【作者】 刘翠清；

【导师】 黄聿； 陆利民； 姚泰；

【作者基本信息】 复旦大学 ， 生理学， 2008， 博士

【摘要】 环核苷酸包括环-磷酸腺苷（cAMP,或称环腺苷酸）和环-磷酸鸟苷（cGMP,或称环鸟苷酸）。作为第二信使物质,它们在机体组织细胞的生物活性中起着重要作用。在血管,其胞内浓度的改变直接影响血管平滑肌的反应性。cAMP由ATP通过腺苷酸环化酶的作用而生成。腺苷酸环化酶既可以通过与之偶联的β肾上腺素受体激活（异丙肾上腺素为该受体的激动剂）,又可以被其激活剂（forskolin）直接激活,从而影响细胞内cAMP的水平。作为一种强效β肾上腺素受体激动剂,异丙肾上腺素（isoprenaline）不仅使血管平滑肌细胞内的cAMP浓度增加,还可以作用于内皮细胞,通过cAMP的作用最终激活eNOS-NO-cGMP信息通路,产生内皮依赖性作用,使血管舒张。cGMP要依靠鸟苷酸环化酶的作用,由GTP生成。在血管,鸟苷酸环化酶的激活通常需要内、外源性NO的作用,也就是说,内皮细胞的eNOS可以被乙酰胆碱（acetylcholine）或其他内皮依赖性舒血管物质激活,使NO生成增加;另外,硝普钠、硝酸甘油等作为NO供体,可以直接释放NO。NO扩散至血管平滑肌细胞,激活可溶性鸟苷酸环化酶（sGC）,进而增加cGMP在胞内的水平。cAMP和cGMP浓度增加的结果是降低胞内钙离子的浓度或其敏感性,或者通过其他机制最终引起血管舒张。同时,二者浓度也受其水解酶磷酸二酯酶（PDE）的影响。研究发现,某些生理病理情况会导致PDE活性的异常,从而直接改变血管的舒张状态。目前,NO供体被广泛用于心血管疾病的治疗,诸如动脉粥样硬化、高血压、冠心病等。但是,治疗过程中出现的硝酸甘油耐受大大限制了该药的临床应用。因此,深入了解耐受产生的机制并探寻合理有效的防治措施,成为亟待研究的课题。在某些病理生理情况下,血栓素（TxA₂）的生成增加,该物质会通过TP受体（thromboxane-prostanoid receptor）激活Rho/Rho激酶通路,引发钙敏感化或抑制NO产生,使血管功能发生异常改变。但是,这种异常改变对cAMP调节的血管舒张作用有何影响还未见报道。因此,在本实验中,我们选取大鼠双侧颈总动脉和胸主动脉为观察对象,分别用myograph和organ bath的方法对血管张力进行测定;用激光共聚焦方法对原代培养的大鼠胸主动脉内皮细胞胞内一氧化氮和钙离子浓度进行测定;用ELISA试剂盒检测血管组织内环核苷酸的水平;并用免疫印迹方法对蛋白水平进行检测,旨在观察:激活TP受体对cAMP调节的内皮依赖性和内皮非依赖性血管舒张的影响并探讨Rho激酶和PDE从中的作用;新型PDE5抑制剂T 0156对硝酸甘油耐受的影响以及作用机制是否和增加血管组织内cGMP水平有关。本课题研究对于进一步揭示某些病理生理情况下血管功能损伤的机制和预防具有重要的意义;并为PDE5抑制剂用于防治硝酸酯类耐受,提高抗缺血药应用的临床效果提供理论依据。本实验研究证实:1)Isoprenaline引起内皮依赖性血管舒张,用L-NAME或ODQ阻断NO通路后,isoprenaline的内皮依赖性舒血管作用消失;U46619(3*10^-8M,1*10^-7M,3*10^-7M)浓度依赖性地抑制isoprenaline引起的血管舒张,并且这一抑制作用被TP受体阻断剂S18886（0.3μM）完全阻断;在内皮功能完好的血管,Rho激酶抑制剂Y27632和HA 1077部分翻转了U46619对isoprenaline引发血管舒张的抑制作用;但是,在去除内皮的血管,两种药物却对U46619的抑制血管舒张作用无明显影响;当把NO通路（L-NAME或者ODQ）阻断后,U46619对血管舒张的抑制作用不再能被Rho激酶抑制剂（Y27632,HA 1077）所翻转。通过原代培养的大鼠主动脉内皮细胞研究证实,isoprenaline(1*10^-7M)可以引起内皮细胞NO产生增加和细胞内钙离子浓度增加,isoprenaline促进NO生成的作用被L-NAME阻断;U46619(1*10^-7M)对isoprenaline引起的NO产生和钙增加均有抑制作用,并且这一抑制作用被TP受体阻断剂S18886阻断;给予Y27632和U46619预孵20分钟,然后再给isoprenaline,显示U46619抑制isoprenaline引起NO生成增加和钙增加的作用被Y27632不同程度翻转。通过免疫印迹实验证明,U46619(1*10^-7M)对isoprenaline(1*10^-7M)引起的e-NOS磷酸化有抑制作用,并且这一作用被Y27632部分翻转。2)在去除内皮的血管,isoprenaline和forskolin（两种增加细胞内cAMP水平的药物）均可引起血管浓度依赖性舒张;U46619(3*10^-8M,1*10^-7M,3*10^-7M)浓度依赖性地抑制上述两种药物的舒血管作用,并且这种抑制作用均被TP受体阻断剂S18886（0.3μM）阻断;Rho激酶抑制剂Y27632显著增强了被U46619抑制的舒血管效应;PDE3和PDE4抑制剂（cilostazol和rolipram）,非选择性PDE抑制剂（IBMX）均部分翻转了U46619对血管舒张的抑制作用;但是,在没有U46619激活TP受体和Rho激酶的情况下,cilostazol和rolipram不能影响isoprenaline引发的血管舒张。cAMP测量结果显示,isoprenaline和forskolin均可引起血管组织内cAMP水平升高;U46619预处理抑制了这两种药物引起的cAMP浓度增加;并且这种抑制作用被PDE3、PDE4和Rho激酶抑制剂（cilostazol、rolipram和Y27632）所翻转。3)将内皮完好和去除内皮的大鼠离体主动脉环在硝酸甘油中预孵90分钟,均可以诱导血管产生“硝酸甘油耐受”,即血管对硝酸甘油的舒张反应减弱;新型PDE5抑制剂T 0156浓度依赖性地抑制了硝酸甘油耐受的形成,使产生耐受的血管舒张反应显著增强;在未产生耐受的血管,T 0156也能使舒张反应增强,但改变幅度要明显弱于产生耐受的血管;cGMP测量结果也显示,当血管发生耐受时,与非耐受情况下相比,cGMP浓度显著降低;T 0156作用结果使cGMP浓度明显增加,但耐受情况下cGMP浓度增加约为11.6倍,非耐受情况下增加约为6.6倍;在我们的实验条件下,TxA₂受体拮抗剂S18886,内皮素受体拮抗剂BQ610以及非选择性环氧合酶抑制剂indomethacin对硝酸甘油耐受的形成都没有影响。综上所述,通过本研究课题,可得出以下结论:1)TP受体激活可抑制isoprenaline的内皮依赖性舒张血管作用,这种抑制作用可能与Rho激酶被激活减少血管内皮细胞钙离子内流和磷酸化eNOS的水平,使eNOS活性降低,NO产生受抑有关;2)TP受体激活可抑制cAMP调节的非内皮依赖性血管舒张作用,这种抑制作用可能与Rho激酶被激活增强cAMP水解酶磷酸二酯酶的活性,降低cAMP浓度有关;3)PDE5活性升高使cGMP水平降低,这可能是硝酸甘油耐受情况下硝酸甘油舒张血管作用减弱的原因之一,新型PDE 5抑制剂T0156对硝酸甘油耐受的产生具有预防作用。更多还原

【Abstract】 The second messengers,cyclic adenosine monophosphate（cAMP） and cyclic guanosine monophosphate（cGMP）,are cyclic nucleotides and they play a key role in mediating a variety of cellular functions.In blood vessels,the intracellular level of both messengers is directly related to the relaxation of vascular smooth muscle cells（SMCs）. The production of cAMP is catalyzed by adenylyl cyclase from ATP.Stimulation of theβ-adrenoceptor,a G-protein-coupled receptor on cell membrane,or an adenylyl cyclase activator forskolin leads to an increased accumulation of intracellular cAMP.Isoprenaline is a potentβ-adrenoceptor agonist which stimulates increase in the cAMP level and thus relaxes vascular smooth muscle of blood vessels.On the other hand,growing evidence shows that vascular endothelium cells also expressβ-adrenoceptors and their activation results in cAMP accumulation causing endothelium-dependent vascular relaxation through stimulation of endothelial NO biosynthesis.Similar to cAMP,guanylyl cyclase is the enzyme which catalyses the formation of cGMP from GTP and can be activated by endogenous NO and NO donors.In conduit arteries,NO production is mediated by endothelial NO synthase and NO is released from endothelial cells in response to acetylcholine and other endothelium-dependent dilators.Endothelium-derived NO diffuses readily to the adjacent vascular smooth muscle cells,where it stimulates soluble guanylyl cyclase and hence increases cGMP levels.NO donors such as sodium nitroprusside（SNP） and nitroglycerin（NTG） can release NO directly.Both cAMP and cGMP decrease the intracellular calcium concentration or calcium sensitivity in vascular smooth muscle cells, resulting in vasorelaxation.Phosphodiesterases（PDEs） are a diverse family of enzymes that hydrolyze cyclic nucleotides and inhibition of the PDE activity enhances vasodilatation mediated by cyclic nucleotides.NO donors are widely used to treat cardiovascular diseases such as arteriosclerosis, hypertension,coronary artery disease.However,the development of nitrate tolerance limits therapeutic value of nitrate drugs.Therefore,it is necessary to understand the mechanisms involved in the development of nitrate tolerance in order to attenuate or prevent nitrate tolerance.It is known that the production of thromboxane A₂ can be greatly enhanced in response to both physiological and pathological stimuli and this prostanoid activates the Rho/Rho kinase pathway through TP receptor,leading to a robust calcium-sensitizing effect or reduced NO release and hence increased vascular tone.However,it is unclear whether the activation of TP receptor could also negatively affect the cAMP-mediated vasodilatation.In the present study,rat carotid arteries from both sides were isolated and suspended in myograph for the measurement of isometric force and rat aortas were suspended in organ baths for recording changes in vessel tension.Primary culture of rat aortic endothelial cells was performed and changes in NO production and intracellular calcium concentration were measured using fluorescent dyes.Levels of relevant proteins in vascular tissues were detected by Western blot analysis.The tissue content of cyclic nucleotides in arteries were chemically assayed by using ELISA kits.We aimed to examine:（1） whether activation of TP receptors attenuates endothelium-derived NO-dependent relaxation induced by theβ-adrenoceptor isoprenaline in rat carotid arteries via a Rho kinase-dependent mechanism, （2） whether the activation of TP receptors inhibits endothelium-independent relaxation in response to cAMP-elevating agents in rat carotid arteries via the increased activity of type 3 or type 4 phosphodiesterase（PDE3,PDE4）,and（3） whether the acute development of nitrate tolerance in isolated rat aortic rings exposed to NTG can be ameliorated by a new potent PDE5 inhibitor,TO 156 via increase of intracellular cGMP.The present study may be helpful to probe into the mechanism of vascular dysfunction in response to both physiological and pathological stimuli and provide theoretical significance for PDE5 inhibitors to increase therapeutic value of nitrate drugs by prevention of nitrate tolerance.The present study demonstrated that:1) Isoprenaline induces endothelial-dependent vasorelaxation,which is blocked by eNOS inhibitor（L-NAME） and sGC inhibitor（ODQ）. The relaxation to isoprenaline was concentration-dependently attenuated in rings pre-contracted with U46619(3*10^-8M,1*10^-7TM,3*10^-7M).Treatment with TP receptor antagonist（S18886,0.3μM） completely normalized isoprenaline-induced relaxation in the presence of U46619.In rings with endothelium contracted by phenylephrine plus U46619, treatment with Y27632 or HA 1077 partially,but significantly improved isoprenaline-induced relaxation.However,the inhibitors failed to influence relaxation in endothelium-denuded rings.Both inhibitors（Y27632 and HA 1077） failed to influence isoprenaline-evoked relaxation of rings with endothelium after inhibition of NO production by L-NAME or inhibition of sGC by ODQ.Imaging results with rat aortic endothelium cells showed that exposure to isoprenaline(1*10^-7 M) caused a slow but constant rise in dichlorofluorescein fluorescence intensity which was almost abolished after pre-exposure to L-NAME.However,exposure to isoprenaline(1*10^-7 M) caused a quicker rise in dichlorofluorescein fluorescence intensity and it was totally abolished in Ca2+ free PSS solution.But it increased sharply and significantly when CaC12（lmM） were administered. The rise in fluorescence intensity of both NO and Ca2+ was significantly inhibited by pretreatment with U46619(1*10^-7 M),which were totally abolished by S18886（0.3μM） pretreatment.Pre-stimulation with Rho kinase inhibitor,Y27632 reversed the inhibitory effect on the dichlorofluorescein fluorescence intensity of NO and Ca2+ by U46619 in endothelial cells.Western blotting data demonstrated isoprenaline(1*10^-7 M) induced phosphorylation of e-NOS at Ser1177 site which was significantly inhibited by U46619 (1*10^-7 M).Pre-exposure to Y27632 partially reversed the inhibited phosphorylation.2) Cyclic AMP-elevating agents,isoprenaline and forskolin,induce concentration-dependently relaxation in carotid artery rings without endothelium.The relaxation to both agents was concentration-dependently attenuated in rings pre-contracted with U46619 compared to phenylephrine-constricted artery rings.The attenuated relaxation was totally blocked by pretreatment with TP receptor antagonist,S 18886（0.3μM）.In rings contracted by phenylephrine plus U46619,treatment with Y27632 partially,but significantly improved cAMP-elevating agents-induced relaxation.Similarly,both PDE3 inhibitor cilostazol and PDE4 inhibitor rolipram and nonselective PDEs inhibitor IBMX improved the vascular relaxation in response to isoprenaline or forskolin.Inhibition of PDE5 with T0156 did not affect relaxation induced by the two cAMP-elevating agents. However,pretreatment with cilostazol or rolipram failed to alter the vasodilation to isoprenaline in endothelium-denuded rings contracted with only phenylephrine,cAMP level was much lower in U46619-pretreated carotid arteries in response to both isoprenaline and forskolin.PDE3,PDE4 and Rho kinase inhibitor（cilostazol,rolipram and Y 27632 respectivley） reversed the inhibited cAMP production by U46619.3) A vascular tolerance（attenuated relaxation） to the NO donor,nitroglycerin can be readily induced by pre-exposure to NTG in rat isolated thoracic arteries with and without endothelium.The inhibition of PDE5 with T0156 improved the reduced relaxation to NTG in tolerant arteries with much higher extent than in non-tolerant arteries,cGMP level was much lower in tolerant arteries than in non-tolerant arteries and T0156 increased cGMP level in response to 1μM NTG,11.6-fold in tolerant rings,6.6-fold in non-tolerant rings. Nitrate tolerance was not associated with changes in the activity of thromboxane A₂ receptors,COX-derived prostanoids,or endothelin A receptor under current experimental condition.In summary,the above-mentioned results demonstrate that:1) Activation of TP receptor attenuated the cAMP-mediated endothelium-dependent relaxation through stimulation of Rho kinase,which inhibited the activity of eNOS through reducing calcium influx and eNOS phosphorylation,thus leading decreased production of NO.2) Stimulation of TP receptor impairs vascular endothelium-independent relaxation to cAMP-elevating agents.This inhibited relaxation induced appeared to be through activation of Rho kinase by increasing activity of cAMP-hydrolyzing PDEs,leading to decreased cAMP level.3) The present pharmacological results also showed a significant contributing role of the increased PDE5 activity in the acute development of nitrate tolerance in rat aortas in vitro.T0156 was proved to be a useful drug in maintaining vasodilator responses to nitroglycerin,the effect of which are frequently attenuated upon continuous administration.更多还原