新型一氧化氮合酶抑制剂对心肌细胞内游离钙浓度的影响
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摘要
一、实验背景在哺乳动物体内存在三种形式的一氧化氮合酶(nitric oxidesynthase，NOS)，即神经元型一氧化氮合酶(neuronal NOS，nNOS or NOS1)、诱导型一氧化氮合酶(inducible NOS，iNOS or NOS2)，内皮型一氧化氮合酶(endothelial NOS，eNOS or NOS3)，三者均以左旋精氨酸为底物，生成一氧化氮(nitric oxide，NO)和瓜氨酸。NO参与调整包括神经、泌尿、免疫、消化、呼吸等系统的功能。三种亚型的NOS均可以在心肌中表达，一般认为NOS1位于心肌传导系统及神经元中；NOS2在炎症因子刺激下，心脏各种组织均可以表达；NOS3在血管内皮、心肌细胞、心内膜处表达。NOS产生的NO对维持心脏功能起着重要作用，包括对心肌收缩功能的调整，影响细胞的能量产生、底物代谢，细胞生长及存活。NO可以通过两种方式发挥其作用：环磷酸鸟苷(3',5'-cyclic guanosine monophosphate，cGMP)依赖途径及对蛋白巯基的亚硝基化作用。NO可激活的鸟苷酸环化酶，促进cGMP生成，后者继续激活蛋白激酶G而发挥作用。
NOS亚型的在心肌中的亚细胞定位是调整NO作用的一个重要因素，NOS3位于肌膜的凹穴处，靠近β肾上腺素能受体，NOS3／NO抑制心肌对β肾上腺素能受体激动剂的反应性。而NOS1以肌浆网兰尼碱受体(ryanodine receptor，RyR)为靶组织，可以增强心肌收缩时肌浆网的钙释放。NOS1与NOS3对心肌收缩功能起着相反的调节作用。心肌正常的时候，不表达NOS2，在某些特定的病理状态下，在炎症和细胞因子诱导心肌才丌始表达NOS2，产生大量的NO。心肌中的NOS2位于细胞质的囊泡和内质网中，在缺血再灌注的时候，可以对心脏起到保护作用。但更多的时候，NOS2的表达，严重影响着心肌功能，目前研究较多的是NO对心肌收缩抑制的机制。NOS2的表达可以抑制心肌细胞中钙瞬变、降低心肌收缩力和降低心肌对β受体激动剂反应性。越来越多的证据表明，NO可以对兴奋收缩耦联的多个方面进行调节，包括：受体信号传导、L-型钙通道活性、通过肌浆网RyR进行的钙释放及线粒体呼吸过程。
精氨酸(文中所提到的精氨酸均为左旋精氨酸)是NOS的唯一底物，同时也是精氨酸酶的底物。因此精氨酸酶可以通过对底物的竞争性利用而限制NO的产生。精氨酸酶一般理解为鸟氨酸循环最后一个酶，体内存在两种形式的精氨酸酶，一种是肝脏型精氨酸酶(精氨酸酶Ⅰ)，存在于细胞质中，其主要功能是合成尿素；另一种是线粒体型精氨酸酶(即精氨酸酶Ⅱ)，广泛存在体内的多种组织，参与多胺、脯氨酸、谷氨酸等物质的合成。在心脏组织中两种精氨酸酶均可以被检测出。但是在鼠心肌细胞中，仅发现精氨酸酶Ⅱ表达。以BEC抑制精氨酸酶的活性，可以增加钙依赖的NOS的活性和增加NO的产生。
精氨酸是可以影响NOS的另外一个因素。在内皮细胞中，精氨酸浓度是NOS的Km值的2-3倍，理论上不会因NOS底物耗竭而限制NOS活性和NO的产生。相应的，外源性的精氨酸也不应影响NOS的活性。但是，在一些特定的病理过程中，给予额外的精氨酸能够增加NO的产生。这种现象被成为精氨酸颠倒现象(L-Arginine Paradox)：细胞内精氨酸的浓度对于NOS反应来说是饱和的，但是外源性的精氨酸还是可以增加NO的产生。
二、实验目的液相合成法合成的由两分子精氨酸和一分子赖氨酸组成的三肽(以下均简称：三肽)，以体外培养的脂多糖(lipopolysaccharide，LPS)诱导表达NOS2的巨噬细胞、表达NOS3的脐静脉内皮细胞、表达NOS1和NOS3的SD乳鼠心肌细胞(cardiac myocytes，CMs)为对象，硝酸还原酶法检测三肽对不同细胞的NO合成的抑制效果；白介素6(interleukin-6，IL-6)联合LPS诱导心肌NOS2表达，免疫印记法(western blotting)等技术检测NOS2蛋白的表达。以表达NOS2的心肌细胞为研究对象，进一步研究三肽对β肾上腺素能受体激动剂异丙肾上腺素(isoproterenol，ISO)刺激心肌细胞游离钙浓度变化的影响，给予不同的钙通道阻滞剂，包括肌膜L-型通道、肌浆网兰尼碱受体(ryanodine receptor，RyRs)、肌醇1，4，5-三磷酸受体(inositol 1，4，5,-triphosphate rceptor，IP3R)。明确参与三肽发挥效应的钙通道类型。同时探讨共同以精氨酸为底物的精氨酸酶对正常心肌细胞和表达NOS2的心肌细胞中ISO诱导心肌细胞内游离钙浓度变化的影响，系统地探讨NOS、精氨酸酶、精氨酸三者对心肌功能影响的程度及相互间的影响。
三、实验方法
1、LPS处理巨噬细胞24 h表达NOS2，检测三肽对乳鼠心肌细胞、脐静脉内皮细胞和表达NOS2的巨噬细胞中NO的浓度，明确三肽对相应的NOS亚型的抑制效果。
2、IL-6联合LPS处理心肌细胞24 h，western blouing检测6、12、24 h三个时间点NOS2的蛋白表达情况。
3、表达NOS2的心肌细胞为对象，给予L-NNA、不同浓度的三肽处理12 h，研究三肽对表达NOS2的心肌细胞NO抑制作用，寻找同L-NNA具有相同抑制效果的三肽浓度。
4、正常心肌细胞，给予精氨酸酶抑制剂S-(2-boronoethyl)-L-cysteine(BEC)和NOS1抑制剂vinyl-L-N-5-(1-imino-3-butenyl)-L-omithine(L-VNIO)处理，实验分为BEC组、L-VNIO组和BEC+L-VNIO组．给予相应处理后，取上清检测NO值。明确精氨酸酶对心肌细胞NO产生的影响。
5、表达NOS2的心肌细胞为对象，激光共聚焦显微镜(Laser Scan ConfocalMicroscope，LSCM)检测三肽对ISO刺激的心肌细胞胞内游离钙浓度变化的影响。同时比较对NO具有相同抑制效果的低浓度的三肽同高浓度的L-NNA对ISO刺激的心肌细胞内游离钙浓度变化的影响，明确NOS的细胞定位是细胞功能调整的重要方式。
6、分别以维拉帕米(verapamil)阻滞L-型通道、普鲁卡因(procaine)阻断RyR、肝素(heparin)阻断IP3R后，各组均给予三肽。检测上述处理后心肌细胞钙浓度的变化。明确三肽升高心肌内游离钙浓度的途径。
7、正常的心肌细胞，分为ISO、ISO+BEC、ISO+L-VNIO、ISO+BEC+L-VNIO组，各组给予相应处理后。上机检测细胞内游离钙浓度。明确精氨酸酶及NOS1对心肌功能的影响。
8、表达NOS2的心肌细胞，分别给予三肽、L-VNIO、BCE、三肽+L-VNIO处理。检测各组细胞内钙浓度，明确精氨酸酶对表达NOS2心肌细胞的钙浓度的影响。
四、统计学处理
采用SPSS12．0统计软件进行数据分析，结果以(?)+s表示，多组间均数比较采用单因素方差分析，组间两两比较采用LSD检验，方差不齐时采用Tamhane法，处理组同对照组间的多重比较采用Dunnett法；分析各处理方式的主效应及交互效应采用析因设计资料的方差分析；两组间均数比较采用两独立样本t检验，P<0．05为显著性差异。
五、实验结果
1、三肽对各种细胞NO产生的影响
在表达NOS2的巨噬细胞中，三肽、L-NNA、及对照组的NO值(umol／L)分别为208．2±19．3、279．5±20．7、308．9±17．1，进行单因素方差分析显示，各组间NO浓度差异有显著性意义(F=73．553,P=0．000)。LSD法进行组间比较结果显示：三肽能够明显抑制巨噬细胞NO的产生(P=0．000)，且其效果优于非选择性的NOS抑制剂L-NNA(P=0．000)，三肽显示出对NOS2的良好的抑制效果。
在表达NOS3的内皮细胞中，三肽、L-NNA及对照组的NO值(umol／L)分别为179．5±9．4、157．8±13．9、188．2±11．6，进行单因素方差分析结果显示，三组间NO值差异有显著性意义(F=17．660，P=0．000)。LSD法进行组间比较结果显示：三肽组与对照组间NO浓度的差异没有显著性意义(P=0．110)。L-NNA组同三肽组(P=0．000)及对照组相比(P=0．000)，则降低了NO的产生。三肽对内皮细胞中的NOS3活性影响较小。
在心肌细胞中，三肽、L-NNA、及对照组的NO值(umol／L)分别为55．5±7．5、30．2±5．9、61．4±9．9，单因素方差分析结果显示，三组间NO值的差异有显著性意义(F=41．061，P=0．000)。LSD法进行组间比较结果显示：三肽组与对照组NO浓度的差异没有显著性意义(P=0．101)，但L-NNA组较其他两组明显降低了心肌NO的产生(P=0．000)，三肽对心肌细胞中的NOS3和NOS1活性影响较小。另外，给予正常的心肌精氨酸处理，对心肌NO的产生的影响没有显著性意义(P=0．228)。值得注意的是，三肽组同精氨酸组相比，二者NO值的差异有显著性意义(P=0．006)，精氨酸组的NO值明显高于三肽组。
2、心肌细胞NOS2表达
IL-6联合LPS处理心肌细胞6、12、24 h，6 h后心肌NOS2开始表达增加，并随着处理时间延长，NOS2表达不断增加，三个时间点的NOS2的蛋白表达量分别是100、157．1±9．4、178．1±11．0。单因素方差分析结果显示，各时间点的NOS2蛋白表达的差异有显著性意义(F=187．101，P=0．000)。以Tamhane法进行各组间比较，24h同6h(P=0．000)及12h(P=0．003)相比，24 h后NOS2蛋白表达最为明显。
3、NOS抑制剂对表达NOS2心肌细胞NO浓度的影响
表达NOS2的心肌细胞，给予三肽、L-NNA、精氨酸处理，检测各组中NO值。对照组、三肽组、L-NNA组及精氨酸组的NO值分别是：150．0±13．5、111．0±15．2、130．2±15．2和168．2±11．5。采用单因素方差分析显示，各处理组间NO值的差异有显著性意义(F=29．230，P=0．000)。LSD法进行组间比较结果显示：同对照组相比，三肽(P=0．000)和L-NNA(P=0．001)明显抑制心肌中NO的产生，而精氨酸则促进心肌细胞中NO合成(P=0．003)。三肽对表达NOS2的心肌NO合成的抑制效果明显优于L-NNA(P=0．002)。
心肌细胞给予不同浓度的三肽，检测各组中NO值。0．2、0．4、0．6和0．8mmol／L组的NO值分别是：143．4±11．3、128．6±10．1、119．2±9．2和114．3±12．3。采用单因素方差分析结果显示，各处理组间的NO值的差异有显著性意义(F=20．290，P=0．000)。LSD法进行组间比较结果显示：同对照组相比，三肽0．2mmol／L对心肌细胞NO的合成具有抑制效果(P=0．004)，0．4mmol／L组的NO值，同1mmol／L的L-NNA组相比较，差异没有显著性意义(P=0．490)；
4、BEC和L-VNIO对心肌细胞NO浓度的影响
在正常的心肌细胞中，给予精氨酸酶抑制剂BEC及NOS1抑制剂L-VNIO处理后，对照组、BEC组、L-VNIO组及BEC+L-VNIO组NO值分别为：61．4±9．9、72．8±9．4、44．8±7．9和51．2±7．0。采用析因设计资料的方差分析，结果显示给予BEC可以增加NO的产生(F=10．620，P=0．002)，给予NOS1抑制剂L-VNIO可以显著抑制NO的产生(F=48．909，P=0．000)。同时给予BEC和L-VNIO，两种处理因素的交互效应不显著(F=0．838，P=0．366)。
5、NOS抑制剂对表达NOS2的心肌细胞游离钙浓度的影响
同正常心肌给予ISO刺激时的钙离子浓度改变相比，心肌表达NOS2后，对ISO反应性明显减弱。前者钙离子浓度为172．0±8．0，而后者为125．6±14．0，以两独立样本的t检验进行统计分析，结果显示两者的差异有显著性意义(t=9．099，P=0．000)。
表达NOS2的心肌细胞给予空白对照、精氨酸、三肽、三肽+精氨酸处理后，心肌细胞中游离钙浓度峰值分别为123．6±13．1、114．3±9．3、157．4±15．4、135．5±12．9。以单因素方差分析结果显示，各处理组间的差异有显著性意义(F=40．726，P=0．000)。LSD法进行组间比较结果显示：在表达NOS2的心肌细胞中，同对照组相比，三肽(1mmol／L)做为NOS2抑制剂，可以明显促进ISO刺激的心肌中游离钙浓度的升高(P=0．000)。精氨酸则可以抑制ISO刺激的心肌中游离钙浓度的升高(P=0．007)，精氨酸组同三肽+精氨酸组相比，两组细胞的游离钙浓度的差异有显著性意义(P=0．000)，三肽可以减弱精氨酸对ISO诱导的心肌钙升高的抑制作用，表明精氨酸对心肌钙浓度的影响，可能是通过NOS2途径。分析对心肌细胞NO产生具有相同抑制效果的三肽(0．4mmol／L)组和L-NNA(1mmol／L)组，结果显示，0．4mmol／L三肽对ISO诱导心肌细胞游离钙浓度升高的促进作用明显优于后者(P=0．003)。
6、不同钙通道阻滞剂对三肽效应的影响。
表达NOS2的心肌细胞，给予三肽、三肽+维拉帕米、三肽+肝素及三肽+普鲁卡因处理，ISO刺激心肌，检测各组细胞内钙浓度的变化。上述四组检测钙的结果分别为152．0±13．3、137．1±10．9、150．0±11．0、129．8±8．3。单因素方差分析结果显示，各组问的差异有显著性意义(F=9．273，P=0．000)。以Dunnett法进行处理组同对照组间的多重比较，结果显示，同对照组相比，给予维拉帕米(P=0．009)和普鲁卡因(P=0．000)可以减弱三肽的效应。肝素(P=0．957)组同对照组的差异不具有显著性差异。提示抑制NOS2引起心肌钙浓度升高的机制可能是通过改善多种钙通道的功能而实现。
7、精氨酸酶抑制剂对ISO刺激的正常心肌时胞内游离钙浓变化的影响
给予空白对照、BEC、L-VNIO、BEC+L-VNIO处理，心肌细胞中游离钙浓度峰值四组分别为172．0±8．0、188．5±11．1、143．0±8．9、146．7±8．3。采用析因设计资料的方差分析，结果显示：L-VNIO明显抑制ISO对心肌的钙浓度作用(F=147．008,P=0．000)。BEC明显促进ISO对心肌的钙浓度作用(F=11．460,P=0．002)。同时给予BEC和L-VNIO，两者组合的交互效应显著(F=5．356,P=0．026)，分析BEC促进心肌钙浓度，而L-VNIO抑制钙浓度，表明BEC和L-VNIO存在拮抗效应，表明精氨酸酶对心肌钙的影响机制可能是通过NOS1途径。
8、BEC对ISO刺激的表达NOS2的心肌时胞内游离钙浓变化的影响
表达NOS2的心肌细胞，分别给予三肽、L-VNIO、BCE、三肽+L-VNIO处理。上述各组游离钙浓度分别为：152．0±13．3、109．9±7．6、133．1±10．9和139．2±12．2。检测上述处理对ISO诱导的心肌细胞内钙浓度变化的影响，单因素方差分析结果显示，各处理组间的差异有显著性意义(F=17．633，P=0．000)。LSD法进行组问比较结果显示：在表达NOS2的心肌细胞中，同对照组相比，给予NOS1抑制剂，可以抑制ISO诱导的心肌细胞内游离钙的升高(P=0．017)；而给予精氨酸酶抑制剂，对ISO诱导的心肌的钙改变未见明显影响(P=0．427)；同对照组相比，同时给予L-VNIO和三肽，可以促进ISO诱导的心肌细胞的钙升高(P=0．045)。
五、结论
1、LPS刺激巨噬细胞可以表达NOS2，LPS+IL-6可以诱导心肌细胞表达NOS2。
2、三肽对NOS2具有较好的选择性及抑制性。
3、在正常心肌细胞中，抑制心肌中的精氨酸酶可以促进心肌对ISO的反应性，其机制可能是通过NOS1实现的。
4、表达NOS2的心肌对ISO的反应性降低。三肽抑制NOS2，可以增强心肌对ISO的反应。其机制是通过影响多种钙通道实现的。
5、精氨酸能够降低表达NOS2的心肌对ISO的反应性。其机制可能是通过NOS2的途径实现。
Background: Three nitric oxide synthase(NOS) enzymes are described in mammalian systems, all of which oxidize the terminal guanidino nitrogen of L-arginine to form nitric oxide (NO) and the amino acid L-citrulline. These isoforms-neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3)-play modulatory roles in essentially all organ systems; including (but not limited to) the nervous, immune, respiratory, urologic, gastrointestinal, and cardiovascular systems. All of them are present in the heart: NOS 1 has been detected in cardiac conduction tissue and intracardiac neurons; NOS2 can be expressed by virtually all cells in the heart, often in conjunction with the expression of inflammatory cytokines; and finally, NOS3 is expressed in coronary endothelium, endocardium, and cardiac myocytes. It has been established that NO also has important effects on myocardial function which include the modulation of contractile function, the tone of vascular smooth muscle cells, energetics, substrate metabolism, cell growth and survival. There are at least two distinct downstream signaling modes for NO: 3',5'-cyclic guanosine monophosphate ( cGMP) production and protein thiol nitrosylation (S-nitrosylation). NO activates soluble guanylyl cyclase (S-GC) by binding to its heme moiety, forming an Fe-nitrosyl complex, leading to the production of cGMP, which in turn activates protein kinase G (PKG) and a cascade of biological signaling events.
Subcellular localization of NOS with effector molecules is an important regulatory mechanism for NO signalling.NOS3 localizes to caveolae, where compartmentalization with P-adrenergic receptors and L-type Ca~(2+) channels allows NO to inhibitβ-adrenergic-induced inotropy. NOS1,however, is targeted to cardiac SR. NO stimulation of SR Ca~(2+) release via the ryanodine receptor (RyR) in vitro suggests that NOS1 has an opposite, facilitative effect on contractility. NOS2 is not normally present in myocardium, inflammation and/or cytokine activation cause induction of NOS2 and producing NO. It has been reported that NOS2 induction suppresses myocyte Ca~(2+) transients and contributes to depressed myocardial contractility andβ-adrenergic hyporesponsiveness. There is accumulating evidence that NO participates in all aspects of EC coupling, including receptor signal transduction, L-type Ca~(2+)-channel activity, SR calcium release through the RYR, and mitochondrial respiration.
L-Arginine, the sole substrate for the NOS enzyme in producing NO, is also a substrate for arginase. Thus arginase has a potential role in limiting substrate available for NO production. Arginase, often perceived solely as the last of the now six enzymes of the urea cycle, exists in 2 isoforms, cytosolic form arginase I and mitochondrial form arginaseⅡ. ArginaseⅠis located in the cytosol and thought to be primarily involved in ureagenesis., whereas arginaseⅡis located in the mitochondrial matrix and has been thought to be more widely expressed and to be involved in the biosynthesis of polyamines, the amino acids ornithine, proline, and glutamate and in the inflammatory process, among others. Both arginaseⅠandⅡwere detected in crude myocardial homogenates, but only arginaseⅡwas present in isolated rat myocytes. Arginase inhibition with S-(2-boronoethyl)-L-cysteine (BEC) augmented Ca~(2+)-dependent NOS activity and NO production in myocytes.
Arginine concentration also governs NOS activity. The intracellular concentration of L-arginine in endothelial cells exceeds its K_m for the NOS enzyme by 2- to 3-fold, indicating that L-arginine availability should not limit NOS activity or NO production. Also, exogenous L-arginine administration should not influence NOS activity and NO production. However, in certain conditions, the addition of extracellular L-arginine enhances NO-dependent relaxation, giving rise to the "arginine paradox". The "arginine paradox" refers to the dependence of cellular NO production on exogenous L-arginine concentration despite the theoretical saturation of NOS enzymes with intracellular L-arginine. decreased availability of L-arginine blocked induction of NO production in cytokine-stimulated astrocytes, owing to inhibition of NOS2 protein expression.
Objective: Solusion phase synthesis of tripeptide contained L-Arginine and L-Lysine. Primary cultured Human Umbilical Vein Endothelial Cells (HUVEC), cardiac myocytes, macrophages with different treatment and examined the inhibitory effect of tripeptide on NOS activety. Expression of NOS2 was induced by lipopolysaccharide (LPS) and interleukin-6 (IL-6) for 24 hours before NOS2 protein was assayed by western blotting and cardiac myocyes (CMs) were incubated by new arginine analog for 12h before changes of the fluorescence signal of free calcium caused by isoproterenol (ISO), aβ-AR agonist, were measured under laser scanning confocal microscope (LSCM). The roles of ryanodine receptor (RyRs), inositol 1,4,5,-triphosphate rceptor (IP3R) and L-type calcium channel involved tripeptide effects was examined.
Methods:
1, Macrophages treated with LPS for 24 h to express N0S2. We examined the inhibitory effect of tripeptine on NO production in HUVEC, CMs and Macrophages.
2, CMs were treated with LPS and IL-6 for 6. 12, 24 h. NOS2 expression measured by western blotting.
3, CMs expressed NOS2 and treated with different concentration tripeptide or L-NNA for 12 h, to measure NO production.
4, CMs expressed NOS2 and treated with BEC and/or L-VNIO, then to test the NO levels.
5, CMs expressed NOS2 and treated with different NOS inhibitor for 12 h. LSCM measured the changes of fluorescence singal of free calcium caused by ISO.
6, CMs treated with verapamil(inhibitor of L-type channel), procaine(inhibitor of RyRs) and heparin(inhibitor of IP3R). LSCM measured the changes of fluorescence singal. To study the role of different calcium channel inhibitors in tripeptine effects on free calcium concentration..
7, Normal CMs treated with ISO, ISO+BEC, ISO+L-VNIO, ISO+BEC+L-VNIO, respectively. LSCM measured the changes of fluorescence singal caused by ISO.
8, CMs expressed NOS2 and treated with tripeptide, L-VNIO, BEC, tripeptide+L-VNIO. LSCM measured the changes of fluorescence singal caused by ISO.
Statistical analysis
All data are expressed as means±SD, and differences among groups were analyzed by one-way ANOVA followed by LSD test or Tambane test when equal variance was not assumed. Differences between two groups were analyzed by Independent - Samples T Test. A value of p<0.05 was considered statistically significant.
Results:
1,1, Effects of tripeptide on NO production
In macrophages expressing NOS2, after treated with tripeptide, L-NNA, the NO levels was 208.2±19.3, 279.5±20.7 and 308.9±17.1, respectively. The data was analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F=73.553, P=0.000). The data analyzed by LSD shows that compared with control group, tripeptide significantly inhibited NO production in macrophages (P=0.000) and the effect of tripeptide is better than that of L-NNA (P=0.000).
HUVEC expresses NOS3. The levels of NO (umol/L) in tripeptide group, L-NNA and control group is 179.5±9.4, 157.8±13.9 and 188.2±11.6, respectively. The data was analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F= 17.660, P=0.000). Compared with control group, L-NNA could significantly inhibit NO production (P=0.000) and tripeptide had no the effect (P=0.110). Tripeptide has little influence on NO production in HUVEC.
CMs expressed NOS1 and NOS3. The levels of NO (umol/L) in tripeptide, L-NNA and control group is 55.5±7.5, 30.2±5.9 and 61.4±9.9, respectively. The data was analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F=41.061, P=0.000). Compared with control group, L-NNA could significantly inhibit NO production (P=0.000) and tripeptide had no the effect (P=0.101).
2, NOS2 protein expression in response to different treatment
Cardiac myocytes treated with IL-6 and LPS for 6, 12 and 24 h, NOS2 protein expressions were measured by western blotting. The result showed that NOS2 protein began to express at 6h. The data was analyzed by one-way ANOVA. Significant difference was found in 6h, 12h and 24h group (F=187.101, P=0.000). NOS2 protein expression at 24h was significantly high compared with 6h (P=0.000) and 12h (P=0.003).
3, Effect of NOS inhibtor on NO production in CMs expressed NOS2
In CMs expressed NOS2, The NO values were analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F=29.230, P=0.000). The data analyzed by LSD shows that compared with control group, tripeptide (P=0.000) and L-NNA (P=0.001) significantly inhibit NO production and arginine increased NO production (P=0.003). The effect of tripeptide on inhibiting NO production is better than that of L-NNA (P=0.000).
CMs treated with different concentration tripeptide. The levels of NO were analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F=20.290, P=0.000). Compared with control group, tripeptide (0.2mmol/L) could inhibit NO production significantly (P=0.004). On decrease NO production, there was no significant difference between tripeptide (0.4mmol/L) and L-NNA(lmmol/L) group (P=0.490).
4, Effect of BEC and L-VNIO on NO production in CMs
In normal CMs treated with L-VINO, a NOS1 inhibitor, and BEC, a arginase inhibitor, the levels of NO were measured. The data was analyzed by factorial analyze. BEC increased NO production significantly (F=10.620, P=0.002) and L-VNIO decreased NO production significantly (F=48.909, P=0.000). BEC and L-VNIO had no interaction (F=0.838, P=0.366).
5, Effect of NOS inhibitor on free calcium concentration caused by ISO in myocytes expressed NOS2
Increased NOS2 activity contributes to decreased ISO-induced free calcium concentration. Free calcium concentration induced by ISO in normal myocytes is 172.0±8.0 and that in myocytes expressed NOS2 is 125.6±14.0. The data was analyzed by independent-samples t test. The result showed there is significant difference between two groups (t=9.099, P=0.000).
The free calcium concentrations in every group were analyzed by one-way ANOVA. Significant difference was found in every group (F=40.726, P=0.000). Compared with control group, specific inhibition of NOS2 by Tripeptide (1mmol/L) dramatically increased free calcium concentration induced by ISO (P=0.000) and arginine significantly decreased free calcium concentration induced by ISO (P=0.007). Tripeptide (0.4mmol/L) is better than L-NNA (lmmol/L) on increasing free calcium concentration (P0.003).
6, Effects of some drugs inhibiting Ca~(2+) channels
CMs treated with verapamil-a inhibitor of L-type calcium channel, heparin-a inhibitor of IP3R, and procaine-a inhibitor of RyR, the free calcium concentrations were 152.0±13.3 in control group, 137.1±10.9 in verapamil grouop, 150.0±11.0 in heparin group and 129.8±8.3 in procaine group. The data was analyzed by one-way ANOVA. Significant difference was found in every group (F=9.273, P=0.000). Verapamil (P=0.009) and procaine (P=0.000) could significantly inhibit tripeptide induced increasing in ISO-induced free calcium concentration in myocytes. Heparin has no the effect(P=0.957).
7, Effect of arginase inhibitor on normal myocyte free calcium concentration caused by ISO
CMs treated with BEC, L-VNIO and BEC+L-VNIO, the data of free calcium concentrations were analyzed by factorial analyze. Specific inhibition of NOS1 by L-VNIO decreased the ISO-induced free calcium concentration (F=147.008, P=0.000) and arginase inhibitor increased that in CMs (F=11.460, P=0.002). There was significant interaction between BEC and L-VNIO (F=5.356, P=0.026), L-VNIO could inhibit the effect of BEC on free calcium concentration in CMs.
8, Effect of arginase inhibitor on free Ca~(2+) concentraton caused by ISO in myocyte expressed NOS2.
CMs expressed NOS2, the value of free calcium concentration was analyzed by one-way ANOVA. Significant difference was found in groups (F=17.633, P=0.000). Compared with control group, L-VNIO could significantly decreased free calcium concentration induced by ISO (P=0.017), L-VNIO+tripeptide could significantly increased free calcium concentration induced by ISO (P=0.045) and BEC had no significant influence on free calcium concentration induced by ISO (P=0.427).
Conclusions:
1, macrophages treated with LPS and CMs treated with LPS and IL-6 could express NOS2 protein.
2, Tripeptide has highly selective and inhibitive effect on NOS2 activety.
3, Arginase strongly limited ISO-induced increases in free Ca~(2+) concentration in a NOS1-dependent manner.
4, Increased NOS2 activity contributed to limit ISO-induced myocardial free Ca~(2+) concentration. Tripeptide restored myocardial responsiveness toβ-adrenergic agonist via improving the functions of several calcium channels.
5, Arginine strongly limited ISO-induced increases in free Ca~(2+) concentration via increasing NO production and expression of NOS2.
NOS亚型的在心肌中的亚细胞定位是调整NO作用的一个重要因素，NOS3位于肌膜的凹穴处，靠近β肾上腺素能受体，NOS3／NO抑制心肌对β肾上腺素能受体激动剂的反应性。而NOS1以肌浆网兰尼碱受体(ryanodine receptor，RyR)为靶组织，可以增强心肌收缩时肌浆网的钙释放。NOS1与NOS3对心肌收缩功能起着相反的调节作用。心肌正常的时候，不表达NOS2，在某些特定的病理状态下，在炎症和细胞因子诱导心肌才丌始表达NOS2，产生大量的NO。心肌中的NOS2位于细胞质的囊泡和内质网中，在缺血再灌注的时候，可以对心脏起到保护作用。但更多的时候，NOS2的表达，严重影响着心肌功能，目前研究较多的是NO对心肌收缩抑制的机制。NOS2的表达可以抑制心肌细胞中钙瞬变、降低心肌收缩力和降低心肌对β受体激动剂反应性。越来越多的证据表明，NO可以对兴奋收缩耦联的多个方面进行调节，包括：受体信号传导、L-型钙通道活性、通过肌浆网RyR进行的钙释放及线粒体呼吸过程。
精氨酸(文中所提到的精氨酸均为左旋精氨酸)是NOS的唯一底物，同时也是精氨酸酶的底物。因此精氨酸酶可以通过对底物的竞争性利用而限制NO的产生。精氨酸酶一般理解为鸟氨酸循环最后一个酶，体内存在两种形式的精氨酸酶，一种是肝脏型精氨酸酶(精氨酸酶Ⅰ)，存在于细胞质中，其主要功能是合成尿素；另一种是线粒体型精氨酸酶(即精氨酸酶Ⅱ)，广泛存在体内的多种组织，参与多胺、脯氨酸、谷氨酸等物质的合成。在心脏组织中两种精氨酸酶均可以被检测出。但是在鼠心肌细胞中，仅发现精氨酸酶Ⅱ表达。以BEC抑制精氨酸酶的活性，可以增加钙依赖的NOS的活性和增加NO的产生。
精氨酸是可以影响NOS的另外一个因素。在内皮细胞中，精氨酸浓度是NOS的Km值的2-3倍，理论上不会因NOS底物耗竭而限制NOS活性和NO的产生。相应的，外源性的精氨酸也不应影响NOS的活性。但是，在一些特定的病理过程中，给予额外的精氨酸能够增加NO的产生。这种现象被成为精氨酸颠倒现象(L-Arginine Paradox)：细胞内精氨酸的浓度对于NOS反应来说是饱和的，但是外源性的精氨酸还是可以增加NO的产生。
二、实验目的液相合成法合成的由两分子精氨酸和一分子赖氨酸组成的三肽(以下均简称：三肽)，以体外培养的脂多糖(lipopolysaccharide，LPS)诱导表达NOS2的巨噬细胞、表达NOS3的脐静脉内皮细胞、表达NOS1和NOS3的SD乳鼠心肌细胞(cardiac myocytes，CMs)为对象，硝酸还原酶法检测三肽对不同细胞的NO合成的抑制效果；白介素6(interleukin-6，IL-6)联合LPS诱导心肌NOS2表达，免疫印记法(western blotting)等技术检测NOS2蛋白的表达。以表达NOS2的心肌细胞为研究对象，进一步研究三肽对β肾上腺素能受体激动剂异丙肾上腺素(isoproterenol，ISO)刺激心肌细胞游离钙浓度变化的影响，给予不同的钙通道阻滞剂，包括肌膜L-型通道、肌浆网兰尼碱受体(ryanodine receptor，RyRs)、肌醇1，4，5-三磷酸受体(inositol 1，4，5,-triphosphate rceptor，IP3R)。明确参与三肽发挥效应的钙通道类型。同时探讨共同以精氨酸为底物的精氨酸酶对正常心肌细胞和表达NOS2的心肌细胞中ISO诱导心肌细胞内游离钙浓度变化的影响，系统地探讨NOS、精氨酸酶、精氨酸三者对心肌功能影响的程度及相互间的影响。
三、实验方法
1、LPS处理巨噬细胞24 h表达NOS2，检测三肽对乳鼠心肌细胞、脐静脉内皮细胞和表达NOS2的巨噬细胞中NO的浓度，明确三肽对相应的NOS亚型的抑制效果。
2、IL-6联合LPS处理心肌细胞24 h，western blouing检测6、12、24 h三个时间点NOS2的蛋白表达情况。
3、表达NOS2的心肌细胞为对象，给予L-NNA、不同浓度的三肽处理12 h，研究三肽对表达NOS2的心肌细胞NO抑制作用，寻找同L-NNA具有相同抑制效果的三肽浓度。
4、正常心肌细胞，给予精氨酸酶抑制剂S-(2-boronoethyl)-L-cysteine(BEC)和NOS1抑制剂vinyl-L-N-5-(1-imino-3-butenyl)-L-omithine(L-VNIO)处理，实验分为BEC组、L-VNIO组和BEC+L-VNIO组．给予相应处理后，取上清检测NO值。明确精氨酸酶对心肌细胞NO产生的影响。
5、表达NOS2的心肌细胞为对象，激光共聚焦显微镜(Laser Scan ConfocalMicroscope，LSCM)检测三肽对ISO刺激的心肌细胞胞内游离钙浓度变化的影响。同时比较对NO具有相同抑制效果的低浓度的三肽同高浓度的L-NNA对ISO刺激的心肌细胞内游离钙浓度变化的影响，明确NOS的细胞定位是细胞功能调整的重要方式。
6、分别以维拉帕米(verapamil)阻滞L-型通道、普鲁卡因(procaine)阻断RyR、肝素(heparin)阻断IP3R后，各组均给予三肽。检测上述处理后心肌细胞钙浓度的变化。明确三肽升高心肌内游离钙浓度的途径。
7、正常的心肌细胞，分为ISO、ISO+BEC、ISO+L-VNIO、ISO+BEC+L-VNIO组，各组给予相应处理后。上机检测细胞内游离钙浓度。明确精氨酸酶及NOS1对心肌功能的影响。
8、表达NOS2的心肌细胞，分别给予三肽、L-VNIO、BCE、三肽+L-VNIO处理。检测各组细胞内钙浓度，明确精氨酸酶对表达NOS2心肌细胞的钙浓度的影响。
四、统计学处理
采用SPSS12．0统计软件进行数据分析，结果以(?)+s表示，多组间均数比较采用单因素方差分析，组间两两比较采用LSD检验，方差不齐时采用Tamhane法，处理组同对照组间的多重比较采用Dunnett法；分析各处理方式的主效应及交互效应采用析因设计资料的方差分析；两组间均数比较采用两独立样本t检验，P<0．05为显著性差异。
五、实验结果
1、三肽对各种细胞NO产生的影响
在表达NOS2的巨噬细胞中，三肽、L-NNA、及对照组的NO值(umol／L)分别为208．2±19．3、279．5±20．7、308．9±17．1，进行单因素方差分析显示，各组间NO浓度差异有显著性意义(F=73．553,P=0．000)。LSD法进行组间比较结果显示：三肽能够明显抑制巨噬细胞NO的产生(P=0．000)，且其效果优于非选择性的NOS抑制剂L-NNA(P=0．000)，三肽显示出对NOS2的良好的抑制效果。
在表达NOS3的内皮细胞中，三肽、L-NNA及对照组的NO值(umol／L)分别为179．5±9．4、157．8±13．9、188．2±11．6，进行单因素方差分析结果显示，三组间NO值差异有显著性意义(F=17．660，P=0．000)。LSD法进行组间比较结果显示：三肽组与对照组间NO浓度的差异没有显著性意义(P=0．110)。L-NNA组同三肽组(P=0．000)及对照组相比(P=0．000)，则降低了NO的产生。三肽对内皮细胞中的NOS3活性影响较小。
在心肌细胞中，三肽、L-NNA、及对照组的NO值(umol／L)分别为55．5±7．5、30．2±5．9、61．4±9．9，单因素方差分析结果显示，三组间NO值的差异有显著性意义(F=41．061，P=0．000)。LSD法进行组间比较结果显示：三肽组与对照组NO浓度的差异没有显著性意义(P=0．101)，但L-NNA组较其他两组明显降低了心肌NO的产生(P=0．000)，三肽对心肌细胞中的NOS3和NOS1活性影响较小。另外，给予正常的心肌精氨酸处理，对心肌NO的产生的影响没有显著性意义(P=0．228)。值得注意的是，三肽组同精氨酸组相比，二者NO值的差异有显著性意义(P=0．006)，精氨酸组的NO值明显高于三肽组。
2、心肌细胞NOS2表达
IL-6联合LPS处理心肌细胞6、12、24 h，6 h后心肌NOS2开始表达增加，并随着处理时间延长，NOS2表达不断增加，三个时间点的NOS2的蛋白表达量分别是100、157．1±9．4、178．1±11．0。单因素方差分析结果显示，各时间点的NOS2蛋白表达的差异有显著性意义(F=187．101，P=0．000)。以Tamhane法进行各组间比较，24h同6h(P=0．000)及12h(P=0．003)相比，24 h后NOS2蛋白表达最为明显。
3、NOS抑制剂对表达NOS2心肌细胞NO浓度的影响
表达NOS2的心肌细胞，给予三肽、L-NNA、精氨酸处理，检测各组中NO值。对照组、三肽组、L-NNA组及精氨酸组的NO值分别是：150．0±13．5、111．0±15．2、130．2±15．2和168．2±11．5。采用单因素方差分析显示，各处理组间NO值的差异有显著性意义(F=29．230，P=0．000)。LSD法进行组间比较结果显示：同对照组相比，三肽(P=0．000)和L-NNA(P=0．001)明显抑制心肌中NO的产生，而精氨酸则促进心肌细胞中NO合成(P=0．003)。三肽对表达NOS2的心肌NO合成的抑制效果明显优于L-NNA(P=0．002)。
心肌细胞给予不同浓度的三肽，检测各组中NO值。0．2、0．4、0．6和0．8mmol／L组的NO值分别是：143．4±11．3、128．6±10．1、119．2±9．2和114．3±12．3。采用单因素方差分析结果显示，各处理组间的NO值的差异有显著性意义(F=20．290，P=0．000)。LSD法进行组间比较结果显示：同对照组相比，三肽0．2mmol／L对心肌细胞NO的合成具有抑制效果(P=0．004)，0．4mmol／L组的NO值，同1mmol／L的L-NNA组相比较，差异没有显著性意义(P=0．490)；
4、BEC和L-VNIO对心肌细胞NO浓度的影响
在正常的心肌细胞中，给予精氨酸酶抑制剂BEC及NOS1抑制剂L-VNIO处理后，对照组、BEC组、L-VNIO组及BEC+L-VNIO组NO值分别为：61．4±9．9、72．8±9．4、44．8±7．9和51．2±7．0。采用析因设计资料的方差分析，结果显示给予BEC可以增加NO的产生(F=10．620，P=0．002)，给予NOS1抑制剂L-VNIO可以显著抑制NO的产生(F=48．909，P=0．000)。同时给予BEC和L-VNIO，两种处理因素的交互效应不显著(F=0．838，P=0．366)。
5、NOS抑制剂对表达NOS2的心肌细胞游离钙浓度的影响
同正常心肌给予ISO刺激时的钙离子浓度改变相比，心肌表达NOS2后，对ISO反应性明显减弱。前者钙离子浓度为172．0±8．0，而后者为125．6±14．0，以两独立样本的t检验进行统计分析，结果显示两者的差异有显著性意义(t=9．099，P=0．000)。
表达NOS2的心肌细胞给予空白对照、精氨酸、三肽、三肽+精氨酸处理后，心肌细胞中游离钙浓度峰值分别为123．6±13．1、114．3±9．3、157．4±15．4、135．5±12．9。以单因素方差分析结果显示，各处理组间的差异有显著性意义(F=40．726，P=0．000)。LSD法进行组间比较结果显示：在表达NOS2的心肌细胞中，同对照组相比，三肽(1mmol／L)做为NOS2抑制剂，可以明显促进ISO刺激的心肌中游离钙浓度的升高(P=0．000)。精氨酸则可以抑制ISO刺激的心肌中游离钙浓度的升高(P=0．007)，精氨酸组同三肽+精氨酸组相比，两组细胞的游离钙浓度的差异有显著性意义(P=0．000)，三肽可以减弱精氨酸对ISO诱导的心肌钙升高的抑制作用，表明精氨酸对心肌钙浓度的影响，可能是通过NOS2途径。分析对心肌细胞NO产生具有相同抑制效果的三肽(0．4mmol／L)组和L-NNA(1mmol／L)组，结果显示，0．4mmol／L三肽对ISO诱导心肌细胞游离钙浓度升高的促进作用明显优于后者(P=0．003)。
6、不同钙通道阻滞剂对三肽效应的影响。
表达NOS2的心肌细胞，给予三肽、三肽+维拉帕米、三肽+肝素及三肽+普鲁卡因处理，ISO刺激心肌，检测各组细胞内钙浓度的变化。上述四组检测钙的结果分别为152．0±13．3、137．1±10．9、150．0±11．0、129．8±8．3。单因素方差分析结果显示，各组问的差异有显著性意义(F=9．273，P=0．000)。以Dunnett法进行处理组同对照组间的多重比较，结果显示，同对照组相比，给予维拉帕米(P=0．009)和普鲁卡因(P=0．000)可以减弱三肽的效应。肝素(P=0．957)组同对照组的差异不具有显著性差异。提示抑制NOS2引起心肌钙浓度升高的机制可能是通过改善多种钙通道的功能而实现。
7、精氨酸酶抑制剂对ISO刺激的正常心肌时胞内游离钙浓变化的影响
给予空白对照、BEC、L-VNIO、BEC+L-VNIO处理，心肌细胞中游离钙浓度峰值四组分别为172．0±8．0、188．5±11．1、143．0±8．9、146．7±8．3。采用析因设计资料的方差分析，结果显示：L-VNIO明显抑制ISO对心肌的钙浓度作用(F=147．008,P=0．000)。BEC明显促进ISO对心肌的钙浓度作用(F=11．460,P=0．002)。同时给予BEC和L-VNIO，两者组合的交互效应显著(F=5．356,P=0．026)，分析BEC促进心肌钙浓度，而L-VNIO抑制钙浓度，表明BEC和L-VNIO存在拮抗效应，表明精氨酸酶对心肌钙的影响机制可能是通过NOS1途径。
8、BEC对ISO刺激的表达NOS2的心肌时胞内游离钙浓变化的影响
表达NOS2的心肌细胞，分别给予三肽、L-VNIO、BCE、三肽+L-VNIO处理。上述各组游离钙浓度分别为：152．0±13．3、109．9±7．6、133．1±10．9和139．2±12．2。检测上述处理对ISO诱导的心肌细胞内钙浓度变化的影响，单因素方差分析结果显示，各处理组间的差异有显著性意义(F=17．633，P=0．000)。LSD法进行组问比较结果显示：在表达NOS2的心肌细胞中，同对照组相比，给予NOS1抑制剂，可以抑制ISO诱导的心肌细胞内游离钙的升高(P=0．017)；而给予精氨酸酶抑制剂，对ISO诱导的心肌的钙改变未见明显影响(P=0．427)；同对照组相比，同时给予L-VNIO和三肽，可以促进ISO诱导的心肌细胞的钙升高(P=0．045)。
五、结论
1、LPS刺激巨噬细胞可以表达NOS2，LPS+IL-6可以诱导心肌细胞表达NOS2。
2、三肽对NOS2具有较好的选择性及抑制性。
3、在正常心肌细胞中，抑制心肌中的精氨酸酶可以促进心肌对ISO的反应性，其机制可能是通过NOS1实现的。
4、表达NOS2的心肌对ISO的反应性降低。三肽抑制NOS2，可以增强心肌对ISO的反应。其机制是通过影响多种钙通道实现的。
5、精氨酸能够降低表达NOS2的心肌对ISO的反应性。其机制可能是通过NOS2的途径实现。
Background: Three nitric oxide synthase(NOS) enzymes are described in mammalian systems, all of which oxidize the terminal guanidino nitrogen of L-arginine to form nitric oxide (NO) and the amino acid L-citrulline. These isoforms-neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3)-play modulatory roles in essentially all organ systems; including (but not limited to) the nervous, immune, respiratory, urologic, gastrointestinal, and cardiovascular systems. All of them are present in the heart: NOS 1 has been detected in cardiac conduction tissue and intracardiac neurons; NOS2 can be expressed by virtually all cells in the heart, often in conjunction with the expression of inflammatory cytokines; and finally, NOS3 is expressed in coronary endothelium, endocardium, and cardiac myocytes. It has been established that NO also has important effects on myocardial function which include the modulation of contractile function, the tone of vascular smooth muscle cells, energetics, substrate metabolism, cell growth and survival. There are at least two distinct downstream signaling modes for NO: 3',5'-cyclic guanosine monophosphate ( cGMP) production and protein thiol nitrosylation (S-nitrosylation). NO activates soluble guanylyl cyclase (S-GC) by binding to its heme moiety, forming an Fe-nitrosyl complex, leading to the production of cGMP, which in turn activates protein kinase G (PKG) and a cascade of biological signaling events.
Subcellular localization of NOS with effector molecules is an important regulatory mechanism for NO signalling.NOS3 localizes to caveolae, where compartmentalization with P-adrenergic receptors and L-type Ca~(2+) channels allows NO to inhibitβ-adrenergic-induced inotropy. NOS1,however, is targeted to cardiac SR. NO stimulation of SR Ca~(2+) release via the ryanodine receptor (RyR) in vitro suggests that NOS1 has an opposite, facilitative effect on contractility. NOS2 is not normally present in myocardium, inflammation and/or cytokine activation cause induction of NOS2 and producing NO. It has been reported that NOS2 induction suppresses myocyte Ca~(2+) transients and contributes to depressed myocardial contractility andβ-adrenergic hyporesponsiveness. There is accumulating evidence that NO participates in all aspects of EC coupling, including receptor signal transduction, L-type Ca~(2+)-channel activity, SR calcium release through the RYR, and mitochondrial respiration.
L-Arginine, the sole substrate for the NOS enzyme in producing NO, is also a substrate for arginase. Thus arginase has a potential role in limiting substrate available for NO production. Arginase, often perceived solely as the last of the now six enzymes of the urea cycle, exists in 2 isoforms, cytosolic form arginase I and mitochondrial form arginaseⅡ. ArginaseⅠis located in the cytosol and thought to be primarily involved in ureagenesis., whereas arginaseⅡis located in the mitochondrial matrix and has been thought to be more widely expressed and to be involved in the biosynthesis of polyamines, the amino acids ornithine, proline, and glutamate and in the inflammatory process, among others. Both arginaseⅠandⅡwere detected in crude myocardial homogenates, but only arginaseⅡwas present in isolated rat myocytes. Arginase inhibition with S-(2-boronoethyl)-L-cysteine (BEC) augmented Ca~(2+)-dependent NOS activity and NO production in myocytes.
Arginine concentration also governs NOS activity. The intracellular concentration of L-arginine in endothelial cells exceeds its K_m for the NOS enzyme by 2- to 3-fold, indicating that L-arginine availability should not limit NOS activity or NO production. Also, exogenous L-arginine administration should not influence NOS activity and NO production. However, in certain conditions, the addition of extracellular L-arginine enhances NO-dependent relaxation, giving rise to the "arginine paradox". The "arginine paradox" refers to the dependence of cellular NO production on exogenous L-arginine concentration despite the theoretical saturation of NOS enzymes with intracellular L-arginine. decreased availability of L-arginine blocked induction of NO production in cytokine-stimulated astrocytes, owing to inhibition of NOS2 protein expression.
Objective: Solusion phase synthesis of tripeptide contained L-Arginine and L-Lysine. Primary cultured Human Umbilical Vein Endothelial Cells (HUVEC), cardiac myocytes, macrophages with different treatment and examined the inhibitory effect of tripeptide on NOS activety. Expression of NOS2 was induced by lipopolysaccharide (LPS) and interleukin-6 (IL-6) for 24 hours before NOS2 protein was assayed by western blotting and cardiac myocyes (CMs) were incubated by new arginine analog for 12h before changes of the fluorescence signal of free calcium caused by isoproterenol (ISO), aβ-AR agonist, were measured under laser scanning confocal microscope (LSCM). The roles of ryanodine receptor (RyRs), inositol 1,4,5,-triphosphate rceptor (IP3R) and L-type calcium channel involved tripeptide effects was examined.
Methods:
1, Macrophages treated with LPS for 24 h to express N0S2. We examined the inhibitory effect of tripeptine on NO production in HUVEC, CMs and Macrophages.
2, CMs were treated with LPS and IL-6 for 6. 12, 24 h. NOS2 expression measured by western blotting.
3, CMs expressed NOS2 and treated with different concentration tripeptide or L-NNA for 12 h, to measure NO production.
4, CMs expressed NOS2 and treated with BEC and/or L-VNIO, then to test the NO levels.
5, CMs expressed NOS2 and treated with different NOS inhibitor for 12 h. LSCM measured the changes of fluorescence singal of free calcium caused by ISO.
6, CMs treated with verapamil(inhibitor of L-type channel), procaine(inhibitor of RyRs) and heparin(inhibitor of IP3R). LSCM measured the changes of fluorescence singal. To study the role of different calcium channel inhibitors in tripeptine effects on free calcium concentration..
7, Normal CMs treated with ISO, ISO+BEC, ISO+L-VNIO, ISO+BEC+L-VNIO, respectively. LSCM measured the changes of fluorescence singal caused by ISO.
8, CMs expressed NOS2 and treated with tripeptide, L-VNIO, BEC, tripeptide+L-VNIO. LSCM measured the changes of fluorescence singal caused by ISO.
Statistical analysis
All data are expressed as means±SD, and differences among groups were analyzed by one-way ANOVA followed by LSD test or Tambane test when equal variance was not assumed. Differences between two groups were analyzed by Independent - Samples T Test. A value of p<0.05 was considered statistically significant.
Results:
1,1, Effects of tripeptide on NO production
In macrophages expressing NOS2, after treated with tripeptide, L-NNA, the NO levels was 208.2±19.3, 279.5±20.7 and 308.9±17.1, respectively. The data was analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F=73.553, P=0.000). The data analyzed by LSD shows that compared with control group, tripeptide significantly inhibited NO production in macrophages (P=0.000) and the effect of tripeptide is better than that of L-NNA (P=0.000).
HUVEC expresses NOS3. The levels of NO (umol/L) in tripeptide group, L-NNA and control group is 179.5±9.4, 157.8±13.9 and 188.2±11.6, respectively. The data was analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F= 17.660, P=0.000). Compared with control group, L-NNA could significantly inhibit NO production (P=0.000) and tripeptide had no the effect (P=0.110). Tripeptide has little influence on NO production in HUVEC.
CMs expressed NOS1 and NOS3. The levels of NO (umol/L) in tripeptide, L-NNA and control group is 55.5±7.5, 30.2±5.9 and 61.4±9.9, respectively. The data was analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F=41.061, P=0.000). Compared with control group, L-NNA could significantly inhibit NO production (P=0.000) and tripeptide had no the effect (P=0.101).
2, NOS2 protein expression in response to different treatment
Cardiac myocytes treated with IL-6 and LPS for 6, 12 and 24 h, NOS2 protein expressions were measured by western blotting. The result showed that NOS2 protein began to express at 6h. The data was analyzed by one-way ANOVA. Significant difference was found in 6h, 12h and 24h group (F=187.101, P=0.000). NOS2 protein expression at 24h was significantly high compared with 6h (P=0.000) and 12h (P=0.003).
3, Effect of NOS inhibtor on NO production in CMs expressed NOS2
In CMs expressed NOS2, The NO values were analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F=29.230, P=0.000). The data analyzed by LSD shows that compared with control group, tripeptide (P=0.000) and L-NNA (P=0.001) significantly inhibit NO production and arginine increased NO production (P=0.003). The effect of tripeptide on inhibiting NO production is better than that of L-NNA (P=0.000).
CMs treated with different concentration tripeptide. The levels of NO were analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F=20.290, P=0.000). Compared with control group, tripeptide (0.2mmol/L) could inhibit NO production significantly (P=0.004). On decrease NO production, there was no significant difference between tripeptide (0.4mmol/L) and L-NNA(lmmol/L) group (P=0.490).
4, Effect of BEC and L-VNIO on NO production in CMs
In normal CMs treated with L-VINO, a NOS1 inhibitor, and BEC, a arginase inhibitor, the levels of NO were measured. The data was analyzed by factorial analyze. BEC increased NO production significantly (F=10.620, P=0.002) and L-VNIO decreased NO production significantly (F=48.909, P=0.000). BEC and L-VNIO had no interaction (F=0.838, P=0.366).
5, Effect of NOS inhibitor on free calcium concentration caused by ISO in myocytes expressed NOS2
Increased NOS2 activity contributes to decreased ISO-induced free calcium concentration. Free calcium concentration induced by ISO in normal myocytes is 172.0±8.0 and that in myocytes expressed NOS2 is 125.6±14.0. The data was analyzed by independent-samples t test. The result showed there is significant difference between two groups (t=9.099, P=0.000).
The free calcium concentrations in every group were analyzed by one-way ANOVA. Significant difference was found in every group (F=40.726, P=0.000). Compared with control group, specific inhibition of NOS2 by Tripeptide (1mmol/L) dramatically increased free calcium concentration induced by ISO (P=0.000) and arginine significantly decreased free calcium concentration induced by ISO (P=0.007). Tripeptide (0.4mmol/L) is better than L-NNA (lmmol/L) on increasing free calcium concentration (P0.003).
6, Effects of some drugs inhibiting Ca~(2+) channels
CMs treated with verapamil-a inhibitor of L-type calcium channel, heparin-a inhibitor of IP3R, and procaine-a inhibitor of RyR, the free calcium concentrations were 152.0±13.3 in control group, 137.1±10.9 in verapamil grouop, 150.0±11.0 in heparin group and 129.8±8.3 in procaine group. The data was analyzed by one-way ANOVA. Significant difference was found in every group (F=9.273, P=0.000). Verapamil (P=0.009) and procaine (P=0.000) could significantly inhibit tripeptide induced increasing in ISO-induced free calcium concentration in myocytes. Heparin has no the effect(P=0.957).
7, Effect of arginase inhibitor on normal myocyte free calcium concentration caused by ISO
CMs treated with BEC, L-VNIO and BEC+L-VNIO, the data of free calcium concentrations were analyzed by factorial analyze. Specific inhibition of NOS1 by L-VNIO decreased the ISO-induced free calcium concentration (F=147.008, P=0.000) and arginase inhibitor increased that in CMs (F=11.460, P=0.002). There was significant interaction between BEC and L-VNIO (F=5.356, P=0.026), L-VNIO could inhibit the effect of BEC on free calcium concentration in CMs.
8, Effect of arginase inhibitor on free Ca~(2+) concentraton caused by ISO in myocyte expressed NOS2.
CMs expressed NOS2, the value of free calcium concentration was analyzed by one-way ANOVA. Significant difference was found in groups (F=17.633, P=0.000). Compared with control group, L-VNIO could significantly decreased free calcium concentration induced by ISO (P=0.017), L-VNIO+tripeptide could significantly increased free calcium concentration induced by ISO (P=0.045) and BEC had no significant influence on free calcium concentration induced by ISO (P=0.427).
Conclusions:
1, macrophages treated with LPS and CMs treated with LPS and IL-6 could express NOS2 protein.
2, Tripeptide has highly selective and inhibitive effect on NOS2 activety.
3, Arginase strongly limited ISO-induced increases in free Ca~(2+) concentration in a NOS1-dependent manner.
4, Increased NOS2 activity contributed to limit ISO-induced myocardial free Ca~(2+) concentration. Tripeptide restored myocardial responsiveness toβ-adrenergic agonist via improving the functions of several calcium channels.
5, Arginine strongly limited ISO-induced increases in free Ca~(2+) concentration via increasing NO production and expression of NOS2.
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