腺病毒载体介入的人β2—肾上腺素能受体基因导入心肌细胞的研究

目的：观察重组缺陷型腺病毒即腺病毒载体介导的人β2肾上腺素受体（β2－AR）基因导入大鼠心肌细胞后β2－AR的表达及其功能,方法：构建含人β2－AR基因的腺病毒载体（Adβ2－AR）,转梁大鼠的心肌细胞,检测心肌细胞对人β2－AR的表达及环磷酸腺苷（cAMP)的含量。结果：Northern印迹杂交显示Adβ2－AR转染的心肌表达人β2－ARmRNA;Western免疫印迹杂交分析表明转染的心肌细胞有人β2－AR;放射性配基检测显示转基因心肌细胞的β－AR密度[（234±6．4）fmol/mg蛋白]是未转染的心肌细胞密度[(76±2．8）fmol/mg蛋白]的3倍（P＜0．01）;经10μmol/L异丙肾上腺素作用,转基因的心肌细胞的cAMP量[（124±6．8）pmol/10^6细胞]明显高于对照组[（58．4±4．6）pmol/10^6细胞]（P＜0．01）。结论人β2－AR基因经腺病毒载体导入心肌细胞后能有效地表达并有激活肾上腺素信号的作用。     

腺病毒相关病毒载体介导人β2肾上腺素能受体、增强型绿色荧光蛋白基因导入心肌细胞的表达

目的探讨充血性心力衰竭(心衰)及其他心脏疾病的基因治疗途径.方法构建携带人β2肾上腺素能受体(β2-AR)和增强型绿色荧光蛋白(EGFP)双基因的腺病毒相关病毒(adeno-associatedvirus,AAV)载体,感染大鼠的心肌细胞,检测心肌细胞β2-AR、EGFP的表达及环腺苷酸(cAMP)的含量.结果Northern印迹杂交显示感染的心肌细胞表达人β2-ARmRNA;Western免疫印迹杂交分析表明感染的心肌细胞有人β2-AR;在荧光显微镜下可观察到心肌细胞表达EGFP;放射性配基检测显示感染的心肌细胞β-AR密度[(204.0±6.4)fmol/mg蛋白]明显高于未感染的心肌细胞[(76.0±2.8)fmol/mg蛋白,P＜0.01];经10μmol/L异丙肾上腺素作用,感染的心肌细胞的cAMP含量[(116.2±5.8)pmol/106cells]明显高于对照组[(58.4±4.6)pmol/106cells,P＜0.01].结论人β2-AR、EGFP基因经AAV载体导入心肌细胞后能有效地表达,且表达的人β2-AR具有激活肾上腺素信号的作用.     

基因转染β肾上腺素能受体激酶抑制剂对支气管哮喘小鼠β2肾上腺素能受体和环腺苷酸的影响

目的　研究基因转染β肾上腺素能受体激酶抑制剂 (βARKct)对应用 β2 肾上腺素能受体 (β2 AR)激动剂后支气管哮喘 (简称哮喘 )小鼠肺部 β2 AR和环腺苷酸 (cAMP)的作用 ,并探讨不同基因转染途径对两者的影响。方法　 (1)以 10 %卵清蛋白致敏并激发BALB/c小鼠建立哮喘模型 ,并设正常小鼠为正常对照组 (A组 )。将哮喘小鼠分为 7组 ,每组 5只 ,分别给予生理盐水 (哮喘对照组 ,B组 )肌肉注射、沙丁胺醇肌肉注射 (C、D、E、F、G组 ) ,以建立长期应用 β2 AR激动剂的哮喘动物模型。(2 )构建带有 βARKct基因表达质粒 ,通过小鼠尾静脉注射和气管内滴入的转染途径进行基因转染。D组给予静脉注射转染 βARKct、E组给予静脉注射转染空载质粒、F组给予气管滴注转染 βARKct、G组给予气管滴注转染空载质粒。(3)用Western blot检测基因的表达。放射免疫法检测小鼠肺部的 β2 AR和cAMP的水平。结果　 (1)基因转染 βARKct在小鼠肺部有表达 ,且F组的基因表达量高于D组。(2 )A组 β2 AR和cAMP分别为 (10 9± 11)fmol/mg、(3 5 0± 0 5 0 )pmol/L ;B组 β2 AR和cAMP分别为 (81± 3)fmol/mg、(1 5 0± 0 2 0 )pmol/L ,与A组比较差异有显著性 (P均 <0 0 5 ) ;C组 β2 AR和cAMP分别为(6 3± 3)fmol/mg、(0 90± 0 10     

腺病毒载体介导的人β2肾上腺素能受体基因在心肌细胞特异性表达的研究

目的观察构建的重组腺病毒载体介导的人β2肾上腺素能受体(β2-AR)基因能否在心肌细胞中特异性表达.方法构建含心肌肌凝蛋白轻链(mlc-2v)启动子携带β2-AR基因的重组腺病毒载体(Admlcβ2-AR),用重组病毒感染大鼠心肌细胞、3T3细胞、HFCL细胞及HeLa细胞,检测感染的细胞中人β2-AR基因的表达.结果RT-PCR显示只有感染的心肌细胞表达人β2-ARrRNA;Westem免疫印迹杂交分析表明感染的心肌细胞有人β2-AR表达;放射性配基检测显示感染的心肌细胞β-AR密度(153±5.2)fmol/mg蛋白,明显高于未感染的心肌细胞(76±2.8)fmol/mg蛋白,P＜0.01).结论构建的重组腺病毒载体可有效地将β2-AR基因导入心肌细胞并使其特异性表达.     

选择性β1受体阻滞剂对高血压患者β2受体的调节作用

目的 :研究选择性 β1 受体阻滞剂对高血压患者心肌 β2受体密度与功能的影响。方法 :采用 3H-双氢阿普洛尔 (3H- DHA)放射性配基结合分析法 ,检测 2 0例连续服用选择性β1 受体阻滞剂治疗 4个月以上和 2 0例 4个月内未服用β受体阻滞剂治疗的高血压男性患者外周血淋巴细胞 β2 受体密度 ;采用羟甲叔丁肾上腺素 (选择性β2 受体激动剂 ,静脉注射 )药物试验 ,测定上述两组患者心脏 β2 受体的反应性。结果 :外周血淋巴细胞β2 受体最大结合容量 (Bmax)在服用选择性 β1 受体阻滞剂治疗组为 5 2 8± 10 4fmol/10 7cell,与非 β受体阻滞剂治疗组比较无明显差异 (5 71± 98fmol/10 7cell,P>0 .0 5 ) ;羟甲叔丁肾上腺素静脉注射后 ,测定增加心率 30次/分的变时剂量 (CD30 )在选择性 β1 受体阻滞剂治疗组为 1.8± 0 .3μg/kg,与非 β受体阻滞剂治疗组比较显著降低 (2 .7±0 .2μg/kg,P<0 .0 0 1)。两组患者注药后血压无明显变化。结论 :β1 受体阻滞剂不改变高血压患者外周血淋巴细胞 β2 受体密度 ,但可上调β2 受体的敏感性。这一调节特性可能是临床β受体阻滞剂停药综合征的细胞生物学机制     

老年人膀胱逼尿肌β受体反应性环腺苷酸水平变化及其调节机制

目的　探讨年龄对人膀胱逼尿肌肾上腺素能 β受体反应性环腺苷酸 (cAMP)水平的影响及受体后信号传递途径调节因素的作用。　方法　用放射免疫法检测在异丙肾上腺素、BRL 37344和forskolin作用下 ,老年组 (15例 )和青年组 (7例 )逼尿肌组织中cAMP含量以及百日咳毒素 (PTX)、霍乱毒素 (CTX)、vinpocetine和KT 5 72 0对其影响。　结果　在异丙肾上腺素、BRL 37344和forskolin存在下老年组逼尿肌组织cAMP含量分别为 (15 0± 1 6 )、(12 2± 1 8)pmol mg蛋白和 (5 4 5± 4 0 )pmol mg蛋白 ,青年组显著下降 (P <0 0 1) ;CTX对老年组cAMP水平无显著影响 ,PTX与KT 5 72 0显著提高了 β受体激动剂存在下的cAMP含量 ,对forskolin的作用无明显影响 ,vinpocetine能显著提高 β受体激动剂和forskolin作用后的cAMP水平。　结论　老年人膀胱逼尿肌的 β受体反应性cAMP水平显著下降 ,腺苷酸环化酶活性的下降和抑制性G蛋白Gi的活性提高可能是其生化基础 ;Ⅰ型磷酸二酯酶和蛋白激酶A抑制剂能改善老年人逼尿肌的 β受体反应性 ,可以用于治疗老年人膀胱储尿功能障碍。     

贵州汉族人群支气管哮喘患者β2-肾上腺素能受体基因多态性与cAMP的关系

目的探讨支气管哮喘人群β2-肾上腺素能受体(β2-AR)16、27位点基因多态性与环磷酸腺苷(cAMP)的相关性.方法采用等位基因特异性聚合酶链反应方法,对44例支气管哮喘患者进行β2-AR基因多态性分析,并测定血cAMP水平.结果血cAMP在各基因型间的水平,16位点精氨酸/精氨酸(18.03±4.37)pmol/ml,甘氨酸/精氨酸(18.54±14.22)pmoL/ml,甘氨酸/甘氨酸(13.00±4.07)pmol/ml,三者比较差异有显著性(P《0.01);27位点谷氨酰胺/谷氨酰胺(15.99±4.89)pmol/ml,谷氨酸/谷氨酰胺(16.14±4.34)pmol/ml,谷氨酸/谷氨酸(13.69±7.88)pmol/ml,三者比较差异无显著性(P》0.05).结论β2-AR基因16位点多态性与血cAMP相关联.     

重组腺病毒介导的人一氧化氮合酶基因转染对大鼠颈总动脉球囊损伤后新生内膜的影响

目的 :研究重组腺病毒介导的人内皮型一氧化氮合酶基因 (eNOS)表达生成的一氧化氮合酶 (NOS)对球囊损伤后大鼠颈总动脉新生内膜的抑制作用。方法 :在 2 93细胞内扩增、纯化Ad LacZ和Ad eNOS ,鉴定其是否携带有LacZ和eNOS基因。建立大鼠颈总动脉球囊损伤模型后 ,将磷酸缓冲液 (PBS)、Ad LacZ和Ad eNOS在体内分别转染到损伤血管段 ,以X gal染色、苏木精 伊红染色 ,免疫组化及计算机图像分析处理等方法观察转染动脉节段外源性eNOS蛋白表达及其对新生内膜的影响。结果 :重组腺病毒携带有eNOS基因 ,并且在损伤血管段得到有效表达。转染后PBS组、Ad LacZ组和Ad eNOS组的新生内膜面积分别为 (0 .187± 0 .0 18)、(0 .134± 0 .0 6 1)和 (0 .0 6 3± 0 .0 2 6 )mm 2 ,新生内膜与中膜面积比值 (I/M)分别为 1.5 76± 0 .2 73、1.342± 0 .35 7和 0 .5 6 0± 0 .16 1。与PBS组、Ad LacZ组相比Ad eNOS组无论新内膜面积 ,还是管腔狭窄程度都明显减小。结论 :腺病毒介导的eNOS基因转染能有效抑制球囊损伤后血管内膜的增生 ,可防治血管成形术后再狭窄     

奥氮平与喹硫平对阿尔茨海默病基因双转染N2a细胞分泌淀粉样β蛋白42的影响

目的　观察奥氮平与喹硫平对瑞典APP基因和早老素1基因双转染N2a细胞分泌Aβ4 2的作用。方法　应用MTT方法检测细胞活性,BCA法测定细胞的蛋白质含量,Westernblot检测N2a与双转染N2a细胞APP基因的表达。双转染N2a细胞分别给予奥氮平与喹硫平处理,用酶联免疫吸附法(ELISA)测定双转染N2a细胞分泌到培养液及细胞内的Aβ4 2水平,并与非处理对照组比较。结果　奥氮平处理组细胞外Aβ4 2浓度(4.78±0 .5 4 )nmol/L明显低于对照组(7.6 9±0 .6 2 )nmol/L ,P <0 .0 5 ;喹硫平处理组细胞外Aβ4 2浓度(4.0 9±0 .18)nmol/L明显低于对照组(7.5 0±0 .5 0 )nmol/L ,P <0 .0 5。奥氮平处理组细胞内Aβ4 2浓度(6 1.0 6±3.2 3)pmol/L与对照组(5 8.6 2±3.82 )pmol/L比较,差异无显著性意义(P >0 .0 5 ) ;喹硫平处理组细胞内Aβ4 2浓度(5 9.75±3.0 1)pmol/L与对照组(6 0 .11±2 .0 3)pmol/L比较,差异无显著性意义(P >0 .0 5 )。结论　奥氮平与喹硫平能够减少双转染N2a细胞外Aβ4 2的分泌,推测奥氮平与喹硫平在临床上的应用有可能通过减少神经细胞Aβ4 2的分泌延缓阿尔茨海默病的发展。     

白细胞介素12重组腺病毒气道内应用对哮喘豚鼠治疗作用的研究

目的　探讨激发前气道内应用白细胞介素 12 (IL 12 )重组腺病毒对过敏性气道高反应的调节作用。方法　以C5 7BL/ 6小鼠经鸡卵蛋白 (OVA)免疫建立哮喘模型 ,实验分 6组 ,每组 6只。激发前气管内单次使用IL 12重组腺病毒 (10 8pfu/mouse) ,观察抗原激发后反应的变化。结果　 (1)小鼠气道内应用IL 12重组腺病毒在肺内可有效表达 ,48h血浆及肺泡灌洗液IL 12分别为 (5 40± 6 0 )U/ml和 (470 0± 80 0 )U/ml,对照病毒和PBS组未检出 ,两组比较差异有显著性 (P <0 0 1)。 (2 )在抗原激发阶段使用IL 12重组腺病毒 ,可明显抑制肺内IL 4[(3 5± 2 0 )ng/ml∶85 0± 2 5 0 )ng/ml]和IL 5[(6 5± 4 5 )ng/ml∶(5 4 0± 14 0 )ng/ml];γ干扰素 (IFN γ)的产生增加 [(6 90 0± 32 0 )ng/ml∶(12 5±3 2 )ng/ml];并明显抑制气道高反应性 [(36 0± 30 )cmH2 O∶(810± 5 0 )cmH2 O];抑制外周血 [(0 7±0 1) %∶(9 2± 0 5 ) % ]及肺泡灌洗液 [(3 5± 0 7)∶(2 1 6± 4 7)× 10 4 /ml]中的嗜酸细胞的水平 ;与对照组比较 (t分别 =7 97、7 92、5 1 6、18 9、9 33、47 1,P均 <0 0 1) ;但与总IgE[(6 5± 9) μg/ml∶(6 7± 10 )μg/ml]及抗原特异性IgE[(32± 8)∶(33± 8)U/ml]比较无明显影响 (P均 >0 0 5 )。     

The PDZ-binding motif of the beta2-adrenoceptor is essential for physiologic signaling and trafficking in cardiac myocytes

beta1- and beta2-adrenergic receptors (AR) regulate cardiac myocyte function through distinct signaling pathways. In addition to regulating cardiac rate and contractility, beta1AR and beta2AR may play different roles in the pathogenesis of heart failure. Studies on neonatal cardiac myocytes from beta1AR and beta2AR knockout mice suggest that subtype-specific signaling is determined by subtype-specific membrane targeting and trafficking. Stimulation of beta2ARs has a biphasic effect on contraction rate, with an initial increase followed by a sustained Gi-dependent decrease. Recent studies show that a PDZ domain-binding motif at the carboxyl terminus of human beta2AR interacts with ezrin-binding protein 50/sodium-hydrogen exchanger regulatory factor, a PDZ-domain-containing protein. The human beta2AR carboxyl terminus also binds to N-ethylmaleimide-sensitive factor, which does not contain a PDZ domain. We found that mutation of the three carboxyl-terminal amino acids in the mouse beta2AR (beta2AR-AAA) disrupts recycling of the receptor after agonist-induced internalization in cardiac myocytes. Nevertheless, stimulation of the beta2AR-AAA produced a greater contraction rate increase than that of the wild-type beta2AR. This enhanced stimulation of contraction rate can be attributed in part to the failure of the beta2AR-AAA to couple to Gi. We also observed that coupling of endogenous, wild-type beta2AR to Gi in beta1AR knockout myocytes is inhibited by treatment with a membrane-permeable peptide representing the beta2AR carboxyl terminus. These studies demonstrate that association of the carboxyl terminus of the beta2AR with ezrin-binding protein 50/sodium-hydrogen exchanger regulatory factor, N-ethylmaleimide-sensitive factor, or some related proteins dictates physiologic signaling specificity and trafficking in cardiac myocytes.     

Pulmonary β adrenoceptor density in arrhythmogenic right ventricular cardiomyopathy and idiopathic tachycardia

Objective. In recent in vivo studies using positron emission tomography (PET) our group demonstrated that the myocardial β adrenoceptor (βMAR) density is reduced in arrhythmogenic right ventricular cardiomyopathy (ARVC) and idiopathic right ventricular outflow tract tachycardia (RVO-VT) associated with an increased presynaptic catecholamine washout. It was hypothesised that the reduction of myocardial βAR density is secondary to an increase of local catecholamines in the myocardium resulting from the presynaptic dysfunction since circulating plasma catecholamines were demonstrated to be unchanged in these conditions. To further prove this hypothesis of an organ-limited adrenergic nervous dysfunction of the heart, this study aimed to investigate βAR density in another thoracic organ, the lung. Methods. Pulmonary and myocardial βAR density was measured in 7 ARVC patients, 8 RVO-VT patients and in a group of healthy controls (n = 13) using the non-selective β-blocker [¹¹C]-CGP 12177 and PET. Results. Pulmonary βAR density was similar in controls (12.4 ± 1.7 pmol/g tissue), ARVC (11.6 ± 1.7 pmol/g tissue, p = ns) and RVO-VT (12.8 ± 2.0 pmol/g tissue, p = ns), whereas myocardial βAR density was significantly reduced in ARVC (6.3 ± 1.1 pmol/g tissue, p = 0.006) and RVO-VT (6.8 ± 1.2 pmol/g tissue, p = 0.02) as compared to controls (8.8 ± 1.5 pmol/g tissue). Conclusion. The unchanged pulmonary βAR density in the presence of a previously described significant reduction in myocardial βAR density in the same patient principally supports our pathophysiological hypothesis that the myocardial βAR density may be reduced in ARVC and RVO-VT because of an increase in local synaptic catecholamine levels due to an organ-limited presynaptic adrenergic dysfunction of the heart. Since in the present study only pulmonary βAR density was measured, future functional studies excluding pulmonary βAR desensitisation are required to finally prove the unchanged pulmonary sympathetic innervation in ARVC and RVO-VT. Received: 22 May 2000, Returned for 1. revision: 19 June 2000, 1. Revision received: 5 July 2000, Returned for 2. revision: 2 August 2000, 2. Revision received: 7 August 2000, Accepted: 9 August 2000     

扩张型心肌病患者血清自身抗体、心钠素、一氧化氮含量的变化及意义

目的　探讨扩张型心肌病 (DCM)患者血清抗 β1 肾上腺能受体与 M2 胆碱能受体自身抗体水平、心钠素(ANP)和一氧化氮 (NO)含量的变化及意义。方法　用 EL ISA法检测 62例 DCM患者和 76例健康人血清抗β1 肾上腺能受体与 M2 胆碱能受体自身抗体 ;用放射免疫法和硝酸还原酶法测定 40例 DCM患者和 19例健康人血清ANP、NO水平。结果　 (1) DCM患者血清自身抗体的 P/ N值和阳性率明显高于正常对照组 (P<0 .0 5或 0 .0 1) ;(2 ) DCM患者血清 ANP、NO水平亦高于正常对照组 (P<0 .0 5 ) ;(3 )抗β1 抗体阳性组血清 ANP、NO水平高于阴性组 (P<0 .0 5或 0 .0 1) ;(4)抗 M2 抗体阳性组血清 ANP水平高于阴性组 (P<0 .0 1) ,而 NO水平无显著差异 (P>0 .0 5 )。结论　 (1) DCM患者血清中存在高水平的抗β1 和 M2 受体自身抗体、ANP和 NO;(2 )自身抗体有致心肌细胞肥大的作用 ,可能参与 DCM心室重构 ;(3 )抗 β1 和 M2 受体的自身抗体在心室重构中存在不同的信号通路     

血管紧张素Ⅱ对心肌细胞缝隙连接蛋白Cx43表达的时相性调控

目的研究血管紧张素Ⅱ(AngⅡ)诱导心肌细胞肥大后缝隙连接蛋白Cx43表达的变化规律和机制。方法分离培养大鼠心肌细胞后分为正常对照组、AngⅡ组和缬沙坦组,用Westernblot和免疫荧光法观察不同时间(12、24、48、72h)和不同浓度(1.0×10-9～1.0×10-5mol/L)下心肌细胞Cx43表达的变化,采用免疫荧光法检测3组心肌细胞24和72hCx43的表达。结果AngⅡ组心肌细胞较正常对照组明显肥大,蛋白含量增加。AngⅡ组心肌细胞在24～48hCx43表达上调,72h则明显下调,较正常对照组减少30%,而且在AngⅡ作用下呈浓度依赖性下调,24hAngⅡ组荧光阳性细胞数较正常对照组和缬沙坦组明显上调,72h则较其他组明显下调。缬沙坦可拮抗AngⅡ对Cx43表达的作用。结论AngⅡ诱导心肌细胞肥大过程中Cx43的表达出现一定时相性变化,并和AngⅡ呈明显的量效关系,提示AngⅡ可能通过AT1受体调控Cx43的表达而参与心肌细胞缝隙连接重构。     

重组腺相关病毒介导人血管内皮生长因子165基因在培养大鼠心肌细胞中的表达

目的探讨2型腺相关病毒载体介导的人血管内皮生长因子165基因在离体心肌细胞中的表达及表达产物的生物活性。方法构建携带人血管内皮生长因子的2型腺相关病毒载体(rAAV-2/hVEGF165),感染大鼠原代心肌细胞,采用逆转录聚合酶链反应、免疫印迹法和酶联免疫吸附测定等方法检测心肌细胞人血管内皮生长因子165的表达,应用MTT法检测转染后细胞上清液中血管内皮生长因子165的生物学活性。结果逆转录聚合酶链反应检测结果发现感染的心肌细胞可表达人血管内皮生长因子165 mRNA;免疫印迹法检测结果表明感染的心肌细胞中有人血管内皮生长因子165,酶联免疫吸附测定法检测到血管内皮生长因子蛋白分泌水平为546.5±15.6pg/L,未转染组未检测到血管内皮生长因子蛋白的分泌;还发现感染的心肌细胞上清具有明显的促人内皮细胞增殖作用。结论2型腺相关病毒载体介导的人血管内皮生长因子165可有效转染大鼠心肌细胞,且表达产物具有促内皮细胞增殖作用。     

Functional remodeling in post-myocardial infarcted rats: focus on beta-adrenoceptor subtypes

Cellular electrophysiological remodeling of the infarcted heart may lead to the deterioration of cardiac function and/or to arrhythmias. The present study was designed to characterize the functional expression of the hyperpolarization-activated current (I(f)) and its modulation by beta(1)-, beta(2)- and beta(3)-adrenoceptor (AR) subtypes, in patch-clamped ventricular myocytes isolated from the heart of post-myocardial infarcted (PMI) rats and sham-operated control (SHAM) rats. Maximum specific conductance of I(f) was significantly higher in left ventricular myocytes (LVM) from PMI rats compared to right ventricular myocytes from PMI rats as well as LVM and RVM from SHAM rats. All other basic properties of I(f) were similar. beta(1)AR stimulation with noradrenaline caused a rightward shift of V(H) in LVM from PMI rats which was significantly smaller (52.2%) than in LVM from SHAM rats. Incubation with pertussis toxin (PTX) largely restored the effect of beta(1)AR in PMI cells (86.6% vs. SHAM cells), but did not affect beta(1)AR response in SHAM cells. beta(2)AR response was significantly and equally increased by PTX-pretreatment (by 94% in SHAM and 87% in PMI cells). Conversely, beta(3)AR stimulation by the selective agonist SR 58611A caused a leftward shift of the activation curve which was significantly larger in PMI cells than in SHAM cells (P<0.01). beta(3)AR response was blunted by PTX-pretreatment, by incubation with N(G)-monomethyl-l-arginine acetate or by the selective beta(3)AR antagonist SR 59230A 1 microM. In conclusion, I(f) is significantly overexpressed in LVM from PMI rat hearts. In these cells, I(f) modulation by beta(1)AR is significantly depressed while beta(3)AR modulation is markedly enhanced, probably reflecting the increased activity of PTX-sensitive G(i) proteins in PMI cells.     

Desensitization of myocardial beta-adrenergic receptors during cardiopulmonary bypass. Evidence for early uncoupling and late downregulation.

BACKGROUND. Cardiopulmonary bypass (CPB), a process routinely used during cardiac surgery, is a potent stimulant to the release of endogenous catecholamines. Hence, we tested the hypothesis that CPB results in myocardial beta-adrenergic receptor (beta AR) desensitization. METHODS AND RESULTS. We obtained canine transmyocardial left ventricular biopsies before, during (155 minutes), and after CPB (pre-CPB, CPB, and post-CPB, respectively) and determined beta AR density, proportion of beta 1AR to beta 2AR, and beta AR coupling capacity to adenylyl cyclase. Beta AR density was stable at 112 +/- 14 fmol/mg (pre-CPB) and 103 +/- 9 fmol/mg (CPB) but decreased post-CPB to 84 +/- 7 fmol/mg. The ratio of beta 1AR to beta 2AR (determined by two-site fit for [125I]-iodocyanopindolol competition binding with the beta 1AR selective antagonist ICI89.406) remained constant throughout (60 +/- 3: 40 +/- 3 pre-CPB, 55 +/- 3: 44 +/- 3 CPB, and 61 +/- 2: 39 +/- 2 post-CPB), revealing that both beta 1AR and beta 2AR subtypes were downregulated. A different pattern was noted in the functional properties of these receptors during CPB. Decreased maximal isoproterenol-stimulated adenylyl cyclase activity (252 +/- 14 to 216 +/- 12 pmol/30 min/mg), submaximal isoproterenol-stimulated adenylyl cyclase activity (183 +/- 10 to 157 +/- 11 pmol/30 min/mg), and zinterol-stimulated adenylyl cyclase activity (187 +/- 11 to 159 +/- 11 pmol/30 min/mg, a measure of beta 2AR subtype activation) were noted during CPB, at the time when weaning from CPB takes place. However, this desensitized pattern was found to be completely reversed by 30 minutes post-CPB, with adenylyl cyclase activities returning to pre-CPB levels or slightly higher. Control dogs that did not receive CPB showed no change in beta AR density or adenylyl cyclase activity. CONCLUSIONS. These data suggest that myocardial beta AR desensitization does occur during CPB in healthy, nonischemic canine myocardium and that this pattern is reversed 30 minutes after discontinuation of CPB. In addition, a slower process of beta AR downregulation persists after discontinuation of CPB. Because successful weaning from CPB is a critical process during myocardial surgery, these findings have potentially important implications in the management of such patients.     

Localization of cardiac L-type Ca(2+) channels to a caveolar macromolecular signaling complex is required for beta(2)-adrenergic regulation

L-type Ca(2+) channels play a critical role in regulating Ca(2+)-dependent signaling in cardiac myocytes, including excitation-contraction coupling; however, the subcellular localization of cardiac L-type Ca(2+) channels and their regulation are incompletely understood. Caveolae are specialized microdomains of the plasmalemma rich in signaling molecules and supported by the structural protein caveolin-3 in muscle. Here we demonstrate that a subpopulation of L-type Ca(2+) channels is localized to caveolae in ventricular myocytes as part of a macromolecular signaling complex necessary for beta(2)-adrenergic receptor (AR) regulation of I(Ca,L). Immunofluorescence studies of isolated ventricular myocytes using confocal microscopy detected extensive colocalization of caveolin-3 and the major pore-forming subunit of the L-type Ca channel (Ca(v)1.2). Immunogold electron microscopy revealed that these proteins colocalize in caveolae. Immunoprecipitation from ventricular myocytes using anti-Ca(v)1.2 or anti-caveolin-3 followed by Western blot analysis showed that caveolin-3, Ca(v)1.2, beta(2)-AR (not beta(1)-AR), G protein alpha(s), adenylyl cyclase, protein kinase A, and protein phosphatase 2a are closely associated. To determine the functional impact of the caveolar-localized beta(2)-AR/Ca(v)1.2 signaling complex, beta(2)-AR stimulation (salbutamol plus atenolol) of I(Ca,L) was examined in pertussis toxin-treated neonatal mouse ventricular myocytes. The stimulation of I(Ca,L) in response to beta(2)-AR activation was eliminated by disruption of caveolae with 10 mM methyl beta-cyclodextrin or by small interfering RNA directed against caveolin-3, whereas beta(1)-AR stimulation (norepinephrine plus prazosin) of I(Ca,L) was not altered. These findings demonstrate that subcellular localization of L-type Ca(2+) channels to caveolar macromolecular signaling complexes is essential for regulation of the channels by specific signaling pathways.     

β1肾上腺素能受体阻断剂与β2肾上腺素能受体激动剂联用对心力衰竭大鼠心功能及心肌细胞凋亡的影响

目的 在一定范围内激活β2肾上腺素能受体（AR）可以在不加重心室重构的前提下改善心力衰竭（心衰）大鼠心功能,推测β1AR阻断剂联合β2AR激动剂有可能进一步改善心衰大鼠心功能并减轻心肌细胞凋亡,该实验对此进行了研究并探讨了其机制。方法 随机选取9只雄性Wistar大鼠为对照组。将异丙基肾上腺素诱导的心衰大鼠随机分为美托洛尔组（n=11）,联合治疗组（n=11）,安慰剂组（n=10）。美托洛尔组给予美托洛尔50mg／kg,一日两次灌胃。联合治疗组给予非诺特罗125μg／kg,美托洛尔50mg／kg一日两次灌胃。安慰剂组给予等量生理盐水一日两次灌胃。对照组不予处理。治疗8周后应用超声心动图评价心功能,TUNEL法检测心肌细胞凋亡指数,测定Caspase-3酶活性,Westernblot测定bcl-2及bax蛋白质表达,测定脏器重量／体重,组织病理学测定胶原容积分数（CVF）。结果 （1）美托洛尔组及联合治疗组均较安慰剂组左室舒张末期直径（LVEDd）、左室收缩末期直径（LVESd）、E峰A峰比值（E／A）明显下降,短轴缩短率（FS）、射血分数（EF）则有明显增高。联合治疗组较美托洛尔组LVEDd、LVESd有进一步降低（均为P〈0．05）,FS、EF则有进一步增高（均为P〈0．01）。（2）美托洛尔组及联合治疗组较安慰剂组左室重量体重比（LVW／BW）、肺脏重量体重比（PW／BW）及CVF明显降低（均为P〈0．01）。联合治疗组LVW／BW及PW／BW较美托洛尔组进一步降低（P〈0．01）,但两组之间CVF无显著差异。（3）美托洛尔组及联合治疗组较安慰剂组心肌细胞凋亡指数（AI）及Caspase．3活性均有明显减低。联合治疗组较美托洛尔组有进一步减低（均为P〈0．01）。（4）与安慰剂组相比,美托洛尔组及联合治疗组bax蛋白表达有明显下降而bcl-2／bax显著升高,并且以联合治疗组改善更为显著（均为P〈0．01）。结论 β1AR阻断剂联合β2AR激动剂较单用β1AR阻断剂进一步改善心衰大鼠的心功能,减轻心室重构。明显降低bax蛋白表达及bcl-2／bax,减轻心肌细胞凋亡很可能是其疗效提高的机制之一。