Laboratory studies
Effect of pannexin-1on the release of glutamate and cytokines in
astrocytes
Li Wei a ,b ,1,Hanmiao Sheng a ,1,Long Chen a ,b ,Bin Hao c ,Xiaohong Shi d ,?,Yinghui Chen a ,b
a
Department of Neurology,Jinshan Hospital,Fudan University,Shanghai 201508,PR China b
Department of Neurology,Shanghai Medical College,Fudan University,Shanghai,PR China c
School of Pharmacy,Shanghai Jiaotong University,Shanghai 201508,PR China d
Department of Endocrinology,Jinshan Hospital,Fudan University,Shanghai,PR China
a r t i c l e i n f o Article history:
Received 28February 2015Accepted 1May 2015
Keywords:Astrocytes Cytokine Gap junction Glutamate Pannexin-1
a b s t r a c t
We investigated the effect of pannexin-1(PANX-1)on the release of glutamate and cytokines in U87human malignant glioma (U87-MG)cells using small interfering RNA (siRNA)transfection and adenosine triphosphate/lipopolysaccharide treatment.PANX-1is a new family member of the gap junction proteins,and is permeable to ions,metabolic molecules,second messengers,and neurotransmitters,among other factors.PANX-1plays an important role in the regulation of cell in?ammation.However,the relationship between PANX-1and the secretion of glutamate,interleukin (IL)-6and IL-8in astrocytes is still poorly understood.We found that the levels of IL-6,IL-8and glutamate were lower in PANX-1siRNA transfected cells,while the levels of IL-6,IL-8and glutamate were unaltered in cells without changes in the expres-sion of PANX-1protein.This provides further support for the hypothesis that PANX-1plays an important role in the release of IL-6,IL-8and glutamate in U87-MG cells.
ó2015Elsevier Ltd.All rights reserved.
1.Introduction
Astrocytes are the most abundant non-neuronal cells in the central nervous system (CNS).In addition to their trophic and structural role,astrocytes play an important role in the mainte-nance of homeostatic pH balance,neurotransmitter and ion levels,as well as the regulation of cell communication and in?ammation.Morphological and functional alterations of astrocytes may result in the disruption of brain homeostasis and may induce multiple brain diseases,including epilepsy,stroke,trauma and Alzheimer’s disease [1,2].
Gap junctions (GJ),which were discovered more than ?ve dec-ades ago,can form direct intercellular cytoplasmic connections and are involved in many biological events,including the transfer of ions and small molecules [3,4].Connexins (CXS),the predominant family members of GJ,have been identi?ed as key regulators in the release of in?ammatory mediators in many cell types [5–8].Pannexins (PANX)were described as a new family member of GJ proteins in 2000[9].As one of its subtypes,PANX-1is abundantly expressed in the brain and forms hemi-channels at the plasma membrane of astrocytes.PANX-1channels are permeable to ions,metabolic molecules,second messengers (including calcium ions [Ca 2+]and adenosine triphosphate [ATP])and neurotransmitters,which are smaller than 1kDa [10–12].Animal and clinical studies have shown that there are some overlapping functions between PANX-1and CXS,and most CXS inhibitors demonstrate an inhibi-tory effect on PANX-1[6,13].Therefore,PANX-1may be responsi-ble for the release of cytokines and glutamate in astrocytes.However,the role of PANX-1in the release of glutamate,inter-leukin (IL)-6and IL-8from astrocytes is still poorly understood.In?ammation and the disruption of the balance between excitatory and inhibitory responses are involved in the onset and develop-ment of many neurological disorders,including cerebrovascular diseases,epilepsy,and neurodegenerative diseases [14–16].Therefore,we examined the effect of PANX-1on the release of glu-tamate,IL-1b ,IL-6and IL-8from astrocytes using enzyme-linked immunosorbent assays (ELISA),the Bio-Plex suspension array system (Bio-Rad Laboratories,Hercules,CA,USA)and high perfor-mance liquid chromatography (HPLC).
2.Materials and methods 2.1.Materials
The U87human malignant glioma cell line (U87-MG)was orig-inally obtained from the Chinese Academy of Sciences,and used in this study.PANX-1small interfering RNA (siRNA;catalogue
https://www.doczj.com/doc/9d15997006.html,/10.1016/j.jocn.2015.05.043
0967-5868/ó2015Elsevier Ltd.All rights reserved.
?Corresponding author.Tel.:+8621341899905517;fax:+8602157943141.
E-mail address:shixh80301@https://www.doczj.com/doc/9d15997006.html, (X.Shi).
1
Li Wei and Hanmiao Sheng contributed equally to this work.
number sc-21687)and control siRNA(catalogue number sc-37007) were purchased from Santa Cruz Biotechnology(Dallas,TX,USA). Lipofectamine2000reagent was purchased from Invitrogen (Carlsbad,CA,USA).ATP and lipopolysaccharide(LPS)were obtained from Sigma-Aldrich(St Louis,MO,USA).The Cell Counting Kit8was obtained from Dojindo Laboratories (Kumamoto,Japan).A rabbit polyclonal anti-PANX-1antibody was purchased from Abcam(Cambridge,UK).The PrimeScript RT master mix and SYBR Premix Ex TaqII were purchased from Takara Bio(Shiga,Japan).The human IL-1b/IL-1F2Quantikine ELISA Kit was obtained from R&D Systems(Minneapolis,MN,USA).The Bio-Plex suspension array system was purchased from Bio-Rad Laboratories.
2.2.Cell culture
U87-MG cells were cultured in Dulbecco’s modi?ed Eagle’s medium(DMEM)with10%fetal bovine serum and a mixture of antibiotics and antimycotics in5%carbon dioxide and95%air at 37°C.The cells were passaged every4–5days to maintain expo-nential growth.
2.3.siRNA transfection
U87-MG cells were seeded in6-well plates and cultured in DMEM without antibiotics to improve the transfection ef?ciency. When the cells became50–80%con?uent,they were transfected with75nM PANX-1siRNA or control siRNA using the Lipofec-tamine2000reagent.PANX-1siRNA and Lipofectamine2000 reagent were diluted in serum-free medium(Opti-MEM;Life Tech-nologies,Carlsbad,CA,USA).The media was changed to growth media6h after transfection,and the cells were harvested48h after transfection for subsequent messenger RNA and protein expression analyses.
2.4.Real-time reverse transcription polymerase chain reaction(PCR)
Total cellular RNA was extracted using Trizol reagent followed by isopropanol precipitation.Next,RNA(500ng)was used for complementary DNA synthesis using the PrimeScript RT Master Mix kit,followed by real-time PCR analysis using SYBR Premix Ex TaqII,using an ABI Prism7300system(Applied Biosystems,Foster City,CA,USA).The speci?c PCR primer sequences were designed and synthesized by Bio-TNT(China):PANX-150-AATCTGTGACTTC TGCGACAT-30(sense)and50-CCATTTCCATTAGGGACTCAA-30 (anti-sense);b-actin50-AAGGTGACAGCAGTCGGTT-30(sense)and 50-TGTGTGGACTTGGGAGAGG-30(anti-sense).The initial denatura-tion phase was30min at95°C,followed by40cycles of denatura-tion at95°C for5s,and annealing at60°C for31s.After PCR ampli?cation,5l l of PCR product and a DNA ladder were simulta-neously run on a2%agarose gel to con?rm the PCR products.The relative quanti?cation of the PCR products was performed after normalization against b-actin,using the comparative cycle thresh-old method.
2.5.Western blot analyses
The total cellular proteins were extracted using sodium dodecyl sulfate(SDS)lysis buffer,and the protein amounts were estimated using a BCA Protein Assay kit(Beyotime Institute of Biotechnology, Jiangsu,China).All proteins were boiled for5min in SDS sample buffer.Next,the protein samples(20l g/well)and the molecular weight marker were simultaneously subjected to10%SDS polyacrylamide gel electrophoresis and then electrophoretically transferred onto immobilon-P membranes for105min at 300mA,using a wet electroblotting system(Bio-Rad).The membranes were then blocked with5%fat-free milk in1?Tris buffered saline with Tween-20for1h at room temperature and subsequently incubated with anti-PANX-1(1:1000)or anti-glyceraldehyde3-phosphate dehydrogenase(GAPDH; 1:10000)overnight at4°C.The membranes were then incubated with1:1000diluted goat anti-rabbit or goat anti-mouse immunoglobulin G antibody coupled to horseradish peroxidase (HRP).Immunoreactivity was detected by enhanced chemilumi-nescent detection using the Chemiluminescent HRP Substrate kit (Merck Millipore,Billerica,MA,USA)according to the manufac-turer’s instructions.Finally,the protein expressions were quanti-?ed by densitometry using image analysis software(Tinon Software,Zhongshan,China)and normalized against GAPDH pro-tein expression.
2.6.Stimulation of cells with ATP or LPS
U87-MG cells were seeded on96–well plates and stimulated with different concentrations of ATP or LPS.The cell proliferation was estimated using a Cell Counting Kit8according to the manu-facturer’s instructions.Then U87-MG cells were seeded in6-well plates and stimulated with ATP or LPS for24h.After24h of stim-ulation,the cells were washed with phosphate buffered saline and lysed in SDS lysis buffer.Simultaneously,the cultural supernatants were collected and centrifuged at14,000revolutions per minute for5min at4°C to remove cell debris.Supernatants were stored atà80°C for the detection of cytokines and glutamate.A control sample containing0l mol/l ATP or LPS was also processed.
2.7.HPLC
The levels of glutamate in the supernatants were quanti?ed using the Agilent1100System(Agilent Technologies,Santa Clara, CA,USA)with?uorescence detection after derivatization with o-phthaldialdehyde.The components were separated with a Zor-bax Extend-C18(250mm?4.6mm;5l m)column(Agilent Tech-nologies).The samples were eluted using in40mM sodium phosphate buffer(40mM monosodium phosphate;pH7.2)and acetonitrile.The levels of glutamate were quanti?ed by performing a comparison against a standard curve derived from a standard solution of glutamate.
2.8.ELISA and Bio-Plex suspension array system
The level of immunoreactive IL-1b released from U87-MG cells into the culture media was measured by ELISA,using a commercial human IL-1b/IL-1F2Quantikine ELISA kit.The levels of immunore-active IL-6,IL-8,IL-10and tumor necrosis factor(TNF)-a were measured using the Bio-Plex suspension array system.The assays were performed according to the manufacturer’s instructions.
2.9.Data analyses
The results are presented as the mean±standard error of the mean,unless otherwise stated.For the statistical analyses,each treatment was compared with its respective control,and the statistical signi?cance between the groups was assessed using the Student’s two-tailed t-test.Calculations were performed using GraphPad InStat(version5.0;GraphPad Software,San Diego,CA, USA),and signi?cance was established at p<0.05for all tests.
136L.Wei et al./Journal of Clinical Neuroscience23(2016)135–141
3.Results
3.1.PANX-1gene silencing and protein expression
To investigate the role of PANX-1in U87-MG cells,siRNA was used to knockdown the expression of PANX-1.According to real-time PCR results,the PANX-1knockdown resulted in a79% decrease compared with the control siRNA transfected cells (Fig.1A;p<0.05).The western blotting analyses showed that PANX-1-siRNA had a strong effect on downregulating the expres-sion of the PANX-1protein in U87-MG cells(Fig.1B,C;p<0.05).
3.2.PANX-1silencing reduces the release of glutamate
To determine the relationship between PANX-1and the release of glutamate in human astrocytes,HPLC was used to analyze the glutamate levels in culture media.The level of glutamate in PANX-1siRNA transfected cells was decreased compared to the control siRNA transfected cells,with a decrease of approximately 40%(Fig.2;p<0.05).the supernatants of cells transfected with PANX-1siRNA was decreased(Fig.2;p<0.05).
3.4.Effect of ATP on the release of IL-1b,IL-6,IL-8and glutamate in U87-MG cells
The U87-MG cells were exposed to ATP,which resulted in a strong induction of cell death by ATP in a dose-dependent manner (Fig.3A–C).Because there was obvious cell death when the cells were treated with5l g/ml ATP,we used0.1l g/ml and1l g/ml ATP to stimulate U87-MG cells in the following https://www.doczj.com/doc/9d15997006.html,pared with untreated cells,there were no differences in either the expression of PANX-1protein or the levels of IL-1b,IL-6,IL-8and glutamate in ATP-treated cells(Fig.3;p>0.05).
3.5.Effect of LPS on the release of IL-1b,IL-6,IL-8and glutamate in U87-MG cells
To evaluate the effect of the in?ammatory response on U87-MG cells,we treated U87-MG cells with LPS.In the ATP-treated cells,
(mRNA)and protein expression of pannexin-1(Panx-1)in small interfering RNA(siRNA)and control siRNA(scrambled) reaction(PCR)showed the mRNA quanti?cation of Panx-1in scrambled and siRNA-treated cells.Panx-1mRNA levels
blotting analyses showed the protein quanti?cation of Panx-1in scrambled and siRNA-treated cells.Glyceraldehyde
control.*p<0.05,siRNA versus scrambled.The error bars represent the mean±standard error of the mean and were
L.Wei et al./Journal of Clinical Neuroscience23(2016)135–141137
member of PANX proteins that is expressed in astrocytes.PANX-1is able to form intercellular channels,which are permeable to small molecules such as Ca 2+and ATP [6,10,11].In this study,we investigated the relationship between PANX-1and the in?ammatory immune response in the CNS,and also the effect of PANX-1on the release of glutamate.
We found that the baseline levels of IL-10and TNF-a in the supernatants of untreated U87-MG cells were below the detection limit.Multiple lines of evidence suggest that PANX-1contributes to the release of mature IL-1b via an association with purinergic receptors (P2X7receptor)[17].Therefore,changes in PANX-1pro-tein levels may result in an alteration of IL-1b levels.However,we found that in the PANX-1siRNA transfected cells,the IL-1b levels were lower than in the control siRNA transfected cells,although the difference was not statistically signi?cant.These data suggest that PANX-1expression in U87-MG cells had no effect on IL-1b secretion.We hypothesize that this might be due to the U87-MG cells being a human malignant cell line,therefore,having biologi-cal characteristics that are different from primary astro-cytes obtained from human tissue.
IL-6and IL-8are mainly synthesized in astrocytes and have dual functions in the CNS [18–20].Our ?ndings demonstrated that the siRNA knockdown of PANX-1signi?cantly decreased IL-6and IL-8secretion in U87-MG cells.Previous studies have shown that ATP can upregulate the expression of PANX-1[19].However,we found that there was no statistically signi?cant difference in the expression of the PANX-1protein or the secretion of IL-6and IL-8between the untreated cells and ATP-treated cells.To investi-gate whether U87-MG cells are sensitive to in?ammatory stimuli,we treated U87-MG cells with LPS,a common in?ammatory stim-ulus [20].Interestingly,we found that LPS had the same effect on U87-MG cells as ATP.Consistent with this result,it has been recently reported that LPS has no effect on primary astrocytes,U251and SNB-19malignant glioma cells [21].Moreover,the effect of LPS on the release of cytokines in U87-MG cells supports the hypothesis that there is a strong relationship between PANX-1expression and IL-6and IL-8secretion.PANX-1is an important component of the in?ammatory response in the CNS and accumu-lating experimental evidence indicates that the brain immune response is involved in the initiation and development of many neurological disorders,including stroke and epilepsy.For example,recent studies have highlighted the activation of in?ammatory pathways in epilepsy.Moreover,with regards to epilepsy treat-ment,some anti-epileptic drugs (valproate,carbamazepine,pheny-toin,levetiracetam and others)can modulate immune system activity,and adrenocorticotropic hormone,dexamethasone,hydro-cortisone and human intravenous immunoglobulins can suppress seizure activity [22–26].
Glutamate is an important excitatory neurotransmitter in the CNS and dysregulation of its release and reuptake alters neuronal excitability and contributes to the generation of seizures,Alzhei-mer’s disease and senile dementia [14,15].Elucidating the effect of PANX-1on the release of glutamate from astrocytes will provide further insight into the mechanisms underlying the pathogenesis of various neurological diseases.In our study,we found that silenc-ing PANX-1with siRNA decreased the PANX-1protein levels and attenuated the production of glutamate,demonstrating that PANX-1plays a critical role in glutamate secretion in
astrocytes.
glutamate released from pannexin-1small interfering RNA (siRNA)and control siRNA (scrambled)transfected U87-MG pannexin-1-siRNA and control siRNA for 48h.The levels of (A)interleukin (IL)-1b ,(B)IL-6,(C)IL-8and (D)glutamate were determined the Bio-Plex suspension array system (Bio-Rad Laboratories,Hercules,CA,USA)and high performance liquid chromatography,The error bars represent the mean ±standard error of the mean and were obtained from at least three experiments.
The exogenous application of ATP and LPS had no effect on PANX-1protein expression and glutamate levels,which supports the hypothesis that PANX-1contributes to the release of glutamate.The results of the present study demonstrate that PANX-1has an important role in the release of IL-6,IL-8and glutamate in U87-MG cells,which could play a critical role in glial cell-mediated innate defense and maintenance of glutamate
in
adenosine triphosphate (ATP)on the release of interleukin (IL)-1b ,IL-6,IL-8and glutamate in U87-malignant glioma (MG)cells.(A–C)induction of cell death by ATP on the U87-MG cells.(D–E)Pannexin-1protein expression in U87-MG cells exposed to 0.1l g/ml and 1l g/ml western blotting analyses.Levels of (F)IL-1b ,(G)IL-6,(H)IL-8and (I)glutamate were determined by enzyme-linked immunosorbent system (Bio-Rad Laboratories,Hercules,CA,USA)and high performance liquid chromatography,respectively.The error bars represent and were obtained from at least three experiments.
the CNS,and that it may represent a potential target for therapeu-tic intervention in many brain diseases.However,further studies are necessary to determine the mechanisms that underlie the release of IL-6,IL-8and glutamate by PANX-1in human astrocytes.Con?icts of Interest/Disclosures
The authors declare that they have no ?nancial or other con?icts of interest in relation to this research and its publication.This work was supported by grants obtained from the Shanghai Municipal Health Bureau (No.2012-234)to Yinghui Chen.References
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【摘要】黄自先生是我国杰出的音乐家，他以艺术歌曲的创作最为代表。而黄自先生特别强调了钢琴伴奏对于艺术歌曲组成的重要性。本文是以黄自先生创作的具有爱国主义和人道主义的艺术歌曲《天伦歌》为研究对象，通过对作品分析，归纳钢琴伴奏的弹奏方法与特点，并总结黄自先生的艺术成就与贡献。 【关键词】艺术歌曲；和声；伴奏织体；弹奏技巧 一、黄自艺术歌曲《天伦歌》的分析 （一）《天伦歌》的人文及创作背景。黄自的艺术歌曲《天伦歌》是一首具有教育意义和人道主义精神的作品。同时，它也具有民族性的特点。这首作品是根据联华公司的影片《天伦》而创作的主题曲，也是我国近代音乐史上第一首为电影谱写的艺术歌曲。作品创作于我国政治动荡、经济不稳定的30年代，这个时期，这种文化思潮冲击着我国各个领域，连音乐艺术领域也未幸免――以《毛毛雨》为代表的黄色歌曲流传广泛，对人民大众，尤其是青少年的不良影响极其深刻，黄自为此担忧，创作了大量艺术修养和文化水平较高的艺术歌曲。《天伦歌》就是在这样的历史背景下创作的，作品以孤儿失去亲人的苦痛为起点，发展到人民的发愤图强，最后升华到博爱、奋起的民族志向，对青少年的爱国主义教育有着重要的影响。 （二）《天伦歌》曲式与和声。《天伦歌》是并列三部曲式，为a+b+c，最后扩充并达到全曲的高潮。作品中引子和coda所使用的音乐材料相同，前后呼应，合头合尾。这首艺术歌曲结构规整，乐句进行的较为清晰，所使用的节拍韵律符合歌词的特点，如三连音紧密连接，为突出歌词中号召的力量等。 和声上，充分体现了中西方作曲技法融合的创作特性。使用了很多七和弦。其中，一部分是西方的和声，一部分是将我国传统的五声调式中的五个音纵向的结合，构成五声性和弦。与前两首作品相比，《天伦歌》的民族性因素增强，这也与它本身的歌词内容和要弘扬的爱国主义精神相对应。 （三）《天伦歌》的伴奏织体分析。《天伦歌》的前奏使用了a段进唱的旋律发展而来的，具有五声调性特点，增添了民族性的色彩。在作品的第10小节转调入近关系调，调性的转换使歌曲增添抒情的情绪。这时的伴奏加强和弦力度，采用切分节奏，节拍重音突出，与a段形成强弱的明显对比，突出悲壮情绪。 c段的伴奏采用进行曲的风格，右手以和弦为主，表现铿锵有力的进行。右手为上行进行，把全曲推向最高潮。左手仍以柱式和弦为主，保持节奏稳定。在作品的扩展乐段，左手的节拍低音上行与右手的八度和弦与音程对应，推动音乐朝向宏伟、壮丽的方向进行。coda 处，与引子材料相同，首尾呼应。 二、《天伦歌》实践研究 《天伦歌》是具有很强民族性因素的作品。所谓民族性，体现在所使用的五声性和声、传统歌词韵律以及歌曲段落发展等方面上。 作品的整个发展过程可以用伤感――悲壮――兴奋――宏达四个过程来表述。在钢琴伴奏弹奏的时候，要以演唱者的歌唱状态为中心，选择合适的伴奏音量、音色和音质来配合，做到对演唱者的演唱同步，并起到连接、补充、修饰等辅助作用。 作品分为三段，即a+b+c+扩充段落。第一段以五声音阶的进行为主，表现儿童失去父母的悲伤和痛苦，前奏进入时要弹奏的使用稍凄楚的音色，左手低音重复进行，在弹奏完第一个低音后，要迅速的找到下一个跨音区的音符；右手弹奏的要有棱角，在前奏结束的时候第四小节的t方向的延音处，要给演唱者留有准备。演唱者进入后，左手整体的踏板使用的要连贯。随着作品发展，伴奏与旋律声部出现轮唱的形式，要弹奏的流动性强，稍突出一些。后以mf力度出现的具有转调性质的琶音奏法，要弹奏的如流水般连贯。在重复段落，即“小
我国艺术歌曲钢琴伴奏-精 2020-12-12 【关键字】传统、作风、整体、现代、快速、统一、发展、建立、了解、研究、特点、突出、关键、内涵、情绪、力量、地位、需要、氛围、重点、需求、特色、作用、结构、关系、增强、塑造、借鉴、把握、形成、丰富、满足、帮助、发挥、提高、内心 【摘要】艺术歌曲中，伴奏、旋律、诗歌三者是不可分割的重 要因素，它们三个共同构成一个统一体，伴奏声部与声乐演唱处于 同样的重要地位。形成了人声与器乐的巧妙的结合，即钢琴和歌唱 的二重奏。钢琴部分的音乐使歌曲紧密的联系起来，组成形象变化 丰富而且不中断的套曲，把音乐表达的淋漓尽致。 【关键词】艺术歌曲；钢琴伴奏；中国艺术歌曲 艺术歌曲中，钢琴伴奏不是简单、辅助的衬托，而是根据音乐 作品的内容为表现音乐形象的需要来进行创作的重要部分。准确了 解钢琴伴奏与艺术歌曲之间的关系,深层次地了解其钢琴伴奏的风 格特点,能帮助我们更为准确地把握钢琴伴奏在艺术歌曲中的作用 和地位，从而在演奏实践中为歌曲的演唱起到更好的烘托作用。 一、中国艺术歌曲与钢琴伴奏 “中西结合”是中国艺术歌曲中钢琴伴奏的主要特征之一，作 曲家们将西洋作曲技法同中国的传统文化相结合，从开始的借鉴古 典乐派和浪漫主义时期的创作风格，到尝试接近民族乐派及印象主 义乐派的风格，在融入中国风格的钢琴伴奏写作，都是对中国艺术 歌曲中钢琴写作技法的进一步尝试和提高。也为后来的艺术歌曲写 作提供了更多宝贵的经验，在长期发展中，我国艺术歌曲的钢琴伴 奏也逐渐呈现出多姿多彩的音乐风格和特色。中国艺术歌曲的钢琴
写作中，不可忽略的是钢琴伴奏织体的作用，因此作曲家们通常都以丰富的伴奏织体来烘托歌曲的意境，铺垫音乐背景，增强音乐感染力。和声织体，复调织体都在许多作品中使用，较为常见的是综合织体。这些不同的伴奏织体的歌曲，极大限度的发挥了钢琴的艺术表现力，起到了渲染歌曲氛围，揭示内心情感，塑造歌曲背景的重要作用。钢琴伴奏成为整体乐思不可缺少的部分。优秀的钢琴伴奏织体，对发掘歌曲内涵，表现音乐形象，构架诗词与音乐之间的桥梁等方面具有很大的意义。在不断发展和探索中，也将许多伴奏织体使用得非常娴熟精确。 二、青主艺术歌曲《我住长江头》中钢琴伴奏的特点 《我住长江头》原词模仿民歌风格，抒写一个女子怀念其爱人的深情。青主以清新悠远的音乐体现了原词的意境，而又别有寄寓。歌调悠长，但有别于民间的山歌小曲；句尾经常出现下行或向上的拖腔，听起来更接近于吟哦古诗的意味，却又比吟诗更具激情。钢琴伴奏以江水般流动的音型贯穿全曲，衬托着气息宽广的歌唱，象征着绵绵不断的情思。由于运用了自然调式的旋律与和声，显得自由舒畅，富于浪漫气息，并具有民族风味。最有新意的是，歌曲突破了“卜算子”词牌双调上、下两阕一般应取平行反复结构的惯例，而把下阕单独反复了三次，并且一次比一次激动，最后在全曲的高音区以ff结束。这样的处理突出了思念之情的真切和执著，并具有单纯的情歌所没有的昂奋力量。这是因为作者当年是大革命的参加者，正被反动派通缉，才不得不以破格的音乐处理，假借古代的