心钠素在强啡肽降压效应中的作用

前已报道,吗啡能刺激心钠素的释放。本文报告,静注强啡肽可引起大鼠血压的降低,这一作用可为纳洛酮和抗心钠素IgG预处理所阻断。静注强啡肽可引起大鼠血浆中心钠素水平升高和心房心钠素的下降。实验结果提示,强啡肽的降压效应可能由心房中心钠素释放所致。     

心脏也是一个内分泌器官

1984年以来,加拿大和美国学者从大鼠心房提取物中分离出一类生物活性肽,称心钠素或心房肽(cardinotrin,atriopeptin)。日本学者从人体心房提取物中分离出一种促进尿钠排出的多肽,称α-人心房利尿多     

椎管内注射牛肾上腺髓质22肽差异性翻转吗啡耐受作用

牛肾上腺髓质22肽（bovine adrenal medulla22,BAM22）是脑啡肽原A的一种降解产物,与阿片受体和感觉神经元特异性受体（sensory neuron-specific receptor,SNSR）均有亲合力。本研究的目的是探讨BAM22对吗啡耐受的影响。连续7d对大鼠椎管内注射20μg吗啡形成吗啡耐受后,分为吗啡组、盐水组和BAM22组,第8天三组大鼠椎管内分别注射吗啡、生理盐水和BAM22,第9天三组大鼠椎管内均注射吗啡后,运用撤足反射、福尔马林实验和免疫组织化学等方法观察吗啡的作用效果。结果显示：在撤足反射实验中,BAM22组的吗啡能延长撤足反射潜伏期最大可能作用的48．5％,并持续约1h：在福尔马林实验中,BAM22组的吗啡能分别缩短福尔马林引起的第一期和第二期疼痛行为变化3．2min和24min,比盐水组分别减少45％和82％（P〈0．05,P〈0．001）;此外,在免疫组织化学实验中,BAM22组的吗啡能显著减少热刺激引起的脊髓背角c-Fos蛋白表达,其Ⅰ-Ⅱ层、Ⅲ-Ⅳ层和Ⅴ-Ⅵ层均减少约80％（P〈0．001）。本研究从整体和细胞水平表明,BAM22能翻转吗啡的耐受,这种作用在持续性疼痛模型中的表现要比急性痛中更为明显,显示BAM22对吗啡耐受的差异性调制;同时也提示感觉神经元特异性受体可能参与吗啡耐受的调制。     

吗啡耐受和电针耐受时大鼠脑内血管紧张素原的基因表达加速

我们以往的工作表明,脑内血管紧张素Ⅱ(AⅡ)作为一种抗阿片物质参与吗啡耐受和电针耐受。本工作探讨在此过程中脑内AⅡ的基因表达是否加速。采用酚抽提法提取大鼠脑组织总RNA,使用人工合成的寡脱氧核糖核酸探针进行打点杂交。放射自显影结果表明,多次皮下注射吗啡或连续数小时电针的大鼠脑血管紧张素原(Ang)mRNA含量明显升高。在IBAS图象分析仪上,测量各杂交点自显影曝光斑的积分光密度值(I.O.D.)表明,连续注射吗啡1d或4d使Ang mRNA分别增高1倍或8倍;电针3或6h使Ang mRNA分别增高5倍或13倍。说明大鼠经3—4h吗啡或电针处理,即可引起在转录水平上脑内Ang基因表达加强。     

脊髓GSK-3β信号通路在大鼠慢性吗啡耐受形成中的作用

目的:评价脊髓糖原合成酶激酶3β(GSK-3β)信号通路在大鼠慢性吗啡耐受形成中的作用。方法:健康雄性SD大鼠,体重200~250 g,经枕骨大孔行鞘内置管。取鞘内置管成功的40只大鼠,采用随机数字表法,将大鼠随机分为5组(n=8):生理盐水组(C组)、慢性吗啡耐受组(M组)、吗啡+GSK-3β抑制剂(SB216763)组(MS组)、GSK-3β抑制剂组(S组)和二甲基亚砜组(D组)。皮下注射吗啡10 mg/kg,每天2次,连续5 d,建立吗啡耐受模型。在第6天皮下注射吗啡前30 min MS组、S组和DMSO组分别鞘内注射SB216763(溶于10μl DMSO中)14 pmol、SB216763(溶于10μl DMSO中)14 pmol和DMSO 10μl。于皮下注射吗啡前1 d(基础值)、皮下注射吗啡后30 min第1、2、3、4和5天,第6天鞘内注射后1 h,测定大鼠甩尾潜伏期,以计算最大抗伤害效应百分比(MPAE)。鞘内注射4 h后取8只大鼠,处死后取脊髓组织,采用Western blot法测定p-GSK-3β的表达水平。结果:与第1天比较,M组和MS组第4、5天MPAE明显降低(P0.05);与C组比较,M组和MS组MPAE升高,M组脊髓GSK-3β表达没有变化,MS组脊髓p-GSK-3β表达上调(P0.05),DMSO组和S组上述指标差异无统计学意义;与M组比较,MS组MPAE升高,脊髓p-GSK-3β表达上调(P0.05)。结论:脊髓GSK-3β信号通路可能参与了大鼠吗啡慢性耐受的形成。     

人和大鼠脊髓内的心钠素样物质

应用特异的心钠素免疫金银染色和放射免疫测定法,证明在人和大鼠脊髓内亦存在有心钠素样物质。心钠素免疫金银染色发现在人脊髓各段均有心钠素免疫反应阳性的神经元广泛分布。这些神经元主要位于脊髓腹角,同时脊髓背角和侧角亦有少量分布。应用对照吸收试验,其心钠素免疫反应阳性颗粒便消失或明显减少。心钠素放射免疫测定发现,从大鼠颈髓到胸、腰、骶髓均有心钠素样物质存在,其中以骶髓含量最高,为21.9±4.48ng/g组织;腰髓次之,为3.78±0.74ng/g组织;颈、胸髓含量最低,分别为0.58±0.14和0.46±0.21ng/g组织。应用凝胶过滤和高压液相层析证明,大鼠脊髓中心钠素亦以多分子形式存在,但以28个氨基酸的大鼠心房利纳多肽(rANP)为主。此外,对在体大鼠脊髓蛛网膜下腔灌流研究发现,高钾去极化刺激可使大鼠脊髓心钠素样物质释放。     

慢性吗啡耐受大鼠脑内孤啡肽生成与释放增加

本文彩放射免疫分析法测定了慢性吗啡耐受过程中大鼠脑室灌流液、中脑导水管周围灰质（ＰＡＧ）及杏仁核中孤啡肽（ＯＦＱ）免疫活性的动态变化。结果观察到：（１）大鼠连续５ｄ皮下注射递增剂量的盐酸吗中民慢性吗啡耐受,其脑室灌流中ＯＦＱ－ｉｒ随吗啡注射剂量和注射次数的增加逐渐上升,第５ｄ注射后较对照组升高了５２％;（２）皮下注射吗啡１ｄ、３ｄ、５ｄ的大鼠ＰＡＧ中ＯＦＱ－ｉｒ比对照组分别升高了１７％、４８％和８     

吗啡耐受增加福尔马林致痛大鼠脊髓Fos和NADPH-d阳性神经元的表达

运用Ｆｏｓ免疫组织化学、ＮＡＤＰＨ－ｄ组织化学及Ｆｏｓ／ＮＡＤＰＨ－ｄ双标技术,研究了吗啡耐受对福尔马林致痛大鼠脊髓Ｆｏｓ、ＮＡＤＰＨ－ｄ阳性及Ｆｏｓ／ＮＡＤＰＨ－ｄ双标神经元表达的影响。结果观察到：在非吗啡耐受大鼠,福尔马林诱发的Ｆｏｓ－ｌｉｋｅ ｉｍｍｕｎｏｒｅａｃｔｉｖｉｔｙ（Ｆｏｓ－ＬＩ）主要分布在同侧脊髓背角浅层和颈部,急性静注吗啡可减少Ｆｏｓ－ＬＩ表达;长时间应用吗啡导致福尔马林诱发的     

慢性吗啡给予、戒断及再给药过程中大鼠海马感觉门控的动态变化

药物成瘾被认为是药物长期作用于脑而产生的一种慢性复吸性脑疾病,长期反复的药物（如吗啡）滥用会导致一系列严重后果,如药物依赖、药物耐受、强迫性药物寻求等。本实验利用条件化位置偏好（conditioned place preference,CPP）模型来检测大鼠对吗啡依赖和心理渴求等过程;采用双声刺激听觉诱发电位来研究大鼠在慢性吗啡给予、戒断以及再给药过程中海马感觉门控（N40）的动态变化。吗啡组大鼠注射吗啡（10mg／kg,i．p．）12d,经历第一次戒断12d,再次注射吗啡（2．5mg／kg,i．P．）1d,之后经历第二次戒断2d;对照组大鼠注射同体积生理盐水,其余实验条件与吗啡组相同。CPP实验表明,这种药物给予方法促使大鼠对吗啡产生药物依赖和心理渴求。双声刺激诱发电位实验表明,吗啡组大鼠在吗啡给予期间海马感觉门控受到损伤;第一次戒断期的第1～2天海马感觉门控能力减弱,第3天增强,第4～12天逐渐恢复到正常水平;再次给予吗啡后海马感觉门控能力与对照组相比显著降低,并且随后2d的戒断期内海马感觉门控能力也一直保持较低水平,表明再次给药使大鼠海马感觉门控对吗啡更加敏感化。结果提示,长期反复的吗啡给予及再给药干扰了海马的感觉门控能力,吗啡成瘾对大脑可能产生长期影响。     

缰核痛相关神经元对伤害性刺激和吗啡的反应

目的:观察缰核痛相关神经元对经典镇痛药吗啡的反应,了解缰核的痛觉属性.方法:实验在浅麻醉下的成年大鼠进行.通过脑室插管微量注射,或经五管微电极电泳吗啡、纳络酮、八肽胆囊收缩素(CCK-8)等,并记录缰核内痛相关神经元的单位放电.结果:在内侧缰核、外侧缰核记录的痛相关神经元放电,又可分为痛兴奋性神经元和痛抑制性神经元.在缰核微电泳吗啡后,痛兴奋性神经元以抑制反应为主,痛抑制性神经元以兴奋反应为主.微电泳纳洛酮可以翻转吗啡对缰核的作用.在吗啡耐受大鼠腹腔注射吗啡10 mg/kg,LHb痛相关神经元表现为镇痛效应的数量远大于MHb痛相关神经元的,表明外侧缰核受吗啡的作用程度高于内侧缰核.对吗啡耐受大鼠脑室注入CCK拮抗剂后,再由腹腔注射吗啡,可减弱对吗啡的耐受程度.反之,在腹腔注射吗啡(10 mg/kg)10 min后,侧脑室注射CCK-8(15 ng/10μl),CCK-8可拮抗吗啡对LHb的镇痛作用,但对MHb的拮抗作用不明显.结论:缰核的痛兴奋性神经元和痛抑制性神经元对伤害性(痛)刺激敏感而不易发生适应.其中外侧缰核神经元对吗啡的敏感性高于内侧缰核神经元.     

Synthesis and presence of atrial natriuretic factor in rat ventricle

Rat heart ventricles contained immunoreactive atrial natriuretic factor (irANF) and mRNA for ANF. The size of ANF mRNA in the ventricle was identical with that of the atria. High performance gel filtration chromatography showed that 84% of ventricular irANF elutes at a position corresponding to the low molecular weight form of ANF (99-126) and 16% of irANF elutes at a position corresponding to the precursor form of ANF. The irANF content of the ventricles of spontaneously hypertensive rats was 3 times as much as that of Wistar Kyoto rats. These results suggest that ventricle synthesizes ANF in response to hypertension and processes in a manner different from that in atria.     

Changes in the content of atrial natriuretic factor with the progression of hypertension in spontaneously hypertensive rats

In order to assess possible roles of atrial natriuretic factor (ANF) in spontaneously hypertensive rats (SHR), we examined the content of immunoreactive-ANF in plasma, the atria, hypothalamus and pons of SHR and Wister Kyoto (WKY) rats by radioimmunoassay at different stages of age. With the progression of hypertension, plasma concentration of ANF increased whereas it decreased in the atria in SHR. This suggests ANF is secreted in response to hypertension. On the other hand, at hypothalamus and pons, ANF content was significantly higher in SHR than in WKY rats. This finding suggests possible involvement of ANF in the central regulation of blood pressure.     

Quantification of messenger ribonucleic acid for atrial natriuretic factor in atria and ventricles of Dahl salt-sensitive and salt-resistant rats

The levels of atrial natriuretic factor (ANF) and the mRNA for ANF were measured in the left ventricles of Dahl salt-sensitive (S) and salt-resistant (R) rats. ANF and ANF mRNA were both much higher in ventricular tissue of newborn rats of both strains compared to young adults, which represents the normal developmental pattern. There was no strain difference between S and R when the rats were young (1.5 months of age), but in older animals (8.5 months of age), when S rats were markedly hypertensive, there was a 5- to 10-fold increase in both left ventricular ANF and left ventricular ANF mRNA in S, but not R, rats. Atrial ANF mRNA was not similarly increased in hypertensive S rats. The ANF levels present in ventricles could not be accounted for by contamination with plasma ANF. Moreover, HPLC analysis of the forms of ANF in ventricles of newborn and hypertensive S rats showed that immunoreactive ANF in ventricles was present mainly in the same precursor form found in atria and not the shorter peptide form found in plasma. Northern blot analysis showed that ANF mRNA for atria and ventricles were the same size. It is concluded that in the S rat the heart left ventricle responds to hypertension by increasing production and storage of ANF.     

Capsaicin-sensitive neural pathway mediates atrial natriuretic factor (ANF) release in response to physiological stimuli

Increases in intravascular volume are detected by mechanoreceptors situated at the junctions of the great veins with the atria. We had previously shown that localized distension of the superior vena caval/right atrial junction, simulating increased cardiac preload, elicits release of ANF remotely from the atrial appendage. We proposed that ANF secretion is stimulated via intrinsic neural pathways running from the venoatrial junctions to the appendage. We developed a technique whereby non-adrenergic, non-cholinergic sensory nerves could be selectively destroyed in the heart of adult rats by instilling capsaicin into the pericardial space. Four days later, the animals were killed, and isolated perfused atria were prepared with small balloons positioned so that the superior vena caval/right atrial junction could be discretely stretched. Immunoreactive ANF secretion into the perfusate was measured. Although distension of the venoatrial junction increased ANF secretion from the control atria, there was no such response in the denervated atria. We conclude (A) that local application of capsaicin to the heart of adult rats induces selective functional neural deficits and (B) that information regarding distension of the junction of the great veins and the atria is normally transmitted across the atrium via these nerves to stimulate ANF secretion from peptide stores located in the atrial appendage. We propose that these pathways are crucial to ensure appropriate ANF secretion in response to an increase in circulating blood volume.     

Expression of atrial natriuretic factor gene in heart ventricular tissue

A novel peptide hormone, atrial natriuretic factor (ANF), was recently isolated and characterized in mammalian atria. This hormone has potent natriuretic, diuretic and vasorelaxant activities. Since ANF bioactivity was initially found in atria but not in ventricles, it was assumed that the ANF gene is specifically expressed in atria. We now report that ANF mRNA is present in ventricular tissue as well as in atria. This is clearly demonstrated by in situ hybridization and by Northern blot analysis. Rat ventricular ANF mRNA concentration is a hundred-fold lower than in atria. As in atria, the 126 amino acids precursor form of ANF is predominant in ventricles and it is present at a thousand-fold lower concentration. The ten-fold discrepancy in the ratio of ANF mRNA to immunoreactivity between atria and ventricles could reflect a higher rate of peptide release in the latter. Thus, ventricular ANF production may be physiologically significant in view of the much larger ventricular mass.     

Atrial natriuretic factor: radioimmunoassay and effects on adrenal and pituitary glands

A simple and sensitive radioimmunoassay was developed for measurement of immunoreactive atrial natriuretic factor (IR-ANF) in rat and human plasma and in rat atria. The two atria contain about 20 micrograms ANF per rat. The right atrium contained 2.5 times more ANF than did the left. Ether anesthesia and morphine markedly increased IR-ANF in rat plasma. The concentration of IR-ANF in plasma of clinically normal human subjects was 65.3 +/- 2.5 pg/ml. Paroxysmal tachycardia and rapid atrial pacing significantly increased IR-ANF in human plasma. Two- to seven-fold higher concentrations were found in coronary sinus blood than in the peripheral circulation. In the plasma of rats and humans, circulating ANF is probably a small-molecular-weight peptide. ANF acts on the adrenal and the pituitary. ANF inhibits aldosterone secretion from rat zona glomerulosa and steroid secretion by bovine adrenal zona glomerulosa and fasciculata. ANF stimulated the basal secretion of arginine vasopressin (AVP) in vitro and inhibited KCl-stimulated release of AVP.     

Intra-atrial communication and control of atrial natriuretic factor (ANF) release

Atrial natriuretic factor (ANF) release was studied in isolated perfused atria prepared from rats. When the vein-atrial junction (VAJ) was distended with an inflatable balloon, ANF release into the perfusate was greater in intact atria than in appendectomized atria. It was concluded that distention of the VAJ causes ANF release from the atrial appendage. A cascade experiment was then prepared whereby buffer from one isolated atrium perfused a second atrium. Although the VAJ of the first atrium could be distended by balloon, the atrial appendage was ligated so ANF was not secreted into the perfusate. The second atrium was intact, but no balloon was inserted. Despite the fact that there were no changes in intraluminal pressure, ANF secretion from the second atrium increased when the VAJ of the first atrium was distended. This response was blocked by the endothelin (ET) A receptor antagonist BQ-123. However, no distention-induced changes in ET-1 levels could be found in the perfusate from the first atrium. It is proposed that, in response to changes in distention of the VAJ, ANF is released remotely from the atrial appendage. The mediator does not appear to be ET-1 itself, but rather some factor that stimulates ET-1-induced ANF release within the tissue of the atrial appendage.     

Effects of changes in water-sodium balance on levels of atrial natriuretic factor messenger RNA and peptide in rats

Responses of atrial mRNA, atrial peptide and plasma peptide of atrial natriuretic factor (ANF) to treatments to alter fluid volume were studied in rats using RNA dot hybridization assay and radioimmunoassay. Specific changes in the level of ANF mRNA relative to total atrial RNA were observed in atria from sodium restricted rats and water deprived then sodium loaded rats, demonstrating an association of change in water-sodium balance with the expression of ANF gene. The levels of mRNA and the immunoreactive ANF in plasma decreased to 30% and 15% of controls, respectively, on water-deprivation and then increased again to control levels after administering 1.8% NaCl solution, whereas atrial immunoreactive ANF increased to about twice the control on water-deprivation and decreased again after supplying NaCl solution, in parallel with the level of the hematocrit. These findings suggest that atrial ANF content is dependent more on ANF release than on biosynthesis.     

Effect of atrial natriuretic factor on arterial blood pressure and frequency of heart contraction in rats with genetically determined hypertension and in normotensive rats

An effect of peptide released by the heart atria of mammals and called the atrial natriuretic factor (ANF) on blood pressure and heart contractions was studied in rats with genetically determined arterial hypertension (SHR) and in normotensive Wistar-Kyoto rats (WKY). Nine male SHR rats and 11 male WKY rats, aged between 12 and 16 weeks, were given normal saline infusion for 30 minutes through implanted catheters to both ulnar vein and artery. Then, an infusion of ANF at the rate of 0.3 microgram/kg per hour followed for 35 minutes. An infusion of ANF produces significant decrease in the mean arterial blood pressure, systolic and diastolic pressures without significant effect on pulse pressure and heart contractions. AFN infusion with the same rate did not produce any significant differences in the arterial blood pressure and heart contractions in Wistar-Kyoto rats. The obtained results suggest that ANF may play a role in pathogenesis of the arterial blood hypertension.     

ADP-ribosylation of isolated rat islets of Langerhans

A rapid and reproducible radioimmunoassay method was developed for rat atrial natriuretic factor (ANF)-IV. The method is also applicable to human atrial peptide. ANF was detected in rat hypothalamus (5.03 pmoles/g tissue), right (86.8 pmoles/mg tissue) and left atria (52.5 pmoles/mg tissue), and plasma (156 fmoles/ml). After high salt intake immunoreactive ANF in atria and plasma increased significantly, while a significant decrease was observed in hypothalamus. Gel chromatography revealed high and low molecular weight ANF in atria and hypothalamus while only a low molecular weight form was found in plasma.