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心脉乐片对急性冠脉综合征患者血浆CGRP和ET的影响

【摘要】  目的 研究心脉乐片对急性冠脉综合征(ACS)患者血浆降钙素基因相关肽(CGRP)和内皮素(ET)的影响。方法 100例ACS患者随机分为两组,常规治疗组48例,服用阿司匹林、硝酸异山梨醇酯等药物治疗;联合治疗组52例,服用硝酸异山梨醇酯加心脉乐片治疗。用放射免疫法测定治疗前后血浆CGRP和ET的浓度,并与50例正常人对照。结果 患者较正常人血浆CGRP浓度明显降低(P<0.05),ET明显增高(P<0.05);常规治疗组和联合治疗组均可提高CGRP浓度,降低ET浓度(P<0.05)。与常规治疗组相比较联合治疗组作用更明显(P<0.05)。结论 常规治疗加心脉乐片能显著改善患者的CGRP和ET的代谢失衡。

【关键词】  心脉乐片;急性冠脉综合征;降钙素基因相关肽;内皮素

 降钙素基因相关肽(CGRP)和内皮素(ET)是目前已知具有较强舒血管和缩血管的活性多肽物质,它们对全身各系统尤其是心血管系统有重要的调节作用。目前在心血管疾病方面对血浆CGRP和ET浓度改变的研究较多,但心脉乐片对急性冠脉综合征(ACS)患者血浆降钙素基因相关肽和内皮素浓度影响的报道较少。本文旨在探讨ACS患者血浆CGRP和ET浓度的变化及心脉乐片的影响,以进一步阐明心脉乐的作用机制。

  1 临床资料

  1.1 病例选择

  100例ACS患者,男58例,女42例,年龄45~80岁,平均(67.5±5.2)岁,均符合WHO诊断冠心病的标准,随机分为常规治疗组(48例)和联合治疗组(52例),两组间年龄、性别等无明显差别,对照组为本院健康体查者,男30例,女20例,年龄45~78岁,平均(65.2±4.5)岁。

  1.2 方法

  受检者取空腹时静脉血5ml,注入10%EDTANa2 30μl和抑肽酶40μl的试管中混匀,4℃3000r/min离心10min。分离血浆置冰箱-40℃备测。CGRP、ET采用放射免疫法测定,试剂盒由解放军总医院东亚免疫技术所提供。常规用药阿司匹林300mg,1次/d,氯吡格雷75mg,1次/d,硝酸异山梨醇酯100mg,3次/d;联合治疗组在常规治疗同时加用心脉乐片(解放军第323医院)4片,3次/d。两组疗程均为2周,治疗前后分别测定血浆CGRP和ET浓度各1次。

  1.3 统计学方法

  数据用(x±s)表示,组间用F检验。自身对照用配对t检验。

  2 结果

  ACS患者较正常人血浆CGRP明显降低,ET明显增高(P<0.01),常规治疗和联合治疗均可提高CGRP浓度,降低ET浓度(P<0.01);与常规治疗组相比,联合治疗组作用更明显(P<0.05)。见表1表1 两组ACS患者治疗前、后血浆CGRP和ET浓度与对照组的比较

  3 讨论

  CGRP是调节心血管活动的重要神经递质,是目前发现的最强的舒血管物质。它通过激活腺苷酸环化酶,使细胞内CGRP水平升高,发挥其舒血管效应,从而调节局部血管张力,维持全身循环的稳定,对缺血心肌和血管内皮细胞具有保护功能,并能抑制血管平滑肌细胞增殖[1,2]。ET是内皮细胞分泌的21肽血管活性物质,具有强烈的缩血管作用,是组织缺血缺氧时释放的一种内源性致病因子[3],此外,ET可增加胞浆和溶酶体漏出,促进心肌内钙的聚集和心肌细胞脂质过氧化损伤;还可刺激心肌细胞增生肥大,增加循环阻力,导致冠脉储备能力下降,以致心肌舒缩功能不全[4]。有研究表明,冠心病患者血浆CGRP浓度明显降低。ET浓度增高。经治疗后可见CGRP浓度升高,ET浓度降低,推测CGRP水平下降使心肌细胞内CGRP水平降低,外周血管阻力增加,心肌收缩及舒张性降低,而ET浓度升高促进了心肌缺血的发生和发展[5]。

  本研究所用的心脉乐片是由丹参、三七、冰片、降香等六味中药材按君、臣、佐、使原则科学配方,采用现代提取技术精制而成。主要作用为扩张冠状动脉,提高冠状动脉血流量,降低心肌耗氧量,还可抑制脂质过氧化物,清除氧自由基。抑制血小板聚集,从而保护缺血心肌,改善心脏功能[6]。研究结果提示,在常规治疗的基础上用心脉乐片治疗冠心病、急性冠脉综合征,患者可通过提高机体CGRP的释放,降低ET水平,进一步改善血管内皮功能,改善心肌缺血、缺氧状态,增加冠脉血流量,减轻患者症状。

【参考文献】
   1 Masash,Yanang is awa.A novel Potene Vasoconsticor Peptide Proueed by vasulay endothe(ia)cells,Natar,1998,323:411-415.

  2 Bell D. Mc Demott BJ Calcitionin gene-alatd Peptide in the cardiovascular system:characterization of rcceptor Populations and their Physiotgical.significance Pharmacl Rev,1996,48:253.

  3 Yoshiyu T,Isamu M,Ping sheng Wu,et al.Effects of an endothelin receptor antagonistion rats with cyclosporineinduced hypertension.Hypertension,1995,26:932.

  4 裴建明,李红梅.降钙素基因相关肽和心房肽对犬冠脉的舒张作用.中国应用生理学杂志,1995,11(1):47-50.

  5 裴建明,李红梅.降钙素基因相关肽对正常及心肌缺血时犬心脏收缩时间间期的影响.陕西医学杂志,1994,11(23):699-701.

  6 李红梅,李敏.老年高血压患者血浆内皮素,心钠素及降钙素基因相关肽对心脏功能的影响.中华临床医学杂志,2007,3(8):14-16.

  

日期:2011年6月29日 - 来自[2010年第8卷第9期]栏目
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芫花根质量标准研究

【摘要】 目的 建立芫花根中芫花酯甲的定性鉴别和定量分析方法。方法 采用薄层色谱法对芫花根中芫花酯甲进行定性鉴别,采用高效液相色谱法测定芫花酯甲的含量。色谱条件:Kromasil C18色谱柱(4.6 mm×250 mm,5 μm);流动相为甲醇-水(85∶15),流速1.0 mL/min;柱温40 ℃;检测波长为238 nm;记录时间40 min。结果 芫花根中芫花酯甲的薄层色谱鉴别特征明显、专属性强;含量测定线性范围为0.047~0.282 μg(r=0.999 6);平均回收率为99.2%,RSD=2.00%(n=6)。结论 该方法操作简便,结果准确,能够对芫花根中的芫花酯甲进行定性鉴别和定量分析,提高了该药材的质量控制水平。

【关键词】  芫花根;芫花酯甲;薄层色谱法;高效液相色谱法

Research about Quality Standard on Root of Daphne genkwa Sieb.et Zucc 

  HAN Wei, ZHANG Jun, WU Li-jun, et al

  1.Pharmacy College, Shanxi University of TCM, Xianyang 712046, China;2.Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China;3.School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China

    Abstract:Objective To develop qualitative identification and quantitative analysis method of yuanhuacin in root of Daphne genkwa sieb.et Zucc. Method Yuanhuacin was identified by TLC and its content was determined by HPLC. Kromasil C18 (4.6 mm×250 mm, 5 μm) column was used. The mobile phase was methanol-water (85∶15) at a flow rate of 1.0 mL/min. The column temperature was 40 ℃. The detection wavelength of yuanhuacin were set at 238 nm. The record time was 40 min. Results The linear range of yuanhuacin was 0.047~0.282 μg (r=0.999 6). The average recovery was 99.2% with RSD=2.0% (n=6). Conclusion These analytical methods were simple and accurate, and can be used for the qualitative identification and quantitative analysis of yuanhuacin. It is suitable for quality control of root of Daphne genkwa Sieb.et Zucc.

    Key words:Daphne genkwa Sieb.et Zucc;yuanhuacin;TLC;HPLC
 
    芫花根为瑞香科植物芫花Daphne genkwa Sieb.et Zucc.的干燥根,广泛分布于我国长江流域各省和黄河流域的部分地区。本品味辛、苦,性温,有毒;具有逐水、解毒、散结等功效;用于治疗水肿、乳痈等。本品主要含二萜类、黄酮类和香豆素类成分[1];芫花酯甲为主要的二萜类成分,具有利水作用,能直接兴奋子宫平滑肌,为引产药的有效成分,还能抑制P388癌细胞核酸和蛋白质的合成,有抗白血病作用。该成分具有强烈的刺激性,对皮肤、黏膜系统有较大的毒害作用,是芫花根利水、抗生育作用的有效成分,也是毒性成分。本试验对芫花根中芫花酯甲进行定性定量研究,以期为提高芫花根的质量控制水平提供试验数据。

  1  仪器与试药

    薄层成像系统TRANSILLVMINATOR2020D型(冷泉港科技有
限公司);Merck TLC薄层板(德国Merck公司);Waters高效液相色谱仪(600 Pump;Waters 600 Controller;Waters 2487 Dual λ Absorbance Detector);Millennium32色谱管理软件;CX-250超声波清洗器(北京医疗设备二厂,功率250 W,频率40 kHz)。

    芫花酯甲对照品为自制,经1H NMR和13C NMR鉴定结构,HPLC检测其纯度,面积归一化法计算对照品的含量在98%以上,符合对照品纯度要求。甲醇为色谱纯,水为自制高纯水。其余试剂均为分析纯。

    芫花根采自河南信阳,经笔者鉴定来源于瑞香科植物芫花Daphne genkwa Sieb.et Zucc.的干燥根。

  2  定性鉴别

    取本品粉末10 g,置于100 mL三角烧瓶中,用95%乙醇80 mL回流提取1.5 h,提取液回收至无醇味,加水分散,用石油醚(60~90 ℃)萃取,回收溶剂至干,溶解于1 mL甲醇中,得供试品溶液。另取芫花酯甲加甲醇制成每1 mL约含2 mg的溶液,作为对照品溶液。照薄层色谱法[2005年版《中华人民共和国药典》(一部)附录ⅣB]试验,吸取上述溶液各15 μL,分别点于同一荧光板上,以苯-乙酸乙酯(1∶1)系统展开,取出,晾干,置紫外光灯(254 nm)下检视,样品色谱中,在与对照品色谱相应的位置上,显相同颜色的暗斑。

  3  含量测定

  3.1  色谱条件

    Kromasil C18色谱柱(4.6 mm×250 mm,5 μm);以甲醇-水(85∶15)为流动相;流速1.0 mL/min;柱温40 ℃;检测波长为238 nm。

  3.2  溶液的制备

  3.2.1  对照品溶液的制备
 
  取芫花酯甲适量,精密称定,用甲醇溶解并定容,制成0.023 5 μg/μL的溶液,摇匀,即得对照品溶液。

  3.2.2  供试品溶液的制备 

  取芫花根样品约5 g,置100 mL具塞锥形瓶中,准确加入三氯甲烷50 mL,超声(功率250 W,频率40 kHz)30 min,滤过,用少量三氯甲烷洗涤残渣,洗液并入滤液中蒸干,残渣加50 mL甲醇,称重,回流1 h,放置,待其冷却至室温,称重,用甲醇补足减失的重量,摇匀,滤过,取续滤液过微孔滤膜(0.22 μm),即得供试品溶液。

  3.3  线性关系考察

  取对照品溶液(0.023 5 μg/μL),分别进样2、4、6、8、10、12 μL,以芫花酯甲的进样量为横坐标,以峰面积为纵坐标,绘制标准曲线,得芫花酯甲的回归方程为:Y=2 768 985.32X-
19 820,r=0.999 6,结果表明进样量在0.047~0.282 μg范围内线性关系良好。

  3.4  方法学考察

  3.4.1  精密度试验 

  精密吸取同一样品5 μL,连续进样5次,按上述色谱条件测定峰面积,RSD=1.43%,表明仪器性能良好。

  3.4.2  重复性试验 

  取同一样品5份,按照供试品溶液的制备方法制备,分别进样5 μL,测定芫花酯甲的峰面积并计算含量,平均含量为0.023 6%,RSD=1.69%(n=5),表明本方法重复性较好。

  3.4.3  稳定性试验 

  精密吸取同一样品,分别在样品制备后0、2、4、8、16、24 h进样5 μL,测定芫花酯甲的峰面积,RSD=1.28%,表明样品溶液在24 h内稳定。

  3.4.4  加样回收率试验 

  精密称取样品6份,各约2.5 g,分别精密加入对照品0.60 mg,按“3.2.2”项下方法制备供试液,分别精密吸取5 μL,测定芫花酯甲含量及加样回收率,结果平均回收率为99.2%,RSD=2.00%。见表1。

  3.5  样品测定

  精密吸取按“3.2”项下方法制备的对照品溶液和供试品溶液各5 μL,分别进样测定。含量测定结果见表2,色谱图见图1。表2  芫花根样品含量测定结果(略)

  4  讨论
   
  薄层色谱显色条件的选择:本试验对比了偏钒酸铵-硫酸溶液显色和薄层板用荧光板在254 nm下检视为暗斑点两种显色方法。用荧光板点样量小,分离效果好,容易观察;同时发现用偏钒酸铵-硫酸显色,放置一段时间后,在自然光下芫花酯甲显蓝紫色斑点,效果比直接观察红棕色斑点好。
   
  供试品溶液制备方法的选择:参考文献[2]的方法,经试验确定,选择三氯甲烷超声30 min、溶剂用量为50 mL的甲醇提取为最佳方法。检测波长的确定:采用光电二极管阵列检测器在200~400 nm对芫花酯甲色谱峰进行全波长扫描,发现在238 nm处有最大吸收,因此,选择238 nm为检测波长。
   
  本试验测定了芫花根、根皮、木心、市售根饮片,发现市售根饮片经过浸泡、切制后,芫花酯甲的含量明显降低;在临床应用芫花根的时候可以去除体积较大的木心,直接应用根皮。本方法简便,准确性高,能够提高芫花根药材的质量标准,对于保证其临床药效具有参考价值。

【参考文献】
    [1] 应百川,王成瑞,周炳南,等.芫花根有效成分的研究[J].化学学报,1975, 35(1):103-107.
  [2] 黄兰岚,李娆娆,原思通,等.不同产地芫花药材中芫花酯甲含量比较[J].中成药,2008,30(10):附10-12.

日期:2010年10月7日 - 来自[色谱分析实例]栏目

心可舒对冠心病合并2型糖尿病患者N0、ET的影响

  近年研究表明,内皮功能异常与多种心血管疾病的发生发展密切相关。冠心病和2型糖尿病的发病均与血管内皮功能障碍、血管内炎症反应以及动脉硬化有关。我们通过随机、双盲、对照试验,观察了80例冠心病合并2型糖尿病患者治疗前后血管内皮因子一氧化氮(NO)、内皮素(ET)浓度的变化,以探讨心可舒胶囊对冠心病合并2型糖尿病患者血管内皮功能的影响。

  1资料与方法

  1.1一般资料选择2008年1月至l2月间我科住院的冠心病合并2型

  糖尿病患者80例,入选病例采用随机数字表法分为两组:心可舒胶囊治疗组(心可舒组)40例中男性26例、女性14例;年龄44~72岁,平均(57.2+6.3)岁;病程5~15年,平均(9.4±3.5)年。对照组40例中男性24例、女性16例;年龄46~73岁,平均(59.1±6.2)岁;病程4~14年,平均(8.5±4.8)年。两组一般资料比较差异无统计学意义。治疗前两组间全血黏度高切、全血黏度中切、全血黏度低切、全血还原黏度、红细胞刚性指数、红细胞聚集指数、卡松黏度、卡松曲服应力、三酰甘油、总胆固醇、一氧化氮、内皮素等比较差异无统计学意义。

  1.2诊断标准

  糖尿病诊断依照1999年WH0诊断标准;冠心病诊断参照l979年WH0诊断标准。

  1.2.1病例纳入标准:①符合糖尿病诊断标准;②均有胸闷、胸痛、心慌、乏力等症状,体格检查有心率增快、血压升高、表情焦虑;③经冠状动脉造影检查证实至少1支主要冠状动脉分支存在狭窄≥50%。

  1.2.2病例排除标准:①近6个月有心肌梗死或明显脑卒中史;②心功功能IIl、IV级或有严重心律失常等心脏严重疾病者:③合并有肝肾和造血系统等严重疾病者;④急慢性感染性疾病及酮症酸中毒等。

  1.3治疗方法

   患者均按照冠心病、糖尿病的饮食控制。对照组给予常规治疗:单硝酸异山梨酯20m9/次,2次,d;二甲双胍O.59,次,3次,d。治疗组:在常规治疗基础上口服心可舒胶囊4粒/次,3次,d,疗程为3个月。用放射免疫法检测血液流变学指标及ET;用化学发光法检测NO;用酶法测定血清甘油三酯(TG)、总胆固醇(Tc)、高密度脂蛋白-胆固醇、低密度脂蛋白一胆固醇(LDL.C)、载脂蛋白B(ApoB)、尿酸(UA)和游离脂肪酸(FFA)等。

  1.4统计学方法

  采用SPSSl4.0统计软件,数据比较采用t检验、x检验。P<0.05为有统计学意义。

  3讨论

  N0和ET是两种重要的内皮源性血管活性因子,在维持血管正常结构和功能方面起相反作用。ET-1不仅具有长效缩血管、促血小板、单核巨噬细胞的聚集、黏附作用,还是促平滑肌细胞增生剂;其过量释放可引起冠状动脉痉挛、心肌缺血坏死。相反,N0是内皮合成和分泌的重要扩血管物质,生理情况下N0能影响ET的分泌,二者的动态平衡对维持心血管系统恒定的舒张状态,调节冠状动脉基础张力有重要作用。内皮损伤时,血浆BT水平升高和(或)N0水平下降,二者比例失衡可导致血管舒张功能异常。

  心可舒胶囊由山楂、丹参、葛根、三七、木香组成。药理研究证明,心可舒胶囊可清除氧自由基、减少脂质过氧化物、降低血脂,抑制炎症反应,同时其有效成分丹参素可直接通过调节血管内皮细胞ET-1 mRNA的表达水平,控制内皮素的产生与分泌,提高血中N0的浓度,保护血管内皮功能。本研究发现,两组的N0水平均升高,但治疗组更为明显;治疗后两组ET水平均显著低于治疗前,但治疗组的下降更明显。提示心可舒胶囊可进一步提高冠心病合并2型糖尿病患者的血浆N0水平,降低ET水平,具有保护血管内皮的功能。冠心病合并2型糖尿病患者常伴有高脂血症和血液流变学的异常。本研究表明,治疗组的低切全血黏度、全血还原黏度及卡松黏度均显著降低,与对照组比较有显著性差异,提示心可舒胶囊具有降低血液黏度、改善血流动力学的作用。此外,本研究还发现,心可舒胶囊具有一定的降TG作用。总之,心可舒胶囊可能通过调节N0、ET的平衡对冠心病合并2型糖尿病患者发挥作用。

 

日期:2010年1月14日 - 来自[中医中药]栏目
循环ads

低剂量干扰素-α在治疗ET中的应用

【摘要】    目的 探讨低剂量的干扰素-α在治疗原发性血小板增多症(ET)中的疗效、副作用及对预后的影响。 方法对我院1997年1月~2006年12月间诊断为ET经过干扰素-α治疗的患者进行回顾性分析。 结果 12例患者中男女各6例,就诊时中位年龄52岁(33~82岁)。经过干扰素-α治疗6个月,完全缓解(CR)4例,部分缓解(PR)5例,无效(NR)3例;治疗12个月CR 6例,PR 5例,NR 1例。治疗有效者骨髓巨核细胞均减少,脾脏缩小,出血及血栓均明显控制。随访1.5~2年,CR 6例,PR 5例,复发1例。治疗过程中的副作用主要表现为流感样症状,但均能耐受。 结论低剂量干扰素-α治疗ET能取得很好的疗效及较好的耐受性,但需长期治疗。

【关键词】  血小板增多症;原发性;治疗;干扰素-α

  Clinical application of low-dose interferon alpha treatment in essential thrombocythemia.

  ZHOU  Ming, LI Qing-shan,WANG Shun-qing,et al.

  (Guangzhou Municipal First People's Hospital, Guangzhou 510180, Guangdong, P. R.China )

  Abstract: Objective    To explore the effect of low dose of interferon alpha on efficacy, side-effect and  prognosis of essential thrombocythemia (ET) patients.  Methods   ET patients diagnosed in our hospital from January 1997 to December 2006 were retrospectively analyzed.  Results   Twelve patients include 6 males and 6 females, with a median age of 52 years ranging from 33 to 82 years.After low-dose interferon alpha treatment for 6 months,patients achieved complete remission(CR,n=4),partial remission (PR,n=5),and non response (NR,n=3),respectively.After 12 months,there were CR in 6 cases,PR in 5 cases,NR in l case,respectively. The number of megakaryocyte in bone marrow reduced, spleens withdrew, hemorrhage and thrombosis were significantly controlled in those effectively treated.Twelve patients were followed-up over 1.5 to 2 years, there were CR in 6 cases,PR in 5 cases,relapse in 1 case,respectively. The main side effect was flu-like syndrome and patients could continue to receive interferon therapy.  Conclusion   The patients were well responsive and tolerant to interferon-α,but the patients must be treated for a longer period.

  Key words:  Thrombocythemia;  Treatment; Interferon-α

  原发性血小板增多症(ET)是一种慢性克隆性骨髓增殖性疾病,其特征为血小板持续性增高,血小板的功能及形态的异常。目前尚无国际统一的诊断标准,真性红细胞增多症研究组(PVSG)于1997年第3次修订了本病的诊断标准[1]。传统的治疗方法有细胞毒性药物,如:羟基脲、马利兰及放射性磷等,然而,这些方法均有可能促使疾病向白血病转化。近年来,干扰素IFN-α治疗ET的有效性已日益引起人们的重视。我科应用低剂量干扰素-α治疗ET,获得了较好的疗效,现报告如下。

  1  材料与方法

  1.1  临床资料  所有病例均为我科自1997年1月~2006年12月收治的ET患者,符合诊断标准,共12例。12例患者中,男女各6例,中位年龄52岁(33~82岁)。在用干扰素治疗前其中未经药物治疗者8例,用羟基脲无效者1例(例2),停用羟基脲者1例(例7),妊娠1例(例6),临床及血液学检查见表1。

  表1 10例患者的临床及血液学资料(略)

  1.2  疗效判定标准  完全缓解(CR):临床表现及血小板数恢复正常;部分缓解(PR):血小板计数较治疗前下降50%,临床表现有所减轻;无效(NR):达不到部分缓解者。

  1.3  血小板的单采  所有血小板>800×109/L患者经过CS-3000plus 血细胞分离机单采清除血小板1~3次,使得患者外周血血小板降至800×109/L以下后,进行干扰素-α治疗。

  1.4  干扰素-α治疗  12例均在确诊后经上述血小板单采后接受干扰素-α治疗。具体方案如下:低剂量应用干扰素α,每周2~3次,每次300万U皮下注射。均加用小剂量的阿斯匹林(100mg/d)。

  2  结果

  2.1  疗效观察  经过干扰素-α的12个月治疗,12例患者中外周血血小板及骨髓中的巨核细胞见表2。其中,例2用羟基脲治疗4周无效,采用干扰素治疗2个月后血小板开始下降。6个月后达PR,12个月达CR。例8干扰素治疗1个月后出现血小板下降,6个月达CR,10个月时血小板进行性上升,干扰素治疗无效。

  表2  干扰素治疗ET的血小板及骨髓巨核细胞的变化(略)

  注:CR完全缓解;PR:部分缓解;NR无效。

  2.2  随访情况  患者经过12个月的干扰素治疗,仍然采用上述的干扰素治疗方案。例4及例10随访2年均为CR,均未再出现脑血栓;所有伴有脾肿大者,在治疗有效者均有脾脏缩小。例6在治疗15个月后停用干扰素治疗,血小板有所回升,随访至18个月血小板在500×109/L~600×109/L。例2、3、4、9、12随访1.5~2年维持CR状态;例1、5、7随访18个月维持PR。例8经胃镜检查发现有十二指肠多发性溃疡,在治疗6个月后达到CR,未出现上消化道出血,但在第10个月后血小板进行性上升,第12个月血小板达到900×109/L,再次出现上消化道出血。12例中无1例转变为白血病。

  2.3  干扰素副作用的观察  流感样症状:发热反应,8例注射干扰素后出现发热,体温波动在37.5~39℃,在注射后4~6h发生,注射3~5次后逐渐减轻至消失。有6例分别出现不同程度的肌肉酸痛、疲乏、骨痛、体重下降、嗜睡、抑郁,应用解热镇痛剂(消炎痛)可控制症状。有3例出现消化道反应:食欲下降、腹胀等,1例出现脱发。

  3  讨论

  原发性血小板增多症是一种少见的骨髓增殖性疾病,以往有关ET的治疗多选择羟基脲、马利兰、瘤可宁及放射性磷等药物治疗,血小板过高可有自发性出血倾向及血栓形成的危险,血小板单采仅能短期内减少血小板,骨髓抑制性药物往往引起骨髓抑制且增加转化为急性白血病的风险[1~4]。干扰素在血液方面主要应用于毛细胞白血病、多发性骨髓瘤及低度恶性的淋巴瘤,在骨髓增殖性疾病中可使部分慢性粒细胞白血病患者细胞遗传学获得完全缓解[5],以往干扰素在骨髓增殖性疾病中的原发性血小板增多症的应用较少,近年应用渐增多。本文观察患者干扰素-α用量为每次300万U皮下注射,每周2~3次,剂量相对较小,观察疗效及副作用。
  
  IFN-α对巨核细胞的增殖和分化具有直接和间接的抑制作用,在体外培养体系中,IFN-α使CFU-MK形成率明显减少,且其抑制作用与剂量呈正相关,对纯巨核细胞的研究表明,其不仅使巨核细胞的数量减少,而且巨核细胞的体积变小,高倍体巨核细胞数量减少,提示血小板生成素 (TPO)刺激的巨核细胞分化受阻[3,6],因而其治疗ET的有效性日益受到人们的重视。我们采用血小板单采迅速降低外周血血小板数后,应用低剂量的干扰素-α治疗ET。Sacchi研究212名ET患者,应用IFN-α治疗有效率达90%,开始每天300万U,大多数在2个月内血小板迅速降低,平均缓解时间为大约3个月[7]。且其对于骨髓抑制性药物无效或耐药者同样有效[7~9],本组例2在羟基脲治疗无效后采用干扰素治疗仍然有效。本组研究的12例中,治疗有效者骨髓巨核细胞抑制明显减少,无效者又升,也证明了干扰素对巨核细胞的抑制作用。在治疗6个月后,NR  3例,CR  4例,PR  5例;12个月后NR  1例,CR  6例,PR  5例,表明IFN-α治疗是一个长期的过程,不要在短期内轻易停药。同时也观察到1例(例8)在治疗10个月后无效,可能与患者产生抗IFN-α抗体有关[10]。例6在停止用药治疗后3个月,血小板的回升,但没有达到治疗前水平,说明干扰素治疗后有一定的“后续”效应。对于干扰素的副作用各家报道不一,Sacchi报道的273例 ET患者25%由于不能耐受其副作用而停止继续应用干扰素治疗[7],但本组观察的12例患者主要表现为流感样症状,均能耐受治疗。对于妊娠者及年龄<60岁者推荐使用干扰素治疗[7,11,12]。例1在治疗过程中正常分娩,避免了化疗药的不良反应,同时也提示了干扰素的安全性。
   
  近年越来越多学者推荐ET的治疗按照引起血栓出血性并发症的危险因素进行分层和治疗[12~14],对低危患者(<60岁,无血栓史,血小板计数<1 500×109/L,且无心血管危险因素如吸烟、肥胖、高血压),密切观察,避免使用细胞毒药物;中危患者(<60岁,无血栓史,但血小板计数>1 500×109/L或有心血管危险因素),控制危险因素,予干扰素、阿那格雷或羟基脲治疗;高危患者(≥60岁或有血栓史),首选羟基脲,可同时加用干扰素。可见,干扰素在低、中、高危患者均可使用。本文观察的患者中有6例属高风险患者,但用干扰素治疗均取得了PR或CR。

  总之,低剂量应用干扰素-α治疗ET,患者可获得很好的疗效及耐受性,但需要坚持长期用药。

【参考文献】
    [1] Murphy S,Peterson P,Iland H,et al.Experience of the Polycythemia Vera Study Group with essential thrombocythemia:a final report on diagnostic critera,survial,and leukemic transition by treatment[J].Semin Hematol,1997;34(1):29~39.

  [2] Kiladjian JJ, Rain JD,Bernard JF, et al.Long-term incidence of hematological evolution in three French prospective studies of hydroxyurea and pipobroman in polycythemia vera and essential thrombocythemia[J].Semin Thromb Hemost,2006;32(4 Pt 2):417~421.

  [3] Tefferi A, Murphy S.Current opinion in essential thrombocythemia:pathogenesis, diagnosis,and management[J].Blood Rev,2001;15(3):121~131.

  [4] Gilbert HS.Other secondary sequelae of treatments for myeloproliferative disorders[J].Semin Oncol,2002;29(3 Suppl 10):22~27.

  [5] Kantarjian HM,O’Brien S,Anderlini P,et al.Treatment of myelogenous leukemia:current status and investigational options[J].Blood,1996; 87(8):3069~3081.

  [6] Steensma DP, Tefferi A.Cytogenetic and molecular genetic aspects of essential thrombocythemia[J].Acta Haematol,2002;108(2):55~65.

  [7] Sacchi S.The role of alpha-interferon in essential thrombocythemia, polycythemia vera and myelofibrosis with myeloid metaplasia(mmm):a concise update[J].Leuk Lymphoma, 1995;19(1):13~20.

  [8] Tefferi A,Elliot MA,et al.New drugs in essential thrombocythemia and polcythemiavera[J].Blood Rev,1997;11(1):1~7.

  [9] Barbui T, Finazzi G.Therapy for polycythemia vera and essential thrombocythemia is driven by the cardiovascular risk[J]. Semin Thromb Hemost,2007;33(4):321~329.

  [10] Tornebohm-Roche E,Merup M,Lockner D,et al. Alpha-2a interferon therapy and antibody formation in patients with essential thrombocythemia and polycythemia vera with thrombocytosis[J].Am J hematol,1995;48(5):163~167.

  [11] Martinelli P,Martinelli V,Agangi A,et al.Interferon alfa treatment for pregnant women affected by essential thrombocythemia:case reports and a review[J].Am J Obstet Gynecol,2004;191(6):2016~2020.

  [12] Finazzi G, Harrison C.Essential thrombocythemia[J].Semin Hematol,2005;42(4):230~238.

  [13] Gisslinger H.Update on diagnosis and management of essential thrombocythemia[J].Semin Thromb Hemost,2006;32(4 Pt 2):430~436.

  [14] Barbui T.The leukemia controversy in myeloproliferative disorders: is it a natural progression of disease, a secondary sequela of therapy, or a combination of both[J]. Semin Hematol,2004;41(2 Suppl 3):15~17.


作者单位:

日期:2010年1月13日 - 来自[2008年第8卷第12期]栏目

阿霉素肾病综合征大鼠肾内皮素变化及其影响的研究

【摘要】    目的 观察内皮素对肾血流及蛋白尿的影响,探讨内皮素、肾血流在蛋白尿发生发展过程中的作用。 方法 制备大鼠阿霉素性肾病模型,动态观察不同病期大鼠血压、肾皮质血流、肾内皮素及24h尿蛋白量的变化,并将各指标进行相关分析。 结果 肾皮质内皮素升高发生在尿蛋白升高之前,随内皮素的升高,尿蛋白漏出量也随之增高,两者显著相关。内皮素可导致肾皮质血流的减少,在蛋白尿高峰期及高峰期后,减少的肾皮质血流可加重尿蛋白的发展,两者呈显著负相关。 结论 肾皮质ET的异常增加是导致蛋白尿发生的原因之一,肾血流量减少可加重蛋白尿的产生。

【关键词】  内皮素 肾病综合征 大鼠

  Experimental study on the effect of endothelin (ET) on renal blood flow and albuminuria in rats with Adriamycin induced nephrotic syndrome.

  SONG  bing.

  (Zhanjiang Education College,Zhanjiang 524037,Guangdong, P. R. China)
   
  Abstract:Objective  To observe the influence of endothelin ET on renaJ b1ood flow and albuminuria and discuss the functions of endothelin ET and RBF during the formation and development of albuminuria.  Methods  Adriamycin nephrosis rat model was prepared  and the renocorical blood flow and endothelin ET in different stages,the variations of the amount  of albuminuria in 24 hours were observed . The results of relevant indexes were analyzed.  Results  There is a close relationship between  endothelin  and aIbuminuria.The level of endothelin increased before the increase of level of albuminuria and the amount of secretion of albuminura increased with the rising level of endothelin.Endothelin may reduce the renocortical blood flow.The result also showed the negative correlation between them as the reduced renocortical blood flow may strengthen the development of albuminuria after the level of albuminuria reached its peak.  Conclusion  The abnormal increase of endothelin ET is one of causes for formation of albuminuria and the reduction of renocortical blood flow may accelerate the development of the albuminuria.
   
  Key words:Nephrotic syndrome; Rat; Endothelin

  肾脏是内皮素(Endothelin  ET)合成和分泌的重要场所,也是ET的靶器官,ET以自分泌或旁分泌方式调节肾功能。肾脏ET表达和释放异常是多种肾脏疾病发生发展的原因。ET能引起肾血管收缩,导致肾血流量及肾小球滤过率下降,还可作用于肾小球系膜细胞、内皮细胞、上皮细胞等[1~3],并影响肾内氧自由基及前列腺素系列物质的生成,从而参与肾脏的病理损伤过程,上述各种病理、生理改变均与蛋白尿相关。而肾损伤过程中,肾小球毛细管张力变化以及由此引起的肾血流动力学变化可能是肾功能进行性损伤的决定因素。为此我们观察了不同时期阿霉素肾病综合症大鼠肾ET及肾血流的变化,以探讨其与蛋白尿的关系。

  1  材料和方法

  1.1  实验动物及分组  SD大鼠,体重150~180g/只,随机分为正常对照组及实验组,分别饲养于代谢笼内,随意饮水,块料饲养。

  1.2  模型制备  实验组一次性尾静脉注射0.2%阿霉素7mg/kg,正常对照组同时尾静脉注射等量生理盐水。

  1.3  观察指标及检测方法

  1.3.1  24h尿蛋白量测定  于注射阿霉素后隔日留24h尿,并用磺基水杨酸比浊法测定尿蛋白量。

  1.3.2  血压测定  分别于注射阿霉素后4、8、32、56d将大鼠麻醉(13.3%戊巴比妥钠及0.6%氯醛糖等量混合液,取0.6ml/100g体重,肌注),用PE50管行左颈动脉插管术,插管成功后将大鼠平稳静置10min,然后将插管经三通管连接于惠浦78834C多功能监护仪,测量收缩压和舒张压,连续记录3个数据取其平均值计算平均动脉压。手术过程动物置控温板上,保持直肠温度在37℃左右。

  1.3.3  肾皮质血流  选用瑞典产激光多普勒微血管流量测定仪(Feri Flux4001 Master,Perimed,Sweden He-Ne 780nm)。仪器经调零及校正。血压测定后,作腹部正中切口进入腹腔,用PF413探头轻触左肾上、中、下极,每点取3个读数的均值,最后取肾上、中、下极平均值,以灌注单位(Pu)表示。整个手术保持大鼠直肠温度在37℃左右。肾内血流分配中,肾皮质外层血流量最丰富,占肾总血流量的80%左右[4]。我们所用的激光多谱勒微血管流量测定仪能较为准确地测定大鼠肾皮质外层约1mm深度范围的肾血流量,故能较好地反映肾局部血流情况[5]。

  1.3.4  肾皮质ET  于用阿霉素4、8、32、56d后测血压及肾皮质血流后取肾皮质,将肾皮质组织加入预冷的1ml 0.5mol/L醋酸溶液,稍作研磨后,在100℃水浴中煮10min,用玻璃匀浆器研磨至看不见颗粒为止,4℃ 3 000g离心20min,抽取上清液置低温保存待测。ET-l药盒由301医院东亚免疫技术研究所提供,采取放射免疫分析法直接测定肾皮质ET含量。

  1.4  统计学处理  (1)数据资料均用x±s表示;(2)t检验及显著性检验;(3)相关分析及显著性检验。

  2  结果

  2.1  肾病病征  在实验中观察到大鼠有明显腹水,浮肿,肾生化检验有高胆固醇,低蛋白血症,电镜下肾小球上皮细胞足突广泛融合变平坦,内皮细胞肿胀,窗孔减少,证明大鼠肾病综合征(Ntphrotic syndronce.NS)诊断成立。

  2.2  24h尿蛋白  注射阿霉素后5~6d尿蛋白开始升高,约7~8d差异有显著性,28~32d为最高点,48~56d有所下降,见表1。根据尿蛋白漏出情况,以用阿霉素4、8、32、56d划分为蛋白尿前期、初期、高峰期及高峰后期。

  表1  观察鼠不同时期24h尿蛋白量测定(略)

  注:与正常组比较,为P<0.05,为P<0.01。

  2.3  平均动脉压  如表2所示,平均动脉压在8、32、56d时略有升高,以32d达到高点56d时有所回落,但与正常组比较差异均未有显著性。

  2.4  肾皮质血流变化  如表2所示,肾皮质血流在用阿霉素4d即见下降,但至8d差异才有显著性,32d时肾皮质血流量最低,56d有所回升。

  2.5  肾组织ET变化  如表2所示,注射阿霉素4d肾ET即有显著升高,8d上升更为明显(差异极显著),32d时ET值最高,56d ET值有所下降。

  表2  观察鼠不同时期平均动脉压、肾皮质血流、肾ET值(略)

  注:与正常组比较,为P<0.05,为P<0.01。

  2.6  不同时期肾皮质血流与24h尿蛋白相关分析  肾皮质血流在蛋白尿高峰期及高峰期后与蛋白尿严重程度呈显著负相关,蛋白尿前期及蛋白尿初期相关不显著(见表3)。

  2.7  不同时期肾ET与24h尿蛋白相关分析  肾ET在蛋白尿整个发生发展过程中与尿蛋白显著相关(见表3)。

  2.8  不同时期肾ET、平均动脉压与肾皮质血流相关分析  肾ET在整个蛋白尿发生发展过程中与肾皮质血流呈显著负相关。平均动脉压在整个病程中与肾皮质血流无显著相关(见表3)。

  表3  肾病鼠不同时期各指标间相关分析(略)

  注:为P<0.05。

  3  讨论

  3.1  动物模型  尾静脉一次性注射阿霉素制造大鼠微小病变型肾病综合征(MCNS)模型是一种较为成熟的实验方法[6],我们在实验中观察到大鼠有明显腹水,浮肿,生化检查有高胆固醇,低蛋白血症,电镜下肾小球上皮细胞足突广泛融合变平坦,内皮细胞肿胀,窗孔减少(以上数据未显示),证明大鼠NS诊断成立,尿蛋白的高峰期在注药后28~32d。

  3.2  ET在蛋白尿发生发展中的变化  蛋白尿的产生机制极为复杂,影响因素很多,实验中我们观察到尿蛋白尚未明显升高即有肾皮质ET的显著升高,这可能由于肾脏有多种组织具有产生ET的能力[7],且由于肾脏是ET排泄的主要器官,因而其肾脏是ET含量较高的器官,肾组织ET的测定更直接反映肾局部ET的水平。
   
  ET是一种使血管强烈收缩的生物活性肽,是目前所知的作用最强的长效血管收缩剂,它与高血压的发生关系密切,我们在实验中观察到整个病程中平均动脉压虽有升高,但与正常组比尚未达显著性差异,可见在此病理生理情况下升高的ET对全身的影响尚较小,但对肾脏的影响已十分明显。实验结果显示,肾皮质血流在蛋白尿前期即有下降,从蛋白尿上升期始有显著改变,在尚无大量蛋白尿时,肾皮质血流量已开始减少,始时并未观测到尿量的减少(资料未显示)。这是由于肾血流量和肾小球滤过率变化程度不同步所致。肾皮质血流与ET相关分析表明,肾皮质ET在整个病程中与肾皮质血流呈显著负相关,提示ET可导致肾皮质血流减少。肾皮质血流与蛋白尿相关分析表明,在蛋白尿极期及极期后,也就是在有大量蛋白尿时,肾皮质血流与尿蛋白漏出量呈负相关。有资料表明,大量蛋白尿时,肾局部血流减少更为明显并与尿蛋白排泌量呈显著负相关,在整个蛋白尿发生发展过程中,肾局部血流的改变并非是一个平缓渐进的过程,其大幅度改变的时间正是肾病鼠从轻度蛋白尿向大量蛋白尿发展的时间,而增加肾血流量可使尿蛋白排泌量明显减少[8]。照此推断,肾血流的减少可加重蛋白尿的产生,也就是说,当肾损伤达一定程度后,肾血流的减少是蛋白尿增加的原因之一。
   
  肾脏内有多种细胞能合成ET,有结果表明:肾小球内皮细胞(EC)、肾小球上皮细胞(GEC)、肾小球系膜细胞(MC)和肾小管上皮细胞都能合成ET-1和ET-3,肾小球系膜细胞上ETA、ETB两种受体均存在,其中ETA型受体选择性地与ET-1结合,而ETB型受体与三种ET都能结合,又有资料表明,ET也是强有力的肾脏血管收缩剂,同时又具有压力利尿作用,可通过影响肾小球出、入球小动脉、系膜细胞的收缩减少肾血流量(RBF)及肾小球滤过率(GFR)[9]。但对RBF和GFR的影响并不同步,对GFR和尿钠排泄(URaV)尚无影响的小剂量ET已能引起肾血管阻力的显著增加[10],因而早期肾局部血流减少并不同时伴有尿量的减少。在肾损伤过程中,肾血流的改变、肾小球毛细血管张力的变化及毛细血管内皮所承受的流体切力的变化可能正反馈内皮细胞合成ET,进一步加速肾小球的损伤而导致蛋白尿增加。
   
  在实验中观察到肾ET升高发生在尿蛋白升高之前,且随ET的增加,尿蛋白也随之增加,在整个蛋白尿发生发展的过程中,肾皮质ET与尿蛋白排出量呈显著相关。所以,我们认为ET对肾脏病理生理的影响是导致蛋白尿发生的原因之一。除影响肾血流外,ET作用于肾脏导致蛋白尿发生发展的途径还有:①ET能促进细胞内钙池释放Ca以及开放钙通道,使Ca大量内流。结果造成钙超载和细胞坏死。致使肾小球正常生理功能遭到破坏[11],导致蛋白尿漏出增多;②内皮细胞和上皮细胞受ET作用而发生改变,有报导予健康鼠注入ET 0.5hr后,即可见到诸如肾小球脏层上皮细胞足突融合变平坦,内皮细胞肿胀,窗孔减少等病理改变[2],我们的实验也观察到病鼠肾小球内皮及上皮足突有类似的改变。肾小球上皮及内皮细胞是滤过膜的重要组成部分,上皮足突及内皮细胞的改变破坏了滤过膜的完整性,导致对蛋白通透性增加,是产生大量蛋白尿的重要原因;③ET导致肾组织丙二醇(MDA)升高:孙惠勤等予健康大鼠注入ET 3hr,即见肾组织匀浆丙二醇(MDA)显著升高[2];MDA为自由基代谢产物,其本身及前体均与蛋白尿的产生有极密切的关系[12];因此,ET所致肾组织MDA增加,亦是引起蛋白尿增加的原因之一。④ET还可使肾内前列腺素合成增加[13],尤其是TXA的增加,导致TXA/PGI比例失衡,而TXA/PGI系统参与蛋白尿的形成并与蛋白尿的严重程度呈线性关系,TXA越高或PGI越低蛋白尿的程度越重[14]。
   
  总之,本实验结果提示:肾皮质ET的异常增多是导致阿霉素诱发大鼠肾病蛋白尿发生发展的途径之一,肾皮质ET的改变引起肾皮质血流量的改变,在肾损伤达一定程度时,肾血流量的下降会导致尿蛋白的增加。

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作者单位:湛江教育学院,广东 湛江 524037.

日期:2010年1月13日 - 来自[2007年第7卷第10期]栏目
循环ads

Cloning and Functional Expression of a Novel Gi Protein-Coupled Receptor for Adenine from Mouse Brain

【关键词】  Cloning

    An orphan G protein-coupled receptor from the rat has recently been demonstrated to act as a transmembrane receptor for the nucleobase adenine. The receptor is possibly involved in nociception. Here we report the cloning and functional expression of an additional Gi-coupled receptor for adenine (Genbank accession code DQ386867). mRNA for this receptor was obtained from mouse brain and the mouse neuroblastoma x rat glioma hybrid cell line NG108-15. The new mouse protein sequence shares only 76% identity with that of the rat adenine receptor, suggesting that the receptors are not species homologs but distinct receptor subtypes. In human 1321N1 astrocytoma cells stably expressing the new mouse receptor, adenine and 2-fluoroadenine inhibited the isoproterenol-induced cAMP formation with IC50 concentrations of 8 and 15 nM, respectively. The adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 µM) as well as the P2 receptor antagonist suramin (300 µM) failed to change the responses to adenine. In contrast, pretreatment of cells with pertussis toxin abolished the effect of adenine. When the novel adenine receptor was expressed in Sf21 insect cells, a specific binding site for [3H]adenine was detected. In competition assays, the rank order of potency of selected ligands was identical to that obtained in membranes from NG108-15 cells and rat brain cortex (adenine > 2-fluoroadenine > 7-methyladenine > 1-methyladenine >> N6-dimethyladenine). In summary, our data show that a second mammalian DNA sequence encodes for a Gi-coupled GPCR activated by low, nanomolar concentrations of adenine.

    Adenine was recently identified as the endogenous ligand of an orphan rat G protein-coupled receptor (GPCR) (Bender et al., 2002). The expression of mRNA for this receptor in dorsal root ganglia neurons (Bender et al., 2002), as well as reports on neurotrophic effects of adenine (Watanabe et al., 2003; Yoshimi et al., 2003), points to a role of adenine as a neuronal signaling molecule. Spinally administered adenine has been shown to facilitate electrically evoked neuronal responses in the rat dorsal horn, indicating an enhancement in nociception by the action of adenine (Matthews and Dickenson, 2004). Recent studies demonstrating the specific binding of [3H]adenine to membrane preparations provided further evidence for the expression of the adenine receptor in rat neuronal tissues as well as a number of cell lines of neuronal origin (Gorzalka et al., 2005; Watanabe et al., 2005). The study by Gorzalka et al. (2005) also showed that an adenine receptor endogenously expressed in the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 (Hamprecht, 1977) mediated an inhibition of adenylate cyclase activity. RT-PCR experiments indicated that NG108-15 cells express a mouse rather than a rat mRNA sequence encoding for an adenine receptor (Gorzalka et al., 2005). It is noteworthy that there is evidence for the occurrence of an adenine receptor in human cells (Gorzalka et al., 2005). In healthy humans, plasma concentrations of adenine amount to values of approximately 70 nM; in patients with chronic renal failure, the plasma concentration of adenine even increases to values above 1 µM (Slominska et al., 2002). Therefore, adenine may play important roles in the pathophysiology of chronic renal failure (for a recent report on a GPCR-mediated effect of adenine on the Na+-ATPase activity in the proximal tubule, see Wengert et al., 2007). The human genome, however, seems to encode no direct ortholog of the rat adenine receptor (see also Bender et al., 2002), indicating that one has to search for a distinct sequence to identify human adenine receptors. Here, we report the cloning of a novel sequence encoding a GPCR for adenine. The sequence, which was found in mouse brain, mouse spleen, and NG108-15 cells, is clearly distinct from the mouse ortholog (mMrgA10) of the rat adenine receptor. Whether the mouse ortholog mMrgA10 or other members of the Mas-related gene family found in the mouse genome (Choi and Lahn, 2003; Zylka et al., 2003) operate as receptors for adenine is not known. We also demonstrate the functional expression of this second member of the family of GPCRs for adenine, which couples via a pertussis toxin-sensitive G protein to inhibition of adenylate cyclase, and characterize the pharmacological properties of the receptor. The presented results may facilitate the identification of a human GPCR for adenine. Some of these results have been presented at a meeting of the German Society of Pharmacology and Toxicology (von Kügelgen et al., 2007).

    Molecular Biological Experiments. Poly A+ mRNA was isolated from homogenized brain tissue, the liver, the kidney and the spleen of adult male MNRI mice (Charles River WIGA, Sulzfeld, Germany) as well as from NG108-15 cells cultured as described previously (Kaulich et al., 2003) using the Oligotex Direct mRNA Mini Kit (QIAGEN, Hilden, Germany). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using either the primer pair (sense, CACCATGGGGGAAAGCAGCACCGGTGCAG; antisense, TGGCTCTGCTTTGCTTTTTGACATCTCC) or, for expression analysis, primers for a gene-specific sequence of the new mouse gene (240 bp of Genbank accession code DQ386867; sense, CTCCTTTGCCTTTACACCTTCAGG; antisense, ACACCCATAGTCATTTACATATTTGG) and Superscript One Step RT-PCR mix (Invitrogen, Karlsruhe, Germany). Annealing temperature for the PCR reaction was 56.0°C (35-40 cycles). For control experiments, the enzyme reverse transcriptase was omitted [Taq Platinum Polymerase (Invitrogen) was used instead of the RT-PCR enzyme mix]. PCR products were analyzed by ethidium bromide staining after agarose (0.9%) gel electrophoresis. After amplifying the product of one RT-PCR reaction using Platinum Pfx polymerase (Invitrogen), the total coding sequence was cloned into the expression vector pcDNA3.1D/V5-His-TOPO (Invitrogen). The sequence was then identified by cycle sequencing (SequiTherm Exel II DNA sequencing kit; Epicenter Technologies, Madison WI) using a LICOR Gene READIR 4200 sequencer (MWG-Biotech, Ebersberg, Germany). The sequencing was repeated at GATC (Konstanz, Germany) with identical results.

    Expression of Recombinant Adenine Receptors in a Mammalian Cell Line. Human 1321N1 astrocytoma cells (European Cell Culture Collection, Salisbury, UK) were cultured at 5% CO2 and 36.5°C in Dulbecco's modified Eagle medium (Invitrogen) containing GlutaMAX-I (Invitrogen) and 10% fetal bovine serum (Invitrogen). The cells were split once a week by treating with trypsin-EDTA (0.5 g/l; Invitrogen). 1321N1 astrocytoma cells were transfected using the pcDNA3.1 expression vector and Lipofectamine 2000 (Invitrogen) as described by the manufacturer. Cells stably expressing the receptor constructs were selected two days after transfection by culturing in the presence of 800 µg/ml G-418 (Geneticin; Invitrogen). Cells from passages 4 to 20 of the transfected 1321N1 astrocytoma cells were used for further experiments. For some experiments, 1321N1 astrocytoma cells expressing the mouse adenine receptor were pretreated with pertussis toxin 200 ng/ml for approximately 20 h (for control experiments, see Chemicals).

    Generation of Recombinant Baculoviruses. The sequence encoding for the mouse adenine receptor (without vector-encoded epitopes) was cloned into the pVL1393 baculovirus transfer vector (BD Pharmingen, San Diego, CA) between the BamHI and BglII restriction sites using standard techniques. Recombinant baculoviruses were generated using the Baculovirus expression vector system from BD Pharmingen, which is based on the method of Guarino and Summers (1986). In brief, 2 x 106 cells were cotransfected with 0.5 µg BaculoGold DNA (Pharmingen, San Diego, CA) and 2 µg of transfer vector DNA containing the mouse adenine receptor gene using the calcium phosphate transfection method. Six days after transfection, the supernatant was harvested. The transfection supernatant was amplified to produce high titer virus stocks. The titer was determined using a modified endpoint dilution method (Reed and Muench, 1938). In brief, in a 24-well plate, 2 x 105 cells/well were infected with virus dilutions from 10-2 to 10-11. After 3 to 5 days, cells were inspected for signs of infection, and the highest effective virus dilution was used for further transfection experiments.

    Expression of Recombinant Adenine Receptors in Insect Cells. Spodoptera frugiperda (Sf21) cells (gift from J. Höhfeld, Cell Biology, Bonn, Germany) were grown in Insect express protein-free medium (Lonza Verviers SPRL, Verviers, Belgium). Cells were grown at 27°C and subcultured twice a week. Sf21 cells (1.8 x 107) were infected with recombinant baculoviruses at a multiplicity of infection of 1. They were harvested 72 h after infection. Cells were homogenized with 20 strokes in a tight-fitting Dounce homogenizer in 50 mM Tris-HCl buffer, pH 7.4, containing 1 mM phenylmethylsulfonyl fluoride. The cell suspension was centrifuged for 10 min at 1000g, 4°C. The supernatant was then centrifuged at 48,000g, 60 min, 4°C, and the resulting pellet was resuspended in 50 mM Tris-HCl buffer, pH 7.4. The protein concentration was determined by the method of Bradford (1976). Membranes were kept frozen at -80°C until use.

    Expression Analysis. 1321N1 astrocytoma cells stably expressing the mouse adenine receptor were cultured on coverslips in Dulbecco's modified Eagle's medium supplemented with 800 µg/ml G-418 for 2 days. Expression of the recombinant adenine receptor was assessed by direct immunofluorescence staining using a FITC-coupled monoclonal antibody against the V5 receptor epitope encoded by the expression vector (1:500; anti V5-FITC; Invitrogen). Expression was then verified by fluorescence microscopy on a Zeiss Axiovert 100 microscope equipped with an oil immersion 100x objective (Zeiss, Jena, Germany), a charge-coupled device camera, and a Polychrome II monochromator (excitation wavelength, 475 nm; exposure time, 1 sec; TILL Photonics, Planeg, Germany).

    Analysis of Cellular Cyclic AMP Accumulation. Receptor function was assessed by analyzing the activity of cellular adenylate cyclase activity. For this purpose, nontransfected 1321N1 astrocytoma cells and 1321N1 astrocytoma cells stably expressing the recombinant mouse adenine receptor or the human P2Y12 receptor were cultured on 24-well plates for 2 days. After removal of the culture medium, cells were washed with Hanks' balanced salt solution (containing 20 mM HEPES, pH 7.3) and then incubated with Hanks' balanced salt solution for 2 h at 36.5°C. Cellular cAMP production was then stimulated by the addition of 3 nM isoproterenol at 36.5°C. Solvent (control), adenine, 2-fluoroadenine, or 2-methylthio-ADP was added together with isoproterenol. The reaction was stopped after 10 min by removal of the reaction buffer followed by the addition of a hot lysis solution (500 µl; 90°C; 4 mM Na2EDTA, 0.01% Triton X-100; Sigma, Munich, Germany). In some experiments, the receptor antagonists DPCPX and suramin were given 10 min before adenine. The multiwell plates were shaken on ice for 1 h. cAMP levels in the supernatant were then quantified by incubation of an aliquot with cAMP binding protein and [3H]cAMP (cAMP assay; GE Healthcare, Freiburg, Germany) and liquid scintillation counting after removal of the unbound [3H]cAMP by charcoal. cAMP levels per well were calculated by linear regression from a standard curve determined for each experiment. The isoproterenol-induced cAMP production in the presence of agonists was expressed as percentage of the isoproterenol-induced cAMP production in the absence of agonists (percentage of control).

    Radioligand Binding Studies. Saturation and competition assays with [8-3H]adenine (27 Ci/mmol; GE Healthcare) were carried out essentially as described previously (Gorzalka et al., 2005). In brief, 50 µg of protein (NG108-15 membrane preparations) or 100 µg (Sf21 membrane preparations) was incubated with 10 nM [3H]adenine (competition assay) in 50 mM Tris-HCl, pH 7.4, in a total volume of 200 µl. Inhibition curves were determined using six to nine different concentrations of adenine or adenine derivative spanning 3 orders of magnitude. For saturation assays, increasing amounts (0.75-150 nM) of [3H]adenine were incubated with the Sf21 membrane preparations (100 µg) in 50 mM Tris-HCl buffer, pH 7.4, in a total volume of 200 µl. Three separate experiments were performed each in duplicate or triplicate as noted. Nonspecific binding was determined in the presence of 100 µM adenine. Incubations were carried out for 1 h at room temperature and terminated by rapid filtration through GF/B glass fiber filters (Whatman, Dassel, Germany). Filters were washed three times with ice-cold 50 mM Tris-HCl buffer, pH 7.4, 2 ml each. Filter-bound radioactivity was measured by liquid scintillation counting.

    Data Analysis and Statistics. Results are presented as means ± S.E. from n observations. Data were analyzed using Prism 4.03 (Graph Pad Software, San Diego, CA). Differences between means were tested for significance by the t test or (for multiple comparisons with the same control) by an analysis of variance followed by the Bonferroni post test (Prism). p < 0.05 was the significance criterion. IC50 values (concentrations causing half-maximal inhibition) were determined by fitting data to sigmoidal curves.

    Chemicals. The following drugs were used: 7-methyladenine (Acros Organics, Geel, Belgium); adenine, ADP sodium salt, ATP sodium salt, N6-benzyladenine, caffeine, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), N6-dimethyladenine, 2-fluoroadenine, 1-methyladenine, 2-methylthio-ADP, 5'-(N-ethylcarboxamido)adenosine (NECA), and pertussis toxin (Sigma); and suramin hexa-sodium salt (Bayer, Leverkusen, Germany). Stock solutions of drugs were prepared with either distilled water or with dimethyl sulfoxide (N6-benzyladenine, N6-dimethyladenine, DPCPX, 2-fluoroadenine, 7-methyladenine, 1-methyladenine, NECA). The solvents were added to the buffer used for control experiments. For pertussis toxin control experiments, the solvents present in the purchased solution were added to the medium; final concentrations: 0.05% glycerol (Serva, Heidelberg, Germany), 0.5 mM NaCl, 0.05 mM Trizma base (Sigma), and 0.38 mM glycine (Merck, Darmstadt, Germany).

    Cloning of a Novel Receptor for Adenine. Poly A+ mRNA was isolated from mouse brain homogenates as well as from the mouse neuroblastoma x rat glioma hybrid cell line NG108-15. RT-PCR with both mRNA preparations and with primers for the coding sequence of the putative mouse ortholog MrgA10 of the rat adenine receptor revealed products of the expected length of approximately 1000 base pairs (for NG108-15 cells, see also Gorzalka et al., 2005). Control reactions without the enzyme reverse transcriptase showed no products confirming that the mRNA preparations contained no genomic DNA. The RT-PCR products were subcloned in the pcDNA3.1 TOPO expression vector. We were surprised to find that sequencing of the subcloned DNA sequence did not reveal the expected sequence of the mouse ortholog of MrgA10 but a distinct sequence encoding for a protein with 332 amino acids, with only 81.6% identity compared with the mouse ortholog MrgA10 and only 76.3% identity compared with the rat adenine receptor (see Fig. 1A). A high degree of variability was found in the predicted transmembrane region 3, and the predicted third extracellular loop (bold residues in Fig. 1A). The new mouse nucleotide sequence was submitted to the Genbank under accession number DQ386867. This novel gene is located on mouse chromosome 7B3, whereas MrgA10 is localized in the region 7B4 to 7B5 (NCBI database). Additional RT-PCR experiments using specific primers for the new mouse gene (discriminating it from the mouse MrgA10 sequence) revealed products of the expected length of 240 base pairs from poly A+ mRNA isolated from mouse brain (B+; marked signal) and mouse spleen (S+, weak signal), but not from mRNA isolated from mouse liver L, or mouse kidney (K; Fig. 1B). This indicates the expression of mRNA for the mouse adenine receptor in neuronal tissues as well as in the spleen. Again, there were no products in reactions without the enzyme reverse transcriptase ("-" in Fig. 1B) in agreement with the absence of genomic DNA in our mRNA preparations.

    Fig. 1. A, alignment of the predicted amino acid sequences of the novel adenine receptor (Novel seq, mouse adenine receptor, present study, Genbank nucleotide sequence accession number DQ386867; see also XM_001003331), the mouse ortholog of MrgA10 (mMrgA10; Genbank nucleotide sequence accession no. XM_195647, please note that the total sequence of XM_195647 encodes for a protein with an extended N terminus) and the rat adenine receptor (rAdenine-r; Genbank nucleotide sequence accession no. AJ311952). Residues that differ in the mouse and the rat adenine receptor are shown in bold. The seven predicted transmembrane regions (TM) are indicated by lines (IL, intracellular loop; EL, extracellular loop). B, RT-PCR experiment indicating the expression of mRNA for the mouse receptor for adenine in tissues using primers specific for amplification of the sequence DQ386867 and poly A+ mRNA isolated from total brain (B), liver (L), kidney (K), and spleen (S). The figure shows RT-PCR products (+) stained by ethidium bromide after agarose gel electrophoresis. Control experiments without reverse transcriptase (-) revealed no products. One of three independent experiments.

    Expression in Astrocytoma Cells and Functional Analysis. Next, experiments were performed to analyze the pharmacological properties of the novel construct when expressed in mammalian cells. In nontransfected human 1321N1 astrocytoma cells, adenine (0.1 and 1 µM) caused no inhibition in cellular cAMP production in the absence and presence of isoproterenol used to stimulate the cellular cAMP accumulation (data not shown). Moreover, in 1321N1 astrocytoma cells stably expressing the recombinant human P2Y12 receptor, the P2Y12 receptor agonist 2-methylthio-ADP (1 µM) caused the expected inhibition of the isoproterenol (10 nM)-induced increases in cAMP levels (cAMP levels in the presence of 1 µM 2-methylthio-ADP amounted to 52.0 ± 1.7% of those without 2-methylthio-ADP, control; n = 3; p < 0.05 versus control, t test), whereas 10 µM adenine again failed to cause a change (98.9 ± 6.0% of control; n = 3). The lack of any effect of adenine confirms the absence of an endogenous inhibitory receptor for adenine in 1321N1 astrocytoma cells.

    1321N1 astrocytoma cells were then transfected with a pcDNA3 vector containing the sequence for the novel adenine receptor and the V5-epitope. Positive clones were selected in the presence of G-418 (see Materials and Methods). The expression of the receptor was verified by immunofluorescence staining against the receptor epitope (see Materials and Methods). In contrast to nontransfected cells, in 1321N1 astrocytoma cells stably expressing the mouse receptor, adenine (1 nM-1 µM) inhibited the isoproterenol-induced cAMP formation in a concentration-dependent manner with a half-maximal concentration (IC50) of 8 nM (95% confidence interval, 3-23 nM) and a maximal inhibition by approximately 40% (Fig. 2A). Likewise, the analog 2-fluoroadenine caused a concentration-dependent inhibition of cAMP production with an IC50 concentration of 15 nM (95% confidence interval, 7-30 nM) and a maximal inhibition by approximately 60% (Fig. 2B). The adenosine A1 receptor antagonist DPCPX used at a concentration of 1 µM and the P2 receptor antagonist suramin (300 µM) failed to alter the inhibitory effects of adenine in cells expressing the recombinant mouse adenine receptor (Fig. 3). DPCPX and suramin also caused no change in the increases in cellular cAMP levels induced by isoproterenol in the absence of adenine (not shown). Finally, the effect of pretreatment with pertussis toxin was studied in astrocytoma cells expressing the recombinant mouse adenine receptor. Pretreatment with pertussis toxin (200 ng/ml) for 20 h did not alter the isoproterenol-induced cAMP formation (see legend to Fig. 4) but abolished the inhibitory effect of adenine observed in cells pretreated with the solvent used for pertussis toxin (Fig. 4).

    Fig. 2. Inhibition of isoproterenol-stimulated cAMP accumulation by adenine (A, IC50 = 8 nM; 95% confidence interval, 3-23 nM) and 2-fluoroadenine (B, IC50 = 15 nM; 95% confidence interval, 7-30 nM) in 1321N1 astrocytoma cells stably expressing the novel (mouse) adenine receptor. Cellular cAMP production was increased by addition of isoproterenol 3 nM for 10 min at 36.5°C; data are given as percentages of the mean increases in cellular cAMP levels in the presence of isoproterenol alone (percentage of control; average increase in cAMP levels by 15.3 ± 1.3 pmol cAMP per well; n = 17). Means ± S.E. from 5 to 21 experiments. ** indicates significant differences from corresponding control (p < 0.01; ANOVA followed by the Bonferroni post test).

    Fig. 3. Inhibition of isoproterenol-stimulated cAMP accumulation by adenine and interaction with 1 µM DPCPX and 300 µM suramin in 1321N1 astrocytoma cells stably expressing the novel (mouse) adenine receptor. Antagonists or solvent were added 10 min before 10 nM adenine. Data are given as percentages of the mean increases in cellular cAMP levels in the presence of isoproterenol alone (percentage of control). Means ± S.E. from 5 to 21 experiments. * indicates significant differences from corresponding control (p < 0.05; ANOVA followed by the Bonferroni post test). For further details, see legend to Fig. 2.

    Fig. 4. Effect of the pretreatment with pertussis toxin (PTX) on the adenine-induced inhibition of isoproterenol-stimulated cAMP accumulation in 1321N1 astrocytoma cells stably expressing the novel (mouse) adenine receptor. Cells were pretreated with 200 ng/ml PTX or its solvent for 20 h. Data are given as percentages of the mean increases in cellular cAMP levels in the presence of isoproterenol alone (percentage of control; pretreated with solvent: average increase in cAMP levels by 10.9 ± 0.4 pmol of cAMP per well; n = 5; pretreated with PTX: 9.8 ± 0.7 pmol cAMP per well; n = 5). Means ± S.E. from five to seven experiments. **, significant difference from corresponding control (no adenine; p < 0.01;); ##, significant difference from corresponding value obtained with cells pretreated with solvent (p < 0.01; ANOVA followed by the Bonferroni post test). For further details see legend to Fig. 2.

    Expression of the Novel Receptor in Sf21 Cells and Analysis of Its Binding Properties. The novel mouse adenine receptor was subsequently expressed in Sf21 insect cells using the Baculovirus expression system. The adenine receptor sequence was inserted into the Baculovirus transfer vector pVl1393. By cotransfecting the vector and linearized Baculovirus DNA into Sf21 cells, recombinant Baculoviruses were generated (see Materials and Methods). After amplifying the viruses, Sf21 cells were infected for protein expression. Membrane preparations of noninfected Sf21 cells showed no specific binding for [3H]adenine (10 nM) (Fig. 5). In contrast, a specific binding site for [3H]adenine could be detected on membranes of Sf21 cells expressing the mouse adenine receptor (Fig. 5). Saturation experiments with [3H]-adenine revealed a single high-affinity binding site with a KD value of 113 ± 17 nM, and a Bmax value of 1.98 ± 0.39 pmol/mg protein was determined (n = 3) (Fig. 6).

    Fig. 5. [3H]Adenine binding (10 nM) to membrane preparations (100 µg of protein) of noninfected (Sf21) and infected (Sf21 pVI-mAde; novel mouse receptor for adenine) Sf21 cells as well as to glass-fiber filters without protein (samples without protein). Nonspecific binding was determined in the presence of 100 µM unlabeled adenine. Data are means ± S.E. from three (Sf21 pVI-mAde), four (samples without protein) or five (Sf21) independent experiments performed in triplicate; ns = not significant, *** indicates significant difference (p < 0.001; t test).

    Fig. 6. Saturation curve of [3H]adenine binding to membranes prepared from Sf21 insect cells expressing the novel (mouse) adenine receptor. A KD value of 113 ± 17 nM and a Bmax value of 1.98 ± 0.39 pmol/mg protein was determined (n = 3). Experiments were performed as described under Materials and Methods. The result shown represents the mean ± S.E. of three independent experiments, each performed in duplicate.

    Selected adenine derivatives, as well as compounds known to activate (NECA) or block (caffeine) adenosine receptors or to activate P2 receptor subtypes (ADP, ATP), were investigated in competition assays at the novel mouse adenine receptor expressed in Sf21 insect cell membranes. Affinity data were compared with those for the adenine receptor expressed in NG108-15 mouse neuroblastoma x rat glioma cell membranes and those expressed in rat brain cortical membrane preparations (Fig. 7 and Table 1). Unsubstituted adenine showed the highest affinity in all three test systems exhibiting Ki values in the nanomolar range (IC50 = 68.5 nM for the mouse receptor in Sf21 cells, 54.9 nM in NG108-15 cells, Ki = 29.9 nM in rat brain cortex). The rank order of potency for the mouse receptor expressed in Sf21 cells was adenine > 2-fluoroadenine > 7-methyladenine > 1-methyladenine >> N6-benzyladenine  N6-dimethyladenine. The same rank orders of potency were observed for NG108-15 cell membranes (Fig. 7B) and rat brain cortical membranes (Table 1). All of the investigated P1 (adenosine) and P2 receptor ligands showed no or only moderate affinity for adenine binding sites in all three membrane preparations (Table 1).

    Fig. 7. Competition curves for adenine, 2-fluoroadenine, and 7-methyladenine versus 10 nM [3H]adenine obtained with a membrane preparation of Sf21 insect cells recombinantly expressing the novel (mouse) adenine receptor (A) or membranes from NG108-15 cells, natively expressing the novel (mouse) adenine receptor (B). The IC50 values were calculated by nonlinear regression function for one-site competition (IC50 values: Sf21, adenine = 68.5 ± 9.8 nM (n = 5); 2-fluoroadenine = 1.47 ± 0.14 nM (n = 2), and 7-methyladenine = 5.76 ± 1.02 nM (n = 2); NG: adenine = 0.154 ± 0.056 nM (n = 2), 2-fluoroadenine = 0.304 ± 0.097 nM (n = 3), and 7-methyladenine = 1.45 ± 0.44 nM (n = 2)). Data are means ± S.E. from two to five independent experiments performed in duplicate as indicated above.

    TABLE 1 Comparison of affinities of adenine and selected compounds for the novel (mouse) adenine receptor and the native rat adenine receptor determined in radioligand binding studies at membrane preparations (NECA, 5'-(N-ethylcarboxamido)adenosine)

    Results are from three independent experiments performed in triplicate, unless otherwise noted. Values in parentheses are percentage inhibition at 100 µM; all other values are IC50 or Ki values.

    An orphan rat GPCR has previously been shown to operate as a receptor for the nucleobase adenine (Bender et al., 2002) indicating that in addition to P1 receptors for adenosine (Fredholm et al., 2001; Yan et al., 2003) and P2Y receptors for extracellular nucleotides (Brunschweiger and Müller, 2006; von Kügelgen, 2006), a third group of GPCRs exists for these chemically related compounds (tentatively termed P0 receptors; Brunschweiger and Müller, 2006). Human cells also seem to express receptors for adenine (Gorzalka et al., 2005). The human sequence encoding an adenine receptor, however, has yet to be identified. The present study now demonstrates that a novel sequence encodes for an additional GPCR for adenine. This receptor shares only 76% identical amino acid residues with the rat receptor, suggesting that these proteins are not species homologs but distinct members of a family of GPCRs for adenine. This view is underlined by the fact that the predicted transmembrane regions 3 of the novel receptor and the rat adenine receptor differ markedly (see TM3 in Fig. 1), whereas species homologs are known to contain conserved transmembrane regions. Moreover, the tissue distribution of the novel receptor and the rat adenine receptor differ; the latter is predominantly expressed in dorsal root ganglia (Bender et al., 2002), whereas we detected mRNA for the novel sequence in the brain and the spleen, suggesting a broader expression. As discussed below, there are also differences in pharmacological properties. A related mouse DNA sequence encodes for the mMrgA10 orphan receptor (Bender et al., 2002; see Choi and Lahn, 2003; Zylka et al., 2003). It is not known yet whether mMrgA10 also operates as a receptor for adenine. Neither for the rat adenine receptor (Bender et al., 2002) nor for the new sequence could a closely related human ortholog be identified in the genome database. A search for conserved regions of both sequences, however, may facilitate the identification of the human sequence.

    For functional experiments, human 1321N1 astrocytoma cells were used. Nontransfected 1321N1 astrocytoma cells do not respond to adenine with a decrease in cAMP formation (present study). Therefore, they are suitable for a pharmacological characterization of a recombinant inhibitory receptor for adenine (despite the fact that the cells possess binding sites for adenine; Gorzalka et al., 2005). In a previous study by Bender et al. (2002), a clone of Chinese hamster ovary (CHO) cells without endogenous adenine receptors had been used for recombinant expression of the rat adenine receptor. However, the clones of CHO cells studied in our laboratory (CHO Flp-In cells and CHO K1 cells) have a high density of adenine binding sites (S. Gorzalka and C. E. Müller, unpublished observations) and possess endogenous receptors for adenine modulating intracellular cAMP levels (K. Hoffmann and I. von Kügelgen, unpublished results).

    The novel recombinant mouse adenine receptor shows pharmacological properties similar but not identical to those of the adenine receptor endogenously expressed in the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 [which expresses the same mouse sequence (Gorzalka et al., 2005) and, possibly, an additional subtype). Both the native and the recombinant receptor couple to inhibition of adenylate cyclase activity via pertussis toxin-sensitive G proteins. Adenine acted as an agonist at the native (Gorzalka et al., 2005) and the recombinant receptor (present study) with half-maximal concentrations of 21 nM (NG108-15 cell membranes) and 8 nM (recombinant receptors), respectively. Adenine has been shown to activate the rat adenine receptor with a similar potency (3 nM; Bender et al., 2002). In addition to adenine, the adenine derivative 2-fluoroadenine was active at the native receptor in NG108-15 cells (Gorzalka et al., 2005) and at the recombinant receptor (Fig. 2B).

    In contrast to the results obtained at the recombinant receptor expressed in astrocytoma cells (present study) and at NG108-15 membranes (Gorzalka et al., 2005), a higher IC50 value for the adenine-induced inhibition of forskolin-stimulated cAMP production had been determined in intact NG108-15 cells (IC50 2.54 µM; Gorzalka et al., 2005). The difference is likely to be caused by an uptake mechanism for adenine in intact neuroblastoma NG108-15 cells, by which adenine is removed from the extracellular surface. In fact, in a recent study, only rat cortical neurons but not astrocytes were found to take up [3H]adenine, indicating that astrocytes do not express specific transport systems for adenine (Nagai et al., 2006).

    An action of adenine via adenosine receptors was excluded by the lack of any interaction with the adenosine A1 receptor antagonist DPCPX, which completely blocks adenosine A1 and A2B receptors and at least partly blocks A2A and A3 receptors at the concentration used (1 µM; Lohse et al., 1987; Müller, 1996). Moreover, adenine does not act on P2 receptors, as shown by the lack of interaction with suramin (acting as an antagonist at P2Y1, P2Y2, P2Y6, P2Y11, P2Y12, and P2Y13 receptors; Table 3 of von Kügelgen, 2006). Hence, these results confirm the idea of a group of GPCRs for the nucleobase adenine distinct from the known adenosine and P2 receptors.

    In addition to functional studies, we were interested in obtaining a suitable system for radioligand binding assays. NG108-15 cell membranes natively expressing the mouse adenine receptor had previously been shown to exhibit specific binding of [3H]adenine (Gorzalka et al., 2005). However, this cell line may express more than one adenine receptor subtype or other specific binding sites for [3H]adenine. Although we tested a series of cell lines, including CHO, human embryonic kidney, and 1321N1 astrocytoma cells, typically used for heterologous recombinant expression of GPCRs, we could not identify any mammalian cell line that did not natively express a specific binding site for [3H]adenine. Therefore, we turned our attention to nonmammalian cells and found that Sf21 insect cell membranes showed no specific binding of [3H]adenine. After infection with recombinant baculoviruses containing the sequence for the novel adenine receptor, specific binding of [3H]adenine was detected in these Sf21 insect cell membranes (Fig. 5). In homologous competition assays of adenine versus [3H]adenine, concentration-dependent inhibition of [3H]adenine binding was observed (Fig. 7). Because Sf21 cell membranes express no high-affinity adenine binding proteins, they represent an ideal expression system for adenine receptor proteins for binding assays. It has been suggested that [3H]adenine may bind to glass fiber filters in a specific manner (Ye et al., 2006; but see Schiedel et al., 2007, for bacterial proteins binding adenine). However, we observed no specific binding of [3H]adenine to glass fiber filters, only nonspecific binding in the absence of Sf21 membranes, which was somewhat lower than the nonspecific binding observed in the additional presence of Sf21 membranes (Fig. 5). The specific binding of [3H]adenine to Sf21 insect cell membranes expressing the novel adenine receptor was clearly protein-dependent and increased with higher protein concentrations (data not shown).

    In saturation experiments at Sf21 cells expressing the novel adenine receptor, a KD value of 113 nM was determined for [3H]adenine. Thus, the KD value was 4-fold higher than that determined for the rat adenine receptor in brain cortical membranes (27.2 nM; Gorzalka et al., 2005) but still in the same concentration range. A comparison of affinities of adenine and selected derivatives for the novel receptor showed that affinities were similar to but not identical with those determined at the native receptors in NG108-15 cells and the rat brain cortex (Table 1). The rank order of potency was: adenine > 2-fluoroadenine > 7-methyladenine > 1-methyladenine >> N6-benzyladenine = N6-dimethyladenine. Slight differences could be observed. For example, 2-fluoroadenine and 7-methyladenine showed a 4- to 5-fold lower affinity for the recombinant receptor than for the native receptor (Fig. 7 and Table 1). These differences are in agreement with the expression of different receptor subtypes in NG108-15 cells. In addition, different membrane environments may contribute to the differences by changing receptor conformation (Chachisvilis et al., 2006). In radioligand binding studies, the adenosine receptor agonist NECA and the antagonist caffeine showed no affinity for the mouse adenine receptor (Table 1). The same was observed for the P2 receptor agonists ATP and ADP. IC50 values determined in radioligand binding studies were generally severalfold higher than IC50 values obtained in functional cAMP assays because of the amplification of the signal mediated by a GPCR.

    In conclusion, we have identified, cloned, and pharmacologically characterized a novel receptor for the purine nucleobase adenine from mouse brain. The low sequence identity with the previously described rat adenine receptor, particularly in the predicted transmembrane region 3, as well as differences in pharmacology, suggests that the new receptor is not a species homolog but rather a distinct receptor subtype. Because human cells also possess specific binding sites for adenine, the existence of a receptor for adenine in human tissues seems very likely. However, a human sequence encoding for an adenine receptor has yet to be identified. The rat adenine receptor discovered by Bender et al. (2002) and the novel adenine receptor described in the present study seem to be members of a new, possibly larger family of receptors for purine nucleobases. The comparison of the sequences may facilitate the identification of the human sequence.

    Acknowledgements

    We thank Petra Spitzlei for skilful technical assistance and Prof. Dr. Jörg Höhfeld for Sf21 insect cells.

    ABBREVIATIONS: GPCR, G protein-coupled receptor; RT-PCR, reverse transcriptase-polymerase chain reaction; ANOVA, analysis of variance; CHO, Chinese hamster ovary; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; NECA, 5'-(N-ethylcarboxamido)adenosine; NG108-15 cells, mouse neuroblastoma x rat glioma hybrid cell line; PTX, pertussis toxin; Sf21 cells, Spodoptera frugiperda 21 cells.

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Wengert M, Ad?o-Novaes J, Assaife-Lopes N, Le?o-Ferreira LR, and Caruso-Neves C (2007) Adenine-induced inhibition of Na+-ATPase activity: evidence for involvement of the Gi protein-coupled receptor in the cAMP signalling pathway. Arch Biochem Biophys 467: 261-267.[CrossRef]

Yan L, Burbiel JC, Maass A, and Müller CE (2003) Adenosine receptor agonists: from basic medicinal chemistry to clinical development. Expert Opin Emerg Drugs 8: 537-576.[CrossRef][Medline]

Ye K, Mulder-Krieger T, Beukers MW, and IJzerman AP (2006) [3H]Adenine's high filter binding precludes its use as a radioligand for adenine receptors. Purinergic Signalling 2: 71.

Yoshimi Y, Watanabe S, Shinomiya T, Makino A, Toyoda M, and Ikekita M (2003) Nucleobase adenine as a trophic factor acting on Purkinje cells. Brain Res 991: 113-122.[CrossRef][Medline]

Zylka MJ, Dong X, Southwell AL, and Anderson DJ (2003) Atypical expansion in mice of the sensory neuron-specific Mrg G protein-coupled receptor family. Proc Natl Acad Sci U S A 100: 10043-10048.[Abstract/Free Full Text]


作者单位:Department of Pharmacology and Toxicology (I.v.K., K.H.) and Pharmaceutical Institute, Pharmaceutical Chemistry I (A.C.S., B.B.A.A., A.A., C.E.M.), University of Bonn, Bonn, Germany

日期:2009年8月25日 - 来自[2008年第70卷第2期]栏目

充血性心力衰竭患者血浆内皮素和降钙素基因相关肽水平的临床观察

充血性心力衰竭患者血浆内皮素和降钙素基因相关肽水平的临床观察

标记免疫分析与临床 2000年第2期第7卷 简要研究报告

作者:周润锁 徐海峰

单位:第四军医大学唐都医院核医学科 西安 710038

  近年来发现内皮素(ET)和降钙素基因相关肽(CGRP)分别是体内最强的缩血管和舒血管生物活性多肽[1,2],在体内它们广泛分布于中枢、周围神经系统和心血管系统及某些器官组织;目前在充血性心力衰竭(CHF)方面的大多数研究仅局限在ET和CGRP单方面的研究,CHF患者体内ET和CGRP平衡状态研究报道不多,为此本文同期检测了40例CHF患者血浆ET和CGRP水平,以及ET/CGRP比值,报道如下。

  资料和方法

  1 研究对象

  1.1 对照组 随机选择40名通过体检及辅助检查排除器质性疾病的健康人,其中男18名,女22名,年龄45.8±8.2(25~65)岁。

  1.2 CHF组 随机选择按NYHA心功能分级为Ⅲ~Ⅳ级的心内科住院的CHF患者40例,其中风心病12例、高心病10例、冠心病10例、扩张型心肌病5例、肺源性心脏病3例;男24例、女16例,年龄55.6±9.2(28~72)岁。

  2 方法

  CHF组和对照组均在清晨采血,分别将外周静脉血2.5mL缓慢注入有10%EDTA-Na230μL和抑肽酶400IU的塑料管中,混匀后全血在-4℃离心(3000r/min)10分钟,分离血浆。标本置-30℃冰箱中保存备测。ET和CGRP放免试剂盒均由北京东亚免疫技术研究所提供。测定仪器为西安262厂XH-6010型放射免疫测定仪,数据由PC机同步自动打印,同时测定ET、CGRP浓度,其单位均以pg/mL表示,并求出ET/CGRP比值。

  3 统计学方法

  采用t检验,数据以

  结果和讨论

  CHF组40例患者血浆ET、CGRP水平分别为70.45±17.23pg/mL、64.37±13.72pg/mL,与对照组(ET、CGRP)水平分别为57.92±14.11pg/mL、53.79±16.8pg/mL比较差异显著(P<0.05)。

  CHF组、对照组的ET/CGRP比值见表。

表 CHF组、对照组的ET/CGRP比值

  CHF组 对照组 P
ET/CGRP 1.51±0.17 1.14±0.29 <0.01

  ET是一种由内皮细胞分泌的由21个氨基酸组成的血管活性物质,具有强烈的动、静脉血管收缩作用,参与血管张力的局部调节,在具有组织缺血、缺氧和血管内皮损伤的疾病中起着重要作用,对心肺功能有抑制作用[3,4]。本文研究结果显示,CHF患者血浆内皮素水平明显升高。这与有关文献报道一致[3]

  CGRP是从人和哺乳动物体内发现的一种神经肽,具有强大的扩张周围血管作用,对缺血心肌细胞有保护作用,对心脏有正性变力和变时效应[5,6]。这说明CGRP增高对CHF患者有血液动力学效应;且心脏功能也能得到改善。近些年研究显示,CHF患者血浆CGRP水平较正常人显著升高[5]。本文的研究结果与之相符,但其增高的详细机理目前还不大清楚,可能与CHF时肾脏功能不全引起CGRP排泄减少有关。

  大多数研究表明,ET和CGRP在体内心血管领域产生着互相拮抗的效应,所以它们在体内的平衡维持显得非常重要。本文的研究表明,CHF患者血浆ET/CGRP比值较正常人明显增高,提示血浆ET和CGRP水平在CHF的病理生 理过程中有一种相互制约、相互影响和动态平衡的关系,CHF患者血浆ET/CGRP这种平衡关系被破坏,这对CHF的发生、发展和预后起一定的作用;当然ET/CGRP的确切关系,尚有待进一步探讨研究。

  依照上述的ET/CGRP的平衡关系,提醒人们在预防和治疗CHF时尽量要恢复ET/CGRP的这种平衡:一方面可采取抑制ET的合成和释放或者阻断其与受体结合的措施;另一方面可促进CGRP的释放,并应用CGRP制剂以提高其体内浓度以拮抗ET,纠正CHF。作者遐想:随着CGRP药理机理和临床的深入研究以及拮杭ET效应药物的开发应用,必然对CHF的防治产生令人鼓舞的影响。

参考文献

  1,Yanagisawa M,Kurihara H, Kimura S et al. A novel potent vasoconstrictor peptide by vascular endothelial cell. Nature,1998;334:411

  2,Rosefeld M G, Mermod J J,Amara S G et al. Novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature,1983;304:129

  3,Haynes W G, Welb D J.The endothelin family of peptides: local hormones with diverse roles in health and disease? Clin Sci, 1993;84:485

  4,唐朝柜,李兆萍,樊贵等.内皮素对离体大鼠心肌细胞的影响.生理学报,1992;44:15

  5,Shekhar Y C,Anand I S, Sarma R et al. Effects of prolonged infusion of human alpha calcitonin gene-related peptide on hemodynamics, renal blood flow and hormone levels in congestive heart failure, Am J Cardiol, 1991;67:732

  6,Crossman D, McEwan T, Macdermot J et al. Human calcitonin gene-related peptide activates adenylate cyclase and releases prostacyclin from human umbilical vein endthelial cells.Br J Pharmacol, 1987;92:695

(1999-04-09收稿)


日期:2009年2月21日 - 来自[检验医学]栏目
循环ads

肺栓塞:诊断和治疗策略

Pulmonary Embolism: Latest Diagnostic & Management Strategies
Michael D. McGoon, MD
The VTE Continuum and Goals of Intervention
Proximal DVT
Risk of VTE
Distal (calf) DVT
Pulmonary Embolism
Death
Recurrence
Post-thrombotic syndrome
CTEPH
25%
50%
3.8% after 2 years Pengo, NEJM 2004
7% in 6 months
17% in 1 year; 28% in 5 years
See also Kearon Cl: Natural History of Venous Thromboembolism; Circulation 2003;107:I-22 - I-30
Venous Thromboembolism: PE - The Scope of the Problem (2004)
30% mortality if untreated (vs 2-8%)
10 -15% of deaths in acute care hospitals
100 - 200,000 deaths yearly (US)
> 50% are undiagnosed
<30% of fatal PE cases have prior DVT suspicion
Venous Thromboembolism: Risk Factors (Relative Risk)
Obesity, smoking, hypertension (2-3)
Oral contraceptives, estrogen modulators (5)
Cancer, acute illness in hospital (5-10)
Thrombophilia
Antithrombin III deficiency (5-50)
Homozygous factor V Leiden mutation (80)
Major trauma or surgery (200)

Venous Thromboembolism: Demographics of Risk (2004)
Brest District, France
Olmsted County, MN
US Hospital Survey (2004)
Afr Am, Whites
Population
VTE Incidence
1.22 – 1.83/1000
DVT 0.48 – 1.24
PE 0.34 – 0.69
US Hospital Survey (2004) AJC 2004;93:1194-1197
Asians, PI’s
Hong Kong 2004
0.23/1000
DVT 0.17 - 0.20
PE 0.039 – 0.05
Lower prevalence of Factor V Leiden and other thrombophilias in Asians
Deep Vein Thrombosis Evaluation of Suspected DVT (2004)
Clinical Probability
Low
High
D-dimer
No DVT
DVT
Duplex
 US
Duplex
 US
D-dimer
No DVT
Serial
 US
Based on Circulation 2004, 109, I-9
pos
neg
pos
neg
neg
pos
neg
pos
neg
pos
Pulmonary Embolism Diagnosis - Ventilation Perfusion Scintigraphy
Normal scan essentially rules out PE
Sensitivity of “high-probability” scan is 41%
Everything in between is indeterminate
By 2001, less used than CT angio
Identifies PE in 14 - 44% of indeterminate VQ scans
Accurate to the segmental level
Can diagnose other intrathoracic diseases; can be combined with leg CT venography
Excellent outcome without anticoagulation after negative CT angio
Swenson et al, Mayo Clinic Proceedings 77:130-138, 2002
Faster, cheaper, less invasive, more widely available than pulmonary angio
Pulmonary Embolism Diagnosis - CT Angiography
Pulmonary Embolism Diagnosis - MR Angiography
No need for IV iodinated contrast material
Pulmonary vascular imaging may be combined with MR venography of the legs and pelvis (DVT)
Lower spatial resolution, longer acquisition time, more costly, less available than CT
Pulmonary Embolism Diagnosis - Pulmonary Angiography
Gold standard for the diagnosis of PE
Problems
Mortality <1%, morbidity 2-5%.
Disagreements in interpretation of results
False-positive results from non-embolic disorders, e.g., mediastinal disorder
Incomplete or nondiagnostic result in 4%.       

Establish Pretest Probability; Image
CT or VQ normal
CT or VQ equivocal;
Prob low or mod
Duplex US
PE
No PE
CT or VQ equivocal; Prob high
CT or VQ high; prob...
Duplex US
PE
Pulm Angio
No PE
pos
neg
pos
neg
pos
neg
low
Mod or
high
Pulmonary Embolism Diagnosis (2004) - Algorithm
Treatment of VTE Mechanical Prophylaxis
Graduated compression stockings reduce DVT 72% after non-orthopedic surgery
Intermittent pneumatic compression devices prevent venous stasis and stimulate endogenous fibrinolysis
Treatment of VTE Traditional Anticoagulants
Unfractionated Heparin
Indirect thrombin inhibitor (requires antithrombin)
Accelerates inhibition of factor Xa and thrombin >1000X
Coumarins
Reduce Vitamin K dependent thrombin substrates, e.g. prothrombin
“Direct thrombin inhibitor”
Treatment of VTE Low Molecular Weight Heparins
Generally as effective as heparin
Less protein and endothelial binding
More predictable, longer half-life
Less bleeding
One/day dose without lab monitoring
Types
Enoxaparin (Lovenox)     Dalteparin (Fragmin)
Ardeparin (Normiflo)  Nadroparin (Fraxiparin)
Tinzaparin (Innohep)  Reviparin (Clivarine)
Treatment of VTE Factor X Inhibitors
Fondaparinux (Arixtra)
Synthetic heparin pentasaccharide
Low risk of bleeding and HIT
=LMWH in DVT Buller: AIM 140:867;2004
= UFH in PE Matisse Inv: NEJM 349:1695;2003
FDA approved: prevention of DVT after hip fx (more effective than LMWH), knee/hip replacement

Treatment of VTE Direct Thrombin Inhibitors
Hirudin (lepirudin), Argatroban, Bivalirudin
Independent of antithrombin
For use in patients with heparin-induced thrombocytopenia
Ximelagatran (Exanta)
Oral, with rapid onset of action
= LMWH + warf after DVT JACC 2000;36:1336 (THRIVE)
Reduces recurrent VTE when used for 18 months after 6 months warfarin NEJM 349;1713,2003 (THRIVE III)
Risk of liver toxicity (4-10%)
Treatment of VTE Pharmacologic Prophylaxis
Post-operative patients
Fondaparinux (ortho) > LMWH > low dose UFH
Medical patients (acutely ill or long hospitalization)
LMWH – reduces risk for DVT or PE at 14, 21 and 110 days
   Samama et al: NEJM 1999;341:793-800 (MEDENOX Study)
   Leizorovicz et al: Circulation 2004;110:874-9 (PREVENT Study)
Treatment of VTE 2004
Anticoagulation contraindicated?
IVC Filter
Hx of HIT?
DTI (lepirudin)
Factor X Inhib (Fondaparinux)
Hospitalize for DVT?
Extensive iliofemoral DVT
Risk of bleeding
Admit
Heparin/LMWH or DTI, and warfarin
Long-term anticoagulation
Suspect PE?
High Risk PE?
Thrombolysis or Embolectomy
yes
no
yes
no
yes
no
yes
End
Recent Useful Reviews
Circulation, vol 109, 12 (supplement); March 30, 2004 Diagnosis of VTE
Circulation, vol 110, 9 (supplement); August 31, 2004 Treatment of VTE
CHEST, vol 126, 3 (supplement); September 2004 Antithrombotic and Thrombolytic Therapy Evidence-based Guidelines
Treatment of VTE IVC Filter
Indications
When anticoagulation cannot be used or doesn’t work
With surgical embolectomy or pulmonary endarterectomy
14% of DVT’s receive filter in US
Goldhaber et al, AJC 2004;93:259
5% Risk of PE; RR 2.6 rehospitalization for DVT recurrence
Consider retrievable filter for temporary need

Treatment of VTE Thrombolytic Treatment
Indications
Massive PE/hemodynamic instability
Free-floating RV thrombus or PFO
Limb-threatening DVT
More rapid thrombolysis than heparin alone
Reduces need to escalate therapy
No proven mortality benefit except in highest risk
1-3% risk of intracranial bleed


Treatment of VTE Pulmonary Embolectomy
Indications
Massive PE/Hemodynamic instability
Failure or contraindication for thrombolysis
Experienced cardiac surgical team available
      
Venous Thromboembolism: Demographics of Risk
450 - 600,000 episodes/year in US
Stein et al: Regional Differences in Rates of Diagnosis and Mortality of Pulmonary Thromboembolism; AJC 2004;93:1194-1197
Pulmonary Embolism Presentation
Asymptomatic (5%)
About 50% of DVT patients with PE - asymptomatic
70-90% of post-op patients with PE - asymptomatic
No symptoms of VTE in 1/2 of pts with chronic thromboembolic pulmonary hypertension
Syndrome of “uncomplicated” PE (22%)
Often atypical, s

日期:2006年2月21日 - 来自[心血管相关]栏目
共 38 页,当前第 1 页 9 1 2 3 4 5 6 7 8 9 10 11 :

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