or with 10-6mol/L calphostin C for 60 min followed by stimulation with 500 μg/L GH for 10 min
图5 GH激活心肌细胞ERK不依赖PKC
双蛋白激酶MEK(mitogen-activated protein kinase kinase)磷酸化ERK的苏氨酸和酪氨酸残基使ERK活化。而MEK可被几种蛋白激酶(mitogen-activated protein kinase kinase kinases, MAPKKKs)磷酸化丝氨酸残基而激活，形成MAPKKK→MEK→ERK瀑布激活链。对一些细胞株的研究表明Raf-1在丝裂素原信号链中可能起着MAPKKK的作用。本研究发现GH刺激后，心肌细胞Raf-1活性迅速增高，提示Raf-1在GH的心脏效应中可能起了重要作用。
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Raf-1-ERK级联反应的上游调控机制因细胞类型和刺激物的不同而有高度差异性。据报道AⅡ激活心肌细胞ERK通过PKC，而不依赖Ras[4]；相反，AⅡ激活心脏成纤维细胞ERK以及H2O2激活心肌细胞ERK均依赖上游Ras和Src家族酪氨酸激酶[3，9]。Winston等发现GH激活肾胚胎细胞ERK需要Ras[10]。而GH诱导的前脂肪细胞ERK激活与PKC密切相关。本研究经质粒转染试验发现D.N.Ras的过度表达明显抑制了GH对HA-ERK2的激活作用。Ras抑制剂manumycin也显著阻断GH的ERK激活作用。两个实验均表明GH诱导的心肌细胞ERK激活对上游Ras有较强的依赖性。而心肌细胞PKC的下调或抑制，对GH的ERK激活作用无影响。虽然本研究不能排除GH对PKC的激活作用，但可以认为GH诱导心肌细胞Raf-1-ERK途径激活不依赖PKC，而Ras可能起了关键的作用，关于心肌细胞GH受体信号与Ras激活因子之间的偶联过程尚需进一步的研究。
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*国家教委留学回国人员科研基金和日本文部省心血管研究基金资助
参考文献
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4 Zou Y, Komuro I, Yamazaki T, et al. Protein kinase C, but not tyrosine kinase or Ras, plays a critical role in angiotensin Ⅱ-induced protein kinases in cardiac myocytes. J Biol Chem, 1996, 271:33592.
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7 Anderson NG. Growth hormone activates mitogen-activated protein kinase and S6 kinase and promotes intracellular tyrosine phosphorylation in 3T3-F442A preadipocytes. Biochem J, 1992, 284:649.
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收稿日期：1998年5月18日
修稿日期：1998年8月28日
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