尼古丁对血管功能和胰岛素敏感性的影响及机制研究
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摘要
【目的】
尼古丁是一个众所周知的经典化合物。它是烟草的主要成份之一。吸烟普遍被认为是动脉粥样硬化、高血压、糖尿病等多种心血管内分泌疾病的危险因素[1]。但这些不良作用是否是经由尼古丁产生的呢?现在看来,很多对尼古丁的研究结果由于研究方法、对象的不同仍存在矛盾、片面的地方,很多机制尚未阐明,因此不能单纯的断定吸烟的不良作用就是尼古丁产生的。相反,近期研究还发现了尼古丁的许多有益作用,比如抗炎、神经保护、改善肥胖大鼠的胰岛素抵抗等[2]。在本课题组前期建立的尼古丁长期治疗大鼠模型上,我们首次发现尼古丁治疗组大鼠的主动脉对硝普钠(一种一氧化氮供体药物)的内皮非依赖的舒张功能特异性的增强了。此外,该模型的胰岛素抵抗指数显著降低。这提示,尼古丁不仅可使血管中膜平滑肌一氧化氮信号通路增敏,还增强胰岛素敏感性。
尼古丁主要经由尼古丁受体发挥作用。尼古丁受体属配体门控离子通道型受体,由5个亚基组成五聚体,亚基的种类有17种(α1-10,β1-4,γ/ε,δ),配体结合位点在α亚基上[3-6]。传统意义上,尼古丁受体分为肌肉型和神经型,前者介导神经肌肉接头传递,后者介导或调节神经系统的突触传递。最近研究显示,神经型尼古丁受体也可在许多非神经细胞中表达[5, 6],而且,非神经细胞上尼古丁受体及其功能的发现,已经吸引不同领域科学家对这一经典受体产生新的研究兴趣。例如,免疫细胞上α7尼古丁受体的阐明,触发人们研究尼古丁受体激动剂对感染和炎症性疾病的治疗学意义。平滑肌细胞上,慢性尼古丁暴露可通过上调α7受体来增强胰岛素刺激的丝裂原信号通路。当前,在非神经组织(细胞)上考察尼古丁受体及其功能活性,将揭示尼古丁受体的新功能。而确定特定的尼古丁功能受体,对于发挥尼古丁有益的治疗作用意义重大。
综上所述,本课题拟深入研究尼古丁长期治疗对血管功能和胰岛素敏感性的影响和机制,这不仅有利于明确尼古丁对心血管和内分泌系统的作用,揭示尼古丁受体的新功能和血管舒张新通路,也对寻找治疗血管疾病和胰岛素抵抗的新靶点具有重要意义。
【方法】
第一部分:尼古丁对血管功能的影响及机制研究。该部分研究首先在本课题组前期建立的大鼠尼古丁长期治疗模型上考察主动脉外膜、内膜抗收缩功能;内膜依赖的血管舒张功能;中膜收缩和内膜非依赖的舒张功能。接着,进一步考察尼古丁增强中膜一氧化氮舒张反应机制。一方面在大、小鼠尼古丁治疗模型上考察尼古丁对中膜舒张反应影响的时间过程以明确该作用是急性作用还是慢性作用,另外还观察了尼古丁治疗后对胰岛素引起的中膜舒张反应的影响以初步判断尼古丁致舒张反应增强作用是否具有普遍意义。然后在离体血管实验中运用抑制剂和拟似物,检测关键蛋白表达和活性的方法明确该作用是否经由一氧化氮信号通路的3’, 5’-环鸟苷一磷酸(cGMP)依赖的信号通路。随后,通过制备基因芯片分析预测和经典通路上逐步排查的方法,寻找尼古丁改变的一氧化氮通路下游可能的靶点,如环氧合酶-2、内质网钙泵、AMPK、Rho等,再通过Western-blot,以及血管功能实验等方法进行验证,最终确认靶点。另一方面制备尼古丁受体阻断剂和尼古丁同时治疗的动物模型并使用α7尼古丁受体敲除小鼠进行尼古丁长期治疗,再进行血管功能实验从而寻找介导该作用的尼古丁受体。最后运用清醒测压技术,检测尼古丁模型组与生理盐水组血压等指标的差别。
第二部分:尼古丁对胰岛素敏感性的影响及机制研究。首先在尼古丁长期治疗的正常大鼠模型上通过血清学胰岛素抵抗指数、整体胰岛素耐量实验和糖耐量实验等多个指标验证尼古丁对胰岛素增敏的作用。接着针对机制研究,一方面在整体水平进行尼古丁给药模型的各部位脂肪称重,另一方面,在第一部分制备的尼古丁给药组和生理盐水组的主动脉基因芯片上寻找胰岛素敏感性相关基因的改变。同时,检测尼古丁治疗大鼠模型的四种胰岛素敏感组织(骨骼肌、肝脏、内脏脂肪和皮下脂肪)的关键蛋白(PPAR-γ、COX-2、AMPK)表达水平,从而寻找尼古丁影响的靶蛋白。另一方面,制备外周尼古丁受体阻断剂和尼古丁同时治疗的动物模型,以确认尼古丁致胰岛素增敏的中枢或外周受体机制。
【结果】
第一部分:尼古丁对血管功能的影响及机制研究
1.尼古丁长期治疗不影响主动脉外膜和内膜的抗收缩功能;不影响内膜依赖的舒张功能;不影响中膜平滑肌对新福林的收缩作用,而特异性的增强硝普钠(一氧化氮供体)引起的内皮非依赖性的中膜舒张反应。
2.尼古丁治疗后Sprague-Dawley (SD)大鼠和C57BL/6J小鼠中膜收缩反应不改变,但硝普钠引起的内皮非依赖性的中膜舒张反应都显著增强。大鼠治疗3周出现该现象,6周稳定出现该现象。小鼠治疗4周出现该现象,6周稳定出现该现象。尼古丁治疗后SD大鼠中膜对胰岛素引起的舒张反应也显示增强。这说明该增强作用是长期慢性的作用结果而非急性作用。
3.硝普钠引起的生理盐水和尼古丁组的舒张反应可被可溶性鸟苷酸环化酶抑制剂ODQ完全阻断,可被cGMP模拟物模拟,可被cGMP依赖的蛋白激酶(PKG)抑制剂阻断,而PKG蛋白表达在两组间不改变,蛋白活性没有明显差异。这提示该舒张增敏机制是经由PKG信号通路,且发生在PKG的下游靶位。
4.主动脉芯片结果显示与一氧化氮通路舒张相关基因中尼古丁治疗组内质网钙泵基因sarco(endo)plasmic reticulum calcium-ATPase 3 (SERCA 3)较生理盐水组上调1.56倍,热休克蛋白27(Hsp 27)较生理盐水组下调0.51倍。另外与一氧化氮通路尚无报道关联的COX-2基因表达显著下调0.09倍,我们也将它作为可能影响舒张通路的备选基因。
5.根据芯片结果进行验证:COX-2蛋白表达在尼古丁组主动脉平滑肌显著下调,而COX-2非选择性抑制剂Indomethacin和选择性抑制剂Meloxicam都不能取消尼古丁组和生理盐水组间血管舒张反应的差别;内质网钙泵抑制剂Thapsigargin不能取消尼古丁组和生理盐水组间血管舒张反应的差别;尼古丁组主动脉与收缩相关蛋白Hsp 27表达不变。这些结果排除了SERCA 3、Hsp 27和COX-2参与舒张增敏的机制。
6.进一步使用Rho激酶抑制剂Y-27632不能取消尼古丁组和生理盐水组间血管舒张反应的差别,这排除了尼古丁经由抑制Rho通路从而使钙收缩脱敏以增强舒张的可能。
7.运用AMPK抑制剂Compond C不能取消尼古丁组和生理盐水组间血管舒张反应的差别,又排除了最新提出的AMPKα1舒张通路的参与。
8.使用非选择性阻断剂20 mM的Tetraethylammonium(TEA)阻断钙激活钾通道和电压依赖性钾通道可完全取消尼古丁组和生理盐水组的舒张反应差别,而选择性阻断ATP敏感钾通道(KATP)和内向整流钾通道(Kir)都不能取消两组间血管舒张反应的差别。进一步选择性阻断大电导钙激活钾通道(BKCa)以及阻断4-AP敏感的电压依赖性钾通道却都不能取消尼古丁组和生理盐水组的舒张反应差别。而等渗高钾液(80 mM)可取消硝普钠引起的尼古丁组和生理盐水组的舒张反应差别。这说明尼古丁致硝普钠舒张增敏的机制是经由TEA敏感的钾通道,而不是的BKCa、KATP、Kir和4-AP敏感的电压依赖性钾通道。
9.外周神经型尼古丁受体阻断剂Hexamethonium和尼古丁同时治疗可以取消尼古丁组和生理盐水组间硝普钠引起的血管舒张反应差别。而尼古丁治疗的α7尼古丁受体敲除小鼠,硝普钠引起的血管舒张反应增强现象仍然存在,说明尼古丁使舒张增敏的机制是经由外周非α7尼古丁受体产生。
10.尼古丁长期给药后不影响基础状态的收缩压、舒张压和平均动脉压但延长心动间期;不影响硝普钠(3μg/kg, 10μg/kg, 30μg/kg, 100μg/kg)急性给药引起的血压下降。
第二部分:尼古丁对胰岛素敏感性的影响及机制研究
1.尼古丁长期治疗后不影响血清血糖水平,但显著减少胰岛素和甘油三酯水平,显著减少HOMA法的胰岛素抵抗指数,说明基础状态下的胰岛素增敏。
2.尼古丁治疗3周和6周后胰岛素耐量实验和葡萄糖耐量实验都显示尼古丁组血糖降低的速度和幅度较生理盐水组和生理盐水限食组都更为显著,说明刺激状态下的胰岛素增敏。
3.尼古丁长期治疗后皮下脂肪和内脏脂肪都明显减少,且内脏脂肪减少更为显著。
4.主动脉基因芯片显示尼古丁给药后脂代谢相关基因如脂酶、肾上腺素受体β3显著上调;能量消耗相关基因解耦联蛋白1显著上调;胰岛素敏感性相关基因过氧化物酶增殖体受体(PPARγ)显著上调。
5.尼古丁治疗不影响PPARγ蛋白在骨骼肌、肝脏、内脏脂肪和皮下脂肪的表达。尼古丁治疗显著减少骨骼肌COX-2表达,而不改变肝脏、内脏脂肪和皮下脂肪的表达。尼古丁治疗显著增加内脏脂肪和下丘脑AMPK表达,而不改变骨骼肌、肝脏和皮下脂肪的表达。
6.外周尼古丁受体阻断剂合并尼古丁治疗并不阻断尼古丁增强胰岛素敏感性的作用,但阻断尼古丁减少血清甘油三酯,说明尼古丁致胰岛素增敏主要经由中枢机制。
【结论】
本课题主要发现并探讨了尼古丁两个全新的作用和机制:
一、尼古丁长期治疗可特异性增强主动脉中膜对一氧化氮的舒张反应。该增敏作用是尼古丁经由非α7的外周神经型尼古丁受体影响了PKG下游途径产生。SERCA 3、Hsp 27和COX-2不参与该下游增敏途径; Rho和AMPK也不参与该下游增敏途径。该增敏途径是通过增强PKG下游的TEA敏感钾通道开放,引起细胞膜超极化从而抑制外钙内流,导致舒张反应增强,并且该钾通道不是平滑肌上目前已知的可被PKG直接激活的BKCa和KATP ,也不是平滑肌上另外两种主要的4-AP敏感的电压依赖性钾通道和Kir通道,有待于进一步确认。
二、尼古丁可增强正常大鼠的胰岛素敏感性。该作用主要是通过中枢尼古丁受体产生,可能的下游机制有上调下丘脑和内脏脂肪AMPK表达,抑制COX-2在骨骼肌的表达发挥,并且该增敏作用与调节PPARγ表达无关。
【Objective】
Smoking is a leading cause of atherosclerosis, hypertension, diabetes and other cardiovascular and endocrine diseases. Nicotine is one of the major components of cigarette with complex effects on cardiovascular systems, and many mechanisms still left unkown. Up to now, most studies showed that nicotine impaired vascular fuction and insulin action, for it reduced endothelium-independent vascular relaxation and led to insulin resistance. However, in chonic nicotine treatment rat model established in our department, aortic reactivity showed that endothelium-dependent relaxation was not altered, but endothelium-independent relaxation induced by sodium nitroprusside (a donor of nitrc oxide) was significantly enhanced. Besides, the insulin resistance index of this model was reduced. These indicated that nicotine not only enhanced the sensitivity of nitric oxide pathway in vascular smooth muscle but also insulin sensitivity.
Nicotine produces most of its effects through nicotine acetylcholine receptors (nAChRs). Nicotine acetylcholine receptors are ligand-gated ion channels, they form five aggregates composed of five subunits. There are seventeen subunits (α1-10,β1-4,γ/ε,δ), and ligands binding site are onαsubunit. Traditionally, Receptors of the nicotinic subclass can be distinguished as“muscle”or“neuronal”. The former mediates nerve-muscle connector transmission, and the latter mediates or modulates nerve system synapsis transmission. Recent studies pointed out that nAChRs are also expressed in many non-neuro cells. Moreover, the fact that nicotine receptors and their function were discovered in non-neuro cells has already attracted scientists in different fields. For example, the clarification ofα7 nicotinic receptor in immune cells triggered the studies on therapeutics significance of nicotinic receptor agonist treating infectious and imflammatory diseases. In smooth muscle cells, chronic nicotine expoture enhances insulin-induced mitogenic signaling via up-regulation ofα7 Nicotinic receptors. Currently, study nAChRs and their function in non-neuro cells will reveal new fuction of nAChRs. Meanwhile, determining specific nicotinic receptor is of most importance for therapeutic use of nicotine.
Above all, this study was to investigate the effects and mechanisms of nicotine on vascular fuction and insulin sensitivity. This will not only contribute to the understanding of the complex effect of nicotine on cardiovascular and endocrine systems, but also has significance in reveal the new fuction of nicotinic receptors and new targets in nitric oxide signaling pathway.
【Methods】
PartⅠ: Effect of nicotine on vascular fuction and its mechanism. First, in nicotine treated rat, anticontraction effect of adventitia and endothelium were tested; endothelium-dependent relaxation effect was tested; as well as smooth muscle contraction and endothelium-independent relaxation were tested. Then, we further investigated the mechanism of nicotine enhancing nitroxide induced relaxation. On the one hand, time course effect of nicotine treatment on aortic reactivity was first studied in SD rats and C57 mice to find out whether this effect of nicotine was generally and chronic. We also observe the relaxation effect of aorta smooth muscle induced by insulin in nicotine treated and untreated groups. Next, we studied its mechanism. In organ bath experiment, we used sGC inhibitor, PKG inhibitor and cGMP analog. Meanwhile, we detected PKG expression and activity to identify whether this effect was through cGMP dependent PKG pathway. Thirdly, we tried to find out the possible target nicotine altered in the down stream of nitric oxide signaling pathway by gene chip analysis accompanied with gradually excluding from old pathway. After selecting the possible targets, such as cyclooxygenase-2, sarcoplamic reticulium Ca2+-ATPase, amp-activated protein kinase, Rho, etc., we carried out western-blot and organ bath experiment to verify and confirm the targets. On the other hand, we made rat model treated at the same time with nicotine and hexamethonium that blocked nicotinic receptors exceptα7 receptor. We also treated alpha 7 knockout mice with nicotine. These two experiment were to find out the possible nicotinic receptor mediated this effect. At last, we investigated the blood pressure indicators in nicotine treated and untreated groups by conscious blood pressure detection technique.
PartⅡ: Effect of nicotine on insulin sensitivity and its mechanism. First, we confirm the insulin sensitivity enhancement effect of nicotine on normal rats through HOMA-index, insulin tolerance test and glucose tolerance test. Then, we studied its mechanism. On the one hand, we weighed the fat tissue from various parts of rats. On the other hand, we analysis the gene chip from PartⅠto find out insulin sentivity related gene alteration. Meanwhile, we detected key protein expression (PPAR-gamma, COX-2, AMPK) in four insulin sensitive tissue (skeletal muscle, liver, visceral fat and subcutaneous fat) in nicotine treated rat model. Moreover, we made hexamethonium plus nicotine treated rat model to study the central or periphery receptor mechanism of nicotine on insulin sensitiviy.
【Results】
PartⅠ: Study on effects and mechanisms of nicotine on vascular function
1. Chronic nicotine treatment did not affect anticontraction effect of aorta adventitia and endothelium; did not affect endothelium-dependent relaxation effect; did not affect aorta smooth muscle contraction effect induced by phenylephrine but had slight inhibition effect on contraction effect induced by low dose KCl; significantly enhanced endothelium independent smooth muscle relaxation.
2. Nicotine did not alter phenyllphrine induced aortic contraction but significantly enhanced sodium nitroprusside (donor of nitroxide) induced endothelium-independent relaxation in Sprague-Dawley (SD) rats and C57BL/6J mice. This phenomenon emerged after three weeks nicotine treatment in rats and four weeks in mice and was stable after 6 weeks nicotine treatment in rats and mice. Nicotine treatment also enhanced insulin induced smooth muscle relaxation. These indicated that the effect of nicotine is chronic rather than acute.
3. The relaxation induce by sodium nitroprusside was totally blocked by sGC inhibitor ODQ and PKG inhibitor RP-8-bromo-cGMP. Further the relaxation was mimicked by cGMP analog. PKG expression between nicotine and saline group was the same, and so was the PKG activity. This indicated the alteration was in the PKG downstream pathway.
4. Analysis on aorta gene chip showed two nitroxide relaxation pathway related gene altered. Sarcoplamic reticulium Ca2+-ATPase mRNA up regulated by 1.56 folds and heat shock protein 27 mRNA downregulated by 0.51 folds. Besides, another candidate gene COX-2 significant down regulated by 0.09 folds.
5. COX-2 protein expression was significantly reduced in aorta smooth mucle in nicotine group. But indomethacin, a nonselective inhibitor of cox-2 and meloxicam, a selective inhibitor of COX-2 could not abolish the relaxation difference between nicotine and saline group. Sarcoplamic reticulium Ca2+-ATPase inhibitor thapsigargin could not cancel the relaxation difference between nicotine and saline group. Hsp 27 protein expression was not altered in nicotine treated rats. These results eliminated SERCA 3、Hsp 27 and COX-2.
6. Rho kinase inhibitor Y-27632 could not abolish the relaxation difference between two groups. This indicated Rho was not involved in this mechanism.
7. AMPK inhibitor compound C could not abolish the relaxation difference between nicotine and saline groups. This eliminated the involvement of AMPK pathway.
8. Non-selective Kca and Kv inhibitor, 20 mM Tetraethylammonium (TEA), could totally abolish the relaxation difference between saline and nicotine group, while selective block KATP and Kir could not affect the difference between two groups. Selective block BKCa and 4-AP sensitive potassium channel could not abolish the relaxation difference between two groups either. In aorta preconstricted by 80 mM KCl, the relaxation difference between two groups induced by SNP was not present.
9. Hexamethonium abrogated the relaxation difference between saline and nicotine groups, while in alpha 7 knockout mice, the relaxation difference between two goups was still exist.
10. Chronic nicotine treatment did not affect basal systolic blood pressure, diastolic blood pressure and mean blood pressure but extended heart period; did not affect acute sodium nitroprusside (3μg/kg, 10μg/kg, 30μg/kg, 100μg/kg) injection induced blood pressure lowering effect.
PartⅡ:
1. Chronic nicotine treatment did not affect serum glucose level, but significantly reduce insulin and triglycride levels and significantly reduced HOMA-insulin resistant index.
2. Insulin tolerance test and glucose tolerance test in 3 weeks and 6 weeks nicotine and saline treated rats all showed that blood glucose lowering percentage was greater in nicotine treated rats.
3. Chronic nicotine treatment significantly reduced subcutaneous fat and visceral fat, especially visceral fat.
4. Aorta gene chip showed that after chronic nicotine treatment lipid metabolism related genes (lipase, adrenergic receptorβ3 ) significantly upregulated; energy expenditure related gene ( uncoupling protein 1) significantly upregulated; insulin sensitivity related gene (peroxisomal proliferators activated receptorγ, PPARγ) significantly upregulated.
5. Nicotine treatment did not affect PPARγexpression in skeletal muscle, liver, visceral fat and subcutaneous fat.Nicotine treatment significantly reduced COX-2 expression in skeletal muscle, but did not affect COX-2 expression in liver, visceral fat and subcutaneous fat.Nicotine treatment significantly increased AMPK expression in visceral fat, but did not affect AMPK expression in skeletal muscle, liver and subcutaneous
6. Periphery nicotinic receptor blocker plus nicotine treatment did not abolish the insulin sensitivity enhancing effect of nicotine, but abolished the triglyceride lowering effect of nicotine. These indicated that nicotine enhance insulin sensitivity major through central mechanism.
【Conclusion】
In this study, two main new effects and mechanisms were discussed: 1. Chronic nicotine treatment can enhance aortic smooth muscle relaxation induced by nitroxide. Nicotine can affect the down stream target of cGMP dependent protein kinase pathway through non-α7 nicotinic receptor. SERCA 3, Hsp 27 and COX-2 are not involved in this mechanism; Rho and AMPK are not involved either. Nicotine acts through enhancing opening of TEA sensitive potassium channel, which leads to hyperpolarization of the membrane, closing calcium channel and relaxation enhancement. This potassium channel is non-classic PKG activated BKCa or KATP channel, and is not 4-AP sensitive Kv potassium channel or Kir channel either, which is still need to be identified.
2. Chronic nicotine treatment can enhance insulin sensitivity. This effect is mainly mediated by central nicotinic receptors. The possible downstream mechanisms are that nicotine could upregulate AMPK protein expression in visceral and hypothalamus; nicotine reduces COX-2 expression in muscle, but it has no relationship with regulating PPARγprotein expression.
尼古丁是一个众所周知的经典化合物。它是烟草的主要成份之一。吸烟普遍被认为是动脉粥样硬化、高血压、糖尿病等多种心血管内分泌疾病的危险因素[1]。但这些不良作用是否是经由尼古丁产生的呢?现在看来,很多对尼古丁的研究结果由于研究方法、对象的不同仍存在矛盾、片面的地方,很多机制尚未阐明,因此不能单纯的断定吸烟的不良作用就是尼古丁产生的。相反,近期研究还发现了尼古丁的许多有益作用,比如抗炎、神经保护、改善肥胖大鼠的胰岛素抵抗等[2]。在本课题组前期建立的尼古丁长期治疗大鼠模型上,我们首次发现尼古丁治疗组大鼠的主动脉对硝普钠(一种一氧化氮供体药物)的内皮非依赖的舒张功能特异性的增强了。此外,该模型的胰岛素抵抗指数显著降低。这提示,尼古丁不仅可使血管中膜平滑肌一氧化氮信号通路增敏,还增强胰岛素敏感性。
尼古丁主要经由尼古丁受体发挥作用。尼古丁受体属配体门控离子通道型受体,由5个亚基组成五聚体,亚基的种类有17种(α1-10,β1-4,γ/ε,δ),配体结合位点在α亚基上[3-6]。传统意义上,尼古丁受体分为肌肉型和神经型,前者介导神经肌肉接头传递,后者介导或调节神经系统的突触传递。最近研究显示,神经型尼古丁受体也可在许多非神经细胞中表达[5, 6],而且,非神经细胞上尼古丁受体及其功能的发现,已经吸引不同领域科学家对这一经典受体产生新的研究兴趣。例如,免疫细胞上α7尼古丁受体的阐明,触发人们研究尼古丁受体激动剂对感染和炎症性疾病的治疗学意义。平滑肌细胞上,慢性尼古丁暴露可通过上调α7受体来增强胰岛素刺激的丝裂原信号通路。当前,在非神经组织(细胞)上考察尼古丁受体及其功能活性,将揭示尼古丁受体的新功能。而确定特定的尼古丁功能受体,对于发挥尼古丁有益的治疗作用意义重大。
综上所述,本课题拟深入研究尼古丁长期治疗对血管功能和胰岛素敏感性的影响和机制,这不仅有利于明确尼古丁对心血管和内分泌系统的作用,揭示尼古丁受体的新功能和血管舒张新通路,也对寻找治疗血管疾病和胰岛素抵抗的新靶点具有重要意义。
【方法】
第一部分:尼古丁对血管功能的影响及机制研究。该部分研究首先在本课题组前期建立的大鼠尼古丁长期治疗模型上考察主动脉外膜、内膜抗收缩功能;内膜依赖的血管舒张功能;中膜收缩和内膜非依赖的舒张功能。接着,进一步考察尼古丁增强中膜一氧化氮舒张反应机制。一方面在大、小鼠尼古丁治疗模型上考察尼古丁对中膜舒张反应影响的时间过程以明确该作用是急性作用还是慢性作用,另外还观察了尼古丁治疗后对胰岛素引起的中膜舒张反应的影响以初步判断尼古丁致舒张反应增强作用是否具有普遍意义。然后在离体血管实验中运用抑制剂和拟似物,检测关键蛋白表达和活性的方法明确该作用是否经由一氧化氮信号通路的3’, 5’-环鸟苷一磷酸(cGMP)依赖的信号通路。随后,通过制备基因芯片分析预测和经典通路上逐步排查的方法,寻找尼古丁改变的一氧化氮通路下游可能的靶点,如环氧合酶-2、内质网钙泵、AMPK、Rho等,再通过Western-blot,以及血管功能实验等方法进行验证,最终确认靶点。另一方面制备尼古丁受体阻断剂和尼古丁同时治疗的动物模型并使用α7尼古丁受体敲除小鼠进行尼古丁长期治疗,再进行血管功能实验从而寻找介导该作用的尼古丁受体。最后运用清醒测压技术,检测尼古丁模型组与生理盐水组血压等指标的差别。
第二部分:尼古丁对胰岛素敏感性的影响及机制研究。首先在尼古丁长期治疗的正常大鼠模型上通过血清学胰岛素抵抗指数、整体胰岛素耐量实验和糖耐量实验等多个指标验证尼古丁对胰岛素增敏的作用。接着针对机制研究,一方面在整体水平进行尼古丁给药模型的各部位脂肪称重,另一方面,在第一部分制备的尼古丁给药组和生理盐水组的主动脉基因芯片上寻找胰岛素敏感性相关基因的改变。同时,检测尼古丁治疗大鼠模型的四种胰岛素敏感组织(骨骼肌、肝脏、内脏脂肪和皮下脂肪)的关键蛋白(PPAR-γ、COX-2、AMPK)表达水平,从而寻找尼古丁影响的靶蛋白。另一方面,制备外周尼古丁受体阻断剂和尼古丁同时治疗的动物模型,以确认尼古丁致胰岛素增敏的中枢或外周受体机制。
【结果】
第一部分:尼古丁对血管功能的影响及机制研究
1.尼古丁长期治疗不影响主动脉外膜和内膜的抗收缩功能;不影响内膜依赖的舒张功能;不影响中膜平滑肌对新福林的收缩作用,而特异性的增强硝普钠(一氧化氮供体)引起的内皮非依赖性的中膜舒张反应。
2.尼古丁治疗后Sprague-Dawley (SD)大鼠和C57BL/6J小鼠中膜收缩反应不改变,但硝普钠引起的内皮非依赖性的中膜舒张反应都显著增强。大鼠治疗3周出现该现象,6周稳定出现该现象。小鼠治疗4周出现该现象,6周稳定出现该现象。尼古丁治疗后SD大鼠中膜对胰岛素引起的舒张反应也显示增强。这说明该增强作用是长期慢性的作用结果而非急性作用。
3.硝普钠引起的生理盐水和尼古丁组的舒张反应可被可溶性鸟苷酸环化酶抑制剂ODQ完全阻断,可被cGMP模拟物模拟,可被cGMP依赖的蛋白激酶(PKG)抑制剂阻断,而PKG蛋白表达在两组间不改变,蛋白活性没有明显差异。这提示该舒张增敏机制是经由PKG信号通路,且发生在PKG的下游靶位。
4.主动脉芯片结果显示与一氧化氮通路舒张相关基因中尼古丁治疗组内质网钙泵基因sarco(endo)plasmic reticulum calcium-ATPase 3 (SERCA 3)较生理盐水组上调1.56倍,热休克蛋白27(Hsp 27)较生理盐水组下调0.51倍。另外与一氧化氮通路尚无报道关联的COX-2基因表达显著下调0.09倍,我们也将它作为可能影响舒张通路的备选基因。
5.根据芯片结果进行验证:COX-2蛋白表达在尼古丁组主动脉平滑肌显著下调,而COX-2非选择性抑制剂Indomethacin和选择性抑制剂Meloxicam都不能取消尼古丁组和生理盐水组间血管舒张反应的差别;内质网钙泵抑制剂Thapsigargin不能取消尼古丁组和生理盐水组间血管舒张反应的差别;尼古丁组主动脉与收缩相关蛋白Hsp 27表达不变。这些结果排除了SERCA 3、Hsp 27和COX-2参与舒张增敏的机制。
6.进一步使用Rho激酶抑制剂Y-27632不能取消尼古丁组和生理盐水组间血管舒张反应的差别,这排除了尼古丁经由抑制Rho通路从而使钙收缩脱敏以增强舒张的可能。
7.运用AMPK抑制剂Compond C不能取消尼古丁组和生理盐水组间血管舒张反应的差别,又排除了最新提出的AMPKα1舒张通路的参与。
8.使用非选择性阻断剂20 mM的Tetraethylammonium(TEA)阻断钙激活钾通道和电压依赖性钾通道可完全取消尼古丁组和生理盐水组的舒张反应差别,而选择性阻断ATP敏感钾通道(KATP)和内向整流钾通道(Kir)都不能取消两组间血管舒张反应的差别。进一步选择性阻断大电导钙激活钾通道(BKCa)以及阻断4-AP敏感的电压依赖性钾通道却都不能取消尼古丁组和生理盐水组的舒张反应差别。而等渗高钾液(80 mM)可取消硝普钠引起的尼古丁组和生理盐水组的舒张反应差别。这说明尼古丁致硝普钠舒张增敏的机制是经由TEA敏感的钾通道,而不是的BKCa、KATP、Kir和4-AP敏感的电压依赖性钾通道。
9.外周神经型尼古丁受体阻断剂Hexamethonium和尼古丁同时治疗可以取消尼古丁组和生理盐水组间硝普钠引起的血管舒张反应差别。而尼古丁治疗的α7尼古丁受体敲除小鼠,硝普钠引起的血管舒张反应增强现象仍然存在,说明尼古丁使舒张增敏的机制是经由外周非α7尼古丁受体产生。
10.尼古丁长期给药后不影响基础状态的收缩压、舒张压和平均动脉压但延长心动间期;不影响硝普钠(3μg/kg, 10μg/kg, 30μg/kg, 100μg/kg)急性给药引起的血压下降。
第二部分:尼古丁对胰岛素敏感性的影响及机制研究
1.尼古丁长期治疗后不影响血清血糖水平,但显著减少胰岛素和甘油三酯水平,显著减少HOMA法的胰岛素抵抗指数,说明基础状态下的胰岛素增敏。
2.尼古丁治疗3周和6周后胰岛素耐量实验和葡萄糖耐量实验都显示尼古丁组血糖降低的速度和幅度较生理盐水组和生理盐水限食组都更为显著,说明刺激状态下的胰岛素增敏。
3.尼古丁长期治疗后皮下脂肪和内脏脂肪都明显减少,且内脏脂肪减少更为显著。
4.主动脉基因芯片显示尼古丁给药后脂代谢相关基因如脂酶、肾上腺素受体β3显著上调;能量消耗相关基因解耦联蛋白1显著上调;胰岛素敏感性相关基因过氧化物酶增殖体受体(PPARγ)显著上调。
5.尼古丁治疗不影响PPARγ蛋白在骨骼肌、肝脏、内脏脂肪和皮下脂肪的表达。尼古丁治疗显著减少骨骼肌COX-2表达,而不改变肝脏、内脏脂肪和皮下脂肪的表达。尼古丁治疗显著增加内脏脂肪和下丘脑AMPK表达,而不改变骨骼肌、肝脏和皮下脂肪的表达。
6.外周尼古丁受体阻断剂合并尼古丁治疗并不阻断尼古丁增强胰岛素敏感性的作用,但阻断尼古丁减少血清甘油三酯,说明尼古丁致胰岛素增敏主要经由中枢机制。
【结论】
本课题主要发现并探讨了尼古丁两个全新的作用和机制:
一、尼古丁长期治疗可特异性增强主动脉中膜对一氧化氮的舒张反应。该增敏作用是尼古丁经由非α7的外周神经型尼古丁受体影响了PKG下游途径产生。SERCA 3、Hsp 27和COX-2不参与该下游增敏途径; Rho和AMPK也不参与该下游增敏途径。该增敏途径是通过增强PKG下游的TEA敏感钾通道开放,引起细胞膜超极化从而抑制外钙内流,导致舒张反应增强,并且该钾通道不是平滑肌上目前已知的可被PKG直接激活的BKCa和KATP ,也不是平滑肌上另外两种主要的4-AP敏感的电压依赖性钾通道和Kir通道,有待于进一步确认。
二、尼古丁可增强正常大鼠的胰岛素敏感性。该作用主要是通过中枢尼古丁受体产生,可能的下游机制有上调下丘脑和内脏脂肪AMPK表达,抑制COX-2在骨骼肌的表达发挥,并且该增敏作用与调节PPARγ表达无关。
【Objective】
Smoking is a leading cause of atherosclerosis, hypertension, diabetes and other cardiovascular and endocrine diseases. Nicotine is one of the major components of cigarette with complex effects on cardiovascular systems, and many mechanisms still left unkown. Up to now, most studies showed that nicotine impaired vascular fuction and insulin action, for it reduced endothelium-independent vascular relaxation and led to insulin resistance. However, in chonic nicotine treatment rat model established in our department, aortic reactivity showed that endothelium-dependent relaxation was not altered, but endothelium-independent relaxation induced by sodium nitroprusside (a donor of nitrc oxide) was significantly enhanced. Besides, the insulin resistance index of this model was reduced. These indicated that nicotine not only enhanced the sensitivity of nitric oxide pathway in vascular smooth muscle but also insulin sensitivity.
Nicotine produces most of its effects through nicotine acetylcholine receptors (nAChRs). Nicotine acetylcholine receptors are ligand-gated ion channels, they form five aggregates composed of five subunits. There are seventeen subunits (α1-10,β1-4,γ/ε,δ), and ligands binding site are onαsubunit. Traditionally, Receptors of the nicotinic subclass can be distinguished as“muscle”or“neuronal”. The former mediates nerve-muscle connector transmission, and the latter mediates or modulates nerve system synapsis transmission. Recent studies pointed out that nAChRs are also expressed in many non-neuro cells. Moreover, the fact that nicotine receptors and their function were discovered in non-neuro cells has already attracted scientists in different fields. For example, the clarification ofα7 nicotinic receptor in immune cells triggered the studies on therapeutics significance of nicotinic receptor agonist treating infectious and imflammatory diseases. In smooth muscle cells, chronic nicotine expoture enhances insulin-induced mitogenic signaling via up-regulation ofα7 Nicotinic receptors. Currently, study nAChRs and their function in non-neuro cells will reveal new fuction of nAChRs. Meanwhile, determining specific nicotinic receptor is of most importance for therapeutic use of nicotine.
Above all, this study was to investigate the effects and mechanisms of nicotine on vascular fuction and insulin sensitivity. This will not only contribute to the understanding of the complex effect of nicotine on cardiovascular and endocrine systems, but also has significance in reveal the new fuction of nicotinic receptors and new targets in nitric oxide signaling pathway.
【Methods】
PartⅠ: Effect of nicotine on vascular fuction and its mechanism. First, in nicotine treated rat, anticontraction effect of adventitia and endothelium were tested; endothelium-dependent relaxation effect was tested; as well as smooth muscle contraction and endothelium-independent relaxation were tested. Then, we further investigated the mechanism of nicotine enhancing nitroxide induced relaxation. On the one hand, time course effect of nicotine treatment on aortic reactivity was first studied in SD rats and C57 mice to find out whether this effect of nicotine was generally and chronic. We also observe the relaxation effect of aorta smooth muscle induced by insulin in nicotine treated and untreated groups. Next, we studied its mechanism. In organ bath experiment, we used sGC inhibitor, PKG inhibitor and cGMP analog. Meanwhile, we detected PKG expression and activity to identify whether this effect was through cGMP dependent PKG pathway. Thirdly, we tried to find out the possible target nicotine altered in the down stream of nitric oxide signaling pathway by gene chip analysis accompanied with gradually excluding from old pathway. After selecting the possible targets, such as cyclooxygenase-2, sarcoplamic reticulium Ca2+-ATPase, amp-activated protein kinase, Rho, etc., we carried out western-blot and organ bath experiment to verify and confirm the targets. On the other hand, we made rat model treated at the same time with nicotine and hexamethonium that blocked nicotinic receptors exceptα7 receptor. We also treated alpha 7 knockout mice with nicotine. These two experiment were to find out the possible nicotinic receptor mediated this effect. At last, we investigated the blood pressure indicators in nicotine treated and untreated groups by conscious blood pressure detection technique.
PartⅡ: Effect of nicotine on insulin sensitivity and its mechanism. First, we confirm the insulin sensitivity enhancement effect of nicotine on normal rats through HOMA-index, insulin tolerance test and glucose tolerance test. Then, we studied its mechanism. On the one hand, we weighed the fat tissue from various parts of rats. On the other hand, we analysis the gene chip from PartⅠto find out insulin sentivity related gene alteration. Meanwhile, we detected key protein expression (PPAR-gamma, COX-2, AMPK) in four insulin sensitive tissue (skeletal muscle, liver, visceral fat and subcutaneous fat) in nicotine treated rat model. Moreover, we made hexamethonium plus nicotine treated rat model to study the central or periphery receptor mechanism of nicotine on insulin sensitiviy.
【Results】
PartⅠ: Study on effects and mechanisms of nicotine on vascular function
1. Chronic nicotine treatment did not affect anticontraction effect of aorta adventitia and endothelium; did not affect endothelium-dependent relaxation effect; did not affect aorta smooth muscle contraction effect induced by phenylephrine but had slight inhibition effect on contraction effect induced by low dose KCl; significantly enhanced endothelium independent smooth muscle relaxation.
2. Nicotine did not alter phenyllphrine induced aortic contraction but significantly enhanced sodium nitroprusside (donor of nitroxide) induced endothelium-independent relaxation in Sprague-Dawley (SD) rats and C57BL/6J mice. This phenomenon emerged after three weeks nicotine treatment in rats and four weeks in mice and was stable after 6 weeks nicotine treatment in rats and mice. Nicotine treatment also enhanced insulin induced smooth muscle relaxation. These indicated that the effect of nicotine is chronic rather than acute.
3. The relaxation induce by sodium nitroprusside was totally blocked by sGC inhibitor ODQ and PKG inhibitor RP-8-bromo-cGMP. Further the relaxation was mimicked by cGMP analog. PKG expression between nicotine and saline group was the same, and so was the PKG activity. This indicated the alteration was in the PKG downstream pathway.
4. Analysis on aorta gene chip showed two nitroxide relaxation pathway related gene altered. Sarcoplamic reticulium Ca2+-ATPase mRNA up regulated by 1.56 folds and heat shock protein 27 mRNA downregulated by 0.51 folds. Besides, another candidate gene COX-2 significant down regulated by 0.09 folds.
5. COX-2 protein expression was significantly reduced in aorta smooth mucle in nicotine group. But indomethacin, a nonselective inhibitor of cox-2 and meloxicam, a selective inhibitor of COX-2 could not abolish the relaxation difference between nicotine and saline group. Sarcoplamic reticulium Ca2+-ATPase inhibitor thapsigargin could not cancel the relaxation difference between nicotine and saline group. Hsp 27 protein expression was not altered in nicotine treated rats. These results eliminated SERCA 3、Hsp 27 and COX-2.
6. Rho kinase inhibitor Y-27632 could not abolish the relaxation difference between two groups. This indicated Rho was not involved in this mechanism.
7. AMPK inhibitor compound C could not abolish the relaxation difference between nicotine and saline groups. This eliminated the involvement of AMPK pathway.
8. Non-selective Kca and Kv inhibitor, 20 mM Tetraethylammonium (TEA), could totally abolish the relaxation difference between saline and nicotine group, while selective block KATP and Kir could not affect the difference between two groups. Selective block BKCa and 4-AP sensitive potassium channel could not abolish the relaxation difference between two groups either. In aorta preconstricted by 80 mM KCl, the relaxation difference between two groups induced by SNP was not present.
9. Hexamethonium abrogated the relaxation difference between saline and nicotine groups, while in alpha 7 knockout mice, the relaxation difference between two goups was still exist.
10. Chronic nicotine treatment did not affect basal systolic blood pressure, diastolic blood pressure and mean blood pressure but extended heart period; did not affect acute sodium nitroprusside (3μg/kg, 10μg/kg, 30μg/kg, 100μg/kg) injection induced blood pressure lowering effect.
PartⅡ:
1. Chronic nicotine treatment did not affect serum glucose level, but significantly reduce insulin and triglycride levels and significantly reduced HOMA-insulin resistant index.
2. Insulin tolerance test and glucose tolerance test in 3 weeks and 6 weeks nicotine and saline treated rats all showed that blood glucose lowering percentage was greater in nicotine treated rats.
3. Chronic nicotine treatment significantly reduced subcutaneous fat and visceral fat, especially visceral fat.
4. Aorta gene chip showed that after chronic nicotine treatment lipid metabolism related genes (lipase, adrenergic receptorβ3 ) significantly upregulated; energy expenditure related gene ( uncoupling protein 1) significantly upregulated; insulin sensitivity related gene (peroxisomal proliferators activated receptorγ, PPARγ) significantly upregulated.
5. Nicotine treatment did not affect PPARγexpression in skeletal muscle, liver, visceral fat and subcutaneous fat.Nicotine treatment significantly reduced COX-2 expression in skeletal muscle, but did not affect COX-2 expression in liver, visceral fat and subcutaneous fat.Nicotine treatment significantly increased AMPK expression in visceral fat, but did not affect AMPK expression in skeletal muscle, liver and subcutaneous
6. Periphery nicotinic receptor blocker plus nicotine treatment did not abolish the insulin sensitivity enhancing effect of nicotine, but abolished the triglyceride lowering effect of nicotine. These indicated that nicotine enhance insulin sensitivity major through central mechanism.
【Conclusion】
In this study, two main new effects and mechanisms were discussed: 1. Chronic nicotine treatment can enhance aortic smooth muscle relaxation induced by nitroxide. Nicotine can affect the down stream target of cGMP dependent protein kinase pathway through non-α7 nicotinic receptor. SERCA 3, Hsp 27 and COX-2 are not involved in this mechanism; Rho and AMPK are not involved either. Nicotine acts through enhancing opening of TEA sensitive potassium channel, which leads to hyperpolarization of the membrane, closing calcium channel and relaxation enhancement. This potassium channel is non-classic PKG activated BKCa or KATP channel, and is not 4-AP sensitive Kv potassium channel or Kir channel either, which is still need to be identified.
2. Chronic nicotine treatment can enhance insulin sensitivity. This effect is mainly mediated by central nicotinic receptors. The possible downstream mechanisms are that nicotine could upregulate AMPK protein expression in visceral and hypothalamus; nicotine reduces COX-2 expression in muscle, but it has no relationship with regulating PPARγprotein expression.
引文
1. Smith SC, Jr. Multiple risk factors for cardiovascular disease and diabetes mellitus. Am J Med, 2007, 120 (3 Suppl 1): S3-S11.
2. Liu RH, Mizuta M, and Matsukura S. Long-term oral nicotine administration reduces insulin resistance in obese rats. Eur J Pharmacol, 2003, 458 (1-2): 227-234.
3. Liu RH, Mizuta M, and Matsukura S. The expression and functional role of nicotinic acetylcholine receptors in rat adipocytes. J Pharmacol Exp Ther, 2004, 310 (1): 52-58.
4. Gahring LC and Rogers SW. Neuronal nicotinic acetylcholine receptor expression and function on nonneuronal cells. Aaps J, 2005, 7 (4): E885-894.
5. Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, Li JH, Wang H, Yang H, Ulloa L, Al-Abed Y, Czura CJ, and Tracey KJ. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature, 2003, 421 (6921): 384-388.
6. de Jonge WJ and Ulloa L. The alpha7 nicotinic acetylcholine receptor as a pharmacological target for inflammation. Br J Pharmacol, 2007, 151 (7): 915-929.
7. Hanna ST. Nicotine effect on cardiovascular system and ion channels. J Cardiovasc Pharmacol, 2006, 47 (3): 348-358.
8. Ikushima S, Muramatsu I, and Fujiwara M. Nicotine-induced response in guinea-pig aorta enhanced by goniopora toxin. J Pharmacol Exp Ther, 1982, 223 (3): 790-794.
9. Xiao D, Huang X, Lawrence J, Yang S, and Zhang L. Fetal and neonatal nicotine exposure differentially regulates vascular contractility in adult male and female offspring. J Pharmacol Exp Ther, 2007, 320 (2): 654-661.
10. Xiao D, Xu Z, Huang X, Longo LD, Yang S, and Zhang L. Prenatal gender-related nicotine exposure increases blood pressure response to angiotensin II in adult offspring. Hypertension, 2008, 51 (4): 1239-1247.
11. Eliasson B, Smith U, and Lonnroth P. No acute effects of smoking and nicotine nasal spray on lipolysis measured by subcutaneous microdialysis. Eur J Clin Invest, 1997, 27 (6): 503-509.
12. Swislocki AL, Tsuzuki A, Tait M, Khuu D, and Fann K. Smokeless nicotine administration is associated with hypertension but not with a deterioration in glucose tolerance in rats.Metabolism, 1997, 46 (9): 1008-1012.
13. Eliasson B, Taskinen MR, and Smith U. Long-term use of nicotine gum is associated with hyperinsulinemia and insulin resistance. Circulation, 1996, 94 (5): 878-881.
14. de Jonge WJ, van der Zanden EP, The FO, Bijlsma MF, van Westerloo DJ, Bennink RJ, Berthoud HR, Uematsu S, Akira S, van den Wijngaard RM, and Boeckxstaens GE. Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway. Nat Immunol, 2005, 6 (8): 844-851.
15. Wada T, Naito M, Kenmochi H, Tsuneki H, and Sasaoka T. Chronic nicotine exposure enhances insulin-induced mitogenic signaling via up-regulation of alpha7 nicotinic receptors in isolated rat aortic smooth muscle cells. Endocrinology, 2007, 148 (2): 790-799.
16. Wanstall JC, Homer KL, and Doggrell SA. Evidence for, and importance of, cGMP-independent mechanisms with NO and NO donors on blood vessels and platelets. Curr Vasc Pharmacol, 2005, 3 (1): 41-53.
17. Hofmann F, Feil R, Kleppisch T, and Schlossmann J. Function of cGMP-dependent protein kinases as revealed by gene deletion. Physiol Rev, 2006, 86 (1): 1-23.
18. Begum N. Insulin signaling in the vasculature. Front Biosci, 2003, 8: s796-804.
19. Horman S, Morel N, Vertommen D, Hussain N, Neumann D, Beauloye C, El Najjar N, Forcet C, Viollet B, Walsh MP, Hue L, and Rider MH. AMP-activated protein kinase phosphorylates and desensitizes smooth muscle myosin light chain kinase. J Biol Chem, 2008, 283 (27): 18505-18512.
20. Gao YJ, Holloway AC, Zeng ZH, Lim GE, Petrik JJ, Foster WG, and Lee RM. Prenatal exposure to nicotine causes postnatal obesity and altered perivascular adipose tissue function. Obes Res, 2005, 13 (4): 687-692.
21. Murrin LC, Ferrer JR, Zeng WY, and Haley NJ. Nicotine administration to rats: methodological considerations. Life Sci, 1987, 40 (17): 1699-1708.
22. Gerzanich V, Zhang F, West GA, and Simard JM. Chronic nicotine alters NO signaling of Ca(2+) channels in cerebral arterioles. Circ Res, 2001, 88 (3): 359-365.
23. Rangemark C and Wennmalm A. Endothelium-dependent and -independent vasodilation andreactive hyperemia in healthy smokers. J Cardiovasc Pharmacol, 1992, 20 Suppl 12: S198-201.
24. Lincoln TM, Dey N, and Sellak H. Invited review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol, 2001, 91 (3): 1421-1430.
25. Carvajal JA, Germain AM, Huidobro-Toro JP, and Weiner CP. Molecular mechanism of cGMP-mediated smooth muscle relaxation. J Cell Physiol, 2000, 184 (3): 409-420.
26. Tanaka Y, Tang G, Takizawa K, Otsuka K, Eghbali M, Song M, Nishimaru K, Shigenobu K, Koike K, Stefani E, and Toro L. Kv channels contribute to nitric oxide- and atrial natriuretic peptide-induced relaxation of a rat conduit artery. J Pharmacol Exp Ther, 2006, 317 (1): 341-354.
27. Sorensson J, Jodal M, and Lundgren O. Involvement of nerves and calcium channels in the intestinal response to Clostridium difficile toxin A: an experimental study in rats in vivo. Gut, 2001, 49 (1): 56-65.
28. Ericson M, Engel JA, and Soderpalm B. Peripheral involvement in nicotine-induced enhancement of ethanol intake. Alcohol, 2000, 21 (1): 37-47.
1. Eliasson B, Smith U, and Lonnroth P. No acute effects of smoking and nicotine nasal spray on lipolysis measured by subcutaneous microdialysis. Eur J Clin Invest, 1997, 27 (6): 503-509.
2. Swislocki AL, Tsuzuki A, Tait M, Khuu D, and Fann K. Smokeless nicotine administration is associated with hypertension but not with a deterioration in glucose tolerance in rats. Metabolism, 1997, 46 (9): 1008-1012.
3. Eliasson B, Taskinen MR, and Smith U. Long-term use of nicotine gum is associated with hyperinsulinemia and insulin resistance. Circulation, 1996, 94 (5): 878-881.
4. Liu RH, Mizuta M, and Matsukura S. Long-term oral nicotine administration reduces insulin resistance in obese rats. Eur J Pharmacol, 2003, 458 (1-2): 227-234.
5. Murrin LC, Ferrer JR, Zeng WY, and Haley NJ. Nicotine administration to rats: methodological considerations. Life Sci, 1987, 40 (17): 1699-1708.
6. Kawaguchi T, Ide T, Taniguchi E, Hirano E, Itou M, Sumie S, Nagao Y, Yanagimoto C, Hanada S, Koga H, and Sata M. Clearance of HCV improves insulin resistance, beta-cell function, and hepatic expression of insulin receptor substrate 1 and 2. Am J Gastroenterol, 2007, 102 (3): 570-576.
7. Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, Li JH, Wang H, Yang H, Ulloa L, Al-Abed Y, Czura CJ, and Tracey KJ. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature, 2003, 421 (6921): 384-388.
8. Murrin LC, Ferrer JR, Zeng WY, and Haley NJ. Nicotine administration to rats: methodological considerations. Life Sci, 1987, 40 (17): 1699-1708.
9. Lee S, Muniyappa R, Yan X, Chen H, Yue LQ, Hong EG, Kim JK, and Quon MJ. Comparison between surrogate indexes of insulin sensitivity and resistance and hyperinsulinemic euglycemic clamp estimates in mice. Am J Physiol Endocrinol Metab, 2008, 294 (2): E261-270.
10. Sztalryd C, Hamilton J, Horwitz BA, Johnson P, and Kraemer FB. Alterations of lipolysis and lipoprotein lipase in chronically nicotine-treated rats. Am J Physiol, 1996, 270 (2 Pt 1): E215-223.
11. An Z, Wang H, Song P, Zhang M, Geng X, and Zou MH. Nicotine-induced activation ofAMP-activated protein kinase inhibits fatty acid synthase in 3T3L1 adipocytes: a role for oxidant stress. J Biol Chem, 2007, 282 (37): 26793-26801.
12. Muoio DM, Dohm GL, Fiedorek FT, Jr., Tapscott EB, and Coleman RA. Leptin directly alters lipid partitioning in skeletal muscle. Diabetes, 1997, 46 (8): 1360-1363.
13. Chubb SA, Davis WA, and Davis TM. Interactions among thyroid function, insulin sensitivity, and serum lipid concentrations: the Fremantle diabetes study. J Clin Endocrinol Metab, 2005, 90 (9): 5317-5320.
1. Thomas DD, et al. The chemical biology of nitric oxide: implications in cellular signaling. Free Radic Biol Med, 2008, 45:18~31.
2. Hanafy KA, et al. NO, nitrotyrosine, and cyclic GMP in signal transduction. Med Sci Monit, 2001, 7:801~819.
3. Alderton WK, et al. Nitric oxide synthases: structure, function and inhibition. Biochem J, 2001, 357:593~615.
4. Schild L, et al. Nitric oxide produced in rat liver mitochondria causes oxidative stress and impairment of respiration after transient hypoxia. FASEB J, 2003, 17:2194~2201.
5. Low SY. Application of pharmaceuticals to nitric oxide. Mol Aspects Med, 2005, 26:97~138.
6. Poulos TL. Soluble guanylate cyclase. Curr Opin Struct Biol, 2006 16:736~743.
7. Becker EM, et al. NO-independent regulatory site of direct sGC stimulators like YC-1 and BAY 41-2272. BMC Pharmacol, 2001, 1~13.
8. Hofmann F, et al. Function of cGMP-dependent protein kinases as revealed by gene deletion. Physiol Rev, 2006, 86:1~23.
9. Biel M, et al. Structure and function of cyclic nucleotide-gated channels. Rev Physiol Biochem Pharmacol, 1999, 135:151~171.
10. Lincoln TM, et al. cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol, 2001, 91:1421~1430.
11. Carvajal JA, et al. Molecular mechanism of cGMP-mediated smooth muscle relaxation. J Cell Physiol, 2000, 184:409~420.
12. Yang J, et al. Mathematical modeling of the nitric oxide/cGMP pathway in the vascular smooth muscle cell. Am J Physiol Heart Circ Physiol, 2005, 289:H886~H897.
13. Schlossmann J, et al. Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase I beta. Nature, 2000, 404:197~201.
14. Tang KM, et al. Regulator of G-protein signaling-2 mediates vascular smooth muscle relaxation and blood pressure. Nat Med, 2003, 9:1506~1512.
15. Koller A, et al. Association of phospholamban with a cGMP kinase signaling complex. Biochem Biophys Res Commun, 2003, 300:155~160.
16. Salinthone S, et al. Small heat shock proteins in smooth muscle. Pharmacol Ther, 2008, 119:44~54.
17. Wanstall JC, et al. Evidence for, and importance of, cGMP-independent mechanisms with NO and NO donors on blood vessels and platelets. Curr Vasc Pharmacol, 2005, 3:41~53.
18. Adachi T, et al. Reduced sarco/endoplasmic reticulum Ca2+ uptake activity can account for the reduced response to NO, but not sodium nitroprusside, in hypercholesterolaemic rabbit aorta. Circulation, 2001, 104:1040~1045.
19. Sogo N, et al. S-Nitrosothiols cause prolonged, nitric oxide-mediated relaxation in human saphenous vein and internal mammary artery: therapeutic potential in bypass surgery. Br J Pharmacol, 2000, 131: 1236~1244.
20. Kloss S, et al. Aging and chronic hypertension decrease expression of rat aortic soluble guanylate cyclase. Hypertension, 2000, 35:43~47.
21. Ruetten H, et al. Downregulation of soluble guanylyl cyclase in young and aging spontaneously hypertensive rats. Circ Res, 1999, 85:534~541.
22. Scott WS, et al. Eschericia coli lipopolysaccharide downregulates soluble guanylate cyclase in pulmonary artery smooth muscle. J Surg Res, 1998, 80:309~314.
23. Courtois E, et al. Lead-induced downregulation of soluble guanylyl cyclasse in isolated rat aortic segments mediated by reactive oxygen species and cyclooxygenase-2. J Am Soc Nephrol, 2003, 14: 1464~1470.
24. Kroncke KD, et al. Inactivation of zinc finger transcription factors provides a mechanism for a gene regulatory role of nitric oxide. FASEB J, 2000, 14:166~173.
25. Kimura H, et al. Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide. Blood, 2000, 95:189~197.
2. Liu RH, Mizuta M, and Matsukura S. Long-term oral nicotine administration reduces insulin resistance in obese rats. Eur J Pharmacol, 2003, 458 (1-2): 227-234.
3. Liu RH, Mizuta M, and Matsukura S. The expression and functional role of nicotinic acetylcholine receptors in rat adipocytes. J Pharmacol Exp Ther, 2004, 310 (1): 52-58.
4. Gahring LC and Rogers SW. Neuronal nicotinic acetylcholine receptor expression and function on nonneuronal cells. Aaps J, 2005, 7 (4): E885-894.
5. Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, Li JH, Wang H, Yang H, Ulloa L, Al-Abed Y, Czura CJ, and Tracey KJ. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature, 2003, 421 (6921): 384-388.
6. de Jonge WJ and Ulloa L. The alpha7 nicotinic acetylcholine receptor as a pharmacological target for inflammation. Br J Pharmacol, 2007, 151 (7): 915-929.
7. Hanna ST. Nicotine effect on cardiovascular system and ion channels. J Cardiovasc Pharmacol, 2006, 47 (3): 348-358.
8. Ikushima S, Muramatsu I, and Fujiwara M. Nicotine-induced response in guinea-pig aorta enhanced by goniopora toxin. J Pharmacol Exp Ther, 1982, 223 (3): 790-794.
9. Xiao D, Huang X, Lawrence J, Yang S, and Zhang L. Fetal and neonatal nicotine exposure differentially regulates vascular contractility in adult male and female offspring. J Pharmacol Exp Ther, 2007, 320 (2): 654-661.
10. Xiao D, Xu Z, Huang X, Longo LD, Yang S, and Zhang L. Prenatal gender-related nicotine exposure increases blood pressure response to angiotensin II in adult offspring. Hypertension, 2008, 51 (4): 1239-1247.
11. Eliasson B, Smith U, and Lonnroth P. No acute effects of smoking and nicotine nasal spray on lipolysis measured by subcutaneous microdialysis. Eur J Clin Invest, 1997, 27 (6): 503-509.
12. Swislocki AL, Tsuzuki A, Tait M, Khuu D, and Fann K. Smokeless nicotine administration is associated with hypertension but not with a deterioration in glucose tolerance in rats.Metabolism, 1997, 46 (9): 1008-1012.
13. Eliasson B, Taskinen MR, and Smith U. Long-term use of nicotine gum is associated with hyperinsulinemia and insulin resistance. Circulation, 1996, 94 (5): 878-881.
14. de Jonge WJ, van der Zanden EP, The FO, Bijlsma MF, van Westerloo DJ, Bennink RJ, Berthoud HR, Uematsu S, Akira S, van den Wijngaard RM, and Boeckxstaens GE. Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway. Nat Immunol, 2005, 6 (8): 844-851.
15. Wada T, Naito M, Kenmochi H, Tsuneki H, and Sasaoka T. Chronic nicotine exposure enhances insulin-induced mitogenic signaling via up-regulation of alpha7 nicotinic receptors in isolated rat aortic smooth muscle cells. Endocrinology, 2007, 148 (2): 790-799.
16. Wanstall JC, Homer KL, and Doggrell SA. Evidence for, and importance of, cGMP-independent mechanisms with NO and NO donors on blood vessels and platelets. Curr Vasc Pharmacol, 2005, 3 (1): 41-53.
17. Hofmann F, Feil R, Kleppisch T, and Schlossmann J. Function of cGMP-dependent protein kinases as revealed by gene deletion. Physiol Rev, 2006, 86 (1): 1-23.
18. Begum N. Insulin signaling in the vasculature. Front Biosci, 2003, 8: s796-804.
19. Horman S, Morel N, Vertommen D, Hussain N, Neumann D, Beauloye C, El Najjar N, Forcet C, Viollet B, Walsh MP, Hue L, and Rider MH. AMP-activated protein kinase phosphorylates and desensitizes smooth muscle myosin light chain kinase. J Biol Chem, 2008, 283 (27): 18505-18512.
20. Gao YJ, Holloway AC, Zeng ZH, Lim GE, Petrik JJ, Foster WG, and Lee RM. Prenatal exposure to nicotine causes postnatal obesity and altered perivascular adipose tissue function. Obes Res, 2005, 13 (4): 687-692.
21. Murrin LC, Ferrer JR, Zeng WY, and Haley NJ. Nicotine administration to rats: methodological considerations. Life Sci, 1987, 40 (17): 1699-1708.
22. Gerzanich V, Zhang F, West GA, and Simard JM. Chronic nicotine alters NO signaling of Ca(2+) channels in cerebral arterioles. Circ Res, 2001, 88 (3): 359-365.
23. Rangemark C and Wennmalm A. Endothelium-dependent and -independent vasodilation andreactive hyperemia in healthy smokers. J Cardiovasc Pharmacol, 1992, 20 Suppl 12: S198-201.
24. Lincoln TM, Dey N, and Sellak H. Invited review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol, 2001, 91 (3): 1421-1430.
25. Carvajal JA, Germain AM, Huidobro-Toro JP, and Weiner CP. Molecular mechanism of cGMP-mediated smooth muscle relaxation. J Cell Physiol, 2000, 184 (3): 409-420.
26. Tanaka Y, Tang G, Takizawa K, Otsuka K, Eghbali M, Song M, Nishimaru K, Shigenobu K, Koike K, Stefani E, and Toro L. Kv channels contribute to nitric oxide- and atrial natriuretic peptide-induced relaxation of a rat conduit artery. J Pharmacol Exp Ther, 2006, 317 (1): 341-354.
27. Sorensson J, Jodal M, and Lundgren O. Involvement of nerves and calcium channels in the intestinal response to Clostridium difficile toxin A: an experimental study in rats in vivo. Gut, 2001, 49 (1): 56-65.
28. Ericson M, Engel JA, and Soderpalm B. Peripheral involvement in nicotine-induced enhancement of ethanol intake. Alcohol, 2000, 21 (1): 37-47.
1. Eliasson B, Smith U, and Lonnroth P. No acute effects of smoking and nicotine nasal spray on lipolysis measured by subcutaneous microdialysis. Eur J Clin Invest, 1997, 27 (6): 503-509.
2. Swislocki AL, Tsuzuki A, Tait M, Khuu D, and Fann K. Smokeless nicotine administration is associated with hypertension but not with a deterioration in glucose tolerance in rats. Metabolism, 1997, 46 (9): 1008-1012.
3. Eliasson B, Taskinen MR, and Smith U. Long-term use of nicotine gum is associated with hyperinsulinemia and insulin resistance. Circulation, 1996, 94 (5): 878-881.
4. Liu RH, Mizuta M, and Matsukura S. Long-term oral nicotine administration reduces insulin resistance in obese rats. Eur J Pharmacol, 2003, 458 (1-2): 227-234.
5. Murrin LC, Ferrer JR, Zeng WY, and Haley NJ. Nicotine administration to rats: methodological considerations. Life Sci, 1987, 40 (17): 1699-1708.
6. Kawaguchi T, Ide T, Taniguchi E, Hirano E, Itou M, Sumie S, Nagao Y, Yanagimoto C, Hanada S, Koga H, and Sata M. Clearance of HCV improves insulin resistance, beta-cell function, and hepatic expression of insulin receptor substrate 1 and 2. Am J Gastroenterol, 2007, 102 (3): 570-576.
7. Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, Li JH, Wang H, Yang H, Ulloa L, Al-Abed Y, Czura CJ, and Tracey KJ. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature, 2003, 421 (6921): 384-388.
8. Murrin LC, Ferrer JR, Zeng WY, and Haley NJ. Nicotine administration to rats: methodological considerations. Life Sci, 1987, 40 (17): 1699-1708.
9. Lee S, Muniyappa R, Yan X, Chen H, Yue LQ, Hong EG, Kim JK, and Quon MJ. Comparison between surrogate indexes of insulin sensitivity and resistance and hyperinsulinemic euglycemic clamp estimates in mice. Am J Physiol Endocrinol Metab, 2008, 294 (2): E261-270.
10. Sztalryd C, Hamilton J, Horwitz BA, Johnson P, and Kraemer FB. Alterations of lipolysis and lipoprotein lipase in chronically nicotine-treated rats. Am J Physiol, 1996, 270 (2 Pt 1): E215-223.
11. An Z, Wang H, Song P, Zhang M, Geng X, and Zou MH. Nicotine-induced activation ofAMP-activated protein kinase inhibits fatty acid synthase in 3T3L1 adipocytes: a role for oxidant stress. J Biol Chem, 2007, 282 (37): 26793-26801.
12. Muoio DM, Dohm GL, Fiedorek FT, Jr., Tapscott EB, and Coleman RA. Leptin directly alters lipid partitioning in skeletal muscle. Diabetes, 1997, 46 (8): 1360-1363.
13. Chubb SA, Davis WA, and Davis TM. Interactions among thyroid function, insulin sensitivity, and serum lipid concentrations: the Fremantle diabetes study. J Clin Endocrinol Metab, 2005, 90 (9): 5317-5320.
1. Thomas DD, et al. The chemical biology of nitric oxide: implications in cellular signaling. Free Radic Biol Med, 2008, 45:18~31.
2. Hanafy KA, et al. NO, nitrotyrosine, and cyclic GMP in signal transduction. Med Sci Monit, 2001, 7:801~819.
3. Alderton WK, et al. Nitric oxide synthases: structure, function and inhibition. Biochem J, 2001, 357:593~615.
4. Schild L, et al. Nitric oxide produced in rat liver mitochondria causes oxidative stress and impairment of respiration after transient hypoxia. FASEB J, 2003, 17:2194~2201.
5. Low SY. Application of pharmaceuticals to nitric oxide. Mol Aspects Med, 2005, 26:97~138.
6. Poulos TL. Soluble guanylate cyclase. Curr Opin Struct Biol, 2006 16:736~743.
7. Becker EM, et al. NO-independent regulatory site of direct sGC stimulators like YC-1 and BAY 41-2272. BMC Pharmacol, 2001, 1~13.
8. Hofmann F, et al. Function of cGMP-dependent protein kinases as revealed by gene deletion. Physiol Rev, 2006, 86:1~23.
9. Biel M, et al. Structure and function of cyclic nucleotide-gated channels. Rev Physiol Biochem Pharmacol, 1999, 135:151~171.
10. Lincoln TM, et al. cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol, 2001, 91:1421~1430.
11. Carvajal JA, et al. Molecular mechanism of cGMP-mediated smooth muscle relaxation. J Cell Physiol, 2000, 184:409~420.
12. Yang J, et al. Mathematical modeling of the nitric oxide/cGMP pathway in the vascular smooth muscle cell. Am J Physiol Heart Circ Physiol, 2005, 289:H886~H897.
13. Schlossmann J, et al. Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase I beta. Nature, 2000, 404:197~201.
14. Tang KM, et al. Regulator of G-protein signaling-2 mediates vascular smooth muscle relaxation and blood pressure. Nat Med, 2003, 9:1506~1512.
15. Koller A, et al. Association of phospholamban with a cGMP kinase signaling complex. Biochem Biophys Res Commun, 2003, 300:155~160.
16. Salinthone S, et al. Small heat shock proteins in smooth muscle. Pharmacol Ther, 2008, 119:44~54.
17. Wanstall JC, et al. Evidence for, and importance of, cGMP-independent mechanisms with NO and NO donors on blood vessels and platelets. Curr Vasc Pharmacol, 2005, 3:41~53.
18. Adachi T, et al. Reduced sarco/endoplasmic reticulum Ca2+ uptake activity can account for the reduced response to NO, but not sodium nitroprusside, in hypercholesterolaemic rabbit aorta. Circulation, 2001, 104:1040~1045.
19. Sogo N, et al. S-Nitrosothiols cause prolonged, nitric oxide-mediated relaxation in human saphenous vein and internal mammary artery: therapeutic potential in bypass surgery. Br J Pharmacol, 2000, 131: 1236~1244.
20. Kloss S, et al. Aging and chronic hypertension decrease expression of rat aortic soluble guanylate cyclase. Hypertension, 2000, 35:43~47.
21. Ruetten H, et al. Downregulation of soluble guanylyl cyclase in young and aging spontaneously hypertensive rats. Circ Res, 1999, 85:534~541.
22. Scott WS, et al. Eschericia coli lipopolysaccharide downregulates soluble guanylate cyclase in pulmonary artery smooth muscle. J Surg Res, 1998, 80:309~314.
23. Courtois E, et al. Lead-induced downregulation of soluble guanylyl cyclasse in isolated rat aortic segments mediated by reactive oxygen species and cyclooxygenase-2. J Am Soc Nephrol, 2003, 14: 1464~1470.
24. Kroncke KD, et al. Inactivation of zinc finger transcription factors provides a mechanism for a gene regulatory role of nitric oxide. FASEB J, 2000, 14:166~173.
25. Kimura H, et al. Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide. Blood, 2000, 95:189~197.