【作者】 赵丁；

【导师】 任雷鸣；

【作者基本信息】 河北医科大学 ， 药理学， 2003， 博士

【摘要】 莫索尼定（moxonidine, Mox）为新型咪唑啉类中枢降压药,与可乐定（clonidine,Clo）相比,降压时嗜睡?口渴等副作用明显减少?利用牛延髓腹外侧头端核团细胞膜进行的放射性配基结合实验证实,Mox与咪唑啉I₁位点和α₂ 位点的亲和力比值αI ₁/α₂)为30,而Clo的比值仅为4?Mox对清醒大鼠缺血性心律失常具有保护作用,但其抗心律失常的受体机制尚不清楚?虽然有研究表明心脏组织存在咪唑啉受体（imidazoline receptors, IR）和α2-受体,由于IR的激动剂或阻断剂同时也对α2-受体具有较高的亲和力,使得心脏IR和α2-受体的生理学和药理学研究面临很大的困难?有关Mox和Clo对兔离体窦房结起搏细胞的研究尚未见报道?因此,我们利用细胞内微电极技术,观察了Mox?Clo?胍丁胺 （agmatine,Agm,I-/α2-受体的内源性配基） 和噻拉嗪（xylazine,Xyl,非咪唑啉类α2-受体激动剂）对兔离体窦房结起搏细胞电生理活动的影响;同时比较了Mox、Clo、Agm和Xyl四个药物对兔离体胸主动脉 （α₁-受体标本）和兔离体耳静脉 （α₂-受体标本） 的药理学效应和药物的效价强度;以便综合分析上述激动药主要通过何种受体影响兔离体窦房结起搏细胞的电生理活动?Mox与Clo抑制交感神经释放去甲肾上腺素（noradrenaline,NA）的作用早有报道,曾认为这种作用完全由突触前α₂-受体调节?近十年的研究表明,突触前IR存在于多种动物（包括人）的各种组织,激动该受体时外周交感神经末梢NA释放减少?三磷酸腺苷（adenosine triphosphate, ATP）作为交感神经的共递质诱发动脉收缩已得到广泛承认,交感嘌呤能神经递质对血管收缩功能的调控具有重要意义?鉴于咪唑啉类化合物调节嘌呤能神经递质ATP释放的研究尚未见报道,为了进一步探索外周IR和α₂-受体对心血管系统功能的调节作用,我们采用交感嘌呤能血管收缩实验法,观察Mox?Clo?Agm和Xyl对兔离体隐动脉神经源性血管收缩反应的影响,以确证突触前IR的存在及突触<WP=5>前IR和α₂-受体对交感嘌呤能缩血管反应的调节作用?第一部分 莫索尼定对兔窦房结起搏细胞的电生理效应Mox为第二代中枢降压药,对咪唑啉I₁-受体的激动作用明显大于α₂-受体?有研究表明,Mox可防止大鼠心肌缺血再灌注导致的心动过速?心室纤颤等心律失常的发生,并认为Mox的抗心律失常作用与中枢α₂-受体和I₁-受体激活引起交感张力降低有关?Mox及其他I₁-受体激动剂在另一些模型中的抗心律失常作用也支持了这种解释?在离体心脏,Mox对局部缺血引起的室颤具有除颤作用?但是,Mox对窦房结起搏细胞的电生理效应尚未见报道?本实验采用细胞内微电极技术,拟研究Mox对兔窦房结细胞的电生理效应,并探讨其作用机制?1. Mox对兔窦房结起搏细胞动作电位的影响标本在持续灌流中平衡1h后,将充以3 mol·L-1 KCl的玻璃微电极插入窦房结主起搏细胞,记录细胞内电信号,放大后经MAP2微机动作电位（AP）分析软件自动测算?分析AP各项参数:最大舒张电位（MDP）,幅值（APA）,0期最大除极速率（Vmax）,AP复极达50 %和90 %的时间（APD50和APD90）,舒张期除极速率（VDD）和自发搏动速率（RPF）?用50 μL微量注射器将Mox注入标本槽,终浓度为0.1?0.3?1和3 mmol·L-1?记录给药前?给药后10 s?20 s?30 s? 45 s? 1 min?2 min? 3 min? 4 min? 6 min? 8 min? 15 min和30 min的细胞AP,观察AP各参数的变化及恢复情况?不同浓度的给药时间间隔为30 min?Mox浓度依赖性地降低兔窦房结起搏细胞AP的VDD（6.3±4.6%-19.0±6.2%）和RPF（4.7±3.3%-12.3±5.0%）,延长APD50 （4.3±2.9%-11.6±2.2%）和APD90（4.9±2.8%-12.4±3.6%）?此外,Mox 1.0和3.0 mmol·L-1显著降低MDP;轻微增加APA和Vmax,但无统计学意义?2. 育亨宾对Mox电生理效应的影响育亨宾（ 1 μmol·L-1 ） 灌流标本20 min对兔窦房结起搏细胞AP各参数均无明显影响? 在育亨宾存在的条件下,分别给予不同浓度的<WP=6>Mox,育亨宾取消了Mox降低兔窦房结起搏细胞VDD以及延长APD50 和APD90的作用;抑制Mox在较低浓度降低RPF和MDP的作用;部分拮抗3 mmol·L-1Mox对MDP的作用;但是对1和3 mmol·L-1Mox降低RPF的作用无明显影响?此外,育亨宾处理标本后,1和3 mmol·L-1 Mox明显增加Vmax 和APA?抗心律失常药物主要通过改变心脏的心肌电生理活动,特别是降低心肌细胞的自律性,延长AP的有效不应期而发挥打断折返的效应?本实验中,Mox（0.3-3 mmol·L-1）浓度依赖性地降低兔窦房结起搏细胞AP的VDD和RPF,延长APD50 和APD90?结果表明,Mox可能通过上述作用而抗心律失常;其延长APD50 和APD90,以及减慢VDD的作用主要由α₂-受体中介;Mox增大MDP和减慢RPF的作用与α₂-受体部分相关?第二部分 I-α₂-受体激动剂对兔窦房结起搏细胞的电生理效应Ren等曾报道,第一代中枢性降压药Clo浓度依赖性诱发犬离体动脉收缩, 但是α₂-受体选择性激动药Xyl无效?利用牛延髓腹外侧头端核团细胞膜进行的放射性配基结合实验证实,Clo与I1位点和α₂ 位点的亲和力比值（I 1α₂）为4,Mox的比值可达30?我们在前述兔离体窦房结起搏细胞AP的实验研究中发现,Mox延长AP时程（APD） 以及降低VDD的作用主要由α₂-受体中介,其增大MDP绝对值和减慢RPF的作用则与α₂-受体部分相关??更多还原

【Abstract】 Moxonidine （Mox）, a centrally acting antihypertensive drug, elicits less side effects （such as sedation or dry mouth） than clonidine （Clo）. In radioligand binding experiments using membranes of the bovine rostral ventrolateral medulla, the affinity ratio of I1 site / α₂ site was reported to be 4 for Clo and even about 30 for Mox. It has been reported that Mox offers significant protection against the development of arrhythmias induced by regionally myocardial ischemia in the conscious rat. However, its mechanism as an antiarrhythmic agent is not clear. Though imidazoline receptors （IR） and α₂-adrenoceptors were reported to exist on hearts, however, the agonists or antagonists acting on IR also have higher affinity to α₂-adrenoceptors, so it is difficult to analyze the physiological and pharmacological effects regulated by IR and α₂-adrenoceptors in the heart. On the other hand, very little is known regarding the effects of Mox and Clo on action potentials （APs） of the rabbit sinoatrial node （SAN） pacemaker cells. Consequently, the present study was designed to investigate the effects of Mox, Clo, agmatine （Agm, an endogenous ligand for I-/α₂-receptors） and xylazine （Xyl, a non-imidazolines selective α₂-adrenoceptor agonist） on APs of the rabbit SAN pacemaker cells using intracellular microelectrode technique, and to compare their （Mox, Clo, Agm and Xyl） potency order for producing vasoconstriction in the rabbit isolated thoracic aorta （α₁-adrenoceptor preparation） and rabbit isolated ear vein （α₂-adrenoceptor preparation） to elucidate the contribution of IR and α_-adrenoceptors to the cardiac electrophysiological action induced by the four agonists. Inhibition of the release of noradrenaline （NA） from sympathetic neurons is a typical response to the imidazoline derivatives Clo and Mox.<WP=13>Originally, this effect was thought to be mediated exclusively by presynaptic α₂-adrenoceptors. However, it has recently been established that presynaptic IR mediating inhibition of NA release from postganglionic sympathetic nerve fibers have been identified in several tissues of various species including human beings. On the other hand, adenosine triphosphate （ATP） has been widely considered as the purinergic cotransmitter released together with the classical transmitter of NA induced vasconstrictive responses, indicated that the sympathetic purinergic neurotransmitter holds an important action in the control of vasoconstriction. However, the modulation effects of imidazoline compounds on the vasoconstrictive responses to purinergic transmitters have not been reported. To investigate the modulating effects on cardiovascular functions by peripheral ?R and α₂-adrenoceptors, in the present study, we observed the effects of Mox and Clo on neurogenic vasoconstriction induced by electrical field stimulation in the rabbit isolated saphenous artery. We simultaneously compared the effects of Xyl （a non-imidazolines agonist of α₂-adrenoceptors） and Agm （an endogenous ligand for I-/α₂-receptors） on neurogenic vasoconstriction in the rabbit isolated saphenous artery to clarify the presynaptic modulation effects of presynaptic IR on the purinergic vasoconstrictive responses. Part I Electrophysiologic effect of moxonidine on action potential in rabbit sinoatrial node pacemaker cellsMox is a second-generation, centrally acting antihypertensive drug, which activates imidazoline-1 receptors （I1-R） more strongly than ??2-adrenoceptors. Mox decreased the incidence of arrhythmias of the myocardial ischemia induced by coronary artery occlusion or reperfusion in the rat. It has been suggested that the antiarrhythmic effects of Mox are mediated by acting on the central ??2-adrenoceptors and I1-R, thereby reducing the activity of sympathetic nervous system. Antiarrhythmic<WP=14>effects of Mox and other agonists of IR have also supported this hypothesis. In the rabbit isolated working heart, Mox facilitated spontaneous defibrillation of the ventricular fibrillation during ischemia. IR更多还原