胰岛素对PC12细胞神经型一氧化氮合酶表达及活性的影响

为探讨胰岛素对神经细胞中神经型一氧化氮合酶（nNOS）的表达及活性的影响,应用流式细胞术、原位杂交、电子自旋共振等技术方法研究胰岛素对PC12细胞中神经型一氧化氮合酶的影响.胰岛素作用PC12细胞9 h 后,神经型一氧化氮合酶的免疫荧光强度显著升高,且呈浓度依赖关系,其最大效应为对照的(155±13)%(P＜0.01, n=3, t-test).加入胰岛素(16 mU/L, 6 h)也能够显著上调nNOS mRNA的表达,为对照的(182±13)%(P＜0.01, n=3, t-test).另外加入胰岛素(16 mU/L)作用9 h后,神经型一氧化氮合酶的活性也显著升高,为对照的(167±15)%(P＜0.01, n=4, t-test).由上述结果可知,胰岛素对PC12细胞的神经型一氧化氮合酶的表达及活性有上调作用.     

牛蛙视网膜诱导型一氧化氮合酶免疫组化定位

用免疫组织化学方法研究了诱导型一氧化氮酶（iNOS)在牛蛙视网膜中的表达。结果显示,在正常状态视网膜中,无长突细胞呈弱阳性反应;节细胞层、双极细胞,水平细胞和光感受器内段呈阴性反应,在暗适应状态下,神经节细胞,内核层的无长突细胞呈强阳性反应;一些双极细胞,水平细胞和光感受器内段呈弱阳性反应,提示NO主要在暗适应状态下参与视网膜的信息传递过程。     

羟基红花黄色素A对脂多糖作用后血管内皮细胞诱导型一氧化氮合酶表达的影响

旨在探讨羟基红花黄色素A(hydroxysafflor yellow A,HSYA)对脂多糖(lipopolysaccharide,LPS)作用后人脐静脉内皮细胞株HUVECs细胞株诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)表达的影响.培养HUVECs细胞株,用 1 mg/L LPS及不同浓度的HYSA处理细胞24 h,MTT法检测细胞增殖情况,硝基还原酶法检测培养液中一氧化氮(NO)含量,RT-PCR及Western blotting检测iNOS表达.结果表明0.01、0.1 mmol/L HYSA对LPS引起的iNOS升高无明显作用,但1 mmol/L HYSA能明显抑制LPS作用后高度表达的iNOS量.因此,HYSA能下调LPS所致iNOS的异常表达,这可能有助于临床治疗血管炎症疾病.     

Caspases Mediate 6-Hydroxydopamine-Induced Apoptosis but Not Necrosis in PC12 Cells

Abstract: The neurotoxin 6-hydroxydopamine (6-OHDA) induces apoptosis in the rat phaeochromocytoma cell line PC12. 6-OHDA-induced apoptosis is morphologically indistinguishable from serum deprivation-induced apoptosis. Exposure of PC12 cells to a low concentration of 6-OHDA (25 µ M ) results in apoptosis, whereas an increased concentration (50 µ M ) results in a mixture of apoptosis and necrosis. We investigated the involvement of caspases in the apoptotic death of PC12 cells induced by 6-OHDA, using a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), and compared this with serum deprivation-induced apoptosis, which is known to involve caspases. We show that zVAD-fmk (100 µ M ) completely prevented the apoptotic morphology of chromatin condensation induced by exposure to either 6-OHDA (25 and 50 µ M ) or serum deprivation. Furthermore, cell lysates from 6-OHDA-treated cultures showed cleavage of a fluorogenic substrate for caspase-3-like proteases (caspase-2, 3, and 7), acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin, and this was inhibited by zVAD-fmk. However, although zVAD-fmk restored total cell viability to serum-deprived cells or cells exposed to 25 µ M 6-OHDA, the inhibitor did not restore viability to cells exposed to 50 µ M 6-OHDA. These data show the involvement of a caspase-3-like protease in 6-OHDA-induced apoptosis and that caspase inhibition is sufficient to rescue PC12 cells from the apoptotic but not the necrotic component of 6-OHDA neurotoxicity.     

Regulation of the Manganese Superoxide Dismutase and Inducible Nitric Oxide Synthase Gene in Rat Neuronal and Glial Cells

Abstract: Bidirectional communication occurs between neuroendocrine and immune systems through the action of various cytokines. Responses to various inflammatory mediators include increases in intracellular reactive oxygen species (ROS), notably, superoxide anion (O₂⁻) and nitric oxide (NO^•). Neurotoxicity mediated by NO^• may result from the reaction of NO^• with O₂, leading to formation of peroxynitrite (ONOO⁻). ROS are highly toxic, potentially contributing to extensive neuronal damage. We, therefore, evaluated the effects of a variety of inflammatory mediators on the regulation of mRNA levels for manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) in primary cultures of rat neuronal and glial cells. To determine age-dependent variation of mRNA expression, we used glial cells derived from newborn, 3-, 21-, and 95-day-old rat brains. Interleukin-1β, interferon-γ (IFN-γ), bacterial lipopolysaccharide (LPS), and tumor necrosis factor-α showed significant induction of MnSOD in both glial and neuronal cells. However, only LPS and IFN-γ increased iNOS mRNA. These data demonstrate that these two genes are similarly regulated in two cells of the nervous system, further suggesting that the oxidative state of a cell may dictate a neurotoxic or neuroprotective outcome.     

Cytokine Induction of Inducible Nitric Oxide Synthase in an Oligodendrocyte Cell Line

Abstract : The induction of inducible nitric oxide synthase (iNOS) by proinflammatory cytokines was studied in an oligodendrocyte progenitor cell line in relation to mitogen-activated protein kinase (MAPK) activation and cytokine-mediated cytotoxicity. When introduced individually to cultures of CG4 cells, the cytokines, i.e., tumor necrosis factor-α (TNFα), interleukin-1 (IL-1), and interferon-γ (IFNγ), had either minimal (TNFα) or no (IL-1 and IFNγ) detectable stimulatory effect on the production of nitric oxide. However, combinations of these factors, in particular, TNFα plus IFNγ, elicited a strong enhancement of nitric oxide synthesis and, as revealed by western blot and RT-PCR analysis, the expression of iNOS. TNFα and IL-1 were able to activate p38 MAPK in a time- and dose-dependent manner and together showed a combinatorial effect. In contrast, IFNγ neither activated on its own nor enhanced the activation of p38 MAPK in response to TNFα and IL-1. However, a specific inhibitor of p38 MAPK, i.e., SB203580, inhibited the induction of iNOS in cytokine combination-treated cells in a dose-dependent manner, thereby suggesting a role for the MAPK cascade in regulating the induction of iNOS gene expression in cytokine-treated cells. Blocking of nitric oxide production by an inhibitor of iNOS, i.e., nitro-L-arginine methyl ester, had a minimal protective effect against cytokine-mediated cytotoxicity that occurred before the elevation of nitric oxide levels, thereby indicating temporal and functional dissociation of nitric oxide production from cell killing.     

Cyclosporin-A Inhibits Constitutive Nitric Oxide Synthase Activity and Neuronal and Endothelial Nitric Oxide Synthase Expressions after Spinal Cord Injury in Rats

Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca²⁺/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.     

糖皮质激素对机械通气大鼠肺组织iNOS、NO表达的影响

目的通过观察糖皮质激素对机械通气大鼠肺组织诱导型一氧化氮合酶（iNOS）及一氧化氮（NO）表达的影响,探讨糖皮质激素对呼吸机所致肺损伤（ventilator induced lung injury,VILI）的干预作用。方法 24只雄性Wistar大鼠随机分为对照组、机械通气组、地塞米松（DXM）干预组。用逆转录-聚合酶链反应（RT-PCR）法检测肺组织iNOS mRNA表达,用免疫组织化学染色法检测肺组织iNOS蛋白表达,用硝酸还原酶法测定肺组织和血浆NO含量。结果机械通气组和DXM干预组大鼠肺组织iNOS mRNA及其蛋白表达水平,以及血浆和肺组织NO含量均明显高于对照组（P〈0.01）;DXM干预组上述指标与机械通气组比较均明显降低（P〈0.01）。结论糖皮质激素可通过抑制肺组织iNOS的表达,减少NO的生成,对机械通气大鼠肺组织具有保护作用。     

Up-Regulation of the Neuronal Form of Nitric Oxide Synthase in Response to Prolonged Muscarinic M1 Receptor Stimulation

Abstract: It is generally believed that the neuronal form of nitric oxide synthase (nNOS) is constitutively expressed and that regulation of this enzyme's activity is mediated solely by changes in cytosolic calcium concentration. Serendipitously, however, we observed that pretreatment of Chinese hamster ovary (CHO) cells, which coexpress muscarinic M₁ receptors and nNOS, with 3.3 µ M or 1 m M carbachol (CCh) for 48 h resulted in marked enhancement of maximal muscarinic receptor-stimulated nNOS activity as determined by l -[³H]citrulline and cyclic [³H]GMP production. This was accompanied by a decrease in the potency of CCh. Muscarinic receptor density was reduced in the agonist-pretreated cells, as determined by specific [ N-methyl -³H]scopolamine methyl chloride binding, whereas competition binding studies revealed no changes in agonist affinity. Both receptor-stimulated inositol phosphate formation and elevation of intracellular calcium concentrations were found to be desensitized in agonist-pretreated cells in a manner dependent on CCh pretreatment concentration. It is interesting that ionomycin-stimulated nNOS activity was greater in CCh-pretreated cells. Also, western analysis revealed increased nNOS immunoreactivity in pretreated cells. A similar increase in nNOS immunoreactivity following agonist treatment was demonstrated in N1E-115 neuroblastoma cells, which endogenously express nNOS and muscarinic M₁ receptors. Thus, the enhancement of maximal receptor-stimulated nNOS activity following agonist pretreatment can be attributed to up-regulation of nNOS. It is interesting that this augmentation of the response takes place in spite of receptor down-regulation and desensitization of multiple steps involved in nNOS activation.     

Nitric Oxide Induction of Neuronal Endonuclease Activity in Programmed Cell Death

Neuronal survival is intricately linked to the maintenance of intact DNA. In contrast, neuronal degeneration following nitric oxide (NO) exposure is dependent, in part, on the degradation of DNA through programmed cell death (PCD). We therefore investigated in primary rat hippocampal neurons the role of endogenous deoxyribonucleases, enzymes responsible for metabolically derived DNA cleavage, during NO-induced neurodegeneration. Twenty-four hours following exposure to the NO generators sodium nitroprusside (300 μM) and SIN-1 (300 μM), neuronal survival was reduced from approximately 88 to 23%. Treatment with aurintricarboxylic acid (1–100 μM), an endonuclease inhibitor, during NO exposure increased neuronal survival from 23 to 80% and decreased DNA fragmentation from 70 to 30% over a 24-h period. Enhancement of endonuclease activity alone with zinc chelation actively decreased neuronal survival from approximately 80% to approximately 34%. DNA digestion assays identified not only two constitutively active endonucleases, an acidic endonuclease (pH 4.0–7.0) and a calcium/magnesium-dependent endonuclease (pH 7.2–8.0), but also a NO-inducible magnesium-dependent endonuclease (pH 8.0). In the absence of endonuclease activity, DNA degradation did not occur during NO application, suggesting that endonuclease activity was a requisite pathway for NO-induced PCD. In addition, NO independently altered intracellular pH in ranges that were physiologically relevant for the activity of the endonucleases responsible for DNA degradation. Our identification and characterization of specific neuronal endonucleases suggest that the constitutive endonucleases may play a role in the initial stages of NO-induced PCD, but the subsequent “downstream” degradation of DNA may ultimately be dependent upon the NO-inducible endonuclease.     

Down-Regulation of Neuronal Nitric Oxide Synthase by Nitric Oxide After Oxygen-Glucose Deprivation in Rat Forebrain Slices

Abstract : The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor ( Z )-1-[(2-aminoethyl)- N -(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.     

Inhibition of Inducible Nitric Oxide Synthase Expression by Endothelin in Rat Glial Cells Prepared from the Neonatal Rat Brain

Abstract: In primary cultured rat glial cells, a combination of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) decreased iNOS expression and nitrite accumulation induced by TNF-α/IL-1β. The inhibitory effect of ET on TNF-α/IL-1β-stimulated iNOS expression appears to be mediated by ET_B receptors, because (1) both ET-1 and ET-3 inhibited the effects of TNF-α/IL-1β on iNOS expression and nitrite accumulation, (2) a selective ET_B receptor agonist, Suc-[Glu⁹,Ala^11,15]-ET-1 (8–21) (IRL1620), decreased the effects of TNF-α/IL-1β, and (3) a selective ET_B receptor antagonist, N-cis -2,6-dimethylpiperidinocarbonyl- l -γ-methylleucyl- d -1-methoxycarbonyltryptophanyl- d -norleucine, abolished the inhibitory effects of ETs and IRL1620. Incubation of glial cells with lipopolysaccharide (LPS) caused an increase in iNOS expression. Simultaneous addition of ET-3 decreased the effects of LPS (10 and 100 ng/ml) on iNOS expression. Furthermore, cyclic AMP-elevating agents (dibutyryl cyclic AMP and forskolin) inhibited TNF-α/IL-1β-induced and LPS-induced iNOS expression and nitrite accumulation. These findings suggest that ETs can decrease TNF-α/IL-1β-induced and LPS-induced iNOS expression via ET_B receptors and that cyclic AMP may be involved in this process.     

Neuronal Nitric Oxide Synthase Immunopositivity in Motoneurons of the Rabbit's Spinal Cord After Transient Ischemia/Reperfusion Injury

Summary 1. Motoneurons in the spinal cord are especially vulnerable to ischemic injury and selectively destroyed after transient ischemia. To evaluate the role of nitric oxide (NO) in the pathophysiology of the spinal cord ischemia, the expression of neuronal nitric oxide synthase (nNOS) in the motoneurons of the lumbosacral spinal cord was examined in the rabbit model of transient abdominal aorta occlusion.2. The aim of the present study was to find if there is any consensus between the duration of transient abdominal aorta occlusion, nNOS positivity of the motoneurons and neurological hind limb impairment.3. According to the degree of neurological damage (i.e., from the group with almost no sign of damage to a group with fully developed paraplegia), the experimental animals were divided into three groups. The respective spinal cord segments of each experimental group were compared to the control group.4. Spinal cord ischemia (15 min) was induced by Fogarty arterial embolectomy catheter occlusion of abdominal aorta with a reperfusion period of 7 days. On seventh day, the sections of lumbosacral segments were immunohistochemically treated and L1–L7, and S1–S2 segment sections were monitored using light microscopy.     

Role of Vanilloid Receptors in the Capsaicin-Mediated Induction of iNOS in PC12 Cells

The vanilloid receptor 1(VR1) is a nonselective cation channel that is activated by pungent vanilloid compound, extracellular protons, or noxious heat. mRNA of VR1 and vanilloid receptor 1-like receptor (VRL1) were expressed in PC12 cells, and only VRI mRNA was detected in glioma and A10 cell lines. VRI protein was demonstrated in PC12 cells by immunocytochemistry and Western blotting. Capsaicin (CPS), the VRI receptor agonist, led to an increase in intracellular calcium ion, and this effect was blocked by pretreatment with VR1 receptor antagonist capsazepin (CPZ). Treatment of PC12 cells with low concentration of CPS (5-50 microM) increased reactive oxygen species (ROS) production, and inducible nitric oxide synthase (iNOS) was expressed after CPS treatment for 24 h. These CPS-induced changes are inhibited by pretreatment of CPZ. These findings suggest that CPS-induced iNOS expression through the VR1 and/or VRL1-mediated pathway, and this may explain the CPS-mediated physiological and pathological effects in neuron system.     

Muscular Dystrophy in mdx Mice Despite Lack of Neuronal Nitric Oxide Synthase

Abstract: Neuronal nitric oxide synthase (nNOS) is a component of the dystrophin complex in skeletal muscle. The absence of dystrophin protein in Duchenne muscular dystrophy and in mdx mouse causes a redistribution of nNOS from the plasma membrane to the cytosol in muscle cells. Aberrant nNOS activity in the cytosol can induce free radical oxidation, which is toxic to myofibers. To test the hypothesis that derangements in nNOS disposition mediate muscle damage in Duchenne dystrophy, we bred dystrophin-deficient mdx male mice and female mdx heterozygote mice that lack nNOS. We found that genetic deletion of nNOS does not itself cause detectable pathology and that removal of nNOS does not influence the extent of increased sarcolemmal permeability in dystrophin-deficient mice. Thus, histological analyses of nNOS-dystrophin double mutants show pathological changes similar to the dystrophin mutation alone. Taken together, nNOS defects alone do not produce muscular dystrophy in the mdx model.     

Enhancing Effect of Manganese on L-DOPA-Induced Apoptosis in PC12 Cells

L-DOPA and manganese both induce oxidative stress-mediated apoptosis in catecholaminergic PC12 cells. In this study, exposure of PC12 cells to 0.2 mM MnCl2 or 10-20 microM L-DOPA neither affected cell viability, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nor induced apoptosis, tested by flow cytometry, fluorescence microscopy, and the TUNEL technique. L-DOPA (50 microM) induced decreases in both cell viability and apoptosis. When 0.2 mM MnCl2 was associated with 10, 20, or 50 microM L-DOPA, a concentration-dependent decrease in cell viability was observed. Apoptotic cell death also occurred. In addition, manganese inhibited L-DOPA effects on dopamine (DA) metabolism (i.e., increases in DA and its acidic metabolite levels in both cell lysate and incubation medium). The antioxidant N-acetyl-L-cysteine significantly inhibited decreases in cell viability, apoptosis, and changes in DA metabolism induced by the manganese association with L-DOPA. An increase in autoxidation of L-DOPA and of newly formed DA is suggested as a mechanism of manganese action. These data show that agents that induce oxidative stress-mediated apoptosis in catecholaminergic cells may act synergistically.     

Nitric oxide mediates laminin-induced neurite outgrowth in PC12 cells

Laminin is a potent stimulator of neurite outgrowth in a variety of primary neurons and neuronal cell lines. Here, we investigate the role of nitric oxide in the signaling mechanism of laminin-mediated neurite outgrowth in the PC12 cell line. Within 8 s of exposure to laminin, PC12 cells produce nitric oxide. Peak laminin-induced nitric oxide levels reach 8 nM within 12 s of exposure to laminin and constitutive nitric oxide production is sustained for 1 min. A neurite outgrowth promoting synthetic peptide (AG73), derived from the laminin-1-alpha globular domain, also stimulated nitric oxide release. The nitric oxide synthase inhibitor, 1-NAME, prevents the formation of nitric oxide and here, 1-NAME inhibited both laminin-mediated and AG73-mediated neurite outgrowth by 88 and 95%, respectively. In contrast, C16, a synthetic peptide derived from the laminin-1-gamma chain, is shown here to promote PC12 cell attachment, but not neurite outgrowth. Interestingly, the C16 peptide did not activate nitric oxide release, suggesting that laminin-induced nitric oxide release in PC12 cells is associated only with neurite outgrowth promoting laminin domains and signals. In addition, the data here show that the nitric oxide released by PC12 cells in response to laminin is required as a part of the mechanism of laminin-mediated neurite outgrowth.