三磷酸腺苷对蟾蜍交感神经节细胞的作用及机制分析

目的　观察ATP所引起的蟾蜍椎旁交感神经节细胞电位变化和有关因素的影响 ,以探讨ATP在神经节内的作用及其机制。方法　采用细胞外微电极技术记录离体灌流神经节电位。结果　外源性ATP(2 0 0 μmol L)可引起去极化 (n =1 1 0 ) ,超极化 (n =33) ,以及去极后伴随一超极过程的双相反应 (n =1 2 )。P2 嘌呤受体拮抗剂ci bacronblue 3GA(30 0 μmol/L)和奎尼丁 (30 0 μmol/L) ,均可抑制ATP所致去极反应 ,其幅值分别减小 (2 4 .2± 1 7.5 ) %(n =1 8,P <0 .0 1 )和 (32 .5± 1 1 .5 ) % (n =2 1 ,P <0 .0 1 )。P1 嘌呤受体拮抗剂氨茶碱 (2 0 0 μmol L) ,可抑制ATP所致超极反应 ,幅值减小 (6 5 .0± 2 2 .9) % (n =9,P <0 .0 1 )。在无钠溶液中 ,ATP的去极幅值变化为 (1 3.6± 1 7.5 ) % (n =1 0 ,P >0 .0 5 ) ,无统计学意义。在无钙溶液中或同时加用EDTA(1mol L)以去除溶液的Ca2 ,可使ATP所致去极幅值减小 (2 3.6± 1 8.3) % (n =1 5 ,P <0 .0 1 )。电压依赖性钾通道阻断剂 4—氨基吡啶 (3mmol L)和ATP敏感性钾通道阻断剂格列苯脲 (1 0 0mol L) ,均可可抑制ATP所致超极反应 ,其幅值分别减小 (6 4 .5± 2 1 .9) % (n =1 1 ,P <0 .0 1 )和(6 6 .4± 2 2 .4 ) % (n =6 ,P <0 .0 1 )。前列腺素合成酶抑制剂吲     

大鼠三叉神经节神经元膜P2X嘌呤受体的特征

采用全细胞膜片钳技术研究了大鼠三叉神经节（trigeminal ganglion,TG）神经元膜P2X嘌呤受体的特征。结果发现：大部分受检细胞（78．9％,142／180）对ATP敏感,ATP．激活电流有明显的浓度依赖性。少数细胞无反应（21．1％,38／180）。在对ATP敏感的142个细胞中,绝大部分引起一内向电流（95．1％,135／142）,少数为外向电流（2．1％,3／142）,另有部分细胞出现双相电流（2．8％,4／142）。引起的内向电流在小直径细胞（〈30μm）上多表现为快去敏感电流,对vanilloid高度敏感;在中等大小的细胞（30～40μm）上多表现为慢去敏感电流,对vanilloid不敏感：绝大多数大细胞（〉40μm）对ATP和vanilloid均不敏感。此外,电流的波形与细胞直径密切相关。无论小细胞还是中等细胞其I-V曲线均表现出明显的内向整流趋势。我们还研究了ATP-激活电流的动力学特征,并观察了P2嘌呤受体激动剂、拮抗剂的效应。结果提示：不同类型的ATP受体．离子通道在不同类型的TG神经元上的表达具有不同的特点。     

交感神经节细胞内P物质受体与5—羟色胺受体的共存及其联系

应用离体神经节细胞内记录,观察到在１３３个鼠腹腔神经节细胞及１２９个肠系膜下社会节细胞中,分别有６６个细胞及４７个细胞对Ｐ物质及５－羟色胺同时敏感,分别有４０个细胞及６５个细胞仅对其中一种递质敏感,另有２７个细胞及１７个细胞对其中一种递质敏感。     

神经系统中的嘌呤信号

三磷酸腺苷(ATP)作用于嘌呤受体(P2受体),引起离子通道开放或通过第二信使调节神经细胞功能,不仅参与了特殊感觉、神经元与神经胶质细胞相互作用等生理活动,而且参与了神经损伤修复和疼痛等病理过程.神经系统中的嘌呤信号系统研究,不仅为解释神经系统生理功能及其病理过程提供了新的思路,而且为治疗神经系统损伤和疼痛等疾病开辟了新的希望.     

β-蝮蛇毒素对蟾蜍交感神经节突触传递的影响

β-蝮蛇毒素(β-agkistrodotoxin简写β-AgTX)对骨胳肌神经肌肉接头的作用已有实验分析,本文则观察了β-AgTX对蟾蜍交感神经节胆碱能性和非胆碱能性突触电位的作用。结果表明,β-AgTX对胆碱能性快兴奋性突触后电位(f-EPSP)和由压力微量注射ACh产生的ACh电位快成分有可逆性抑制作用,且对f-EPSP的幅值抑制率明显大于对ACh电位的抑制率,方差分析显示β-AgTX对f-EPSP和对ACh电位的抑制之间的差异显著(P<0.01)。β-AgTX对非胆碱能性迟慢兴奋性突触后电位(1s-EPSP)无明显作用。本结果提示β-AgTX可能是通过抑制节前神经末梢释放AGh的突触前机制和占据突触后N型胆碱能受体影响ACh的作用之突触后机制,抑制蟾蜍交感神经节的胆碱能性传递过程。     

交感神经节细胞对P物质和5-羟色胺的反应

本工作旨在观察Ｐ物质（ＳＰ）受体与５－羟色胺（５－ＨＴ）受体是否分别还是同时存在于豚鼠腹腔神经节（ＣＧ）与肠系膜下神经节（ＩＭＧ）不同细胞,以及这两种递质之间是否存在相互作用。在１３３个ＣＧ细胞中,６６个（４９．６％）对ＳＰ及５－ＨＴ同时敏感,４０个（３０．１％）仅对其中一种递质敏感,此外２７个（２０．３％）对两都不敏感。     

尖吻蝮蛇毒对蟾蜍交感神经节细胞电活动的影响

用细胞内电位记录技术,以离体蟾蜍交感链为标本,观察了尖吻蝮蛇毒(AAV)的神经毒性作用。结果表明,该蛇毒(10-200μg/ml)对交感神经元的静息电位、膜电阻和膜电容没有显著的作用,对动作电位形状也无可测出的影响,但能使阈电位轻皮升高,即使神经元兴奋性稍降低。AAV(>25μg/ml)对胆碱能性的快兴奋性突触后电位有剂量依从性的,部分可逆性的抑制作用。其作用机制至少应部分归之于AAV对突触后膜上的N型胆碱能受体的阻断作用。本研究发现的AAV神经毒性作用可能有一定实践意义。     

三磷酸腺苷对小鼠骨骼肌成纤维细胞NOR-10迁移作用及分子机制初步研究

目的:探讨三磷酸腺苷(ATP)对小鼠骨骼肌成纤维细胞的迁移作用及其可能机制。方法:细胞划痕实验检测1μM、10μM、100μMATP对NOR-10细胞愈合率的影响;细胞迁移小室实验检测空白对照组、100μM ATP组、30μM PPADS+100μM ATP组、100μM RB2+100μM ATP组细胞的迁移率。结果:细胞划痕实验及迁移实验表明高浓度ATP能够促进NOR-10细胞的迁移能力,100μM ATP促细胞迁移能力最强(P0.05),并且其促迁移作用能被30μM PPADS,100μM RB2所抑制(P0.05),但100μM RB2的抑制作用更强(P0.05)。结论:高浓度ATP(10μM)能够促进NOR-10细胞的迁移能力,并且其促迁移可能通过激活P2Y受体作用大于P2X受体。     

皮质醇对离体牛蛙交感神经节B细胞胆碱能突触传递的快速作用

在离体灌流的牛蛙交感神经节标本上,电刺激节前纤维,细胞内记录Ｂ细胞的电活动,观察给予皮质醇对Ｂ细胞突触传递作用。主要结果有：（１）电刺激节前纤维,细胞内记录１７０个Ｂ细胞的动作电位,给予皮质醇后０．５￣３ｍｉｎ内,５２个Ｂ细胞的突触传递发生脱漏甚至完全阻断,有明显的量效关系。甾体激素胞内受体阻断剂ＲＵ３８４８６可部分阻断这种作用。（２）蛋白合成抑制剂放线菌酮不能阻断皮质醇的快速阻断作用。（３）阿托     

NaHS对ATP诱导大鼠小胶质细胞P2X受体表达的影响

目的：观察硫化氢（H₂S）供体硫氢化钠（NaHS）对ATP致伤的大鼠小胶质细胞细胞活力、细胞膜通透性及P2X7受体表达的影响。方法：实验取对数期形态结构及生长分化良好的大鼠小胶质细胞,随机分4组,每组设3个复孔。①正常对照组：常规培养,不进行ATP处理。②ATP组：接种细胞24 h后ATP处理。③NaHS+ATP组：NaHS预先孵育30 min后再用ATP处理,并且NaHS始终存在于反应体系中。④KN-62（P2X₇受体阻断剂）+ATP组：KN-62预先孵育30 min,其余同NaHS+ATP组。MTT检测各组细胞活力,荧光染料YO-PRO-1检测各组相对荧光单位（RFU）反映膜的通透性,Western blot检测各组P2X₇受体表达水平。结果：①与对照组相比,不同浓度的ATP （1、3、5、10 mmol/L）作用3 h均可明显降低大鼠小胶质细胞活力,NaHS （200 μmol/L）干预后大鼠小胶质细胞活力较ATP组明显增加（P<0.01）,但NaHS达400 μmol/L浓度时,其保护作用未进一步增加。②随着ATP浓度的增加,大鼠小胶质细胞内YO-PRO-1的荧光强度显著增加,NaHS预处理可明显减少细胞对YO-PRO-1的摄取（P<0.01）。③ATP （3 mmol/L）能上调P2X₇受体蛋白表达水平,而NaHS （200 μmol/L）预孵育则可明显抑制ATP引起的P2X₇受体蛋白表达的上调（P<0.01）。结论：NaHS可减少ATP致伤的大鼠小胶质细胞的P2X₇受体表达、降低通透性、增加细胞活力,提示调控P2X₇受体的表达和功能可能是H₂S神经保护作用的重要环节。     

Regulation of Cyclic AMP Accumulation in a Rat Sympathetic Ganglion: Effects of Vasoactive Intestinal Polypeptide

Cyclic AMP accumulation in rat superior cervical ganglia during synaptic activity occurs by a noncholinergic, nonadrenergic process. Both preganglionic nerve stimulation and 4-aminopyridine increase ganglion cyclic AMP levels in the presence of atropine or phentolamine. Of the polypeptides tested as putative transmitters, vasoactive intestinal polypeptide (10(-6) M) causes ganglion cyclic AMP accumulation comparable to that produced by preganglionic nerve stimulation.     

Effect of Increasing Choline, In Vivo and In Vitro, on the Synthesis of Acetylcholine in a Sympathetic Ganglion

Abstract: The experiments described in this paper were designed to test whether increasing choline availability over normal physiological levels increases acetylcholine synthesis in the cat's superior cervical ganglion. When ganglia were perfused with Krebs solution, an increase in the medium's choline concentration over physiological (10⁻³M) levels increased tissue choline but did not increase tissue acetylcholine or the release of acetylcholine from stimulated ganglia. However, increasing plasma choline in the whole animal increased ganglionic acetylcholine levels. The basis for this difference in the effects of in vivo and in Vitro exposure to elevated choline levels on the tissue acetylcholine content was found to involve plasma factor(s), rather than indirect actions of choline, and the acetylcholine content of isolated ganglia was increased when the tissue was perfused with plasma, instead of Krebs solution, containing 10⁻³M-choline. The extra acetylcholine generated by this procedure was associated with a subsequent transient increase in transmitter release during short intervals of stimulation, but most of the extra acetylcholine was not readily available for release from stimulated ganglia. It is concluded that increasing choline available to sympathetic ganglia over physiological concentration does not have a sustained effect on the turnover of releasable transmitter under the conditions of these experiments.     

Effects of Lead Ions on the Synaptic Responses of Neurons of the Rat Sympathetic Ganglion

Using intracellular recording, we studied the effects of lead ions on the synaptic responses of neurons of the superior cervical ganglion (SCG) of the rat. Three groups of SCG neurons were found; they differed from each other in directions of the changes evoked by Pb²⁺ applications and in their sensitivity to this agent. In two groups of the phasic SCG neurons, Pb²⁺ decreased the amplitude of EPSP evoked by stimulation of the cervical sympathetic nerve with EC₅₀ of (2.2 ± 0.2) · 10^-6 M and (3.55 ± 0.29) · 10^-5 M, respectively. In the tonic neurons, 2 · 10^-6 M Pb²⁺ increased the EPSP amplitude by 27 ± 2%, on average, as compared with the control values. We postulate that the specificity of responses of these three groups of SCG neurons to the action of Pb²⁺ is determined by the different subunit composition of the nicotinic cholinoreceptors in the membrane of these cells.     

Effects of Salicylic Acid on Heavy Metal-Induced Membrane Deterioration Mediated by Lipoxygenase in Rice

Deterioration of membranes caused by lipoxygenase (LOX) activity under 10 μM PbCl₂ or 10 μM HgCl₂ was partially alleviated by the exogenous application of 100 μM salicylic acid (SA). In two cultivars of rice (Oryza sativa L. cvs. Ratna and IR 36), the presence of SA ameliorated the increased leakage of electrolytes, injury index, and the content of malondialdehyde caused by these heavy metals. Lead decreased H₂O₂ content whereas Hg increased it in both cultivars. Application of SA increased H₂O₂ in presence of Pb, while decreased it in presence of Hg. Both Pb and Hg decreased superoxide dismutase activity, while increased peroxidase activity. The activity of catalase was decreased by Hg but increased by Pb and SA reversed their effects. Thus, SA ameliorated the damaging effects of Pb and Hg on membranes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of Anesthetics on the Renal Sympathetic Response to Anaphylactic Hypotension in Rats

The autonomic nervous system plays an important role in rat anaphylactic hypotension. It is well known that sympathetic nerve activity and cardiovascular function are affected by anesthetics. However, the effects of different types of anesthesia on the efferent renal sympathetic nerve activity (RSNA) during anaphylactic hypotension remain unknown. Therefore, we determined the renal sympathetic responses to anaphylactic hypotension in anesthetized and conscious rats and the roles of baroreceptors in these responses. Sprague-Dawley rats were randomly allocated to anesthetic groups that were given pentobarbital, urethane, or ketamine-xylazine and to a conscious group. The rats were sensitized using subcutaneously injected ovalbumin. The systemic arterial pressure (SAP), RSNA and heart rate (HR) were measured. The effects of sinoaortic baroreceptor denervation on RSNA during anaphylaxis were determined in pentobarbital-anesthetized and conscious rats. In all of the sensitized rats, the RSNA increased and SAP decreased after antigen injection. At the early phase within 35 min of the antigen injection, the antigen-induced sympathoexcitation in the conscious rats was significantly greater than that in the anesthetized rats. Anaphylactic hypotension was attenuated in the conscious rats compared to the anesthetized rats. The anesthetic-induced suppression of SAP and RSNA was greater in the order ketamine-xylazine >urethane = pentobarbital. Indeed, in the rats treated with ketamine-xylazine, RSNA did not increase until 40 min, and SAP remained at low levels after the antigen injection. The baroreceptor reflex, as evaluated by increases in RSNA and HR in response to the decrease in SAP induced by sodium nitroprusside (SNP), was suppressed in the anesthetized rats compared with the conscious rats. Consistent with this finding, baroreceptor denervation attenuated the excitatory responses of RSNA to anaphylaxis in the conscious rats but not in the pentobarbital-anesthetized rats. RSNA was increased markedly in conscious rats during anaphylactic hypotension. Anesthetics attenuated this antigen-induced renal sympathoexcitation through the suppression of baroreceptor function.     

Circadian Variation in the Response to Experimental Endotoxemia and Modulatory Effects of Exogenous Melatonin

Disturbances in circadian rhythms are commonly observed in the development of several medical conditions and may also be involved in the pathophysiology of sepsis. Melatonin, with its antioxidative and anti-inflammatory effects, is known to modulate the response to endotoxemia. In this paper, we investigated the circadian variation with or without melatonin administration in an experimental endotoxemia model based on lipopolysaccharide (LPS). Sixty male Sprague-Dawley rats were assigned to six groups receiving an intraperitoneal injection of either LPS (5?mg/kg), LPS?+?melatonin (1?mg/kg), or LPS?+?melatonin (10?mg/kg) at either daytime or nighttime. Superoxide dismutase (SOD) was analyzed in liver samples collected after decapitation. Furthermore, inflammatory plasma markers (cytokines interleukin [IL]-6, IL-10) and oxidative plasma markers (ascorbic acid [AA], dehydroascorbic acid [DHA], and malondialdehyde [MDA]) were analyzed before and 5?h after the onset of endotoxemia. There were significant higher levels of SOD (p?<?0.05), IL-6 (p?<?0.01), and IL-10 (p?<?0.05) during nighttime endotoxemia compared with daytime. At daytime, melatonin 1 and 10?mg reduced the levels of MDA and increased SOD, IL-6, IL-10, and DHA (p?<?0.05). At nighttime, melatonin reduced the levels of MDA and increased DHA (p?<?0.05). Additionally, 10?mg melatonin resulted in lower levels of AA during daytime (p?<?0.05). No dose relationship of melatonin was observed. The results showed that the response induced by experimental endotoxemia was dependent on time of day. Melatonin administration modulated the inflammatory and oxidative stress responses induced by endotoxemia and also resulted in higher levels of antioxidants during daytime. The effect of circadian time on the endotoxemia response and possible modulatory effects of melatonin need further investigations in a human endotoxemia model.     

Protective Effects of Adeno-associated Virus Mediated Brain-derived Neurotrophic Factor Expression on Retinal Ganglion Cells in Diabetic Rats

Adeno-associated virus vector plasmid carrying the expression cassette of brain-derived neurotrophic factor (BDNF), pAAV-BDNF, was constructed and packaged into recombinant adeno-associated virus (rAAV-BDNF). The rAAV-BDNF was intravitreally injected into streptzotocin (STZ)-induced diabetic Sprague–Dawley (SD) Rats. Data showed that over-expression of BDNF could increase alive retinal ganglion cell (RGC) number and improve its function in streptzotocin(STZ)-induced diabetic rats, which might be a new method to treat diabetic neuropathy and retinopathy.     

Mobilization of the Readily Releasable Pool of Acetylcholine from a Sympathetic Ganglion by Tityustoxin in the Presence of Vesamicol

The present experiments tested whether preganglionic stimulation and direct depolarization of nerve terminals by tityustoxin could mobilize similar or different pools of acetylcholine (ACh) from the cat superior cervical ganglia in the presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol, AH5183), an inhibitor of ACh uptake into synaptic vesicles. In the absence of vesamicol, both nerve stimulation and tityustoxin increased ACh release. In the presence of vesamicol, the release of ACh induced by tityustoxin was inhibited, and just 16% of the initial tissue content could be released, a result similar to that obtained with electrical stimulation under the same condition. When the impulse-releasable pool of ACh had been depleted, tityustoxin still could release transmitter, amounting to some 10% of the ganglion's initial content. This pool of transmitter seemed to be preformed in the synaptic vesicles, rather than synthesized in response to stimuli, as tityustoxin could not release newly synthesized [3H]ACh formed in the presence of vesamicol, and hemicholinium-3 did not prevent the toxin-induced release. In contrast to the results with tityustoxin, preganglionic stimulation could not release transmitter when impulse-releasable or toxin-releasable compartments had been depleted. Our results confirm that vesamicol inhibits the mobilization of transmitter from a reserve to a more readily releasable pool, and they also suggest that, under these experimental conditions, there might be some futile transmitter mobilization, apparently to sites other than nerve terminal active zones.