【作者】 孔子青；

【导师】 王锐；

【作者基本信息】 兰州大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 速激肽家族是拥有共同C-末端序列：FXGLM-NH2的肽家族,哺乳动物速激肽包括SP, NKA, NKB和最近发现的HK-1。根据其来源,速激肽HK-1包括大鼠／小鼠HK-1,人HK-1及其C-末端片段人HK-1(4-11)。HK-1通过结合速激肽NK1,NK2和NK3受体发挥一定的生物学作用,其中大鼠／小鼠HK-1与人、大鼠和小鼠的速激肽NK1受体有较高的亲和活性,且与SP相当；而人HK-1和人HK-1(4-11)与人速激肽NK1受体的结合能力比SP分别弱14倍和70倍。哺乳动物速激肽SP,NKA和NKB对在体血压和心率以及对离体的心脏功能都有一定的调节作用,自从大鼠／小鼠HK-1,人HK-1和人HK-1(4-11)被发现以来,人们已经报道了它们对痛觉传递,免疫发生,雌性生殖系统功能等方面的调节作用,而对于大鼠／小鼠HHK-1,人HK-1和人HK-1(4-11)的在体心血管调节作用和离体心脏功能调节作用方面的研究还未见报道。因此,本论文的第一部分内容主要集中在对HK-1肽的在体心血管活性调节作用研究,运用在体动物实验模型研究大鼠／小鼠HK-1,人HK-1和人HK-1(4-11)对心血管活性的调节作用并揭示其作用机制和相互联系；本论文的第二部分内容主要是利用离体心脏灌流模型研究在没有在体条件下的神经和体液等因素影响下,大鼠／小鼠HK-1,人HK-1和人HK-1(4-11)对离体心脏功能的调节作用及其机制。另外,研究发现编码大鼠／小鼠HK-1的小鼠TAC4基因在小鼠消化道上有表达,同时最近的研究报道速激肽受体在小鼠结肠上也广泛存在,而大鼠／小鼠HK-1自从被发现以来,其对小鼠结肠运动的调节作用还未知。因此,本论文的第三部分主要研究大鼠／小鼠HK-1对小鼠结肠运动的调节作用及其机制,并通过与SP进行比较,深入探讨二者在受体亲和活性与激动活性方面的区别与联系。实验结果如下：1.静脉注射大鼠／小鼠HK-1 (0.1-30 nmol/kg)能剂量依赖地降低大鼠平均动脉压并产生心动过速效应。其降低平均动脉压作用主要由血管内皮上的NK1受体和内皮舒张因子(NO)介导的,而心动过速反应主要是由交感神经兴奋和肾上腺髓质分泌儿茶酚胺类物质介导。静脉注射低剂量人HK-1 (0.1-3 nmol/kg)会导致大鼠平均动脉压的降低,而高剂量人HK-1 (10-30 nmol/kg)会导致双相平均动脉压效应(包括降压和升压作用)。其中,降压作用主要是人HK-1激动血管内皮NK1受体而释放内皮舒张因子(NO)参与的,迷走反射并没有参与降压作用。升压作用主要通过活化交感神经和肾上腺髓质的NK1受体释放儿茶酚胺产生的。此外,静脉注射人HK-1后也产生心动过速效应,这种心动过速反应也是由交感神经和肾上腺髓质共同调节的。有意思的是,人HK-1的羧基末端片段人HK-1(4-11) (0.1-30 nmol/kg)仅能产生剂量依赖的平均动脉压降低作用,且其降压反应机制与人HK-1相似。人HK-1(4-11)还能够引起剂量依赖的心动过速反应,通过对作用机制的研究发现,人HK-1(4-11)主要是通过刺激交感神经兴奋而产生心动过速效应,这与人HK-1引发心动过速反应的机制略有不同。以上结果表明,新发现的哺乳动物速激肽大鼠／小鼠HK-1,人HK-1和人HK-1对大鼠平均动脉压和心率都分别存在独立的调节作用,其中大鼠／小鼠HK-1产生的平均动脉压和心率调节作用均显著强于人HK-1和人HK-1(4-11);同时人HK-1(4-11)是人HK-1的一个活性片段且两者不同的N-末端氨基酸序列产生了对速激肽NKl受体不同的激动活性。大鼠／小鼠HK-1,人HK-1和人HK-1(4-11)作为速激肽受体激动剂参与了心血管活性的调节,而速激肽受体又是慢性心血管疾病的调节因子,因此上述三种速激肽的心血管作用机制研究将有利于人们认识速激肽及其受体在慢性心血管疾病中的作用。2.大鼠／小鼠HK-1能引起豚鼠离体灌流心脏灌流压的降低,这表明大鼠／小鼠HK-1在离体心脏上产生了冠脉舒张作用。研究发现,该作用主要是通过激活冠脉血管内皮细胞上的速激肽NK1受体释放NO,从而舒张冠脉的。人HK-1可引起双相的灌流压变化即先降低灌流压后升高灌流压。其中,引起灌流压降低的作用机制与大鼠／小鼠HK-1相同;而灌流压升高即冠脉收缩主要是通过激活冠脉交感神经元上的速激肽NK2受体后释放儿茶酚胺介导的。人HK-1(4-11)仅能产生冠脉收缩作用,其作用机制与人HK-1引起血管收缩的机制类似。除此之外,大鼠／小鼠HK-1和人HK-1还可诱导心率的降低,即负变时性效应。这个作用主要是通过激活速激肽NK1受体释放乙酰胆碱,然后激活胆碱能M受体介导的。人HK-1(4-11)也会产生负变时性效应,主要通过速激肽NK2受体和胆碱能M受体来介导的。以上结果表明,大鼠／小鼠HK-1,人HK-1和人HK-1(4-11)在没有在体神经和其他外周因素的影响下都可调节离体豚鼠心脏的心脏功能和冠脉活性。本实验还提出了一些关于速激肽HK-1介导冠脉和心率反应的新机制,由于速激肽与心肌缺血等疾病相关,因此,本实验结果为研究速激肽在心肌缺血等病理条件下的调节作用提供了新的理论参考。3.大鼠／小鼠HK-1能诱导小鼠结肠环形肌的自主收缩,而且它对小鼠结肠的近端,中端,远端的最大收缩作用都各不相同。这表明,大鼠／小鼠HK-1对小鼠结肠环形肌收缩呈现显著的区域性差异。通过机理研究发现,大鼠／小鼠HK-1诱导的结肠环形肌收缩效应主要是速激肽NK1受体和胆碱能神经元共同介导的,少量位于平滑肌上的速激肽NK2受体也参与了环形肌的收缩。另外,大鼠／小鼠HK-1也直接作用于结肠平滑肌细胞引起环形肌收缩。我们还研究了SP对小鼠离体结肠运动功能的影响,结果表明SP能诱导结肠环形肌产生双相变化(自主收缩效应和自主舒张效应)。其中,SP的自主收缩效应与大鼠／小鼠HK-1相似,主要通过神经和肌肉中的速激肽NK1受体发挥作用,但速激肽NK2受体并不参与这些效应。SP诱导的自主舒张效应主要是通过作用于神经元上速激肽NK1受体,释放NO而介导的。上述研究结果表明,大鼠／小鼠HK-1在调节小鼠离体结肠运动的受体参与和受体激动活性方面与SP相比都存在显著差异。我们的研究结果有利于人们评价大鼠／小鼠HK-1和SP在胃肠道疾病方面的调节作用,同时也为胃肠道疾病的药理干预提供了理论支持。更多还原

【Abstract】 The tachykinin SP, NKA, NKB and HK-1 are a family of peptides characterized by the presence of a common C-terminal motif FXGLM-NH2. There are several species-divergent HK-1 peptides, which include rat/mouse hemokinin-1 (r/m HK-1), human hemokinin-1 (h HK-1) and human hemokinin-1 (4-11) (h HK-1 (4-11)). HK-1 exerts their effects through the interaction with three types of tachykinin receptors, termed NK1, NK2 and NK3 receptors. In general, the rank order in potencies of SP and r/m HK-1 in binding to the tachykinin NK1 receptor is as follows:SP=r/m HK-1. H HK-1 and h HK-1 (4-11) bind to tachykinin NK1 receptors with 14-and 70-fold reduced affinities, respectively, relative to SP and r/m HK-1. There are extensive researches performed about the actions of SP, NKA and NKB in cardiovascular responses in vivo and in isolated heart. Meanwhile, since the discovery of r/m HK-1, h HK-1 and h HK-1(4-11), many studies have focused on its biological actions including modulation of pain, immunological regulation, female reproductive function. However, little if anything is known about the effects of r/m HK-1, h HK-1 and h HK-1(4-11) on cardiovascular responses in vivo and cardiac functions on isolated heart. Thus, the aim of the present study were therefore to 1) investigate the effect and mechanism of action of r/m HK-1, h HK-1 and h HK-1 (4-11) in the modulation of cardiovascular activity in anesthetized rats; 2) examine the pharmacologic actions of r/m HK-1, h HK-1 and h HK-1 (4-11) in the isolated guinea pig hearts without the influence of other in vivo organs, nerves and other peripheral factors. Moreover, TAC 4 which encodes r/m HK-1 is expressed in mouse gastrointestinal tract. It has been demonstrated that tachykinin NK1, NK2 and NK3 receptors were present in mouse colon. Based on the knowledge of tachykinin NK receptors distribution and the lack of colonic motility studies of r/m HK-1 in mouse, the present study in part 3 was designed to investigate the effect and mechanism of action of r/m HK-1 in the modulation of colonic motility in mouse by comparing it with that of SP.1. Our data showed that intravenously (i.v.) injection of r/m HK-1 (0.1,0.3,1,3, 10 and 30 nmol/kg) lowered mean arterial pressure (MAP) dose-dependently. This effect was significantly blocked by pretreatment with SR140333 (a selective tachykinin NK1 receptor antagonist) and the NO synthase inhibitor L-NAME, respectively, but was not affected by bilateral vagotomy or the muscarinic receptor blocker atropine, which indicated that the depressor response induced by r/m HK-1 is mediated by tachykinin NK1 receptors and endothelium-derived relaxing factor (NO). R/m HK-1 injected i.v. also produced a dose-dependent tachycardia response, which is mediated by the activation of tachykinin NK1 receptors to stimulate sympathetic ganglia and to release catecholamines from adrenal medulla. Lower doses of h HK-1 (0.1-3 nmol/kg) injected i.v. induced depressor response, whereas higher doses (10 and 30 nmol/kg) caused biphasic (depressor and pressor) responses. The depressor response is primarily due to the action on endothelial tachykinin NK1 receptor to release endothelium-derived relaxing factor (NO) and vagal reflex are absent in this modulation. The pressor response is mediated through the activation of tachykinin NK1 receptor to release catecholamines from sympathetic ganglia and adrenal medulla. Moreover, h HK-1 injected i.v. produced a dose-dependent tachycardia response along with blood pressure responses and the activation of sympathetic ganglia and adrenal medulla are involved in the tachycardia response. H HK-1(4-11) only lowered MAP dose-dependently (0.1-30 nmol/kg) and the mechanisms involved in the depressor response is similar to that of h HK-1. Additionally, h HK-1 (4-11) could also produce tachycardia response dose-dependently and the mechanisms involved in the tachycardia response are similar to that of h HK-1 except that bilateral adrenalectomy could not affect the tachycardia markedly, indicating that the tachycardia induced by h HK-1 (4-11) is primarily due to the stimulation of sympathetic ganglia. In a word, to a certain extent, h HK-1 (4-11) is the active fragment of h HK-1, however, the differences between h HK-1 and HK-1(4-11) involved in the effects of cardiovascular system suggest that the divergent amino acid residues at the N-terminus of h HK-1 produced different activation properties for tachykinin NK1 receptor.2. In the present study, the coronary vascular activities and cardiac functions of r/m HK-1, h HK-1 and h HK-1 (4-11) were investigated in isolated, spontaneously beating guinea pig hearts. Bolus injection of r/m HK-1 caused decrease in perfusion pressure indicative of coronary vasodilation, which was primarily due to the action on tachykinin NK1 receptors on vascular endothelial cells, causing the release of nitric oxide that relaxed the coronary vessels. H HK-1 caused biphasic perfusion pressure changes that were coronary vasodilation followed by vasoconstriction. The mechanism of the vasodilation was similar to that of r/m HK-1 while the coronary vasoconstriction was mediated through the activation of tachykinin NK2 receptors on coronary sympathetic neurons to release catecholamines. H HK-1 (4-11) only produced coronary vasoconstriction and the mechanism involved in this effect was similar to that of h HK-1 in vasoconstriction. Moreover, r/m HK-1 and h HK-1 produced similar decreases in heart rate indicative of negative chronotropic action, which were mainly mediated through the activation of tachykinin NK1 receptors to release ACh acting on muscarinic receptors. H HK-1(4-11) also produced negative chronotropic response, which was mainly mediated through tachykinin NK2 receptors and muscarinic receptors. Our present results provide evidence that all of these tachykinins could influence cardiac function and coronary vascular activity in the heart. Our novel findings should facilitate the analysis of the role of the tachykinins encoded by the newly identified TAC4 gene in pathophysiological conditions.3. R/m HK-1 induced substantial contractions on the circular muscle of mouse colon. The maximal contractile responses to r/m HK-1 varied significantly among proximal-, mid-and distal-colon, suggesting that the action of r/m HK-1 was region-specific in mouse colon. The contractile response induced by r/m HK-1 is primarily via activation of tachykinin NK1 receptors leading to activation of cholinergic excitatory pathways and with a minor contribution of NK2 receptors, which may be on the smooth muscle itself. A direct action on colonic smooth muscles may be also involved. In contrast, SP induced biphasic colonic responses (contractile and relaxant responses) on the circular muscle, in which the contractile action of SP was equieffective with r/m HK-1. SP exerted its contractile effect predominantly through neural and muscular tachykinin NK1 receptors, but unlike r/m HK-1 did not appear to act via NK2 receptors. The relaxation induced by SP was largely due to release of nitric oxide (NO) produced via an action on neural NK1 receptors. These results indicate that the receptors and the activation properties involved in r/m HK-1-induced mouse colonic contractile activity are different from those of SP. Therefore, our present results should facilitate the analysis of the role of r/m HK-1 and SP in gastrointestinal disease and may open novel possibilities for pharmacological interventions.更多还原