【作者】 付琴；

【导师】 向继洲；

【作者基本信息】 华中科技大学 ， 药理学， 2006， 博士

【摘要】 β₂肾上腺素受体激动剂克仑特罗对新生大鼠心肌细胞的作用研究β肾上腺素受体（β-adrenergic receptorβ-AR），是典型的G蛋白偶联受体，至少有3种亚型：β₁-、β₂-、β₃-AR。肾上腺素受体介导的儿茶酚胺效应，在心血管功能调节中起着重要的作用。传统认为，只有β₁-AR才调节心脏功能。β₁-AR与兴奋型G_s蛋白偶联，激活腺苷酸环化酶，促进胞内环磷酸腺苷（cAMP）蓄积，从而引起正性变力效应，正性变时效应和正性变性效应。近来发现，β₁-和β₂-AR共同调节心脏的功能。但近十年的研究表明，β₁-AR仅与G_s偶联，而β₂-AR同时与G_s和Gi蛋白双偶联。无论生理还是病理状况下，β₂-AR／G_i偶联都抑制了由β₂-AR／G_s介导的正性肌力作用和正性舒张作用。因此，β₁-和β₂-AR在介导心脏功能方面存在差异。研究还发现，由于新生大鼠心肌细胞缺乏或很少有β₂-AR／G_i偶联，β₂-AR介导的新生大鼠心脏的生物效应与成年大鼠也有所不同。由于种属、细胞、以及动物年龄等的差异，目前关于β₂-AR对心肌细胞作用的报道也结论不一。本学位论文就β₂-AR激动剂克仑特罗（clenbuterol）在新生大鼠心肌细胞介导的生物效应进行了研究，由三部分组成：（1）β₂-AR激动剂克仑特罗对新生大鼠心肌细胞受磷蛋白磷酸化的作用；（2）β₂-AR激动剂克仑特罗对心肌细胞MAPK信号通路的作用及机制研究；（3）β₂-AR激动剂克仑特罗对心肌细胞一氧化氮合成的作用。第一部分克仑特罗对新生大鼠心肌细胞受磷蛋白磷酸化的作用背景：心肌β-AR被儿茶酚胺激活后可以增加心肌收缩并加快心肌舒张。在成年大鼠心肌细胞β₂ AR激活后可通过cAMP-PKA信号途径促L型钙通道开放，但对受磷蛋白（phospholamban，PLB）磷酸化没有影响。推测在新生大鼠心肌细胞β₂ AR对PLB的磷酸化作用有可能不同于成年大鼠心肌细胞。方法：测定给予克仑特罗后心肌细胞搏动频率和胞内cAMP水平变化的时间曲线。采用Western blo方法，检测受磷蛋白在Serine16（Ser16）和Threonine17（Thr17）的磷酸化水平。结果：克仑特罗以时间依赖性方式增加新生大鼠心肌细胞搏动频率以及细胞内cAMP水平。克仑特罗还可以时间-剂量依赖性的方式促进受磷蛋白在Ser16位点的磷酸化，该作用可被选择性β₂AR阻断剂ICI 118551和PKA抑制剂Rp-cAMP所阻断。G_i蛋白抑制剂PTX具有协同作用，但不显著。Clenbuterol还可促进受磷蛋白在Thr17位点的磷酸化，并呈时间依赖性。结论：在新生大鼠心肌细胞β₂克仑特罗可促进受磷蛋白在Ser16和Thr17位点的磷酸化，可能参与改善心脏舒张功能。第二部分克仑特罗对新生大鼠心肌细胞MAPK信号通路的作用及机制研究§1克仑特罗对新生大鼠心肌细胞ERK1／2的作用及机制研究背景：G蛋白偶联受体可通过G_i蛋白激活胞外信号调节蛋白激酶（extracellular signal-regulated kinase 1／2，ERK1／2）。在不同细胞胞内cAMP积聚可增加或减少ERK1／2的活性。钙动员也参与ERK1／2的磷酸化和去磷酸化的调节。在成年大鼠心肌细胞β₂AR可通过G_i蛋白信号途径磷酸化ERK1／2。而β₂ AR可促进新生大鼠心肌细胞钙动员和cAMP水平增加但对成年大鼠心肌细胞无作用。故推测β₂ AR在新生大鼠心肌细胞对ERK1／2磷酸化的作用不同于成年大鼠心肌细胞。本实验拟观察β₂ AR对新生大鼠心肌细胞ERK1／2的磷酸化作用并阐明其机制。方法：采用Western blot方法，检测经与β₂ AR信号通路有关的试剂处理的培养新生大鼠心肌细胞磷酸化ERK1／2蛋白和ERK2总蛋白表达水平。结果：Clenbuterol对新生大鼠心肌细胞的ERK1／2磷酸化具有双相作用，表现为给药后磷酸化水平迅速升高，2min达到最高值，既而下降，在给药后30min降至基础水平的60％。Clenbuterol对ERK1／2的磷酸化和去磷酸化的作用均呈剂量依赖性。细胞外钙有无对clenbuterol的促磷酸化作用无影响，但兰诺丁受体（ryanodine receptor）激动剂ryanodine对此具有协同作用。而选择性β₂ AR激动剂ICI118551，百日咳毒素（pertussis toxin，PTX），钙螯合剂BAPTA-AM和兰诺丁受体阻断剂rethenium red可显著抑制clenbuterol的促磷酸化作用，而钙拮抗药硝苯地平nifedepine的抑制作用不明显。相反，蛋白磷酸酶抑制剂okadaic acid和钙泵ATP酶抑制剂thapsigargin均可翻转clenbuterol的去磷酸化作用。蛋白激酶A抑制剂Rp-cAMP也可抑制clenbuterol的去磷酸化作用。Caffeine亦对ERK1／2具有磷酸化和去磷酸化的双相作用，thapsigargin可阻断caffeine的去磷酸化作用，而okadaic acid对之无影响。Caffeine和clenbuterol一样可持续激活PLB。结论：β₂受体激活后对新生大鼠心肌细胞ERK1／2磷酸化具有双相作用，其中胞内钙动员扮演重要角色。Clenbuterol激活ERK1／2与G_i蛋白信号途径和钙库释放Ca²⁺有关，而clenbuterol对ERK1／2的去磷酸化作用则可能与受磷蛋白磷酸化后加快钙库摄取Ca²⁺有关。§2克仑特罗对新生大鼠心肌细胞p38 MAPK的作用及机制研究背景：文献报道p38 MAPK可诱导新生大鼠心肌细胞凋亡，而在成年大鼠心肌细胞βAR／G_i通路激活的p38 MAPK对βAR／G_s信号通路诱导的细胞凋亡具有保护作用。本论文前部分研究表明clenbuterol通过G_s和G_i蛋白偶联对新生大鼠心肌细胞ERK1／2磷酸化具有双相作用，而在成年大鼠心肌细胞仅通过G_i蛋白偶联促进ERK1／2磷酸化。因此推测clenbuterol对新生大鼠心肌细胞p38 MAPK磷酸化的作用不同于成年大鼠心肌细胞。方法：采用Western blot方法，检测经各种试剂处理后的培养新生大鼠心肌细胞磷酸化p38 MAPK和总p38 MAPK蛋白表达水平。结果：Clenbuterol可激活p38 MAPK，该作用呈时间和剂量依赖性，并能被选择性β₂ AR阻断剂ICI118551所抑制；G_i蛋白抑制剂PTX对clenbuterol诱导的p38 MAPK磷酸化具有协同作用，而蛋白激酶A抑制剂Rp-cAMP可阻断之；钙通道阻滞药nifedipine和Ca²⁺螯合剂BAPTA-AM均可阻断clenbuterol对p38 MAPK的促磷酸化作用。结论：在新生大鼠心肌细胞β₂ AR激活后可通过G_s／cAMP信号途径促进p38 MAPK磷酸化，该作用和细胞外钙内流增加细胞内钙浓度有关。第三部分克仑特罗对新生大鼠心肌细胞一氧化氮合成的作用背景：一氧化氮（Nitric Oxide，NO）是调节心血管、免疫和神经系统功能的重要信号分子。NO具有舒张血管，对各种细胞的细胞毒性和细胞保护作用等多种生物学效应。血管平滑肌细胞、心肌细胞在受到内毒素和某些细胞因子刺激后诱生型一氧化氮合酶（inducible nitric oxide synthase，iNOS）表达增加并释放大量NO。研究表明，在患有扩张型心肌病、缺血性心肌病和其他心血管疾病的患者发生心力衰竭时，iNOS基因表达通常会增加。在新生大鼠心肌细胞，cAMP可增加细胞因子介导的NO合成。喂食β₂AR激动剂clenbuterol可诱导大鼠血管平滑肌细胞合成NO。而本论文第一部分研究表明，clenbuterol可显著增加新生大鼠心肌细胞胞内cAMP水平。在本实验中，拟观察clenbuterol对培养的新生大鼠心肌细胞的NO合成的影响。方法：采用Griess试剂检测新生大鼠心肌细胞培养液中NO的稳定代谢产物亚硝酸盐nitrite的含量，间接反映NO含量。采用Western blot方法，检测培养新生大鼠心肌细胞iNOS蛋白表达水平。结果：单独给予clenbuterol对于NO合成无任何影响，但能使IL-1β诱导心肌细胞产生的NO显著增加，此协同作用呈时间-剂量依赖性。同时，clenbuterol还对IL-1β促进iNOS蛋白表达具有协同作用。非选择性β-AR阻断剂普萘洛尔propranolol和选择性β₂AR阻断剂ICI 118551均可阻断clenbuterol对IL-1β诱导的NO合成以及iNOS蛋白表达的协同作用。结论：在新生大鼠心肌细胞β₂ AR激活可显著增加IL-1β诱导的iNOS表达和NO合成。更多还原

【Abstract】 It is generally accepted that under physiological conditions catecholamines exert positive chronontropic, inotropic and lustropic response through β₁-adrenergic receptor （AR） selectively coupling to G_s protein, activating adenylyl cyclase and enhanceing cAMP production, leading to protein kinase A（PKA） activation, which phosphorylates a multitude of regulatory proteins.Studies during the past decade have demonstrated that coexisting cardiac β-AR, mainlyβ₁ AR and β₂ AR, activating different signaling pathway and fulfill strikingly distinct physiological and pathological roles in the heart. The role of β₂-AR in neonatal rat cardiac myocytes differs from adult rat cardiac myocyte due to the difference of age. To demonstrate the effect of β₂-AR signaling pathway in neonatal rat cardiomyocytes, this thesis investigated: （l）Effect ofβ₂-AR agonist clenbuterol on phospholamban phosphorylation in neonatal rat cardiomyocytes; （2） Effect of P2-AR agonist clenbuterol on MAPK signaling pathway in neonatal rat cardiomyocytes; （3） Effect of β₂-AR agonist clenbuterol on nitric oxide synthesis in neonatal rat cardiomyocytes. Section 1Effect of clenbuterol on phosphorylation of phospholamban in neonatal rat cardiomyocytesBack ground: Catecholamines hasten cardiac relaxation through βadrenergic receptors by phosphorylation of phospholamban and troponin I. β₂ AR stimulation in adult rat cardiomyocytes augments L-type Ca²⁺ current in a cAMP-dependent protein kinase （PKA）dependent manner but fails to the phosphorylation of phospholamban. We assessed the role of β₂ AR in phosphorylation of phospholamban in neonatal rat cardiomyocytes differed from adult rat cardiomyocytes.Methods: Increase in beat number and intracellular cAMP was measured in cardiomyocytes exposed to clenbuterol at diflferent time incubation. Phosphorylation of phospholamban at serine 16 and threonine 17 were determined by Western blot using phosphospecific antibodies.Results: Clenbuterol increased cardiomyocyte beat and intracellular cAMP accumulation in a time-dependent manner. Phosphorylation of phospholamban at serine 16 was increased by clenbuterol in a time-dose-dependent manner, and which was blocked by selective β₂ AR antagonist ICI 118551 and PKA inhibitor Rp-cAMP. Inhibition of G_i with pertussis toxin （PTX） slightly enhanced the phosphorylation of PLB at serine 16 induced by clenbuterol. Clenbuterol increased phosphorylation of Thr17 of phospholamban in a time-dependent manner. Conclusions: β₂ AR activation promotes phosphorylation of phospholamban at serine 16 and threonine 17, thereby potentially improving diastolic function in neonatal rat cardiomyocytes. Section 2Effect of clenbuterol on MAPKs signaling pathway in neonatal rat cardiomyocytes§1. Effect of clenbuterol on ERK1/2 signaling pathway in neonatal rat cardiomyocytesBackground: Several G protein-coupled receptors activate extracellular signal-regulated kinase 1/2 （ERK1/2） through G_i protein pathway. Accumulation of cAMP can either inhibit or stimulate ERKl/2 phosphorylation. Calcium mobilization is also involved in the phosphorylation and dephosphorylation of ERK1/2. β₂ adrenoceptor （AR） activate ERK1/2 in a G_i-dependent manner in cultured adult rat cardiomyocytes. However, β₂ AR agonist modulates the calcium mobilization and cAMP accumulation in neonatal rat cardiomyocytes but not adult rat cardiomyocytes. Therefore, we assumed that β₂ AR agonist mediated phosphorylation of ERKl/2 in neonatal rat cardiomyocytes differed from that observed in adult rat cardiomyocytes. To test this hypothesis, we investigated the effect of β₂ AR agonist clenbuterol on ERKl/2 phosphorylation in neonatal rat cardiomyocytes and explored underlying mechanisms. Methods: Cultured neonatal rat cardiomyocytes were pretreated with or without various agents of the β₂ AR signaling pathways. The phosphorylation of ERKl/2 was determined by Western blot using phospho-specific antibodies.Results: The addition of clenbuterol to the cells evoked a biphasic effect comprising an initial positive effect peaking at 2min, followed by a sustained negative effect leading to 40% decreases in basal phosphorylation of ERKl/2 after 30 min. Both the positive effect and negative effect are in a dose-dependent manner. The increase in phosphorylation was not altered in the presence or absence of extracellular calcium but was enhanced by ryanodine receptor agonist ryanodine. However, selective β₂ AR antagonist ICI 118551, pertussis toxin （PTX）, calcium chelator BAPTA-AM and ryanodine receptor antagonist ruthenium red significantly inhibited the positive effect of clenbuterol, but nifedipine only slightly inhibited it. Whereas, protein phosphatases inhibitor okadaic acid or sarcoplasmic reticulum Ca²⁺-ATPase （SERCA） inhibitor thapsigargin pretreatment reversed the negative effect of clenbuterol. Rp-cAMP attenuated the effect of clenbuterol on ERK1/2 dephosphorylation. Both the positive and negative effects on ERK1/2 phosphorylation of clenbuterol were reproduced by caffeine. But only thapsigargin reversed the negative effect of caffeine, okadaic acid was failed to abrogate it. Caffeine also mimicked the persistent effect of clenbuterol on phospholamban phosphorylation. Conclusions: The present data provide evidence that clenbuterol has a time- and concentration-dependent biphasic effect on ERK phosphorylation. Intracellular calcium mobilization plays an important role in clenbuterol induced biphasic effect on ERK activation. These results indicate that phosphorylation of ERK induced by clenbuterol is via G_i signaling pathway and is involved with the release of calcium from sarcoplasmic reticulum （SR） Ca²⁺ store, whereas, clenbuterol negatively regulates ERK1/2 through restore calcium into SR via cAMP dependent PLB phosphorylation. §2. Effect of clenbuterol on p38 MAPK signaling pathway in neonatal rat cardiomyocytesBackground: Previous studies suggest an involvement of p38 MAPK （mitogen activated protein kinase, MAPK） in cardiac apoptosis. More recent studies proposed that p38 MAPK is activated by β-AR stimulation via a G_i dependent mechanism, protecting myocytes against P-AR/G_s-mediated apoptosis in cultured adult rat cardiomyocytes. Our previous study demonstrated the biphasic effect of clenbuterol on ERK1/2 phosphorylation via both G_s and G_i coupling in neonatal rat cardiomyocytes, but clenbuterol phosophorylated ERK1/2 via G_i coupling in adult rat cardiomyocytes. Therefore, we assumed that clenbuterol-midiated phosphorylation of p38 MAPK in neonatal rat cardiomyocytes differed from that observed in adult rat cardiomyocytes.Methods: Cultured neonatal rat cardiomyocytes were pretreated with or without various agents. The phosphorylation of p38 MAPK was determined by Western blot using phospho-specific antibodies.Results: Clenbuterol induced p38 MAPK phosphorylation in a time-dose-dependent manner in neonatal rat cardiomyocytes. The phosphorylation of p38 MAPK induced by clenbuterol was inhibited by selectiveβ₂ AR antagonist ICI 118551 and protein kinase A inhibitor Rp-cAMP, and which was enhanced by PTX. Calcium channel blocker nifedipine and calcium chelator BAPTA-AM attenuated the effect of p38 MAPK phosphorylation induced by clenbuterol.Conclusions: These data demonstrated that in neonatal rat cardiomyocytes, β₂ AR activates p38 MAPK via G_s/cAMP-dependent signaling pathway, and which correlates with calcium concentration increased by extracellular calcium entry. Section 3Effect of clenbuterol on nitric oxide synthesis in neonatal rat cardiomyocytesBackgound: Nitric oxide （NO） is currently considered an important intracellular messenger in cardiovascular, immune and neural systems. This messenger causes diverse biological actions including vasodilation and cytotoxic or cytoprotective effect in various cells. The inducible NO synthase （iNOS） is expressed in target tissuses （vascular smooth muscle cells and cardiac myocytes） after stimulation with endotoxin and cytokines, and synthesis of relatively much greater amounts of NO by this way. Previous studies have shown that iNOS gene expression occurs frequently in failing human cardiac myocytes of patients with dilated cardiomyopathy, ischemic heart disease and vascular heart disease.In previous studies, cAMP augments cytokine stimulated nitric oxide synthesis in rat cardiac myocytes and dietary clenbuterol have a persisitnet effect on nitric oxide synthesis in rat cultures smooth muscle cells. Our previous study demonstrates that clenbuterol increased cAMP accumulation in neonatal rat cardiomyocyes. The purpose of this study was to investigate the effect of clenbuterol on nitric oxide synthesis in neonatal rat cardiomyocytes.Methods: Using the Griess reagent, we measured the production of nitrite, a stable metabolite of NO, by cultured neonatal rat cardiomyocytes. The expression of inducible NO synthase （iNOS） protein was assayed by Western blot. Results: Incubation of cardiomyocytes for 24 hours with interleukin 1β（IL-1β） caused a significant increase in NO production. Clenbuterol significantly augmented NO synthesis in IL-1β-stimulated but not in unstimulated cells in a dose-dependent manner. The selective β₂ AR antagonist ICI118551 inhibited the effect of clenbuterol. The clenbuterol-induced NO production by IL-1βstimulated cells was accompanied by increase iNOS protein accumulation. The non-selectiveβ-AR antagonist propranolol and the selective β₂ AR antagonist ICI118551 blocked the effect of clenbuterol.Conclusions: These result indicate that clenbuterol upregulates IL-1β-induced iNOS expression and nitric oxide synthesis in neonatal rat cardiomyocytes, which is mediated throughβ₂ AR.更多还原