Immunohistochemical localization of endothelial and inducible nitric oxide synthase within neurons of cattle with rabies

The expression of constitutive endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) in the brains of cattle with natural rabies was studied. Increased expression of eNOS was detected in neurons of the brain stem and Purkinje cells of cerebellum. By contrast, iNOS was diffusely localized in the cytoplasm of affected neurons, and some inflammatory cells were positive. eNOS and rabies antigen were co-localized in inclusion bodies (Negri bodies) in neurons. The specific localization of eNOS, but not iNOS, in the Negri bodies suggests that eNOS is involved in the formation of rabies virus inclusion bodies.     

Direct measurements of nitric oxide release in relation to expression of endothelial nitric oxide synthase in isolated porcine mitral valves

The aim of this study was to measure the direct release of nitric oxide (NO) from the porcine mitral valve using a NO microelectrode. Furthermore, the expression and localization of endothelial nitric oxide synthase (eNOS) in the mitral valve was studied using immunohistochemistry, Western blotting and RT-PCR. Results show that bradykinin increases NO release from mitral valves (DeltaBradykinin: 33.71 +/- 10.41 nm NO, P < 0.001, n = 10), whereas N-nitro-l-arginine methyl esther (l-NAME) decreases NO release when compared with basal level (Deltal-NAME: 82.69 +/- 15.66 nm NO, P < 0.005, n = 4). Both protein and mRNA expression of eNOS in mitral valves and in isolated valvular endothelial cells suggest that the NO release is mainly associated with the mitral valve endothelium. It is concluded that direct NO release from porcine mitral valves coincides with eNOS expression. This study documents useful techniques for investigations into the role of local NO release in mitral valve diseases.     

Distribution of actin filaments in the seminiferous epithelium of the Habu,Trimeresurus flavoviridis

The distribution of actin filaments was examined in the seminiferous epithelium of the Habu (Trimeresurus flavoviridis; snake), by transmission electron microscopy and fluorescence histochemistry. By transmission electron microscopy, actin filaments were clearly found only at the site between Sertoli cell and spermatid without a lattice‐like structure. Fluorescence histochemistry showed a weak labelling of actin filaments in the seminiferous epithelium, whereas these findings seem to be common among reptiles, they are different from those in mammals. Additionally, the bundles of actin filaments adjacent to the plasma membrane of Sertoli cells, appeared in other reptiles, were not observed in the Habu.     

The endothelial nitric oxide synthase/nitric oxide system is involved in the defective quality of bovine oocytes from low mid-antral follicle count ovaries

In a previous survey concerning cows of reproductive age, we demonstrated that oocytes isolated from ovaries with <10 medium antral follicles of 2 to 6 mm in diameter (low ovaries; Lo) show less developmental competence than oocytes collected from ovaries with >10 medium antral follicles (high ovaries; Hi). The aim of the present study was to evaluate whether a defective endothelial nitric oxide synthase/nitric oxide (eNOS/NO) system and vasculature in healthy medium antral follicles is likely to reduce oocyte competence from Lo ovaries. Thus, experiments were conducted to 1) immunolocalize eNOS protein during folliculogenesis; 2) quantify eNOS protein/vasculature in the follicle wall; and 3) verify if NO donor, S-nitroso acetyl penicillamine (SNAP) administration during in vitro maturation affects developmental competence of oocytes isolated from Lo ovaries. Endothelial nitric oxide synthase protein was detected in granulosa and theca cells, as well as in blood vessels from primordial to antral follicles. Quantitative analysis indicated that in medium antral follicles from Lo ovaries, eNOS protein expression and vasculature were reduced (P < 0.05). The addition of SNAP improved blastocyst and hatching rates of oocytes from Lo ovaries, promoting a percentage similar to oocytes from Hi ovaries, and reduced the percentage of apoptotic nuclei in in vitro-produced blastocysts (P < 0.05). Results from our study suggest that in bovine ovaries with small mid antral follicle number, a defective eNOS/NO system is related to a reduced follicle vasculature and may affect oocyte quality, thus inducing a premature decline of fertility.     

Inducible nitric oxide synthase and nitrotyrosine in listeric encephalitis: a cross-species study in ruminants

Listeria monocytogenes (LM) is a Gram-positive facultative intracellular bacterium that causes fatal meningoencephalitis in humans and ruminants. A current paradigm predicts that intracellular bacteria are controlled by nitric oxide (NO) whose synthesis is catalyzed by inducible nitric oxide synthase (iNOS). The ability of macrophages (Mphi) to express iNOS shows extreme interspecies variability. Here the expression of iNOS and synthesis of NO was studied in listeric encephalitis of cattle, sheep, and goats. iNOS was expressed by a subset of Mphi in cerebral microabscesses in all three species. The level of iNOS expression and the density of cells per lesion expressing iNOS was highest in cattle, intermediate in sheep, and lowest in goats. The accumulation of nitrotyrosine (NT), an indicator of local NO synthesis, was observed in lesions of cattle but not in those of small ruminants. The density of iNOS-expressing cells in lesions was inversely correlated with the number of bacteria. No species differences were observed in regard to reactive oxygen intermediate (ROI) production by stimulated granulocytes, using the flow cytometric dihydrorhodamine-123 (DHR) method indicating ROI generation. Thus, the marked species differences in iNOS expression, NT accumulation, and LM content in lesions of ruminants with listeric encephalitis are explained by different amounts of ROI produced. It suggests that variations in the ability of Mphi to synthesize NO are of pathophysiological significance in listeriosis.     

Birthweight determines intestinal microvasculature development and alters endothelial nitric oxide synthase density in young piglets

Blood supply to enterocytes dictates intestinal health and nutrient absorption. These two aspects are impaired in low birthweight (LBW) piglets, but whether the perfusion to intestinal tissues is implicated as well is still unknown. Thus, structural changes in the microvasculature of LBW and normal birthweight (NBW) piglets were investigated during early postnatal development. Additionally, the presence of endothelial nitric oxide synthase (eNOS) in the intestinal mucosa was assessed given its important role to assure perfusion. A total of 22 pigs (11 LBW and 11 NBW) were sacrificed at days 0, 3, 8 and 19 of life. Body weight and intestinal length were recorded and a piece of the small intestine was sampled for immunohistochemical analysis of von Willebrand Factor (vWF, an endothelial cell marker) and eNOS. LBW piglets had a relatively (to body weight) longer intestine than their NBW counterparts. Age did not affect microvasculature, which was more abundant (85% larger vWF-positive area) in NBW than LBW pigs. However, an interaction age*BW was observed for eNOS-IR, showing that eNOS presence peaked in NBW piglets on the first day of life and subsequently decreased. This pattern was not observed in LBW piglets. The less abundant intestinal endothelial mass and the different pattern of eNOS expression observed in LBW piglets suggests microcirculation as a contributing factor in the impaired digestive functioning and gut health of LBW pigs. However, revealing whether the origin of this alteration is prenatal or postnatal, for example due to a lower milk intake, needs further study.     

奶牛血浆钙离子浓度及胎儿胎盘eNOS表达的研究

通过对血浆中Ca2+浓度的检测及胎儿胎盘内皮型一氧化氮合酶(eNOS)表达量的研究,探讨奶牛胎衣不下的发生与这些物质变化的关系。采用终点法检测血浆中Ca2+浓度变化;分别采用免疫组织化学SP法及荧光定量PCR法检测奶牛胎儿胎盘eNOS蛋白及其mRNA的表达。结果表明,胎衣不下组奶牛血浆中Ca2+浓度在分娩前后均低于正常组;两组eNOS蛋白均在胎盘绒毛滋养层细胞和内皮细胞胞浆内表达,蛋白的表达量差异显著(P0.05);两组胎儿胎盘eNOS mRNA表达量差异显著(P0.05)。说明分娩前后血浆中Ca2+水平对奶牛胎衣不下的发生有重要的调节作用,低浓度的Ca2+容易导致子宫收缩无力,同时影响eNOS在胎儿胎盘绒毛中的表达。     

Expression of constitutive endothelial, neuronal and inducible nitric oxide synthase in the testis and epididymis of horse

The expression of three isoforms of nitric oxide synthase (NOS) were examined in the testis and epididymis of a thoroughbred horse. Immunohistochemical studies demonstrated the presence of eNOS immunostaining in some germ cells in the seminiferous tubules and in vascular endothelial cells in the interstitial tissues. Interstitial cells, most likely Leydig cells, were also intensely immunopositive for eNOS. The pattern of immunostaining for nNOS was similar to that for eNOS in the testis. Weak expression of iNOS was detected in the seminiferous tubules of the testis, but intense expression was found in interstitial cells. Inducible NOS was also strongly detected in stereocilia, sperm, epithelium and connective tissue of the epididymis of normal horses. These findings suggest that three isoforms of NOS are expressed in the testis and epididymis of horse and that they play important roles in the biology of interstitial cells that produce testosterone, as well as in spermatogenesis in the seminiferous tubules.     

Expression of vascular endothelial growth factor and endothelial nitric oxide synthase is increased in the placenta of sheep at high altitude in the Andes

Fetal weight and the placenta of sheep at high altitude (HA) are affected by hypoxia. Placental changes (an increase in placental size and vascularization) are greater in ewes from populations that have lived for several generations at HA than in those exposed during just 1 gestation. This study investigated placental expression of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS), 2 molecules involved in placental angiogenesis that could be upregulated by hypoxia. Two groups of ewes were maintained at HA (3589 m) during pregnancy: HA-native ewes (group HH) and ewes native to lowlands but moved to HA immediately after the diagnosis of pregnancy (group LH). A control group (LL) was kept at sea level. Near term, placentomes were removed, weighed, and processed for immunohistochemical detection of VEGF and eNOS, as well as for vascular area measurement. Placental weight was significantly higher in the HH group than in the LH and LL groups; between the latter 2 groups there was no significant difference. The placental area occupied by vasculature was significantly greater in both the HA groups than in the LH group; the number of placentomes was greatest in the LL group. The density of VEGF and eNOS in the placentome tissue was significantly greater in both HA groups than in the LL group. Although the density of VEGF was significantly lower in the HH group than in the LH group, no differences were observed in eNOS density between the HH and LH animals. These results demonstrate that chronic hypoxia upregulates the expression of placental VEGF and eNOS, suggesting an important role of these molecules in the placental response to HA hypoxia. In addition, an attenuated response to hypoxia in VEGF synthesis may be part of the long-term process of adaptation to HA.     

L-硝基精氨酸对阿尔茨海默症小鼠脑海马nNOS阳性神经元分布、NOS活性及NO含量变化的影响

为了研究D-半乳糖联合铝诱导的小鼠阿尔茨海默症(AD)脑海马一氧化氮合酶(NOS)活性和一氧化氮(NO)含量的变化及L-NNA和盐酸多奈哌齐对其变化的影响,探讨NO在阿尔茨海默症中的作用机制及2种药物对脑神经元的保护作用。选取2月龄健康昆明小鼠160只,体质量(20±2)g,随机分为正常对照组、模型组、盐酸多奈哌齐治疗组及L-NNA治疗组,利用D-半乳糖联合三氯化铝建立小鼠AD模型,应用生化检测技术测定各组脑海马在造模后每周NOS活性及NO含量。结果表明,模型组海马内NOS活性开始呈缓慢升高,从第4周开始呈显著升高,保持较高含量至12w造模结束;两治疗组脑海马NOS活性在各时间点极显著或显著低于模型组(P0.01或P0.05),并且L-NNA在4~8周时降低脑海马NOS活性的程度好于盐酸多奈哌齐治疗组(P0.05);NO含量的变化随着NOS活性的变化而变化;利用SABC免疫组织化学方法检测各组脑海马神经型NOS(nNOS)阳性神经元,发现模型组脑海马nNOS阳性神经元密度降低,细胞着色变淡,胞体截面积和最长突起长度变小,经过治疗后神经元密度增加,胞体截面积和最长突起长度显著改善(P0.05)。结果提示NO参与了AD形成过程,高浓度的NO能发挥神经毒性作用损害脑组织;L-NNA通过抑制NOS的活性,降低了脑海马NO的含量,对阿尔茨海默症中海马神经元具有明显的保护作用。     

一氧化氮合酶阳性神经元在大鼠体内的分布与一氧化氮的功能

一氧化氮 (ＮＯ)是一种最新发现的、哺乳动物中最小、最轻并具有独特理化性质和生物学活性的信息和效应分子 ,能激活靶细胞中的鸟苷酸环化酶 (ＧＣ) ,提高环一磷酸鸟苷(ｃＧＭＰ)浓度 ,发挥一系列生物学作用 ,现已成为研究热点之一。ＮＯ广泛存在于神经系统、心血管系统、免疫系统、消化系统、生殖系统与呼吸系统等的细胞内 ,是传递神经信息、调节血压以及机构防御等一系列生命活动必不可少的生物信使。因此 ,内源性ＮＯ的生物学研究、ＮＯ在动物模型大鼠体内的分布及其功能的确定 ,将有助于在动物医学和人类临床医学领域进一步阐明机体某些…     

Postnatal testicular development and actin appearance in the seminiferous epithelium of the Habu,Trimeresurus flavoviridis

The postnatal testicular development and actin distribution in the seminiferous epithelium were examined by light microscopy, using the testes of the Habu (Trimeresurus flavoviridis; snake) from 0-year-old to 3-year-old. At 0-year-old (about 1 month after birth), the testis was quite small in size, and the seminiferous epithelium was composed of only Sertoli cells and large spermatogonia. Actin immunoreactivity was observed in the peritubular myoid cells, but could not be detected in the seminiferous epithelium. At 1-year-old (about 10 months after birth), the testicular size increased to a great degree. In the seminiferous epithelium, spermatocytes newly appeared. Actin could still not be detected in the seminiferous epithelium. At 2-year-old (about 1 year and 10 months after birth), the testes continued to develop in size. In the seminiferous epithelium, elongate spermatids and round spermatids were frequently seen, in addition to Sertoli cells, spermatogonia and spermatocytes. Thus, active spermatogenesis was clearly recognized at this age. Moreover, the actin distribution in the seminiferous epithelium was observed at the site between Sertoli cells and spermatids, as well as that at adult stage. The immunoreactivity of actin in the peritubular myoid cells gradually increased from 0-year-old to 2-year-old. Conclusively, it seems likely that spermatogenesis in the Habu initiates at 2-year-old, accompanying with the appearance of actin in the seminiferous epithelium.     

Effects of nitric oxide donor and nitric oxide synthase inhibitor on ruminal contractions in conscious sheep

The present study was planned to evaluate a role of nitric oxide (NO) in the regulation of regular ruminal contractions in conscious sheep. Intravenous infusion of S-nitroso-acetyl-DL-penicillamine (SNAP) at doses of 3-30 nmol kg(-1) min(-1)for 30 minutes inhibited both the amplitude and frequency of ruminal contractions in a dose-dependent manner. However, intravenous infusion of Nomega-nitro-L-arginine-methyl ester (L-NAME) at doses of 0.3-3.0 micromol kg(-1) min(-1)did not alter the basal tone of intraruminal pressure and the amplitude of ruminal contractions. The frequency of contractions was slightly inhibited by L-NAME infusion at 1.0 micromol kg(-1)min(-1). The effects of L-NAME were abolished by simultaneous infusion of L -arginine at 30 micromol kg(-1) min(-1). These results suggest that exogenous NO can diminish the ruminal contractions, while endogenous NO is not involved in the regulatory mechanism of basal tone and regular phasic contractions of the rumen in healthy sheep.     

Expression of nitric oxide synthase isoforms in the testes of pigs

This study examined the expression of three isoforms of nitric oxide synthase (NOS) in the testes of pigs. Immunohistochemical studies demonstrated the presence of nNOS, eNOS and iNOS in interstitial cells, primary spermatocytes and spermatids. Positive immunoreactions for eNOS and iNOS were detected in peritubular myoid cells. Some vascular endothelial cells were positive for nNOS and eNOS. The expression of nitrotyrosine was detected in interstitial cells. In addition, the histochemical study revealed that all the interstitial cells were stained positively for NADPH-diaphorase, although some spermatids and vascular endothelial cells displayed moderate enzymatic activity. These findings suggest that three isoforms of NOS are expressed in the testis of pig and that they play important roles in the biology of interstitial cells that produce testosterone, as well as in spermatogenesis in the seminiferous tubules.     

Expression of neuronal nitric oxide synthase in the pancreas of the sheep

In numerous mammals, nitric oxide (NO) influences the activity of the exocrine and endocrine pancreas. In this study, immunocytochemistry was utilized to investigate the expression of neuronal nitric oxide synthase (nNOS) in the pancreas of sheep. In double immunocytochemical staining, the co-localization of nNOS with vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) or substance P (SP) was studied. The presence of nNOS was confined to the intrapancreatic neurones (9.6 +/- 1.3%) as well as to nerve fibres of the endocrine pancreas and intrapancreatic ganglia. nNOS-immunoreactive (IR) neurones were round and oval in shape and predominantly (83.3 +/- 2.6%) belonged to the middle-size group (25-50 mum). Numerous, fine islets supplying nNOS-IR nerve terminals were devoid of VIP, SP or NPY. Moderately numerous, non-varicose nNOS-IR nerve fibres of intrapancreatic ganglia frequently expressed VIP or NPY, but not SP; 2.2 +/- 0.6% of nNOS-IR intrapancreatic neurones displayed lack of VIP, whereas 7.5 +/- 0.8% were VIP-IR. All nNOS-IR neurones were devoid of SP. The frequencies of nNOS-IR/NPY-IR and nNOS-IR/NPY-negative intrapancreatic neurones were 2.2 +/- 0.4% and 6.1 +/- 1.1%, respectively. Comparison with other mammals indicated that nitrergic innervation of the ovine pancreas is species-determined and may be a reflection of the ruminants' digestion specificity. The possible origin of nNOS-IR nerve fibres and functional significance of NO in the pancreas of sheep were discussed.     

Inducible nitric oxide synthase expression in Leishmania-infected dog macrophages

Nitric oxide (NO) production by the inducible NO synthase (iNOS or NOS2) represents one of the main microbicidal mechanisms of murine macrophages, but its role in other animal models is poorly investigated. Therefore, the aim of this work was to evaluate NOS2 expression in dog macrophages infected with Leishmania infantum. Macrophages obtained from peripheral blood of healthy dogs were activated with recombinant human interferon (rhIFN)-γ and bacterial lipopolysaccharide (LPS) and then infected with L. infantum promastigotes, zymodeme MON1. For the immunofluorescence assay fixed macrophages were incubated with polyclonal rabbit anti-NOS2 and then with rhodamine F(ab′)₂ goat anti-rabbit IgG. For immunoblotting, cell lysates were submitted to SDS–PAGE and blots were incubated with polyclonal rabbit anti-NOS2 and then with horseradish peroxidase-conjugated goat anti-rabbit IgG. Results demonstrated that L. infantum-infected cells, after stimulation with rhIFN-γ and LPS, displayed high levels of fluorescence for the NOS2 in their cytoplasm, unlike unstimulated uninfected macrophages. In western blotting, polyclonal anti-NOS2 reacted specifically with a protein band corresponding to 130 kDa. The signal produced in Leishmania-infected cells stimulated with rhIFN-γ and LPS was higher than that produced in Leishmania-infected unstimulated cells. No band was detected in cellular lysates from uninfected unstimulated cells. These results indicate that dog macrophages can express NOS2, and suggest a role for IFN-γ and LPS in NOS2 induction also in this animal model.     

Nitric oxide,inducible nitric oxide synthase and inflammation in veterinary medicine

Inflammation is a process consisting of a complex of cytological and chemical reactions which occur in and around affected blood vessels and adjacent tissues in response to an injury caused by a physical, chemical or biological insult. Much work has been performed in the past several years investigating inducible nitric oxide synthase (NOS, EC 1.14.13.39) and nitric oxide in inflammation. This has resulted in a rapid increase in knowledge about iNOS and nitric oxide. Nitric oxide formation from inducible NOS is regulated by numerous inflammatory mediators, often with contradictory effects, depending upon the type and duration of the inflammatory insult. Equine medicine appears to have benefited the most from the increased interest in this small, inflammatory mediator. Most of the information on nitric oxide in traditional veterinary species has been produced using models or naturally occurring inflammatory diseases of this species.     

日粮铜对肉鸡肾脏一氧化氮产生及一氧化氮合酶活性的影响

铜是动物体内一种必需的微量元素,在多种代谢途径中有重要的生物学作用.早期的研究结果表明,提高饲料中铜的含量,可以促进动物生长.但是过量添加铜也可以损害肝脏、肾脏等器官[1-2].一氧化氮(NO)作为一种强有力的内皮细胞舒血管因子,其功能已越来越受到人们的重视.NO对肾脏具有保护和损伤的双重作用.一方面具有扩张动脉,抑制血小板粘附、聚集,抗血栓形成,疏通微循环,增加供血,保护细胞的作用.     

Metabolic effects of nitric oxide synthase inhibition during exercise in the horse

The effect of nitric oxide synthase (NOS) inhibition during exercise on lactate production was investigated in five Thoroughbred horses. A standard exercise test (SET), consisting of three canters (approximately 55 per cent VO2max), with walking and trotting between each canter, was performed twice (control and test, in random order) by each horse. Nphi-nitro-L-arginine methyl ester (L-NAME; 20 mg kg-1), a competitive inhibitor of NOS, induced a significant increase (P < 0.05) in plasma lactate [5.7 (2.9) vs 11.8 (3.8) mmol L-1], which continued to increase despite administration of L-arginine, the substrate for NOS. There were no differences in cardiac output (Q) or the total body oxygen consumption (VO) between each SET. The results show that non-specific inhibition of NOS isoforms during exercise in the horse increases plasma lactate concentration, although the mechanism/s remain uncertain.