MS23对血管平滑肌收缩、血压和血流动力学的影响

【作者】 刘宇；

【导师】 张明升；

【作者基本信息】 山西医科大学 ， 生理学， 2007， 博士

【摘要】 研究背景:第二信使cAMP在多种组织的多种信号传递中起着极其重要的作用,尤其是在心脏和平滑肌系统。许多生物活性物质通过使细胞内cAMP含量增加,进而通过复杂的生物反应过程,最终引发生物效应。在平滑肌细胞内cAMP浓度增高可引起肌张力降低,在血管表现出血管扩张,外周阻力下降;在呼吸道平滑肌可表现出气道阻力下降;而在心脏则可表现出兴奋。细胞内cAMP的水平受合成速度和降解速度的调控。其合成速度取决于腺苷环化酶活性;而降解速度取决于环核苷磷酸二酯酶（cyclicnucleotide phosphodiesterases,PDEs）。PDEs特异性将cAMP和（或）cGMP的3′,5′-环磷酸核裂解为相应的5′-核苷酸,即分别转化为无活性的5′-GMP和5′-AMP,从而其“第二信使”作用终止。在心血管系统主要分布的PDEs为PDE1、PDE2、PDE3、PDE4和PDE5,它们对心血管系统的调控有着至关重要的作用,因而PDEs抑制剂通过使细胞内cAMP升高,而产生诸多的突出的药理学作用。临床上,PDEs抑制药已广泛应用于治疗慢性心功能低下、外周血管阻力增高性疾病、阳痿、呼吸道阻力增加性疾患如哮喘、过敏性疾患和抑郁症等。MS23为一新合成的化合物,由美国南卡大学药学院合成,后来为减少其不良反应,增强其治疗作用,又对MS23的化学结构进行了一系列的改进,并通过药理学和毒理学研究筛选出类似物MP01。前期研究结果表明,MS23浓度依赖性抑制豚鼠脑组织的cAMP降解速度,而对cGMP的降解无影响。酶学研究提示MS23属于PDE4和PDE3抑制剂,而对其他PDEs无显著性影响。离体实验表明,MS23对血管和气道平滑肌有松弛作用,进一步研究证实这一平滑肌松弛作用与其抑制PDEs相一致。前期研究还提示,MS23及其类似物有望成为抗高血压、治疗慢性阻塞性肺疾患的良药。鉴于此,其对血管平滑肌作用特点、相对作用强度及其作用机制等方面值得进一步研究。研究目的:观察MS23和MP01对不同的收缩剂所致的来自大鼠不同组织脏器的血管平滑技≌帕ι叩挠跋?一氧化氮（NO）合成酶抑制剂和内皮对MS23和MP01作用的影响。本实验中刺激剂选用了去甲肾上腺素（noradrenaline,NA）、高钾（60 mmol/L KCl）、血栓素A₂类似物(9,11-Dideoxy-11α,9α-epoxymethanoprostaglandin F_2α,U46619)和苯肾上腺素（phenylephrine,PE）;动脉标本我们选用了胸主动脉、冠状动脉、大脑中动脉、肾动脉和肠系膜动脉。研究方法与结果:一、药物对大鼠胸主动脉环的舒张作用:成年雄性Wistar大鼠,210～250g,击头致昏,立即开胸取主动脉,迅速置于O₂饱和的4℃生理盐溶液（physiological salt solution,PSS）中。剔除脂肪及周围结缔组织后,剪成3-4mm的血管环。血管环用两根不锈钢微型挂钩贯穿血管管腔,水平横向悬挂在浴管内,下方固定,上方以一细钢丝连于张力换能器（PowerLab）。经PowerLab计算机生物信号采集分析系统记录血管的舒缩变化。浴管内含有通以100%CO₂、37℃的PSS 10 ml。为了使标本处于长度—张力曲线的最适点开始收缩,每个血管环悬挂在浴管后,立即给予2.0 g的前负荷,温育平衡2 h。在平衡稳定期间不断调整张力,使之维持稳定,并每20min更换一次新鲜的PSS。所有动脉环用60mmol/L KCl多次刺激,当标本对刺激稳定时,即相邻连续两次同样的刺激所引起的收缩幅度差别＜10%时;且在此基础上10^-5mol/L乙酰胆碱（Ach）所引起的舒张幅度≥30%时,认为血管环组织结构完整,功能正常,开始正式实验,观察药物干预的作用。结果为,60mmol/L KCl和10^-5mol/L NA可使大鼠胸主动脉环产生的张力分别为2.34±0.94g和1.58±0.62g。MS23、MP01和氨茶碱在10^-6～3×10^-5mol/L浓度范围内对高钾引起的预收缩均表现出浓度依赖性舒张效应。其最大舒张率分别为90.5±8.2%、94.5±14.6%、55.2±27.6%,其EC₅₀分别为9.19×10^-6、7.26×10^-6、2.67×10^-5mol/L。对NA引起的预收缩此三药也表现出浓度依赖性舒张作用,其最大舒张率分别为76.2±15.4%、86.6±28.4%、53.6±19.8%,EC₅₀分别为1.44×10^-5、6.71×10^-6、2.69×10^-5mol/L。从EC₅₀值判断,MS23和MP01的舒张作用强度相近,MP01弱强于MS23,而显著强于氨茶碱。本实验还观察了预先给予MS23或MP01对NA引起大鼠胸主动脉环收缩的浓度—收缩量效曲线的影响。结果为,MS23和MP01在10^-6～3×10^-5mol/L浓度范围内浓度依赖性抑制NA引起的主动脉环收缩,使NA浓度—效应曲线非平行性右移,并抑制最大反应,使最大效应分别下降74.7%和70.4%,IC₅₀值分别为4.84×10^-6mol/L和1.97×10^-6mol/L。这些实验结果显示,MS23、MP01和氨茶碱对高钾和NA引起的主动脉平滑肌痉挛具有直接舒张作用或抑制收缩作用,而对此两种刺激剂无明显的选择性。左旋硝基精氨酸甲酯（L-NAME,NG-nitro-L-arginine methylester ester）为一氧化氮（NO）合成酶抑制剂。0.1 mmol/L的L-NAME可取消Ach的舒张作用,而对MS23的血管舒张作用无显著性影响,提示MS23的舒张血管作用与NO产生无关。本实验结果还表明内皮是否存在对MS23和MP01引起的胸主动脉环舒张作用亦无显著性影响。这些结果佐证了MS23和MP01的血管舒张作用来源于其PDE抑制作用,与前期结果相一致。二、药物对大鼠小动脉环的舒张作用:大鼠脱臼后,立即取出肠系膜、肾脏、心脏和大脑,浸入4℃的PSS液中。将取出的肠系膜和肾脏组织浸入含有4℃PSS液的平皿,用大头针固定动脉主干和周围组织,分离去除动脉周围的脂肪组织,选取动脉的三级分支血管,剪取2mm左右的血管环。大脑中动脉环的制备:钝性分离颈内动脉的延续—大脑中动脉,剥离大脑膜,钝性分离大脑中动脉,在靠近内侧处剪取2mm左右的血管环。冠状动脉血管环的制备:钝性分离冠状动脉,在前降支处剪取2mm的血管环。将两根直径为40gm的钨丝穿入管腔,固定血管环在Multi Myograph System-610M浴槽内传感器上。浴槽内含有5ml PSS,持续通以100%O₂,温度控制在37℃,平衡60min后开始实验。平衡期间每隔15min用预热（37℃）的新鲜PSS液更换浴槽内液体一次。血管环的张力变化通过DMT的换能系统采集,并用Chart 5.3生物信号分析处理软件记录在微机上。为了使血管环处于最佳反应状态,分别调整肠系膜动脉、肾动脉、大脑中动脉和冠状动脉的跨壁压,使其分别保持在相当于100mmHg、80mmHg、80mmHg和80mmHg的基础压力状态。为了检测血管环在离体状态下对血管活性物质的反应性,在开始正式实验之前,用120mmol/L的KCl预收缩,当收缩达稳定的平台时用Ach(10^-5mol/L)舒张,连续三次。当相邻两次刺激的收缩幅度差别不大于10%时;在此基础上当Ach所引起的舒张幅度≥20%时,认为血管环组织结构完整,功能正常,开始正式实验。小动脉环实验结果为,60 mmol/L KCl使冠状动脉、大脑中动脉、肾动脉和肠系膜动脉环产生的收缩张力分别为8.67±6.73 mN,2.16±1.10 mN,4.05±2.13 mN,8.89±4.34 mN。在此基础上,MS23、MP01和氨茶碱浓度依赖性舒张这些小动脉,此四个脏器血管对此三药舒张作用的敏感程度均无显著性差异。MS23、MP01和氨茶碱三药比较,对此四个部位的血管的舒张作用以氨茶碱最弱,MS23和MP01作用强度相当。最大浓度的氨茶碱(3×10^-5mol/L)仅可产生35.2%的舒张,无法计算其舒张EC₅₀。与此三药对胸主动脉环的舒张作用比较,可以看出MS23和MP01对小血管的舒张作用与其对胸主动脉环的舒张作用相当,氨茶碱对小血管的舒张作用弱于其对主动脉环的作用。本实验还在肾血管观察比较了MS23、MP01和氨茶碱对不同刺激剂引起预收缩的舒张效应。在10^-6～3×10^-5mol/L浓度范围内,MS23、MP01和氨茶碱浓度依赖性舒张由高钾（60 mmol/L）、U46619（1gmol/L）和NA（10gmol/L）引起的大鼠肾动脉预收缩。高钾和U46619引起的预收缩对此三药的舒张作用较敏感,而NA引起的预收缩对它们的舒张作用不敏感。对NA引起的收缩,3×10^-5mol/L的MS23、MP01或氨茶碱最大仅产生约30%的舒张效应。提示MS23等PDE抑制药对不同刺激有一定的选择性,其意义有待于进一步研究。最后本实验在肾动脉环还观察了MS23对异丙肾上腺素（isoprenaline,ISO）舒血管作用的影响。结果表明,ISO(3×10^-8mol/L)在大多数大鼠肾动脉环对60 mmol/L KCl引起的预收缩具有剂量依赖性舒张作用,但舒张作用较弱,其最大舒张效应仅为（42.8±16.4）%。但当在自身单独存在情况下不足以产生明显舒张作用的低浓度的MS23（1μmol/L）存在的情况下,ISO的舒张率显著增大为（76.8±19.8）%（P＜0.01）;MS23存在的使ISO舒张效应EC₄₀减小了近20倍,提示MS23可改善血管对ISO的舒张效应。结论:1.在大鼠胸主动脉环,MS23、其类似物MP01和氨茶碱对高钾和NA引起的收缩有显著的舒张作用,对刺激剂无明显的选择性;且其血管舒张作用与NO产生及血管内皮是否存在无关。2.MS23和MP01对高钾引起的大鼠胸主动脉环、冠状动脉、大脑中动脉、肾动脉和肠系膜动脉环收缩均有突出的舒张作用,其舒张强度相当,没有明显的血管来源差异性;而氨茶碱对小血管的舒张作用较其对主动脉的舒张作用为弱。3.在大鼠肾动脉,高钾和U46619引起收缩对MS23和MP01的舒张作用较敏感,而NA引起的收缩对MS23和MP01的舒张作用不敏感。4.MS23增强ISO对大鼠肾动脉的舒张作用。研究背景:第二信使cAMP在心脏和血管平滑肌的功能调控方面具有极其重要的作用。在心脏,cAMP在细胞内增高,继而通过复杂而精妙的一系列级联反应最终表现出兴奋作用,包括心肌收缩力增强,传导速度加快,心跳加速;而在血管平滑肌细胞则表现出收缩受抑,张力下降,血管扩张,外周阻力降低。因此抑制降解细胞内cAMP的环核苷磷酸二酯酶（cyclic nucleotide phosphodiesterases,PDEs）对心血管系统可产生突出的药理学作用,PDE抑制药在临床用于治疗多种疾病。PDE抑制药具有显著的扩张血管、加强心肌收缩力、降低呼吸道阻力以及抗炎作用,临床广泛用于治疗慢性呼吸道阻塞性疾患、血管外周阻力增加性疾患和心功能低下性疾患。大量临床研究表明,短期使用氨茶碱和米力农等PDE抑制药对慢性心衰可产生积极的治疗作用,但长期使用则可产生诸多严重的不良反应,如心肌肥厚、心功能异常、心律失常甚至心衰。如果适当去除或抵消PDE抑制药的兴奋心脏所带来的不利作用,而保留其外周血管扩张作用,也许可增高PDE抑制药的安全性、可用性和有效性。MS23原设计为PDE4抑制剂,拟开发为治疗慢性阻塞性肺疾患的药物,因为PDE4抑制剂具有抑制炎症和扩张支气管平滑肌作用。酶学实验证实MS23为PDE4抑制剂,但对PDE3也有相当抑制作用。离体实验证实MS23具有舒张豚鼠支气管平滑肌和大鼠血管平滑肌作用,膜片钳实验表明它可抑制心肌细胞钙离子通道。但毒理学实验证实MS23具有较强的毒性。为减少减轻其不良反应,增强其治疗作用,又对MS23的化学结构进行了一系列的改进,合成了一系列类似化合物,并最后筛选出MP01。从酶学实验结果看,MP01对PDE4和PDE3的抑制作用强度与MS23相当,但毒性作用显著减少减轻。前期离体实验研究表明,MS23具有松弛平滑肌和钙拮抗作用。那么MS23和MP01对整体动物的心血管系统究竟有何影响有待进一步研究。研究目的:本实验拟在麻醉正常血压大鼠和麻醉高血压大鼠及清醒无拘束高血压大鼠,观察MS23对血压和血流动力学指标的影响,探明其对整体动物心血管系统的药理作用特点,并与MP01进行比较,为其将来申报新药提供临床前药理学研究资料,并为其将来临床应用提供基础实验依据。研究方法与结果:一、对麻醉正常血压大鼠血压及其它血流动力学指标的影响:健康雄性Wistar正常血压大鼠64只,体重240～260g。将动物随机平均分为MS23低（0.1 mg/kg）、中（0.3 mg/kg）、高（1.0 mg/kg）剂量组、MP01低（0.1 mg/kg）、中（0.3 mg/kg）、高（1.0 mg/kg）剂量组、盐水正常对照组和氨茶碱（10 mg/kg）组。25%乌拉坦4ml/kg腹腔注射麻醉后施颈部手术,分别分离两侧颈总动脉,一侧颈外静脉,穿线以备插管。分离右侧颈总动脉时尽量向头端分离,结扎远心端,近心端用动脉夹夹闭,用眼科剪在靠远心端结扎处的动脉上剪一斜口,由此将充满肝素生理盐水的细塑料管（管内排尽气泡）朝心脏方向插入动脉管内,并用丝线将血管及其内的导管打一活结,以防出血。然后,缓慢推进导管,并同时观察压力波形的变化和体会在导管接近主动脉瓣时遇到的血流冲击感。一旦出现典型的左心室内压力变化波形,便停止推进,并将血管及插管结扎牢固。插管室内压力换能器连接至PowerLab生物信号采集与分析系统,记录心脏血流动力学各指标。将连有血压压力换能器的另一导管插入左侧颈总动脉,记录动脉血压。将已分离的一侧颈外静脉远心端用线结扎,在结扎处的近端剪一斜口,将连接2ml注射器已注满肝素生理盐水的静脉套管（排尽管内气泡）向心脏方向插入,用线将插管与静脉结扎,备注射药物用。同时连接导联线,记录心电图。上述所有指标稳定后以微机同步记录一段正常心室内压波、血压波和心电图,然后分别按上述剂量静脉给予各种药物,药后连续记录上述各指标2h,观察药物对麻醉大鼠心脏血流动力学指标。记录的指标有:左心室收缩压（LVSP）、左心室舒张末压（LVEDP）、左心室压上升速率(+dp/dt_max)、左心室压下降速率(-dp/dt_max)、平均动脉压（MAP）、心率（HR）和心电图各指标。结果以给药前（药后0min）为对照,分别计算药后1、5、10、20、30、60、90和120min时各指标与0min时（药前）的差值,以（?）±s表示,t检验比较各组不同时间点各指标之间差异的显著性,单因素方差分析比较各组间同时间点同一指标差异的显著性。麻醉正常血压大鼠实验结果表明,MS23 0.1 mg/kg、0.3 mg/kg、1.0 mg/kg iv剂量依赖性地降低麻醉正常大鼠血压,药后20～30min时血压降低作用达峰值,平均动脉压（MAP）分别降低了22.1±7.3、36.9±20.2、53.1±14.7 mmHg,相当于药前的78.5%,63.8%和48.4%（P＜0.05或P＜0.01）。其血压降低作用约持续90min,药后120min时MAP可恢复至药前的91.8%,87.5%,77.3%。在对血压的影响方面,MP01低、中、高剂量组和氨茶碱组的实验结果与MS23结果相类似。MS23和MP01显著减慢麻醉正常大鼠心率,MS23 1.0 mg/kg和MP01 1.0 mg/kg可使心率从给药前的237.6±26.8、230.2±32.6 beats/min分别最低减慢为204.8±31.5、204.5±22.4 beats/min（P＜0.05）。在心电图上表现为,MS23和MP01显著性延长PR间期,而对心电图QRS波宽、QT间期、T波和ST段均无显著性影响。心率和心电图变化在药后30min最为明显。相反,氨茶碱10mg/kg可使心率加快,从药前的246.0±28.7 beats/min到药后60min增快为274.8±22.7 beats/min（P＜0.05）,其心电图以PR间期缩短为主,对心电图QRS波宽、QT间期、T波和ST段均无显著性影响。MS23 0.1 mg/kg、0.3 mg/kg、1.0 mg/kg iv剂量依赖性降低麻醉正常大鼠LVSP。仅在MS23 1.0 mg/kg剂量组出现+dp/dt_max降低;MP01的作用性质和强度与MS23相近。与MS23和MP01结果不同,氨茶碱显著性增大LVSP和+dp/dt_max。MS23、MP01和氨茶碱对LVEDP和-dp/dt_max均无显著影响。二、对麻醉自发性高血压大鼠血压及其它血流动力学指标的影响:雄性自发性高血压大鼠24只,体重220～250g,平均动脉压为164.2±12.6 mmHg。将动物随机平均分为MS23（0.3 mg/kg）组、MP01（0.3 mg/kg）组、盐水正常对照组和氨茶碱（10 mg/kg）组。麻醉、手术操作和记录指标和基本方法等同上。MS23 0.3mg/kg和MP01 0.3 mg/kg iv显著降低麻醉自发性高血压大鼠的MAP,药后1min开始出现显著性降压作用,药后60min左右时血压降低最显著,MAP分别降低52.3±15.8、54.6±18.6 mmHg,分别相当于药前的68.1%,64.2%（P＜0.05）,降压作用持续时间大于2h。药后120min时血压分别恢复至药前的88.5%和83.1%。氨茶碱10 mg/kg的降压作用峰值为30.3±17.4mmHg,出现在药后10min,其降压作用持续时间显著短于MS23和MP01。MS23 0.3 mg/kg和MP01 0.3 mg/kg iv显著减慢麻醉高血压大鼠心率,心率分别减少39.1±9.8 beats/min、50.2±4.9 beats/min。心电图主要表现为,PR间期延长,而QRS波宽、QT间期、T波和ST段均无显著改变。与MS23和MP01结果相反,氨茶碱则对麻醉高血压大鼠心率有加快作用,药后30min时加快了29.4±4.8 beats/min（P＜0.05）。MS23和MP01对麻醉高血压大鼠心脏LVSP和+dp/dt_max有降低作用,而氨茶碱却显著增大LVSP和+dp/dt_max。MS23、MP01和氨茶碱对LVEDP和-dp/dt_max均无显著影响。三、对清醒无拘束高血压大鼠收缩压和舒张压的影响:雄性清醒无拘束自发性高血压（166.5±10.7mmHg）大鼠的血压记录采用遥感测压监测系统（Data science international,美国DSI公司）,该系统通过一个小型无线电装置（植入子）将受试动物的血压信号发射出来,再经过无线电接受装置接受记录入电脑中。戊巴比妥钠40 mg·kg^-1腹腔注射麻醉后,将与压力换能器相连的血管导管插入腹主动脉,将植入子固定在腹腔。手术后动物恢复7d,当血压稳定后,开始观察药物对血压的作用。该系统可长期不间断监测受试大鼠的动脉血压,血压测定在动物处于完全清醒无打扰的状态下进行。我们观察了灌胃给药MS23和MP01对高血压大鼠收缩压（SBP）和舒张压（DBP）的影响。结果为,MS23 1.0mg/kg灌胃后30min开始出现血压降低作用,约4h时作用达高峰,降压作用约持续6h,对DBP的作用更明显。MP01降压作用和特点与MS23相同。降低动脉压的同时,MS23和MP01对清醒无拘束自发性高血压大鼠的心率无显著性影响,甚至有增加心率的趋势。结论:1.MS23 iv降低麻醉正常血压和高血压大鼠的MAP、LVSP和+dp/dt_max,减慢心率,而对LVEDP、-dp/dt_max和心电图其它指标无显著性影响。2.MP01对麻醉大鼠MAP、HR、LVSP和+dp/dt_max的作用,在作用特点和作用强度方面均与MS23相近;而氨茶碱则不同,氨茶碱降低MAP,但增快心率、增大LVSP和+dp/dt_max。3.MS23和MP01对清醒无拘束高血压大鼠的SBP和DBP均有显著的降低作用,且以对DBP的作用更明显,但对心率无显著性影响。更多还原

【Abstract】 Cyclic adenosine Y,5’-monophosphate（cAMP）is synthesized from ATP by at least 9 closely related isoforms of adenylyl cyclases.Cyclic AMP,as a "second messenger",play a key role in biological signal transmissions in many living cells,especially in the myocardial and various smooth muscles.A variety of biological active substances elicit an intracellular cAMP elevation followed by a cascade of special and sophisticated biological reactions,and eventually produce corresponding observed effects.In response to intracellular elevation of cAMP,vascular and respiratory smooth muscles relax,while the myocardial cells exhibit excitation.Cyclic AMP is degraded by at least 11 closely related isoforms of cyclic nucleotide phosphodiesterases （PDEs）.These PDEs specifically cleave the 3’,5’-cyclic phosphate moiety of cAMP and/or cGMP to produce the corresponding inert 5’-nucleotide and terminate second messenger activity of cyclic nucleotides.In the cardiovascular system,the main isoforms of PDEs are PDE1,PDE2, PDE3,PDE4 and PDES.An overwhelming and increasing amount of studies show that these PDEs play crucial roles in the control and regulation of the cardiovascular system.Therefore, agents which inhibits PDE or PDEs may exert a succession of pronounced effects on various cells.PDEs inhibitors have been clinically used to treat chronic heart failure,conditions with increased peripheral vascular resistance,chronic obstructive pulmonary diseases,anandria, allergy and depressive disorders.MS23 is a brand new compound,synthesized by Department of Basic Pharmaceutical Science of Pharmacy College of University of South Carolina.MP01 is an analog of MS23,with fewer and milder toxical effects.Previous studies indicated that MS23 concentrationdependently inhibited cAMP degradation,without effect on cGMP in guinea pig brain. Enzymogical studies verified that MS23 specially inhibited PDE4 and PDE3 without significant effect on other PDEs.Previous studies also suggested that MS23 has relaxation effect on vascular and respiratory smooth muscle,and that it might be used to treat hypertension and chronic obstructive pulmonary diseases.Therefore,it is necessary to clear out pharmacological profiles of MS23 on vascular smooth muscle.The present studies were designed to observe and compare the effects of MS23 and MP01 on the contractions induced by noradrenaline,60 mmol/L KCl,thromboxane A₂ analog U46619 and phenylephrine in rat isolated thoracic aortic rings and coronary,middle cerebral,renal and mesenteric artery rings.Relaxation on precontractions in rat isolated thoracic aortic rings.Male Wistar rats weighing 200～250g were killed by cervical dislocation and thoracic aortas were removed and placed in chilled（4℃）physiological salt solution（PSS）which was gassed with 100%O₂.The blood vessels were cleaned of connective tissue and fat,and cut into 3-4mm long rings.Extreme care was exercised during preparation of the rings to preserve the integrity of the endothelium.The rings were mounted in water-jacketed tissue baths containing 10.0 ml of PSS（bubbled with 100%O₂ and maintained at 37℃）for measurement of isometric tension. Passive tension was adjusted to 2.0 g and all subsequent measurements represent force generated above this baseline.A 2h equilibration period was allowed before any experimental intervention, and during equilibration,the bath was flushed every 20 minutes with the fresh PSS.After equilibration,the rings were activated 2～5 times with PSS containing 60 mmol/L KC1 for 10 minutes.The integrity of endothelium was presumed by observation that the relaxation induced by 10^-5mol/L acetylcholine on 60 mmol/L KC1 contraction was greater than 30%.When the contraction induced by 60 mmol/L KC1 and the relaxation induced by 10^-5mol/L acetylcholine were repeatable（the change was less than 10%between successive contractions or relaxation）, the effects of the drugs to be tested were observed.The contraction tensions induced by 60mmol/L KCl and 10^-5mol/L NA were 2.34±0.94g and 1.58±0.62g,respectively.In concentration range of 10^-6～3×10^-5mol/L, MS23,MP01 and aminophylline concentration- dependently relaxed the precontraction induced by 60 mmol/L KCI in rat isolated thoracic aortic rings.The maximum relaxation were 90.5±8.2 %,94.5±14.6%,55.2±27.6%;the calculated EC5o were 9.19×10^-6,7.26×10^-6,2.67×10^-5 mol/L respectively.The maximal relaxation of MS23,MP01 and aminophylline in the precontraction induced by noradrenaline(10^-5mol/L)were 76.2±15.4%,86.6±28.4%, 53.6±19.8%;EC₅₀were 1.44×10^-5,6.71×10^-6,2.69×10^-5mol/L respectively. Preincubation with MS23 or MP01 shifted the noradrenaline concentration- contraction curve in a nonparallel manner to the right with the maximal response depressed.The calculated IC₅₀for MS23 and MP01 were 4.84×10^-6mol/L and 1.97×10^-6mol/L respectively.These results showed that MS23,MP01 and aminophylline have direct relaxation effects on KCl- and noradrenaline- induced contractions without any preference between these two stimulators;and that the relaxation potency of MS23 is nearly equal to MP01,but stronger than aminophylline.0.1 mmol/L NG-nitro-L-arginine methylester ester（L-NAME）,a nitric oxide（NO） synthetase inhibitor,had no effects on MS23 or MP01 induced relaxation,while abolished acetylcholine induced relaxation.Deprivation of endothelium did not effect the relaxation induced by MS23 or MP01.These results indicated that NO production and endothelium are not involved in the mechanism of relaxation induced by MS23 or MP01;and these results are also agree with the previous conclusion that MS23 is a PDE inhibitor.Relaxation of MS23 on precontractions in rat isolated coronary,middle cerebral,renal,and mesenteric artery ringsTo study and compare the relaxation effects of MS23,MP01 and aminophylline on precontractions in rat isolated small artery with different origin,rat coronary,middle cerebral, renal,and mesenteric arteries were isolated.The third-order branches of the superior mesenteric artery and renal arteries,middle cerebral artery and coronary artery were isolated and cut into 2mm-long rings.The rings were mounted on a wire myograph（Multi Myograph System-610M,Danish Myo Technology A/S,Denmark）.The rings were normalized according to standard procedures and stretched to a state equal to 100mmHg,80mmHg,80mmHg, 80mmHg,respectively.The rings were equilibrated for at least for 1h,and then contracted and relaxed as above mentioned in the experiments with thoracic aortic rings before use of any relaxants to be tested.First,we studied and compared the effects of MS23,MP01 and aminophylline on the precontraction induced by 60 mmol/L KCl in rat isolated coronary,middle cerebral,renal, and mesenteric artery rings.The contraction tensions induced by 60 mmol/L KCl in rat isolated coronary,middle cerebral,renal,and mesenteric artery rings were 8.67±6.73 mN, 2.16±1.1 mN,4.05±2.13 mN,8.89±4.34 mN,respectively.MS23,MP01 and aminophylline concentration- dependently relaxed this contraction,and these three relaxants showed no preference among the four arteries with different origin.The relaxation values of EC₅₀on 60 mmol/L KCl induced contraction in these small arteries with different origin were around 10^-5 mol/L for MS23 and MP01.The maximal relaxation response produced by aminophylline（3×10⁵ mol/L）was only 35.2%.By comparison,it was found that the relaxation effects of both MS23 and MP01 on these small artery rings were comparably potent to their effects on aortic rings, while the relaxation effect of aminophylline on these small artery rings were much weaker than its effects on aortic tings.Secondly,we compared the effects of MS23,MP01 and aminophylline on the precontraction induced by 60 mmol/L KCl,noradrenaline（10μmol/L）and thromboxane A₂ analog U46619（1μmol/L）in rat isolated renal artery rings.The contraction tensions induced by 60 mmol/L KCl,noradrenaline and U46619 in rat renal artery rings were 4.05±2.12mN,2.42±2.55mN,7.02±3.45mN,respectively.60 mmol/L KCl and U46619 induced contractions were more sensitive to the relaxation of MS23,MP01 and aminophylline,while noradrenaline-induced contraction was more resistant to these three relaxants.These three relaxants,at maximal concentration(3×10^-5mol/L),only produced relaxations of less than 30%on noradrenaline-induced contraction.These results suggested that relaxation effects of MS23,MP01 and aminophylline have selectivity over the stimulators.The significance of the selectivity needs further clarification.Finally,we observed the effect of MS23 on isoprenaline induced relaxation in rat isolated renal artery rings.Isoprenaline(3×10^-8mol/L)concentration dependently relaxed the 60 mmol/L KCl induced contraction in most of rat isolated renal artery rings,with the maximal relaxation response being（42.8±16.4）%.MS23（1μmol/L）itself produced no significant relaxation.However,the relaxation response of isoprenaline was significantly increased（76.8±19.8 vs 42.8±16.4）%（P＜0.01）in presence of MS23（1μmol/L） Presence of MS23 decreased EC₄₀of isoprenaline by nearly 20 fold.This result suggested that MS23 ameliorate vascular relaxation effects of isoprenaline,and also given us a clue that MS23 may improve vascular relaxation state.CONCLUSIONS1.MS23,its analog MP01 and aminophylline concentration dependently relax the precontractions induced by 60 mmol/L KCl and noradrenaline in rat isolated thoracic aortic rings.The relaxation effects of the three tested relaxants manifest no selectivity between the two stimulators.NO production and presence of endothelium are not involved in mechanism of the relaxation.2.MS23 and MP01 have profound relaxation effects on 60 mmol/L KC1 induced contraction in rat isolated thoracic aortic rings,and coronary,middle cerebral,renal and mesenteric artery rings,without any preference among these five vascular beds tested;while the relaxation potency of aminophylline on the small arteries is much weaker than its effect on thoracic aortic rings.3.60 mmol/L KCl and U46619 induced contractions are more sensitive to the relaxation of the three tested PDE inhibitors,while noradrenaline- induced contractions is more resistant to them in rat isolated renal artery rings.4.MS23 enhances vascular relaxation effects of isoprenaline in rat isolated renal artery rings. BACKGROUND:Cyclic AMP,as an important intracellular second messenger,plays key role in regulating the functions of the myocytes and vascular smooth muscle.Increased intracellular cAMP levels are followed by a cascade of biological sophisticated,subtle and refined reactions,and eventually result in observed effects.For instance,cardiac excitations,such as enhanced contractility,accelerated heart rate and other positive effects are exhibited,whereas vascular contraction inhibition or relaxation are manifected in response to increased intracellular cAMP. The effects of cAMP is terminated by degradation which is carried out by cyclic nucleotide phosphodiesterases（PDEs）,and therefore,inhibition of PDEs would produce marked pharmacological effects to the cardiovascular system.Actually,PDE inhibitors are now widely used clinically to relieve various diseases such as chronic heart failure,diseases with increased peripheral vascular resistance,chronic obstructive pulmonary diseases,anandria etc.Clinical studies reveal that short term use of PDE inhibitors such as aminophylline and milrinone produces beneficial therapeutic effects in treating chronic heart failure,but chronic use of them may bring about severe adverse effects such as myocardial hypertrophy,arrhythemia,cardiac dysfunctions.Elimination or extenuation of the harmful stimulatory effects and reservation of the beneficial effects of PDE inhibitors on the heart would make PDE inhibitor safer,more applicable and more effective.MS23 was designed to inhibit PDE4 and to cure chronic obstructive pulmonary diseases, because most of PDE4 inhibitors have anti-inflammatory effects and relax bronchial smooth muscle.Enzymological studies showed that MS23 selective inhibited PDE4,and also had some inhibitory effects on PDE3.MS23 was found to relax bronchial and vascular smooth muscle,and to inhibit myocardial Ca²⁺influx in previous studies.Toxicological studies showed that MS23 could cause severe toxic effects.A series of modifications on the molecule of MS23 were made, and systemic screening on the synthesized analog candidates gave birth to MP01.Enzymological studies showed that the inhibitory effects of MP01 on PDE4 and PDE3 were comparable to those of MS23,but toxic effects were much fewer and milder.Previous studies revealed that MS23 and MP01 had remarkable relaxation effects on rings of arteries isolated from various organs,and had inhibitory effects on myocardial Ca²⁺;influx,and therefore,their effects of cardiovascular system in intact animal deserve further investigation.AIM:To study and compare effects of MS23,MP01 and aminophylline on artery blood pressure and hemodynamic parameters in anaesthetized normotensive and spontaneous hypertensive rats; to observe the effects of MS23 and MP01 on systolic and diastolic artery blood pressure in conscious unrestrained spontaneous hypertensive rats.METHODS AND RESULTS:①Hemodynamical effects in anaesthetized normotensive rats:Male normotensive Wistar rats weighing 240～260g were anaesthetized with intraperitoneal injection of urethane 1.0g/kg.Catheters were inserted into left ventricle and common carotid artery to record left ventricle pressure and artery blood pressure.The pressures were recorded by Powerlab system through the pressure transducers.The recorded or calculated parameters were left ventricle systolic pressure（LVSP）,left ventricle end diastolic pressure（LVEDP）,the maximal rise rate of left ventricle pressure(+dp/dt_max),the maximal decline rate of left ventricle pressure(-dp/dt_max),mean artery pressure（MAP）and heart rate（HR）.The electrocardiogram was recorded also.The differences of above parameters between before the injection and 1,5,10, 20,30,60,90,120 min after the drug administration were calculated,respectively.MAP was dose-dependently reduced following intravenous administration（iv）of MS23 0.1～1.0 mg/kg.The maximal decline of MAP was 53.1±14.7 mmHg,appeared at 20～30min after intravenous injection,and the hypotensive effect persisted for about 90min.MP01 and aminophylline had similar hypotensive effects.MS23 1.0 mg/kg iv and MP01 1.0 mg/kg iv markedly reduced HR,237.6±26.8 vs 204.8±31.5 beats/min（P＜0.05）for MS23,and 230.2±32.6 vs 204.5±22.4 beats/rain（P＜0.05）for MP01,respectively.The PR interval was significantly prolonged in electrocardiogram in MS23- or MP01- treated rats.Contrarily,aminophylline 10 mg/kg iv accelerated the HR with shortened PR interval,and the maximal HR increase was 28.8±5.7 beats/min.MS23,MP01 and aminophylline showed no significant effects on LVEDP and -dp/dt_max. ②Hemodynamical effects in anaesthetized spontaneous hypertensive rats:Male spontaneous hypertensive rats weighing 220-260g,with MAP being 164.2±12.6 mmHg were used.The operation and parameter recording procedures were same as above mentioned.In anaesthetized spontaneous hypertensive rats,MS23 0.3mg/kg and MP01 0.3mg/kg iv significantly lowered MAP.The hypotensive effect was appeared at 1min, culminated at around 60min after the injection.MAP was reduced by 52.3±15.8 mmHg for MS23 and 54.6±18.6 mmHg for MP01,respectively.The hypotensive effect persisted for nearly 120min.The maximal MAP reduction induced by aminophylline 10mg/kg was 30.3±17.4mmHg, appeared at 10min after the medication.The persistence time of hypotensive effects in aminophylline-treated rats was much shorter,as compared with MS23 or MP01.MS23 0.3 mg/kg iv and MP01 0.3 mg/kg iv significantly reduced HR by 39.1±9.8 beats/min for MS23,and 50.2±4.9 beats/min for MP01,respectively.Contrarily,aminophylline 10 mg/kg iv increased the HR by 29.4±4.8 beats/min,with shortened PR interval.MS23 and MP01 decreased,but aminophylline increased LVSP and +dp/dt_max.These three phosphodiesterases showed no significant effects on LVEDP and -dp/dt_max.③Effects of MS23 and MP01 on SBP,DBP and HR in conscious unrestrained spontaneous hypertensive rats.Effects of MS23 and MP01 on arterial systolic blood pressure（SBP）,arterial diastolic blood pressure（DBP）and HR were observed in conscious unrestrained male spontaneous hypertensive rats,with MAP being 166.5±10.7mmHg,weighing 230～250g.The animals were anaesthetized with pentobarbital 40mg/kg iv and a fine catheter was inserted into abdominal aorta and connected to a pressure transducer implanter which transduces blood pressures into an electric signals and wirelessly emit the signals.The implanter was fixed in the abdominal cavity.The animals were allowed to recover for 7 days from the operation.The signals of SBP and DBP were received and analyzed with a telemetry blood pressure measure system（Data Science International,DSI）.When the SBP and SBP were stable,MS23（1.0mg/kg）or MP01（1.0mg/kg） or equal volume of normal salinewere intragastrically administrated.Declines of SBP and DBP began at 30min,culminated at about 4h after medications,and hypotensive effects of both MS23 and MP01 persisted for about 6h.DBP was more pronounced reduced as compared with SBP. Neither MS23 nor MP01 showed any significant effects on HR.CONCLUSION: 1.MS23 reduces MAP,LVSP,and +dp/dtmax,and decrease HR,but had no significant effects on LVEDP and -dp/dt_maxin anaesthetized normotensive and spontaneous hypertensive rats.2.In anaesthetized normotensive and spontaneous hypertensive rats,the pharmacologic features and effect potency of MP01 are Similar to MS23;however,aminophylline has different pharmacologic profile in that it reduces MAP,but accelerates HR,enlarged LVSP and +dp/dt_max,with no significant effects on LVEDP and -dp/dt_max.3.MS23 and MP01 lower SBP and DBP,and have more pronounced hypotensive effect on DBP,without significant effect on the HR.更多还原