兔室旁核对血量扩张引起促钠排泄与利尿的作用

在室旁核(PVN)假损毁兔与PVN损毁兔血量扩张(VE)引起尿流量增加,峰值分别为0.59±0.09与0.31±0.03 ml/min (P<0.01),排钠量增加峰值分别为66.76±6.74与36.05±3.44μmol/min (P<0.01),而在PVN假损毁兔与PVN完好兔对VE的反应无显著差别(P>0.05),表明PVN损伤可明显减弱 VE 引起的促钠排泄与利尿效应.颈迷走神经切断并不能改变 PVN损伤的上述作用.双侧肾神经切断兔损毁 PVN对VE引起促钠排泄效应无显著影响,但显著减弱其利尿效应 (P<0.02).PVN损毁对VE时肾小球滤过率(GFR)与肾血浆流量(RPF)无显著影响.结果表明PVN参与VE通过迷走传入神经引起促钠排泄与利尿反应的调节,而肾交感传出神经参与其中促钠排泄的作用.     

大鼠脑胆碱能系统对血量扩张引起利尿与尿钠排泄...

The role of brain cholinergic system on diuresis and natriuresis induced by volume expansion was studied in conscious rats. In a series of experiments, the diuretic, natriuretic and kaliuretic responses induced by volume expansion were compared in three groups of conscious rats pretreated respectively with intracerebroventricular (icv) injection of artificial cerebrospinal fluid (ACSF), atropine and hexamethonium. The natriuretic, kaliuretic and diuretic responses induced by volume expansion were much less in the animals with icv injection of atropine than in the control group with injection of ACSF (P less than 0.01). While the group pretreated with icv injection of hexamethonium showed no significant decrease in these responses of volume expansion than that of the control (P greater than 0.05). Volume expansion produced no change in insulin and PAH clearance in both the atropine and the ACSF group. Thus the atropine suppressed diuresis, natriuresis and kaliuresis are independent of changes in GFR and RPF. It is inferred from the results of the present investigation that volume expansion induced diuresis and natriuresis appear to be due to inhibition of water and sodium reabsorption in the renal tubules and regulated by certain brain cholinergic system.     

兔脊髓α受体对血量扩张引起肾交感神经活动抑制和促钠排泄的作用

在窦主动脉去神经麻醉兔观察阻断脊髓α受体对血量扩张引起肾交感神经活动（RSNA）抑制和促钠排泄反应的影响。兔脊髓蛛网膜下腔注射a肾上腺素能受体阻断剂酚妥拉明与人工脑脊液后,血量扩张引起RSNA抑制分别为（－25．4±5．4）％与（－42．5±5.2）％（P＜0.05）;兔脊髓蛛网膜下腔注射α1受体阻断剂哌唑嗪与人工脑脊液后血量扩张引起RSNA抑制分别为（－29．3±6.1）％与（－42．5±5.2）％（P＜005）。结果表明,阻断脊髓α受体或α1受体均可减弱血量扩张引起RSNA抑制。脊髓注射哌唑嗪后血量扩张引起促钠排泄与利尿反应也显著减弱（P＜005）。     

大鼠脑胆碱能系统对血量扩张引起利尿与尿钠排泄的作用

本工作在清醒大鼠侧脑室注射胆碱能药物,观察脑胆碱能系统对血量扩张引起利尿与尿钠排泄的作用。侧脑室注射人工脑脊液后进行血量扩张引起尿流量、排钠量和排钾量显著增加(P<0.01)。侧脑室注射胆碱能 M 受体阻断剂阿托品后,血量扩张引起尿流量、排钠量和排钾量增加的效应比注射人工脑脊液组的均显著减弱(P<0.01);而侧脑室注射胆碱能 N 受体阻断剂六烃季胺后,血量扩张引起尿流量、排钠量和排钾量增加的效应与注射人工脑脊液组的相比无显著差异(P>0.05)。侧脑室注射人工脑脊液或阿托品大鼠的肾小球滤过率(GFR)与肾血浆流量(RPF)在血量扩张后均无显著变化(P>0.05)。上述结果表明:大鼠脑胆碱能M 受体参与血量扩张引起利尿与尿钠排泄反应的调节。脑 M 受体的这种作用不是通过改变GFR 和 RPF,而可能是通过未明神经液递机制直接影响肾小管对水钠的重吸收。     

肾多巴胺受体对血量扩张和脑室高张盐溶液注射引起促钠排泄的作用

本工作观察麻醉兔用氟哌啶醇阻断肾多巴胺受体对血量扩张（ＶＥ）和脑室高张盐溶液注射（ＩＣＨＮａ）引起促钠排泄反应的影响。在ＶＥ实验中。对照组与氟哌啶醇组排销量增加峰值分别为６５．６±１５．０与１９．０±５．５μｍｏｌ／ｍｉｎ（Ｐ＜０．０２）;在ＩＣＨＮａ实验中,对照组与氟哌啶醇组排钠量增加峰值分别为２８．９±４．６与２９．０±５．８μｍｏｌ／ｍｉｎ（Ｐ＞０．０５）,在肾多巴胺受体完好兔实验中,ＶＥ＋ＩＣＨＮａｇ;起的促钠排泄大于ＶＥ（Ｐ＜０．０１）或ＩＣＨＮａ（Ｐ＜０．０１）引起的反应。肾多巴胺受体阻断也可显著减弱ＶＥ＋ＩＣＨＮａ引起促钠排泄反应。结果表明：肾多巴胺受体阻断显著减弱ＶＥ以及ＶＥ＋ＩＣＨＮａ引起促钠排泄,对ＩＣＨＮａ的反应无效;在正常兔ＶＥ＋ＩＣＨＮａ引起促钠排泄显著大于各单一刺激的反应。     

兔室旁核－脊髓径路对血量扩张引起肾交感神经活动抑制的作用

在室旁核（ＰＶＮ）完好或ＰＶＮ注射抗坏血酸的双侧窦主动脉去神经的麻醉兔,血量扩张引起肾交感神经活动（ＲＳＮＡ）抑制均约４８％。然而在红藻氨酸损毁ＰＶＮ后的３—４ｈ和３—４ｄ,这种ＲＳＮＡ抑制分别减弱到－２８．０±４．５％和－２５．７±４．１％（Ｐ＜０．０５）,同时其抑制时程也显著缩短（Ｐ＜０．０１）。此ＲＳＮＡ抑制也可被脊髓Ｔ１０—Ｔ１２节段蛛网膜下腔注射血管升压素能Ｖ１受体阻断剂而显著地减弱。在血量扩张时对照组与实验组血压均呈小而短暂升高,无显著差别。上述结果表明血量扩张引起的ＲＳＮＡ抑制,部分是通过迷走传入神经触发ＰＶＮ－脊髓径路介导的。     

AT1受体在脑内胆碱能刺激引起的钠水排泄反应中的作用

目的和方法 :本工作通过整体实验和免疫组化的方法 ,观察脑血管紧张素能AT1受体在侧脑室注射氨甲酰胆碱引起的促钠排泄反应中的作用和下丘脑室旁核TH IR的变化。结果 :用脑血管紧张素能AT1受体阻断剂Losar tan( 2 0 μg)预处理 ,可部分阻断侧脑室注射氨甲酰胆碱引起的促钠排泄反应和利尿作用 (P <0 .0 5)。免疫组化实验显示侧脑室给予氨甲酰胆碱后 40min ,下丘脑室旁核 (PVH)、室周核 (Pe)、弓状核 (Arc)和下丘脑前区后部 (AHP)的酪氨酸羟化酶 (TH )免疫反应活性明显增强。Losartan预处理后侧脑室再注射氨甲酰胆碱 ,除PaPo的免疫反应活性未发生明显变化外 ,上述其余神经核团的TH免疫反应活性明显下降。结论 :在脑胆碱能刺激引起的钠水排泄反应中有AT1受体的参与 ;阻断脑血管紧张素能AT1受体对胆碱能刺激引起Arc、Pe和AHP的儿茶酚胺能神经元兴奋性有下调作用。提示脑血管紧张素能和儿茶酚胺能神经通路在下丘脑室旁核等脑区共同参与介导了脑内胆碱能刺激引起的促钠排泄反应 ,同时血管紧张素能神经元还影响儿茶酚胺能神经元的功能活动     

脑胆碱能刺激引起的促钠排泄反应和蓝斑ChAT-IR的变化

目的：观察大鼠侧脑室注射胆碱能激动剂氨甲酰胆碱（CBC）后蓝斑胆碱能神经元活性变化及其与促钠排泄反应的关系。方法：选用SD雄性大鼠通过整体实验和免疫组化方法,观察侧脑室给予氨甲酰胆碱（0．5μg）和俄阿托品（30μg）后肾排钠量的变化及蓝斑胆碱乙酰转移酶（CHAT）免疫反应活性的变化。结果：侧脑室给予氨甲酰胆碱后40min,肾排钠量显著增加,蓝斑的CHAT-IR明显增强（P〈0．05）;阿托品预处理后可明显抑制上述反应。结论：蓝斑的胆碱能神经元参与侧脑室注射氨甲酰胆碱引起的促钠排泄反应。     

高密度脂蛋白促进内皮衍生的一氧化氮抑制血小板聚集的作用

用培养的小牛主动脉内皮细胞与兔水洗血小板直接相互作用的模型 ,探讨高密度脂蛋白对内皮衍生的一氧化氮抗血小板聚集作用的影响。培养的小牛主动脉内皮细胞预先用 10 0 μmol/L阿斯匹林处理 ,抑制细胞内的环氧化物酶活性。凝血酶 ( 0 1U/ml)可诱导兔血小板 ( 2× 10 8/ml) 67 3 3± 7 5 2 %的聚集反应。内皮细胞 ( 1× 10 5～ 1× 10 6 /ml)能抑制凝血酶诱导的血小板聚集 ,抑制强度与内皮细胞的数目正相关。且此作用可被 1mmol/L硝基精氨酸完全取消。表明内皮细胞对凝血酶诱导血小板聚集的抑制作用都是由内皮衍生的一氧化氮所致。在加凝血酶之前加入高密度脂蛋白 ( 1mg/ml)可增强内皮细胞 ( 1× 10 5/ml)的这种作用。高密度脂蛋白 ( 1mg/ml)与内皮细胞共同孵育 1h后 ,将高密度脂蛋白离心弃去 ,内皮细胞对凝血酶诱导血小板聚集的抑制作用不受影响。高密度脂蛋白及内皮细胞对静息血小板均无直接作用。结果表明 ,高密度脂蛋白增强内皮细胞抗凝血酶诱导的血小板聚集反应的作用是通过直接作用于内皮衍生的一氧化氮而产生的     

S-亚硝基谷胱甘肽诱导脐带血CD34+细胞来源的巨核细胞产生血小板

为了探讨一氧化氮供体S-亚硝基谷胱甘肽（S—nitrosoglutathione,GSNO）对脐带血CD34^＋细胞分化来源的巨三核细胞产生血小板的可能作用,我们采用免疫磁珠法从8例健康产妇足月顺产的胎儿脐带血中分选CD34^＋细胞,并在含血小板生成素（thrombopoietin,TPO,50ng／ml）、白细胞介素-3（IL-3,10ng／ml）、干细胞因子（stem cell factor,SCF,50ng／ml）和重组人粒-巨噬细胞刺激因子（rHu GM—CSF,20ng／ml）的无血清培养基中培养14d。随后,用免疫磁珠法分选CD61^＋细胞。CD61^＋细胞在含有（实验组）和缺乏（对照组）GSNO（20mg／ml）的无血清培养基[含TPO（50ng／ml）、IL-3（10ng／ml）、SCF（50ng／ml）]中培养不同时间（30min、2h）。采用流式细胞仪检测培养体系中的血小板数;电子显微镜观察巨核细胞的形态学;倒置显微镜和流式细胞仪观察凝血酶诱导的血小板凝集;ELISA方法检测巨核细胞中cGMP的含量。结果显示,与对照组比较,实验组血小板数量明显增加（P〈0．05）;电子显微镜下可见巨核细胞有明显伪足形成和突出;凝血酶诱导后在倒置显微镜和流式细胞仪上均可观察到血小板凝集现象;GSNO作用后巨核细胞中的cGMP明显升高（P〈0．05）。这些结果提示,GSNO可以促进脐带血CD34^＋细胞来源的巨核细胞产生具有一定功能的血小板,其产生的机制可能部分与cGMP途径有关。     

Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis

Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis.In the present investigation,we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis.The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver.Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis.Platelet aggregation was also measured in the presence of the eNOS inhibitor,diphenylene iodinium chloride (DIC).The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects.During the course of platelet aggregation,concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples,whereas the elevation was not significant in platelets of patients with cirrhosis.A parallel increase was observed in the levels of NO and NOS activity.In the presence of the eNOS inhibitor,platelet aggregation was enhanced and accompanied by an elevated calcium level.The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS.Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis.The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance,and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.     

Combination of hyperglycaemia and hyperlipidaemia induces endothelial dysfunction: Role of the endothelin and nitric oxide systems

Endothelial dysfunction (ED) is a key feature of diabetes and is a major cause of diabetic vasculopathy. Diabetic patients who also exhibit hyperlipidaemia suffer from accelerated vascular complications. While the deleterious effects of high glucose levels (HG) and hyperlipidaemia alone on ED are well established, the effects of combined hyperlipidaemia and HG have not been thoroughly studied. Therefore, the current study examines whether HG and hyperlipidaemia exert synergistic ED, and explores the mechanisms underlying this phenomenon. We applied multi-disciplinary approaches including cultured HUVECs and HMEC-1 as well as knockout mice CByJ.129S7(B6)-Ldlrtm1Her/J (LDLR^−/−) to investigate the mechanisms underlying combined HG and hyperlipidaemia-induced ED. Incremental doses of glucose in the presence or absence of OxLDL were added to HUVECs and HMEC-1. After 5 days, the status of nitric oxide (NO) and endothelin (ET)-1 systems as well as their signal transduction were assessed using Western blot, ELISA and immunoreactive staining. The effects of chronic combination of HG and hyperlipidaemia on endothelial integrity and function as well as alterations in circulatory NO and ET-1 systems were examined in knockout mice LDLR^−/− and their wild-type. HUVEC cells exposed to HG and OxLDL displayed enhanced ET-1 production, more than HG or OxLDL when added alone. Overproduction of ET-1 stems from up-regulation of endothelin converting enzyme (ECE)-1 as observed under these conditions. In contrast, combination of HG and OxLDL dramatically decreased both total endothelial NO synthase (eNOS) by 60%, and activated eNOS (peNOS) by 80%. Moreover, NRF2 decreased by 42% and its active form (pNRF2) by 56%, as compared to baseline. Likewise, ET_B levels decreased by 64% from baseline on endothelial cells. Furthermore, diabetic LDLR^−/− mice displayed a higher blood pressure, plasma triglycerides, cholesterol, ET-1 and NO2/NO3 levels, when compared with normoglycemic LDLR^−/− and BALB mice. Combined hyperglycaemia and hyperlipidaemia activates the ET system and attenuates the nitric oxide system with the Nrf2 signalling pathway. These findings suggest that perturbations in these paracrine systems may contribute to ED.     

Role of the paraventricular nucleus in renal excretory responses to acute volume expansion: role of nitric oxide

Acute volume expansion (VE) produces a suppression of renal sympathetic nerve discharge (RSND) resulting in diuresis and natriuresis. Recently, we have demonstrated that the endogenous nitric oxide (NO) system within the paraventricular nucleus (PVN) produces a decrease in RSND. We hypothesized that endogenous NO in the PVN is involved in the suppression of RSND leading to diuretic and natriuretic responses to acute VE. To test this hypothesis, we first measured the VE-induced increase in renal sodium excretion and urine flow with and without blockade of NO, with microinjection of NG-monomethyl-L-arginine (L-NMMA; 200 pmol in 200 nl), within the PVN of Inactin-anesthetized male Sprague-Dawley rats. Acute VE produced significant increases in urine flow and sodium excretion, which were diminished in rats treated with L-NMMA within the PVN. This effect of NO blockade within the PVN on VE-induced diuresis and natriuresis was abolished by renal denervation. Consistent with these data, acute VE induced a decrease in RSND (52% of the baseline level), which was significantly blunted by prior administration of L-NMMA into the PVN (28% of the baseline level) induced by a comparable level of acute VE. Using the push-pull perfusion technique, we found that acute VE induced a significant increase in NOx concentration in the perfusate from the PVN region. Taken together, these results suggest that acute VE induces an increase in NO production within the PVN that leads to renal sympathoinhibition, resulting in diuresis and natriuresis. We conclude that NO within the PVN plays an important role in regulation of sodium and water excretions in the volume reflex via modulating renal sympathetic outflow.     

胰岛素促进血管内皮细胞产生一氧化氮的实验研究

目的：探讨胰岛素对血管内皮细胞增殖、NO产生和NOS基因表达的影响。方法：培养牛主动脉内皮细胞,测定培养上清液中NO氧化产物NO2^－的水平并应用定量RT－PCR技术检测内皮细胞NOS mRNA的表达水平。结果：①胰岛素对大血管内皮细胞无细胞毒作用,也不影响细胞增殖;②在1－15μg/ml浓度范围内,胰岛素加强内皮细胞释放NO,且呈剂量依赖的方式,NOS特异性抑制剂L－NAME可阻抑之;③胰岛素轻度增加NOS mRNA表达水平,但无统计学意义。结论：胰岛素既不影响大血管内皮细胞增殖,也不影响内皮细胞NOS mRNA表达水平,但以剂量依赖的方式加强内皮细胞产生NO,推测其诱导NO产生的机制可能是通过酶活性的诱导,加速NO的合成。     

Enhancing amplification of late‐outgrowth endothelial cells by bilobalide

Transfusion of autologous late‐outgrowth endothelial cells (OECs) is a promising treatment for restenosis after revascularization. Preparing cells by in vitro amplification is a key step to implement the therapy. This study aimed to demonstrate that bilobalide, a terpenoid, enhances the OEC amplification. Human‐, rabbit‐ and rat OECs and a mouse femoral artery injury model were used. Expanding OECs used endothelial growth medium‐2 as the standard culture medium while exploring the mechanisms used endothelial basal medium‐2. Proliferation assay used MTT method and BrdU method. Migration assay used the modified Boyden chamber. Intracellular nitric oxide, superoxide anion, hydroxyl radical/peroxynitrite and H₂O₂ were quantified with DAF‐FM DA, dihydroethidium, hydroxyphenyl fluorescein and a H₂O₂ assay kit, respectively. Activated ERK1/2 and eNOS were tested with the Western blot. Bilobalide concentration‐dependently enhanced OEC number increase in vitro. Transfusion of bilobalide‐based human OECs into femoral injured athymia nude mouse reduced the intimal hyperplasia. Bilobalide promoted OEC proliferation and migration and increased the intracellular nitric oxide level. L‐NAME, a NOS inhibitor, inhibits but not abolishes OEC proliferation, migration and ERK1/2 activation. Bilobalide concentration‐dependently enhanced the eNOS Ser‐1177 phosphorylation and Thr‐495 dephosphorylation in activated OECs. Bilobalide alleviates the increase in hydroxyl radical/peroxynitrite, superoxide anion and H₂O₂ in proliferating OECs. In conclusion, nitric oxide plays a partial role in OEC proliferation and migration; bilobalide increases OEC nitric oxide production and decreases nitric oxide depletion, promoting the OEC number increase; Bilobalide‐based OECs are active in vivo. The findings may simplify the preparation of OECs, facilitating the implementation of the autologous‐OECs‐transfusion therapy.