一氧化氮对过氧化氢所致听力损失的保护作用

通过全耳蜗灌流法在体观察一氧化氮(N0)能否通过一氧化氮/环磷酸鸟苷(NO/cGMP)途径对抗过氧化氢这种氧自由基所致的听力损失。实验选用耳廓反射灵敏、无耳毒性药物使用史的健康杂色豚鼠(250-350 g)50只,雌雄不拘,随机分为5组,每组10只动物,分别行全耳蜗灌流人工外淋巴液;过氧化氢(H2O2);L-精氨酸(合成NO的底物);H2O2+L-精氨酸;H2O2+L-精氨酸+L-NNA(一氧化氮合成酶的抑制剂),均灌流2 h。通过圆窗龛电极,每隔30 min记录复合动作电位(compound action potential,CAP:由短声Click诱发)阈值,耳蜗微音器电位(cochlear microphonic,CM;由短纯音Tone Burst诱发)幅度,了解耳蜗功能的变化,并分离取出耳蜗基底膜并制备基底膜硬铺片,通过碘化毗啶(PI)和Hoecbst双染色方法,观察耳蜗组织各类细胞损伤情况。结果显示,灌流H2O2+L-精氨酸组的CAP阈移和CM下降幅度值明显低于单独灌流H2O2组,差异有显著性(氏P<0.05);前者形态学观察未见明显的细胞损伤,后者可见大量坏死红染的细胞。H2O1+L-精氨酸+L-NNA组CAP阈移和CM下降幅度与单独灌流H2O2组比较无统计学差异。实验结果提示NO可能通过NO/cGMP途径部分对抗过氧化氢所致的听力损失。     

用共聚焦显微镜观察ACh及ATP对豚鼠耳蜗外毛细胞Ca^2＋的调控

用激光扫描共聚焦显微镜研究了一般公认的耳蜗传出神经递质乙酰胆碱（ＡＣｈ）和三磷酸腺苷（ＡＴＰ）对豚鼠耳蜗外毛细胞（ＯＨＣｓ）胞内游离Ｃａ＾２＋浓度（Ｃａ＾２＋）的作用,ＯＨＣｓ用Ｃａ＾２＋敏感荧光染料Ｆｌｕｏ－３着色,胞内Ｃａ＾２＋的分布以细胞底部稍强。ＡＣｈ在ＯＨＣ底部引起Ｃａ＾２＋的缓慢上长并维持在一个较高水平。ＡＴＰ在整个ＯＨＣ引起一个急剧的Ｃａ＾２＋升高,升高幅度在ＯＨＣ顶部最大。随着ＡＴ     

GABA对大鼠海马谷氨酸受体/NO/cGMP神经通路的调控作用

海马内谷氨酸受体 /ＮＯ/ｃＧＭＰ神经通路在许多病理生理过程中产生非常重要的作用 ,如学习记忆、神经退行性病变等。但关于海马内兴奋性神经环路如何被调控尚不清楚。前期工作表明ＧＡＢＡ可能对海马内兴奋性神经环路起着调控作用 ,关于该方面的研究尚未见报道。本实验应用脑微透析技术探讨ＧＡＢＡ对大鼠海马谷氨酸受体 /ＮＯ/ｃＧＭＰ神经通路的调控作用。1　材料和方法(1 )试剂　 2 ,3 ｄｉｈｙｄｒｏｘｙ 6 ｎｉｔｒｏ 7 ｓｕｌｆａｍｏｙｌ ｂｅｎｚｏ(ｆ) ｑｕｉｎｏｘａｌｉｎｅ(ＮＢＱＸ ,英国ＴｏｃｒｉｓＣｏｏｋｓｏｎ公司…     

卡那霉素耳慢性中毒对豚鼠耳蜗毛细胞中Bcl-2表达的影响

目的:探讨卡那霉素耳慢性中毒对豚鼠耳蜗毛细胞中Bcl-2表达的影响。方法:取20只豚鼠随机分为2组,实验组连续14d肌肉注射硫酸卡那霉素,200mg/(kg.d),对照组连续14d等量肌肉注射生理盐水,停药14d处死动物后制作耳蜗标本,处死前检测其ABR的变化,免疫组化及原位杂交法测定Bcl-2的表达。结果:豚鼠卡那霉素耳慢性中毒后,ABR阈值较对照组明显上升,Bcl-2阳性表达减低,与对照组比较差异有显著性(P<0.05)。结论:卡那霉素耳慢性损害可能与抑制Bcl-2的表达有关。     

庆大霉素对豚鼠耳蜗一氧化氮合酶活性的影响

目的：研究庆大霉素（ＧＭ） 致聋豚鼠耳蜗一氧化氮合酶（ＮＯＳ） 的分布及其变化,探讨一氧化氮（ＮＯ） 与ＧＭ 耳毒机制的关系。方法：ＮＡＤＰＨｄ 酶组化方法及显微图像分析技术。结果：ＧＭ 致聋后,ＮＯＳ 阳性反应物密度在血管纹、内毛细胞、外毛细胞、螺旋神经节、听神经纤维的分布均比对照组明显增高（ Ｐ＜ ０ ．００１）;上述部位的ＮＯＳ平均灰度值降低与对照组相比有显著差异（ Ｐ＜ ０ ．００１）;ＡＢＲ 听阈变化与ＮＯＳ平均密度及灰度值变化高度相关（ 上述各部位ｒ 值范围,ｒ密度听阈：０ ．９１４０～０．８６９８;ｒ灰度听阈：－０ ．７８９２ ～－ ０．９３４７;Ｐ＜ ０．０１）。结论：①ＮＯ 可能与ＧＭ的耳毒作用机制有关,是耳毒作用的重要因素之一;②ＧＭ 耳毒作用部位不仅发生在血管纹和外毛细胞,而且还涉及到内毛细胞、螺旋神经节及听神经纤维     

传出神经递质ACh及耳蜗活性物质ATP对耳蜗外毛细胞的作用

本文综述了耳蜗传出神经对耳蜗外毛细胞的调控作用。从解决和组织化学等形态学角度分析了传出神经纤维在耳蜗的分布特点，并从形态和生理两方面进一步证实了ＡＣＨ是耳蜗传出神经递质之一。近年来，应用离体耳蜗毛细胞的膜片钳和荧光测钙技术，人们对于ＡＣＨ，ＡＴＰ对外毛细胞的调控作用，Ｃａ＾２＋在其中的介导作用及其ＡＣＨＲ和Ｐ２Ｒ的药理分型有了更多的认识，但尚未有统一明确的结论。     

硒的抗癌机制研究——亚硒酸钠对环磷鸟苷的作用

 AFB_1和NDEA与正常小鼠肝切片培养5min即可使cGMP分别升高一倍及一倍半(p<0.001);当有Na_2SeO_3(1μg/mL培养液)存在时,此作用可被消除,单独加入Na_2SeO_3对cGMP无影响。培养介质中存在磷酸二酯酶抑制剂茶碱时,除cGMP基础水平提高外,上述作用均相同。荷腹水型肝癌(HepA)小鼠腹水细胞中cGMP比正常肝中cGMP水平高三倍,连续4天腹腔给硒(Na_2SeO_3、1mg/kg体重)可使癌细胞中cGMP下降一倍。此剂量对正常小鼠肝中cGMP无影响;Na_2SeO_3(1μg/mL)与腹水细胞在体外短时间培养时,对细胞存活无影响,却可使cGMP含量下降。     

缓激肽对大鼠背根神经节分离神经元ATP激活电流的调制作用

在新鲜分离大鼠背根神经节（ＤＲＧ）的５６个细胞标本上,应用全细胞膜片箝技术进行记录。胞外加缓激肽（ＢＫ,１０＾－６￣１０＾－４ｍｏｌ／Ｌ）引坊的ＤＲＧ细胞膜反应结果如下：（１）７１．４％的细胞为内向电流,其电流反应的幅值具有明显的浓度信赖性;（２）１２．５％的细胞为外向电流;（３）１６．１％的细胞未引起可检测的膜反应,单独给予ＡＴＰ（１０＾－６￣１０＾－３ｍｏｌ／Ｌ）在大多数受栓细胞（５４／５６）     

噪声刺激后豚鼠耳蜗抗氧化能力的变化和α-硫辛酸对声损伤的保护作用

实验探讨了声刺激后豚鼠血清总抗氧化能力(total antioxidant capacity,TAC)和耳蜗组织一氧化氮(nitricoxide,NO)含量的变化及α-硫辛酸的抗氧化和对声损伤的保护作用。将豚鼠(350-400 g)随机分为无噪声对照组(n=20)、噪声+生理盐水组(n=20)和噪声+α-硫辛酸组(n=20)。噪声刺激(4.kHz倍频程,115 dB SPL 5 h)结束后立即测试脑于诱发电位(auditory brainstem responses,ABRs),取血清测TAC,制备耳蜗组织匀浆测NO的水平。所得结果如下:(1)无噪声对照组,动物听阈无明显变化;噪声刺激后生理盐水组,听阈上升的幅度明显高于α-硫辛酸组(P<0.05)。(2)噪声+生理盐水组,TAlC明显低于无噪声对照组(P<0.05);噪声+α-硫辛酸组同噪声+生理盐水组相比,TAC明显升高(P<0.05),同无噪声对照组相比,无显著性差异(P>0.05)。(3)噪声+生理盐水组,NO含量高于无噪声对照组(P<0.05);噪声+α-硫辛酸组同噪声+生理盐水组相比,NO含量明显减少(P<0.05),同无噪声对照组相比,差异无显著性(P>0.05)。上述结果提示,噪声刺激后血清TAC降低,耳蜗组织内NO含量增加;α-硫辛酸可通过抗氧化机制对噪声性听力损伤(noise induced hearing loss,NIHL)发挥保护作用。     

骨骼肌中肌浆网/内质网钙ATP酶的功能及其作用机制

骨骼肌是机体生命活动和能量代谢的重要场所,其代谢紊乱会诱发一系列肌肉疾病。Ca²⁺作为肌肉收缩过程的重要调节器,在骨骼肌的功能行使中发挥重要作用。骨骼肌细胞中Ca²⁺浓度主要受肌浆网/内质网钙ATP酶(sarcoplasmic/endoplasmic reticulum Ca²⁺ATPase, SERCA)的调节。SERCA利用ATP水解产生的能量介导胞质Ca²⁺进入肌浆网内腔,维持胞质Ca²⁺平衡。SERCA功能的失调会引发一系列骨骼肌疾病,而SERCA活性受部分肌浆网蛋白质的调控,跨膜蛋白质PLN、SLN、MRLN、DWORF和sAnk1以及胞质蛋白质THADA和SAR,其通过磷酸化,进而调控SERCA的功能。本文对骨骼肌中SERCA的功能、调控SERCA的相关功能蛋白质的结构及其作用机制进行了总结,以期为骨骼肌相关疾病的治疗提供最新的思路和方法。     

Exosomes derived from smooth muscle cells ameliorate diabetes‐induced erectile dysfunction by inhibiting fibrosis and modulating the NO/cGMP pathway

Erectile dysfunction (ED) is a major health issue among men with diabetes, and ED induced by diabetes mellitus (DMED) is particularly difficult to treat. Therefore, novel therapeutic approaches for the treatment of DMED are urgently needed. Exosomes, nanosized particles involved in many physiological and pathological processes, may become a promising tool for DMED treatment. In this study, we investigated the therapeutic effect of exosomes derived from corpus cavernosum smooth muscle cells (CCSMC‐EXOs) on erectile function in a rat model of diabetes and compared their effect with that of exosomes derived from mesenchymal stem cells (MSC‐EXOs). We incubated labelled CCSMC‐EXOs and MSC‐EXOs with CCSMCs and then observed uptake of the exosomes at different time points using laser confocal microscopy. CCSMC‐EXOs were more easily taken up by CCSMCs. The peak concentration and retention time of labelled CCSMC‐EXOs and MSC‐EXOs in the corpus cavernosum of DMED rats after intracavernous injection were compared by in vivo imaging techniques. Intracavernous injection of CCSMC‐EXOs was associated with a relatively high peak concentration and long retention time. Our data showed that CCSMC‐EXOs could improve erectile function in DMED rats. Meanwhile, CCSMC‐EXOs could exert antifibrotic effects by increasing the smooth muscle content and reducing collagen deposition. CCSMC‐EXOs also increased the expression of eNOS and nNOS, followed by increased levels of NO and cGMP. These findings initially identify the possible role of CCSMC‐EXOs in ameliorating DMED through inhibiting corporal fibrosis and modulating the NO/cGMP signalling pathway, providing a theoretical basis for a breakthrough in the treatment of DMED.     

In Vivo NO/cGMP Signalling in the Hippocampus

In the hippocampus of freely-moving rats, basal extracellular levels of cGMP are inhibited by L-NARG or ODQ whereas they are increased by NO donors or phosphodiesterase inhibitors. Activation of NMDA receptors also augments cGMP dialysate levels in a MK-801 and L-NARG sensitive manner, an effect dramatically diminished during ageing. Experiments with AMPA, AMPA receptor antagonists and cyclothiazide revealed complex relationships with GABAergic circuits that potently control the NO/cGMP pathway. Furthermore, the activity of this neurochemical cascade is also modulated by hippocampal nicotinic receptors via enhancement of endogenous glutamate release and stimulation of NMDA receptors. From a behavioural point of view, increased hippocampal excitation leads to the appearance of epileptic-like manifestations that, however, seem unrelated to the increase of NO/cGMP formation.     

Nitric oxide/cGMP pathway components in the Leydig cells of the human testis

In this study we sought to determine whether the main components of the nitric oxide (NO) pathway are localized within the Leydig cells of the human testis and whether the soluble guanylyl cyclase (sGC), the enzyme that accounts for NO effects, is functionally active in these cells. Using an amplified immunocytochemical technique, immunoreactivity for nitric oxide synthase (NOS-I), sGC and cyclic guanosine monophosphate (cGMP) was detected within the cytoplasm of human Leydig cells. Distinct differences in staining intensity were found between individual Leydig cells, between cell groups and between Leydig cells of different patients. By means of a specific cGMP-RIA, a concentration-dependent increase in the quantity of cGMP was measured in primary cultures of human Leydig cells following exposure to the NO donor sodium nitroprusside. In addition, NOS-I immunoreactivity was seen in Sertoli cells, whereas cGMP and sGC immunoreactivity was found in Sertoli cells, some apically situated spermatids and residual bodies of seminiferous tubules. Dual-labelling studies and the staining of consecutive sections showed that there are several populations of Leydig cells in the human testis. Most cells were immunoreactive for NOS-I, sGC and cGMP, but smaller numbers of cells were unlabelled by any of the antibodies used, or labelled for NOS-I or cGMP alone, for sGC and cGMP, or for NOS-I and sGC. These results show that the Leydig cells possess both the enzyme by which NO is produced and the active enzyme which mediates the NO effects. There are different Leydig cell populations that probably reflect variations in their functional (steroidogenic) activity. Received: 27 March 1996 / Accepted: 14 July 1996     

Endogenous nitric oxide/cGMP signalling in the guinea pig bladder: evidence for distinct populations of sub-urothelial interstitial cells

We have examined structures that may operate by using nitric oxide (NO)/soluble guanylyl cyclase (sGC) signalling in the lamina propria of the guinea pig bladder. Cells on the luminal surface of the urothelium and sub-urothelial interstitial cells (SU-ICs) responded to NO with a rise in cGMP. The distribution of these different cells varied between the base, lateral wall and dome. In the base, two regions were identified: areas with sparse surface urothelial cells and areas with a complete covering. A layer of cGMP-positive (cGMP⁺) cells (up to 10 cells deep) was found in the base. cGMP⁺/SU-ICs were also observed in the lateral wall. However, here, the cGMP⁺ cells were confined to a layer of only 1–2 cells immediately below the basal urothelial layer (basal cGMP⁺/SU-ICs). Below these cGMP⁺/SU-ICs lay cells that had a similar structure but that showed little cGMP accumulation (deep cGMP^–/SU-ICs). Both basal and deep SU-ICs expressed the β1 subunit of sGC and the cGMP-dependent protein kinase I (cGKI), suggesting that the deep SU-ICs can sense NO and signal via cGMP. By using BAY 41-2272, a sensor of endogenous NO production, NO-dependent cGMP synthesis was observed primarily in the basal SU-ICs. A third population of cGKI⁺/cGMP⁻ cells was seen to lie immediately below the basal urothelial layer. These cells (“necklace” cells) were less numerous than SU-ICs and extended linking processes suggesting a network. The specific functions of these structures are not known but they may contribute to the emerging multiple roles of the urothelium associated with the generation of bladder sensation.     

丁苯酞对豚鼠平喘作用的初步药效学观察

目的：初步探讨丁苯酞对豚鼠的平喘作用及机制。方法：本次研究分为离体气管平滑肌实验与动物整体实验两部分。在离体实验中制备豚鼠离体气管片,使用Ach、Hist使气管平滑肌收缩达到最大值后,按累积加药法加入丁苯酞,记录1、10、100 mg/L丁苯酞对痉挛气管平滑肌的解痉作用（n=10）。在整体实验中,取经筛选的豚鼠分为正常对照组、模型对照组、地塞米松组及丁苯酞高、低剂量组（n=8）,在吸入低浓度激发液（1% ACh：0.05% Hist=1：1）激发6次（每次10 s）的基础上,第7次吸入高浓度激发液（2% ACh：0.1% Hist=1：1）10 s,观测90 mg/kg、30mg/kg丁苯酞对哮喘豚鼠行为学以及血清NO、支气管肺泡灌洗液（BALF）中ET-1的影响。结果：1、10、100 mg/L的丁苯酞对ACh、Hist引起的离体痉挛状态的豚鼠气管平滑肌有显著的解痉作用（对Ach的解痉率为15.08 ±7.68、42.41 ±13.54、77.56 ±24.82;对Hist的解痉率为19.40 ±7.60、56.84 ±11.72、76.35 ±19.40）且呈一定的量效关系（P< 0.05,0.01）;在乙酰胆碱与组胺的引喘下,90 mg/kg、30 mg/kg丁苯酞能明显延长豚鼠的哮喘潜伏期（53.3 ±13.2、33.1 ±13.0）,改善哮喘行为学,降低血清NO（78.71 ±19.40、84.75 ±20.97）、支气管肺泡灌洗液（BALF）中ET-1（24.30 ±5.80、28.50 ±6.31）的含量（P < 0.05,0.01）。结论：丁苯酞具有一定的平喘作用,而缓解NO、ET-1异常升高是其作用机制之一。     

丹参注射液对庆大霉素耳中毒豚鼠耳蜗氧自由基生成的影响

目的:研究丹参注射液(SM)对庆大霉素(GM)耳中毒豚鼠耳蜗氧自由基生成的影响,探讨SM对GM耳毒性损伤的保护作用及其机制.方法:检测豚鼠耳蜗组织中超氧化物歧化酶(SOD)活力和丙二醛(MDA)含量,结合听性脑干反应(ABR)测试及透射电镜技术.结果:经GM处理的耳蜗组织中SOD活力明显下降,MDA含量则明显增加(P＜0.01),且与ABR阈值升高高度相关(|r|＞0.8,P＜0.05).同时接受SM的动物,其耳蜗组织中SOD活力明显升高(P＜0.01),MDA含量则明显减少(P＜0.05),且听功能显著改善.电镜观察显示耳蜗形态学改变与听力变化相一致.结论:氧自由基及其引发的脂质过氧化参与了GM耳中毒过程,SM可通过提高耳蜗组织中SOD活力,防止脂质过氧化,减轻GM的耳蜗毒性,改善听功能.     

Modulation of cAMP level by tedisamil in guinea pig heart

Tedisamil is antiarrhythmic class III drug with antifibrillating/defibrillating potency linked to enhancement of intermyocyte gap junctional electrical coupling most likely via its sympathomimetic cAMP-related mechanisms. This study was designed to examin the effect of tedisamil on cAMP level in guinea pig hearts in vivo and in vitro in Langendorff preparation. The drug was administered either as a bolus into vena jugularis in dosage 1.0 and 1.5 mg/kg or into the perfusion solution at a concentration of 1.5 × 10^-6 mol/l. In additional experiments, this period was followed by brief 10 min global ischemia, induced by clamping of the aorta or perfusion. After 10 min from the onset of tedisamil administration as well as after 10 min of ischemia the ventricular tissue was immediately frozen for cAMP immunoassay Tedisamil caused in normal heart small but significant dose-dependent increase of myocardial cAMP (pmol/mg) level in vivo 1.8 and 2.5 vs. 1.4 as well as in vitro 1.1 vs. 0.8 (p < 0.05) conditions. Ischemia itself induced accumulation of cAMP in both, in vitro and in vitro experiments, 2.6 vs. 1.4 and 1.3 vs. 0.8, respectively. The preischemic elevation of cAMP by tedisamil was not potentiated by following ischemia, on the contrary, decline of the cyclic nucleotide was detected comparing to ischemia itself. In conclusion, tedisamil increased cAMP level in normal heart and prevented additional ischemia-related elevation of this nucleotide. The results indicate modulation of myocardial cAMP level by tedisamil, which may account for its protective effect on gap junctional electrical coupling.