去甲肾上腺素对大鼠肝星状细胞作用的实验研究

目的 探讨交感神经系统在肝纤维化发生和发展中的作用. 方法采用免疫荧光和RT-PCR检测体外培养的肝星状细胞(HSC)中α1、β2-肾上腺素能受体的表达;用四甲基偶氮唑盐法检测不同浓度的去甲肾上腺素(NE)对HSC增殖活性的影响.同时用RT-PCR检测受NE作用后HSC的活化指标胶原蛋白-1、转化生长因子β(TGF β)及α-平滑肌动蛋白(α-SMA)的表达.用高效液相色谱-电化学法测定活化的HSC中交感神经递质NE的水平. 结果α1和β2-肾上腺素能受体表达于HSC的胞膜和胞质内;NE可呈剂量依赖性地促进HSC增殖,在浓度为100μmol/L时达到最大效应,F=140.464,P<0.05,差异有统计学意义.以NE 100 μmol/L作用细胞24h后,可显著促进反应HSC活化的指标上升,胶原蛋白-1表达为0.3022±0.0610,TGF β表达为2.2080±0.2151,α-SMA mRNA表达为0.5469±0.0108,与对照组胶原蛋白-1(0.1040±0.0556)、TGF β(1.1190±0.0070)、α-SMA mRNA表达(0.0759±0.0449)比较,t值分别为-4.160、-8.763和-17.651,P值均<0.05,差异均有统计学意义.HSC可以合成并释放NE,且受血小板衍生生长因子(10ng/ml)刺激后HSC中NE含量为(14.24±0.21)ng/ml,对照组为(11.34±0.15)ng/ml,两组比较,t=-32.907,P<0.05,差异有统计意义.结论 抑制交感神经系统使HSC活性降低对临床上治疗肝纤维化有一定的指导意义.     

肝纤维化过程中去甲肾上腺素各受体亚型表达的动态变化

目的 研究肝纤维化过程中去甲肾上腺素各受体亚型表达的动态变化.方法 胆总管结扎法建立大鼠肝纤维化模型,HE,Masson染色观察肝脏病理形态学变化;免疫组织化学法检测肝星状细胞活化指标α-平滑肌肌动蛋白(α-SMA)的表达情况;Western blot和实时荧光定量PCR检测α-肾上腺素受体(AR)、β1-AR、β2-AR蛋白和mRNA的表达水平.对数据进行单因素方差分析,LSD检验及相关分析.结果 HE及Masson三色染色显示:随着造模时间延长肝纤维化程度逐渐加重.α-SMA免疫组织化学染色显示:随着肝纤维化的发展,大鼠肝组织中α-SMA阳性细胞明显增多,造模1、2、3,4周时分别为10.58%±1.75%,24.14%±2.02%、29.74%±2.59%,34.28%±2.01%,而假手术组为4.12%±1.51%,P＜0.01.Western blot检测结果显示,造模1～4周大鼠肝组织α-AR、β1-AR,β2-AR蛋白表达量均明显升高(P＜0.05).实时荧光定量PCR结果证实,随着肝纤维化的发展,α-AR、β1-AR、β2-AR mRNA表达水平均逐渐上升(P＜0.01).α-SMA与α-AR、β1-AR、β2-AR蛋白表达水平的相关分析显示,α-SMA与α-AR、β1-AR及β2-AR呈正相关,r值分别为0.564,0.753和0.606.结论 胆总管结扎法成功建立胆汁淤积性大鼠肝纤维化模型,在肝纤维化形成过程中肝星状细胞表达α-SMA增加.随着肝纤维化的进展,α-AR、β1-AR、β2-AR蛋白及mRNA水平明显增加,且与α-SMA呈正相关.     

日本血吸虫诱导小鼠肝纤维化进程中TGF-β1和HSP47的作用机制研究

目的　观察在日本血吸虫诱导的小鼠肝纤维化进程中转化生长因子?β1（Transforming growth factor?β1, TGF?β1）和热休克蛋白47（Heat shock protein 47, HSP47）的动态表达,探讨其在日本血吸虫病肝纤维化发生发展中的作用。方法　将50只雌性ICR小鼠随机分成感染组和正常对照组,每组25只。感染组每只小鼠经腹部皮肤感染日本血吸虫尾蚴（20 ± 1）条,对照组以不含尾蚴的去氯水处理。分别于感染后4、6、8、10周和12周等5个时间点,各取5只小鼠肝组织。应用酶联免疫吸附试验检测各小鼠血清中HSP47和TGF?β1含量,肝组织HE染色观察病理学改变,Masson染色观察胶原增生情况,应用实时荧光定量PCR检测肝组织TGF?β1、HSP47、I型胶原（α1）链COL1A1基因mRNA表达水平。结果　在日本血吸虫诱导小鼠肝纤维化过程中,小鼠血清HSP47和TGF?β1浓度和肝组织中TGF?β1 mRNA、HSP47 mRNA、COL1A1 mRNA表达水平均随纤维化进展而渐次升高。小鼠感染后6周,小鼠血清HSP47和TGF?β1含量分别为（179.26 ± 29.87） pg/mL和（22.37 ± 5.21） ng/mL,显著高于同期正常对照组小鼠的（150.29 ± 34.91） pg/mL和（18.54 ± 7.78） ng/mL（P均< 0.05）。肝组织HSP47 mRNA、COL1A1 mRNA、TGF?β1 mRNA表达水平分别为（0.86 ± 0.04）、（1.17 ± 0.06）和（0.64 ± 0.13）,均高于同期正常对照组小鼠的（0.23 ± 0.03）、（0.20 ± 0.02）和（0.38 ± 0.02）（P均< 0.01）。 结论　TGF?β1和HSP47在日本血吸虫诱导的小鼠肝纤维进程中的表达与肝纤维化进程一致,且随I型胶原的增多呈升高趋势;HSP47有望成为日本血吸虫病肝纤维化的新诊断标志物和治疗靶点。     

日本血吸虫诱导小鼠肝纤维化进程中TGF-β1和HSP47的作用机制研究

目的　观察在日本血吸虫诱导的小鼠肝纤维化进程中转化生长因子?β1（Transforming growth factor?β1, TGF?β1）和热休克蛋白47（Heat shock protein 47, HSP47）的动态表达，探讨其在日本血吸虫病肝纤维化发生发展中的作用。方法　将50只雌性ICR小鼠随机分成感染组和正常对照组，每组25只。感染组每只小鼠经腹部皮肤感染日本血吸虫尾蚴（20 ± 1）条，对照组以不含尾蚴的去氯水处理。分别于感染后4、6、8、10周和12周等5个时间点，各取5只小鼠肝组织。应用酶联免疫吸附试验检测各小鼠血清中HSP47和TGF?β1含量，肝组织HE染色观察病理学改变，Masson染色观察胶原增生情况，应用实时荧光定量PCR检测肝组织TGF?β1、HSP47、I型胶原（α1）链COL1A1基因mRNA表达水平。结果　在日本血吸虫诱导小鼠肝纤维化过程中，小鼠血清HSP47和TGF?β1浓度和肝组织中TGF?β1 mRNA、HSP47 mRNA、COL1A1 mRNA表达水平均随纤维化进展而渐次升高。小鼠感染后6周，小鼠血清HSP47和TGF?β1含量分别为（179.26 ± 29.87） pg/mL和（22.37 ± 5.21） ng/mL，显著高于同期正常对照组小鼠的（150.29 ± 34.91） pg/mL和（18.54 ± 7.78） ng/mL（P均< 0.05）。肝组织HSP47 mRNA、COL1A1 mRNA、TGF?β1 mRNA表达水平分别为（0.86 ± 0.04）、（1.17 ± 0.06）和（0.64 ± 0.13），均高于同期正常对照组小鼠的（0.23 ± 0.03）、（0.20 ± 0.02）和（0.38 ± 0.02）（P均< 0.01）。 结论　TGF?β1和HSP47在日本血吸虫诱导的小鼠肝纤维进程中的表达与肝纤维化进程一致，且随I型胶原的增多呈升高趋势；HSP47有望成为日本血吸虫病肝纤维化的新诊断标志物和治疗靶点。     

去甲肾上腺素对人肝细胞系表达瘦素及瘦素受体的影响

目的探讨交感神经递质去甲肾上腺素(NE)对体外培养人肝细胞系L02表达瘦素及瘦素受体的影响。方法用不同浓度的NE作用于体外培养的人肝细胞系L02,分别于24、48及72 h收集细胞,Western印迹法检测其表达瘦素及瘦素受体蛋白的影响;RT-PCR检测NE对肝细胞系L02瘦素及瘦素受体mRNA的影响。结果①Western印迹结果显示,1、10、100μmol/L NE作用于L02细胞24 h后,瘦素蛋白表达明显高于对照组(1.02±0.08,2.24±0.09,2.35±0.12 vs 0.62±0.09,P<0.05);瘦素受体蛋白表达亦明显高于对照组(1.35±0.13,2.37±0.12,2.39±0.15 vs 0.85±0.13,P<0.05)。②RT-PCR检测L02瘦素以及瘦素受体mRNA的表达情况,1、10、100μmol/L NE作用于L02 24 h后,瘦素mRNA表达明显高于对照组(1.54±0.08,2.37±0.09,2.72±0.12 vs 1.00±0.07,P<0.05);瘦素受体mRNA表达亦明显高于对照组(1.61±0.08,2.27±0.10,3.39±0.11 vs1.00±0.06,P<0.01)。结论交感神经递质NE对体外培养的肝细胞系瘦素以及瘦素受体的表达均有促进作用,从而参与了肝纤维化进程。     

去甲肾上腺素对肝星状细胞表达瘦素以及瘦素受体的影响

目的:探讨交感神经递质去甲肾上腺素(norepinephrine,NE)对体外培养肝星状细胞(hepatic stellate cells,HSC)系表达瘦素以及瘦素受体的影响.方法:用不同浓度的NE作用于HSC,分别于24、48及72 h收集细胞,免疫组织化学方法检测活化HSC表达α-肌动蛋白(α-smooth muscle actin,α-SMA)情况;Western blot法检测其表达瘦素以及瘦素受体蛋白的影响;real-time PCR检测NE对HSC瘦素以及瘦素受体m RNA的影响.结果:(1)免疫组织化学结果显示,1、10、100μm o l/L浓度N E作用于H S C 24 h,α-SMA表达明显增加(14.1±4.4,17.5±5.2,19.8±4.1 vs 11.3±4.5;P0.05);提示NE可以活化HSC,并促进HSC增殖.10μmol/L N E作用H S C 24、48、72 h后,α-S M A表达逐渐增强(17.5±5.2;18.5±5.4;19.2±6.2,P0.05);提示N E对H S C的促增殖作用具有时间依赖性;(2)Western blot结果显示,1、10、100μmol/L NE作用于HSC 24 h后,瘦素蛋白表达明显高于对照组(1.54±0.08,2.72±0.09,2.84±0.18 vs 0.85±0.12,P0.05);瘦素受体蛋白表达亦明显高于对照组(1.57±0.18,2.51±0.17,2.89±0.19 vs0.98±0.15,P0.05);(3)Real-time PCR检测HSC瘦素以及瘦素受体m RNA的表达增加,1、10、100μmol/L NE作用于HSC 24 h后,瘦素m RNA表达明显高于对照组(1.51±0.08,2.58±0.09,3.63±0.12 vs 1.00±0.07,P0.05);瘦素受体m RNA表达亦明显高于对照组(1.71±0.08,2.87±0.10,4.01±0.14 vs1.00±0.08,P0.05).结论:交感神经递质NE对体外活化的HSC瘦素以及瘦素受体的表达均有促进作用,从而参与了肝纤维化的进程.     

肝炎平对肝纤维化大鼠肝组织中M1型胆碱能受体和β2型肾上腺素能受体表达的影响

[目的]研究肝炎平对肝纤维化大鼠肝细胞神经递质受体的作用，探讨其抗肝纤维化的可能机制。[方法]将38只Wistar大鼠分为3组：正常对照（N）组8只、肝炎平（G）组15只及模型（M）组15只。采用光镜及电镜观察肝组织病理学改变；免疫组织化学SP法检测各组肝组织M1型胆碱能受体（M1-AChR）和β2肾上腺素能受体（β2-AR）的表达水平。[结果]G组病理改变较M组明显改善，G组M1-AChR的表达较M组降低而β2-AR的表达较M组显著升高（P〈0．05）。[结论]肝炎平能通过抑制肝组织M1-AChR的表达同时增加β2-AR的表达，从而调节神经递质在肝纤维化中的作用。     

罗格列酮对血吸虫病肝纤维化小鼠肝脏TGF-β1和α-SMA表达的影响

目的 研究过氧化物酶体增殖物激活受体γ (PPARγ)的配体罗格列酮对日本血吸虫病肝纤维化小鼠肝脏转化生长因子β1(TGF-β1)、α-平滑肌肌动蛋白(α-SMA)含量的影响.方法 将30只日本血吸虫病肝纤维化小鼠均分3组对照组,吡喹酮治疗组和罗格列酮治疗组.对照组不作任何治疗.吡喹酮治疗组用吡喹酮500 mg/(kg·d)灌胃治疗2 d,罗格列酮治疗组在吡喹酮治疗2 d后再用罗格列酮4 mg/(Kg·d)灌胃治疗6周.应用HE染色,免疫组化法及多媒体病理图文定量分析,观察罗格列酮治疗血吸虫病肝纤维化小鼠肝脏的病理改变及TGF-β1和α-SMA表达的变化.结果 罗格列酮能显著抑制血吸虫病小鼠肝脏纤维组织的增生,降低肝脏TGF-β1及α-SMA的表达.结论 PPARγ配体罗格列酮有明显的抗日本血吸虫病肝纤维化作用,其抗纤维化机制与其抑制肝星状细胞(HSC)活化、抑制其表达α-SMA及分泌TGF-β1密切相关.     

芦荟大黄素和吡喹酮对小鼠日本血吸虫病肝纤维化模型的影响

目的 探讨芦荟大黄素对血吸虫肝纤维化小鼠的疗效及可能的作用机制.方法 采用日本血吸虫尾蚴感染小鼠建立血吸虫性肝纤维化模型,用芦荟大黄素0.3 mg/kg·d-1灌胃治疗8周.免疫组化检测肝组织转化生长因子β1(TGF-β1)、血小板源性生长因子(PDGF)、Ⅰ型胶原和Ⅲ型胶原的表达.RT-PCR检测细胞周期蛋白依赖性激酶4(CDK 4)mRNA和细胞周期蛋白DI(cyclin D1)mRNA.结果 实验对照组小鼠肝脏TGF-β1、PDGF、Ⅰ型胶原和Ⅲ型胶原的平均吸光度值分别为0.2775±0.0810、0.2140±0.0069、0.2234±0.0248和0.1849±0.0231,较正常对照组升高(P<0.01).CDK4和cyclinD1基因表达增加.芦荟大黄素干预后,小鼠肝脏TGF-β1、PDGF、Ⅰ型胶原和Ⅲ型胶原的平均吸光度值分别为0.1615±0.0326、0.1324 ±0.0201、0.1652±0.0216和0.1409±0.0206,较实验对照组降低(P<0.01).CDK4和cyclin D1基因表达受到抑制.结论 芦荟大黄素对血吸虫性肝纤维化有治疗作用,其作用机制可能与影响肝星状细胞周期有关.     

交感神经递质去甲肾上腺素与肝脏缺血再灌注损伤的研究进展

本文概述交感神经递质去甲肾上腺素在肝脏缺血再灌注损伤中的作用及相关机制。在肝脏缺血再灌注期间,交感神经兴奋去甲肾上腺素释放的增加具有双向作用,不仅可以促进炎症的进展,而且还有一定的肝脏保护作用,促进肝细胞再生。这一双向作用可能和去甲肾上腺素的释放量以及其作用于不同受体有关。进一步研究这双向机制对临床治疗肝脏缺血再灌注损伤有十分重要的意义。     

肝炎平对肝纤维化肝组织中去甲肾上腺素和多巴胺的影响

目的：探讨肝炎平在四氯化碳（CCl4）诱导的肝纤维化模型中对肝脏中去甲肾上腺素（NE）和多巴胺（DA）的影响及其抗肝纤维化的可能机制。方法：CCl4诱导大鼠慢性肝纤维化模型。随机分为正常组、肝纤维化模型组和肝炎平治疗组。用高效液相色谱-电化学（HPLC-ECD）法检测肝组织中NE和DA水平。结果：肝炎平组和模型组大鼠肝组织内NE含量明显低于正常组（P〈0．01），且模型组NE含量低于肝炎平组（P〈0．05）；而DA在3组中的含量差异无显著性意义（P〉0．05）。结论：肝炎平可增加肝纤维化肝组织中NE的含量，可能主要通过调节肝纤维化大鼠肝脏中的NE含量而达到抗肝纤维化的作用。     

Serotonin release mechanisms in bovine pineal gland: stimulation by norepinephrine and dopamine

An assessment of serotonin (5HT) release was made in bovine pineal gland. Bovine pineal fragments took up [3H]5HT by a Na+-dependent process exhibiting two apparent Km, i.e. a high affinity uptake system (Km = 220 nM) and a low affinity uptake system (Km = 197 microM). A significant release of [3H]5HT was elicited by increasing K+ concentrations in the medium (20-80 mM). Exposure of bovine pineal fragments to varying doses of catecholaminergic agonists indicated that a significant [3H]5HT release was elicited at the following threshold concentrations: 10(-6) M norepinephrine (NE), 10(-7) M dopamine (DA), 10(-6) phenylephrine and 10(-6) M isoproterenol. By employing specific receptor agonists and antagonists, the 5HT release activity of adrenergic agonists was found to be mediated by alpha 1-adrenoceptors, while that of DA by D2-dopaminergic receptors. 5HT release elicited by NE or DA, as well as that by 30 mM K+, was Ca2+-dependent. Both NE and DA increase 45Ca2+ uptake in a dispersed cell preparation of bovine pineal glands. As in the case of 5HT release, the effect of NE and DA on calcium uptake was mediated by alpha 1-adrenoceptors and D2-dopaminergic receptors, respectively. These results indicate that both NE and DA control 5HT release in bovine pineal gland.     

Dopamine and L-dopa: inhibition of thyrotropin-stimulated thyroidal thyroxine release

Previous studies had suggested that norepinephrine (NE) and its precursors dopamine (DA) and L-DOPA acted similarly on iodine metabolism of isolated thyroid cells. Present studies indicate that this similarity extends to the inhibition by catecholamines of TSH-stimulated T4 release by mouse thyroids incubated in vitro. DA (5 X 10(-4) M), like NE, shown previously, inhibits TSH-stimulated T4 release. This inhibition was reversed by the alpha-blockers phentolamine, prazosin, and yohimbine, but not by the beta-blocker L-propranolol. DU-18288 and diethyldithiocarbamate, inhibitors of DA beta-hydroxylase, did not reduce DA inhibition, suggesting that prior conversion to NE was not a condition for DA activity. Apomorphine, a dopaminergic agonist but not a NE precursor, acted like DA, and its inhibition was also reversed by alpha-blockers. Furthermore, sulpiride, a dopaminergic blocker, reversed DA and apomorphine inhibition of TSH stimulation. These results suggest that DA inhibits TSH-stimulated T4 release through both adrenergic and dopaminergic receptors. On the other hand, L-DOPA, exerting an inhibition like that of DA, was also reversed by alpha-blockers, but its activity was greatly diminished by carbidopa, an inhibitor of aromatic L-amino acid decarboxylase, the enzyme converting L-DOPA to DA. This indicated that L-DOPA had to be converted to DA for activity. Both DA and L-DOPA inhibited stimulation of T4 release induced by (Bu)2cAMP, suggesting that their effect was exerted at a locus distal to cAMP generation. Indirect confirmation of a cAMP-independent pathway was obtained when DA inhibited TSH-stimulated cAMP formation, but, contrary to T4 release, this inhibition was not reversed by dopaminergic or adrenergic blockers. Presumably, therefore, DA inhibition of TSH-stimulated cAMP production was not related to T4 release. We conclude that 1) DA inhibits TSH-stimulated T4 release in mouse thyroids via alpha-adrenergic and dopaminergic receptors; 2) L-DOPA has to be converted to DA to produce inhibition; and 3) cAMP is unlikely to be an intermediary in DA inhibition.     

Hepatic fibrogenesis requires sympathetic neurotransmitters

BACKGROUND AND AIMS: Hepatic stellate cells (HSC) are activated by liver injury to become proliferative fibrogenic myofibroblasts. This process may be regulated by the sympathetic nervous system (SNS) but the mechanisms involved are unclear. METHODS: We studied cultured HSC and intact mice with liver injury to test the hypothesis that HSC respond to and produce SNS neurotransmitters to promote fibrogenesis. RESULTS: HSC expressed adrenoceptors, catecholamine biosynthetic enzymes, released norepinephrine (NE), and were growth inhibited by alpha- and beta-adrenoceptor antagonists. HSC from dopamine beta-hydroxylase deficient (Dbh(-/-)) mice, which cannot make NE, grew poorly in culture and were rescued by NE. Inhibitor studies demonstrated that this effect was mediated via G protein coupled adrenoceptors, mitogen activated kinases, and phosphatidylinositol 3-kinase. Injury related fibrogenic responses were inhibited in Dbh(-/-) mice, as evidenced by reduced hepatic accumulation of alpha-smooth muscle actin(+ve) HSC and decreased induction of transforming growth factor beta1 (TGF-beta1) and collagen. Treatment with isoprenaline rescued HSC activation. HSC were also reduced in leptin deficient ob/ob mice which have reduced NE levels and are resistant to hepatic fibrosis. Treating ob/ob mice with NE induced HSC proliferation, upregulated hepatic TGF-beta1 and collagen, and increased liver fibrosis. CONCLUSIONS: HSC are hepatic neuroglia that produce and respond to SNS neurotransmitters to promote hepatic fibrosis.     

Regulation of coronary blood flow by counteraction of coronary vascular alpha and beta adrenergic activation during experimental pliable coronary stenosis

In order to evaluate the role of adrenergic receptor-mediated vasomotions of large epicardial coronary arteries in changing coronary blood flow (CBF), the effects of intracoronary norepinephrine (NE), 1.0 microgram/min, were examined in dogs with coronary stenosis which preserved stenosis vasomobility. In untreated dogs, NE caused no significant changes in CBF and stenosis resistance (SR). In dogs treated with propranolol, NE decreased CBF by 65 +/- 7.0% (mean +/- SE) and produced 12-fold intensification of SR followed by LV dP/dt reduction. Similar detrimental responses to NE were observed in dogs treated with atenolol. In dogs treated with phentolamine, NE increased CBF by 33 +/- 5.6% and decreased SR by 65 +/- 7.1%. When NE was administrated directly distal to the stenosis to exclude responses of the stenosed coronary segment, NE failed to affect CBF and SR. These results indicated that alpha receptor stimulation intensified stenosis severity, profoundly decreased CBF and evoked myocardial ischemia, whereas beta stimulation dilated coronary stenosis and increased CBF. The net effects of NE were due to balanced alpha and beta stimulation. Thus, disproportionate activation of alpha and beta (probably beta 1) adrenergic receptors in large coronary arteries with pliable stenosis could modulate their tone and plays an important role in the regulation of CBF.     

Effect of age on upregulation of the cardiac adrenergic beta receptors

Radioligand binding studies were performed to determine whether upregulation of postjunctional beta receptors occurs in sympathectomized hearts of aged animals. Fischer 344 rats 6, 12, and 24 months of age (n = 10) were used in these experiments. To produce sympathectomy, rats were injected with 6-hydroxydopamine hydrobromide (6-OHDA; 2 x 50 mg/kg iv) on days 1 and 8; the animals were decapitated on day 15. The depletion of norepinephrine in the heart was about 86% in each age group. 125I-Iodopindolol (IPIN), a beta adrenergic receptor antagonist, was employed to determine the affinity and total number of beta adrenergic receptors in the ventricles of the rat heart. The maximal number of binding sites (Bmax) was significantly elevated by 37%, 48%, and 50% in hearts from sympathectomized 6-, 12-, and 24-month-old rats, respectively. These results indicate that beta receptor mechanisms in older hearts can respond to procedures that cause upregulation of the beta adrenergic receptors.     

Effects of the polyamines putrescine, spermidine and spermine on the hypertensive action of norepinephrine in anesthetized dogs

In anesthetized and vagotomized dogs, we investigate the associative effects on blood pressure of norepinephrine (NE) with the three polyamines putrescine (Pt), spermidine (Sd) and spermine (Se). Experimental series were performed under beta adrenergic blockade (propranolol 0.5 mg/Kg, iv.), alpha adrenergic blockade (phenoxybenzamine 15 mg/Kg, iv.), and under calcium antagonistic action (verapamil 0.3 mg/Kg, iv.). The three polyamines induced a potentiation on the hypertensive effect of NE, they change the dose/response curve to the left side in a potency rank of Se greater than Sd greater than Pt. Such potentiation was not different when a beta adrenergic blockade or calcium antagonism was present; however phenoxybenzamine neutralized it. On the other side, polyamines had a hypotensive effect when were administered alone to the animals. Such effect is related to a histamine releasing properties of the polyamines, and was abolished by previous administration of anihistaminic agents chlorpheniramine (5 mg/kg, iv.) and cimetidine (20 mg/Kg, iv.). In conclusion our results indicate that the potentiation of the hypertensive effect of NE by polyamines, could be attained through a mechanism which involves the alpha adrenergic receptors of the vascular smooth muscle but is not related to the calcium channels that show voltage dependence.     

Sympathetic nervous system regulates bone marrow-derived cell egress through endothelial nitric oxide synthase activation: role in postischemic tissue remodeling

OBJECTIVE: Catecholamines have been shown to control bone marrow (BM)-derived cell egress, yet the cellular and molecular mechanisms involved in this effect and their subsequent participation to postischemic vessel growth are poorly understood. METHODS AND RESULTS: Tyrosine hydroxylase mRNA levels, as well as dopamine (DA) and norepinephrine (NE) contents, were increased in the ischemic BM of mice with right femoral artery ligation. Angiographic score, capillary density, and arteriole number were markedly increased by treatments with DA (IP, 50 mg/kg, 5 days) or NE (IP, 2.5 mg/kg, 5 days). Using chimeric mice lethally irradiated and transplanted with BM-derived cells from green fluorescent protein mice, we showed that DA and NE enhanced by 70% (P<0.01) and 62% (P<0.001), respectively, the number of green fluorescent protein-positive BM-derived cells in ischemic tissue and promoted their ability to differentiate into cells with endothelial and inflammatory phenotypes. Similarly, both DA and NE increased the in vitro differentiation of cultured BM-derived cells into cells with endothelial phenotype. This increase was blunted by the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester. DA and NE also upregulated the number of CD45-positive cells in blood 3 days after ischemia and that of macrophages in ischemic tissue 21 days after ischemia. Of interest, DA and NE increased BM endothelial nitric oxide synthase (eNOS) mRNA levels and were unable to promote BM-derived cell mobilization in chimeric eNOS-deficient mice lethally irradiated and transplanted with BM-derived cells from wild-type animals. Furthermore, administration of a β2 adrenergic agonist (clenbuterol, IP, 2 mg/kg, 5 days) and that of a dopaminergic D1/D5 receptor agonist (SKF-38393, IP, 2.5 mg/kg, 5 days) also enhanced BM-derived cell mobilization and subsequently postischemic vessel growth. CONCLUSION These results unravel, for the first time, a major role for the sympathetic nervous system in BM-derived cell egress through stromal eNOS activation.     

Norepinephrine increases glucose transport in brown adipocytes via beta3-adrenoceptors through a cAMP, PKA, and PI3-kinase-dependent pathway stimulating conventional and novel PKCs

To identify the signaling pathways that mediate the adrenergic stimulation of glucose uptake in brown adipose tissue, we used mouse brown adipocytes in culture. The endogenous adrenergic neurotransmitter norepinephrine (NE) induced 2-deoxy-D-glucose uptake 3-fold in a concentration-dependent manner (pEC50 approximately 6.5). The uptake was abolished by high doses of propranolol. The NE effect was mimicked by isoprenaline (pEC50 approximately 6.9), BRL 37344 (pEC50 approximately 8.6), CL 316243 (pEC50 approximately 9.7) and CGP 12177 (pEC50 approximately 7.3) and was thus mediated by beta3-adrenergic receptors. The NE-induced effect on 2-deoxy-D-glucose uptake was mediated by adenylyl cyclase and cAMP because responses were inhibited by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and the protein kinase A inhibitor 4-cyano-3-methylisoquinoline. Cholera toxin and 8-bromoadenosine cAMP were both able to increase 2-deoxy-D-glucose uptake. Involvement of other adrenergic signaling pathways (alpha1-and alpha2-adrenergic receptors) were excluded. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, abolished beta-adrenergic- or 8-bromoadenosine cAMP-stimulated 2-deoxy-D-glucose uptake, demonstrating that a cAMP-dependent PI3K-mediated pathway is positively connected to glucose uptake. Inhibition of the beta-adrenergically stimulated response with protein kinase C (PKC) inhibitors (G? 6983, which inhibits (alpha, beta, gamma), (delta), and (zeta) isoforms and Ro-31-8220, which inhibits (alpha, beta1, beta2, gamma) and (epsilon) but not atypical isoforms) indicated that cAMP-mediated glucose uptake is stimulated via conventional and novel PKCs. These results demonstrate that adrenergic stimulation, through beta3-adrenergic receptors/cAMP/protein kinase A, recruits a PI3K pathway stimulating conventional and novel PKCs, which mediate glucose uptake in brown adipocytes.