Norepinephrine transporter: a candidate gene for initial ethanol sensitivity in inbred long-sleep and short-sleep mice

BACKGROUND: Altered noradrenergic neurotransmission is associated with depression and may contribute to drug abuse and alcoholism. Differential initial sensitivity to ethanol is an important predictor of risk for future alcoholism, making the inbred long-sleep (ILS) and inbred short-sleep (ISS) mice a useful model for identifying genes that may contribute to alcoholism. METHODS: In this study, molecular biological, neurochemical, and behavioral approaches were used to test the hypothesis that the norepinephrine transporter (NET) contributes to the differences in ethanol-induced loss of righting reflex (LORR) in ILS and ISS mice. RESULTS: We used these mice to investigate the NET as a candidate gene contributing to this phenotype. The ILS and ISS mice carry different DNA haplotypes for NET, showing eight silent differences between allelic coding regions. Only the ILS haplotype is found in other mouse strains thus far sequenced. Brain regional analyses revealed that ILS mice have 30 to 50% lower [3H]NE uptake, NET binding, and NET mRNA levels than ISS mice. Maximal [3H]NE uptake and NET number were reduced, with no change in affinity, in the ILS mice. These neurobiological changes were associated with significant influences on the behavioral phenotype of these mice, as demonstrated by (1) a differential response in the duration of ethanol-induced LORR in ILS and ISS mice pretreated with a NET inhibitor and (2) increased ethanol-induced LORR in LXS recombinant inbred (RI) strains, homozygous for ILS in the NET chromosomal region (44-47 cM), compared with ISS homozygous strains. CONCLUSIONS: This is the first report to suggest that the NET gene is one of many possible genetic factors influencing ethanol sensitivity in ILS, ISS, and LXS RI mouse strains.     

早期微量泵控制输注去甲肾上腺素对失血性休克兔血流动力学和心肌功能的影响

目的:探讨失血性休克早期微量泵控制输注去甲肾上腺素,对家兔血流动力学、心肌和心功能的影响。方法:实验采用健康成年新西兰兔,随机分为失血性休克去甲肾上腺素治疗组(NE组,n=7)和失血性休克生理盐水治疗组(NS组,n=7);股动脉放血法制备失血性休克模型成功后,NE组立即给予10min去甲肾上腺素的微量泵泵入,速度为0.2-1mg/(kg·h),同时静脉输注与丢失血量等量的生理盐水;NS组立即给予10min生理盐水补液治疗,补液量为3倍的丢失血量,同样采取微量泵匀速补液。10min强化治疗结束,记录为0h。随后维持并记录0,0.5,1,2,4h各时间点血流动力学指标,并留取血清进行心肌酶学(CK和CKMB)和肌钙蛋白(cTnI)分析。结果:NE组较NS组心率明显提高;NS仅在治疗前期(0h和1h)改善平均动脉压(MAP),NE则可在各时间点持续有效改善MAP;NE在一定的使用时程内(0-2h)可改善失血性休克动物的左室平均压(LVAP);NE较NS可明显提升失血性休克的左心室收缩功能;NE较NS从0.5h起可明显改善左心室舒张功能;心肌酶学和TnI结果显示NE组与NS组整体存在明显差异:CK[(23.87±16.85)vs.(13.22±2.50)ng/ml,P<0.05],CKMB[(8.80±5.68)vs.(5.27±1.14)ng/ml,P<0.05]和cTnI[(109.09±37.81)vs.(84.87±10.73)pg/ml,P<0.05]。结论:早期微量泵控制给予去甲肾上腺素治疗失血性休克,有助于改善早期血流动力学和心功能指标,但可能增加失血性休克心肌的损伤。     

复方丹参滴丸对应急作训战士血液流变学及血浆去甲肾上腺素影响的研究

目的：探讨复方丹参滴丸对应急作训战士血液流变学及血浆去甲肾上腺素(NE)调理的作用。方法：选新入伍战士80人，随机分为正常组、应急对照组、复方丹参滴丸组、谷维素组，每组20人，口服药物5d后应急作训，观察应急作训后血浆NE、血液流变学的变化。结果：复方丹参滴丸组应急作训后战士血浆NE含量较其他组低．可调整全血黏度。结论：复方丹参滴丸可不同程度地对抗应急作训所致的血浆去甲肾上腺素升高，改善血液流变性。     

1型多聚二磷酸腺苷核糖合成酶对去甲肾上腺素诱导心脏成纤维细胞表达基质金属蛋白酶的影响

目的:探讨1型多聚二磷酸腺苷核糖合成酶(PARP-1)对去甲肾上腺素(NE)诱导培养的大鼠心脏成纤维细胞基质金属蛋白酶(MMP)-1,MMP-9及金属蛋白酶组织抑制剂(TIMP)-1表达的调节作用及其机制。方法:①培养乳鼠心脏成纤维细胞,10μmol/LNE刺激细胞24h,使用实时定量PCR法检测MMP-1、MMP-9及TIMP-1的基因表达水平;使用PARP抑制剂3-氨基苯甲酰胺(3-aminobenzamide,3AB)后,观察PARP-1对上述基因表达的影响。②检测心脏成纤维细胞内活性氧(ROS)水平、PARP酶活性的变化。③采用凝胶阻滞实验检测心脏成纤维细胞内转录因子AP-1的DNA结合能力,研究PARP-1对AP-1DNA结合能力的影响。结果:NE诱导心脏成纤维细胞内MMP-1,MMP-9及TIMP-1的基因表达水平明显增加。细胞内ROS产生增加,PARP酶被激活。核内转录因子AP-1的DNA结合能力明显增强。PARP抑制剂3AB可明显减少NE诱导的MMP-1、MMP-9及TIMP-1的基因表达水平,同时显著抑制AP-1的DNA结合能力。使用抗氧化剂vitC减少ROS产生,抑制了NE诱导的PARP-1活性增加及AP-1的DNA结合,进而显著降低了NE诱导的MMP-1、MMP-9及TIMP-1的基因表达水平。结论:NE刺激心脏成纤维细胞内ROS产生明显增多,大量的ROS激活了PARP使其酶活性显著增高,PARP通过调节转录因子AP-1的DNA结合调控了MMP-1,MMP-9及TIMP-1的基因表达。PARP可能是心脏纤维化过程中的重要调节机制之一。     

交感神经递质及其受体在小鼠血吸虫肝纤维化中的作用

目的 研究交感神经递质及其受体在正常小鼠及血吸虫病肝纤维化小鼠体内的变化情况.方法 应用腹部敷贴尾蚴法制备血吸虫病小鼠模型.30只昆明小鼠随机分为正常对照组和肝纤维化模型组.采用HE和Van Gieson染色,光镜观察肝组织病理学改变及纤维化程度,免疫荧光组织化学染色结合激光共聚焦扫描检测肝组织中α1A、β2肾上腺素能受体表达情况,同时用高效液相色谱-电化学法检测血浆中去甲肾上腺素和多巴胺的水平. 结果 正常组α1A、β2肾上腺素能受体散在表达于肝细胞胞质和肝窦内,模型组在门静脉和虫卵结节周围肝细胞胞质中阳性表达明显增加(α1A受体的平均灰度值分别为18.28±7.56、30.53±8.88,β2受体的平均灰度值分别为28.67±6.42、42.29±4.56,t值分别为-2.888和-6.648,P值均＜0.05);血吸虫肝纤维化小鼠和正常小鼠去甲肾上腺素含量分别为(7.83±4.36)、(2.72±0.94)ng/ml,多巴胺的含量分别为(6.97±4.33)、(0.74±0.34)ng/ml,t值分别为-3.372和-4.428,P值均＜0.05.结论 交感神经递质及其受体表达的上调可能是血吸虫肝纤维化发展的作用机制之一.     

Effects of 5,7–Dihydroxytryptamine Lesions of the Prefrontal Cortex on Consumption of Sucrose-Ethanol Solutions: Relationship to Prefrontal Monoamines

Thirty adult male Wistar rats received 8 μg bilaterally of 5,7–dihy-droxytryptamine into the medial prefrontal cortex (mPFC). Rats were then trained, via a sucrose fading paradigm, to consume increasing concentrations of alcohol. After death, dopamine (DA), norepineph-rine (NE), serotonin (5–HT), and their metabolites were measured in the mPFC, nucleus accumbens (NA), and raphe nucleus. The le-sioned group demonstrated a reduction in 5–hydroxyindoleacetic acid (5–HIAA), DA, and NE in the mPFC ( p < 0.05), and a trend toward reduction of 5–HT in the NA. In comparison with controls, lesioned animals consumed less of all solutions containing sucrose and alcohol. On regression analyses, monoamines in the mPFC (i.e., 5–HIAA, dihydrophenylacetic acid and NE) predicted consumption of the 5% ethanol solution ( p = 0.00S), 10% ethanol solution ( p = 0.0006), and the 5% sucrose solutions ( p = 0.0006), but not the 20% sucrose solutions. In each case, monoamine levels were positively correlated with consumption. No relationships were seen between monoamine levels in the NA and raphe, and in consummatory behavior.     

肌球蛋白轻链磷酸化与心肌损伤的实验研究

应用水平平板等电聚焦(电压2000V,时间2.5h、pH 4～6)、银染和凝胶光密度扫描相结合的一种新方法,研究去甲肾上腺素性心肌损伤兔心肌肌球蛋白轻链磷酸化(P-MLC/MLC×100%)的改变及α、β受体阻滞剂对该改变的影响。结果表明,去甲肾上腺素(NE,2mg/kg)显著降低心肌的P-MLC/MLC(P-MLC/MLC为35.40±5.37%;对照组为61.07±6.97%,P<0.01),酚妥拉明(10mg/只)可保护心肌P-MLC 66.43±8.43%,而普萘洛尔则无此作用(P-MLC/MLC为36.54±4.05%)。     

干预G蛋白βγ亚基介导的信号转导途径对心肌细胞肥大的影响

目的 研究G蛋白βγ亚基在心肌细胞肥大中的作用.方法 采用细菌内同源重组方法制备重组腺病毒载体rAd-βARKct 50 MOI(multiplicity of infection)感染经去甲肾上腺素处理肥大乳鼠心肌细胞,荧光倒置显微镜下观察心肌细胞形态及绿色荧光蛋白的表达,EMAIL图像分析软件测定心肌细胞的表面积,RT-PCR检测βARKct、α-MHC和β-MHC的mRNA表达水平.结果 经测序鉴定证实rAd-β ARKct构建成功,对心肌细胞的感染率40%～50%,与去甲肾上腺素组相比,重组腺病毒组α-MHC定量比值显著上升,β-MHC定量比值显著降低.结论 编码βARKct的重组腺病毒表达载体可以体外感染乳鼠心肌细胞并在心肌细胞中表达βARKct;抑制Gβγ蛋白的功能,可显著逆转去甲肾上腺素介导的肌球蛋白重链表型的改变(α-MHC/β-MHC),逆转心肌细胞肥大.     

Hypoxia does not activate ATP-sensitive K+ channels in arteriolar muscle cells

OBJECTIVE: To test the hypothesis that hypoxia activates ATP-sensitive K+ (KATP) channels in cremasteric arteriolar muscle cells, resulting in membrane hyperpolarization and inhibition of norepinephrine-induced contraction. METHODS: Arteriolar muscle cells were isolated enzymatically from second- and third-order arterioles that were surgically removed from hamster cremaster muscles. The effects of hypoxia (PO2 = 12-15 mm Hg) were then examined on norepinephrine-induced contraction, membrane currents, and membrane potential in these cells at room temperature. Whole-cell currents and membrane potential were recorded using the perforated patch technique. RESULTS: Hypoxia (12-15 mm Hg PO2) reversibly inhibited norepinephrine-induced contraction to 52 +/- 6% of the response in normoxic solutions (156 mm Hg, n = 12 digests, p < 0.05). These effects of hypoxia could be prevented by superfusion of the cells with either solutions containing the KATP channel antagonist glibenclamide (1 microM) or solutions containing 35 mM K+ to reduce the electrochemical gradient for K+ diffusion. Cromakalim, an activator of KATP channels, also inhibited norepinephrine-induced contraction to a similar extent as hypoxia, and in a glibenclamide and 35 mM K(+)-sensitive manner. These results are consistent with the KATP channel hypothesis. In contrast, hypoxia had no effect on estimated whole-cell membrane conductance between -40 and -90 mV in voltage-clamp experiments; on holding current measured at -60 mV in cells superfused with 143 mM K+ under voltage-clamp conditions; or on membrane potential in current-clamp experiments, despite positive effects of cromakalim in all three protocols. These electrophysiological data lead to rejection of the hypothesis that hypoxia activates KATP channels. CONCLUSIONS: Hypoxia inhibits norepinephrine-induced contraction of cremasteric arteriolar muscle cells by a mechanism that does not involve KATP channels. It is speculated that the inhibitory effects of glibenclamide and 35 mM K+ on the effects of hypoxia on contraction resulted from depolarization induced by these treatments rather than specific inhibition of KATP channels.