APOPTOTIC AN IN AN IN VITRO MODEL OF NEURONAL OXIDATIVE STRESS

1. Oxidative stress is believed to be an important mediator of neuronal cell death but the precise mechanism by which this accurs is unknown. 2. We have developed an in vitro model of neuronal oxidative stress to study the pathways by which free radicals kill neurones. 3. We have shown that oxidative stress, cystine deprivation and glutathione depletion results in cell death with the morphological and biochemical features of apoptosis. 4. Neuronal apoptosis induced by oxidative stress can be inhibited by macromolecular synthesis inhibitors. 5. This in vitro model will be a valuable tool for defining the molecular targets of toxic free radicals in neurones and, in turn, in designing rational new therapies for free radical mediated diseases.     

INVOLVEMENT OF CYTOSKELETAL PROTEINS IN THE MECHANISMS OF ORGANOPHOSPHORUS ESTER-INDUCED DELAYED NEUROTOXICITY

1. Organophosphorus ester-induced delayed neurotoxicity (OPIDN) is a neurodegenerative disorder characterized by the presence of swellings in the distal parts of large axons in the central and peripheral nervous systems with subsequent axonal degeneration and paralysis. 2. An early change in OPIDN is enhanced activity and autophosphorylation of Ca²⁺/calmodulin-dependent kinase II. 3. In OPIDN, there is also a dose- and time-dependent increase in Ca²⁺/calmodulin-dependent kinase mediated phosphorylation of the cytoskeletal proteins, α- and β-tubulin, microtubule associated protein-2, neurofilament triplet proteins and myelin basic protein. 4. Anomalous hyperphosphorylation of neurofilaments decreases their transport rate down the axon relative to their rate of entry resulting in their accumulation. 5. Consistent with the neurochemical results is the presence of anomalous aggregations of phosphorylated neurofilaments in early stages of OPIDN. 6. These findings suggest that aberrant hyperphosphorylation of cytoskeletal proteins is a post-translational modification involved in the pathogenesis of OPIDN.     

NITRIC OXIDE: ROLE IN NEUROTOXICITY

1. Nitric oxide (NO) is a novel neuronal messenger molecule which interacts with surrounding neurones, not by synaptic transmission but by diffusion between cells. 2. No is produced following stimulation of the enzyme, NO synthase (NOS). After synthesis, NO exerts its biological actions by diffusion to the site of action. Therefore, the way to regulate the physiological actions of NO is to regulate NOS. 3. NOS is activated by the influx of calcium from glutamate-activated N-methyl-D-aspartate receptors. Overactivation of these receptors leads to overproduction of NO and neuronal cell death. 4. NOS can be regulated at the catalytic site, at the flavoproteins, at the calmodulin site and by phosphorylation. 5. In excess, NO is toxic to neurones. This toxicity is mediated largely by an interaction with the superoxide anion, presumably through the generation of the oxidant, peroxynitrite. 6. NO or peroxynitrite-mediated neuronal injury involves the activation of the nuclear protein, poly(ADP-ribose)synthetase.     

THE THERMAL POTENTIATION OF ACETAMINOPHEN-INHIBITED PMN OXIDATIVE METABOLISM IN VITRO

The effect of high temperatures (39, 41, and 43 °C) on acetaminophen (AM-) induced inhibition of the oxidative respiratory burst of polymorphonuclear leukocytes (PMNs) in vitro has been examined. Whole blood or isolated human PMNs were exposed to various temperatures in vitro in the presence or absence of AM for 0–90 min. Phagocyte membrane-bound NADPH oxidase was studied using the luminol chemiluminescence (CL) response and the superoxide dismutase inhibitable reduction of ferricytochrome C. The NADPH oxidase was stimulated by phorbol myristate acetate (PMA). The results showed that high temperatures (39–43 °C) potentiate the AM inhibitory effect on CL peak response of phagocytes in a temperature-dependent manner. Furthermore, the inhibition of superoxide (O⁻₂) production induced by AM was potentiated by incubating the cells at 39 or 43 °C at different time intervals. These studies suggest that high temperatures significantly potentiate the AM inhibitory effect on oxidative metabolism of PMNs in vitro. These actions of AM may influence the outcome in patients with infectious febrile conditions.     

慢性甲基苯丙胺中毒大鼠和人血清中硝基化损伤水平及其神经毒性的研究

目的:通过观察慢性甲基苯丙胺中毒大鼠及长期甲基苯丙胺依赖人类血清中3-NT的含量变化,间接反映体内ONOO-的含量变化,探讨甲基苯丙胺导致蛋白质硝基化参与的神经毒性机制。方法:建立慢性甲基苯丙胺中毒大鼠模型,收集甲基苯丙胺长期依赖者血清与未吸毒的正常人血清;ELISA检测大鼠及人血清内3-NT的含量。结果:与对照组相比,甲基苯丙胺组大鼠和人血清中3-NT含量明显增多,差异有统计学意义(P<0.001)。结论:慢性甲基苯丙胺中毒动物和长期毒品依赖者血清的3-NT含量增高,表明其所代表的关键蛋白的硝基化参与了甲基苯丙胺的神经毒性作用。     

NEUROTOXICITY OF BETA-AMYLOID PROTEIN: CYTOCHEMICAL CHANGES AND APOPTOTIC CELL DEATH INVESTIGATED IN ORGANOTYPIC CULTURES

1. This study is an attempt to examine in vitro the cyto chemical changes in hippocampal neurones induced by beta-amyloid protein (β-AP). 2. The mechanism of cell death, and the vulnerability of different regions of the hippocampus to b-AP toxicity, has also been explored using TUNEL staining to locate fragmented DNA in both dissociated and organotypic cultures. 3. Apoptotic cell profiles and the detection by immunocytochemistry of ubiquitin and tau protein confirmed the acute neurodegenerative effects of b-AP, and organotypic cultures revealed the dentate gyrus to be especially vulnerable. 4. A scrambled sequence of b-AP, a peptide with similar hydrophobic groups to b-AP, and islet pancreatic amyloidogenic peptide also showed neurodegenerative effects, although less severely than b-AP. 5. It is concluded that organotypic cultures provide a valuable in vitro model with which to observe and characterize the neurotoxic effects of b-AP. These effects, however, may be non-specific and related more to the general amyloidogenicity of the b-AP molecule.     

REGIONAL DIFFERENCES IN THE MODULATORY ROLE OF THE EPITHELIUM IN SHEEP AIRWAY

• 1 The airway epithelium may modulate smooth muscle responsiveness via the release of biologically active substances, such as nitric oxide (NO) and prostaglandins. Based on regional differences in structure and function described for the airway epithelium, we performed a comparative study on the responsiveness of sheep isolated, epithelium‐intact or ‐denuded, first‐ to fourth‐order bronchi to acetylcholine (ACh).
• 2 We performed contractility studies using KCl or cholinergic stimuli in the presence or absence of NO or prostaglandin‐related drugs in epithelium‐intact and epithelium‐denuded bronchial strips obtained from all four airway regions. We also studied the expression of NO synthase (NOS), using the NADPH‐diaphorase staining technique, and the effect of airway epithelium removal on the synthesis of NO metabolites in the different bronchi orders.
• 3 There was no difference in the response of first‐ to fourth‐order epithelium‐intact bronchi to ACh (1 nmol/L–100 mmol/L) or KCl (5–100 mmol/L). Removal of the epithelium had no effect on ACh‐induced contractions of first‐ and second‐order bronchi, but increased responses of third‐ and fourth‐order bronchi to ACh. The NO synthase inhibitor N^G‐nitro‐l‐ arginine methyl ester (100 µmol/L) increased ACh‐induced contractions of fourth‐order epithelium‐intact bronchi only. The NO donor sodium nitroprusside (1 nmol/L–1 mmol/L) equally relaxed 1 µmol/L carbachol‐precontracted epithelium‐denuded first‐ and fourth‐order bronchi.
• 4 Although NAPDH–diaphorase staining demonstrated no regional differences in NOS expression, basal levels of NO metabolites were 4.5‐fold greater in fourth‐ compared with second‐order epithelium‐intact bronchi.
• 5 The cyclo‐oxygenase inhibitor indomethacin (10 µmol/L) had no effect on ACh‐induced contractions of first‐ to fourth‐order epithelium‐intact bronchi, but decreased responses of fourth‐order epithelium‐denuded bronchi to ACh. The contractile effect of the thromboxane A₂ mimetic U‐46619 (1 nmol/L–10 µmol/L) was greater in fourth‐ compared with first‐order epithelium‐denuded bronchi.
• 6 In conclusion, the sheep airway epithelium exhibits regional differences in its modulatory role and this is particularly apparent in small bronchi.

     

EFFECTS OF DRUGS ON UTEROPLACENTAL BLOOD FLOW AND THE HEALTH OF THE FOETUS

1. The placental vascular bed is normally fully dilated. Therefore, changes in vascular resistance elsewhere in the body can affect uteroplacental blood flow (UBF). For example, antihypertensive drugs, such as diazoxide, hydralazine and the angiotensin-converting enzyme inhibitor captopril, cause falls in arterial pressure and, hence, in UBF. 2. Angiotensin II (AngII), prostacyclin and nitric oxide (NO) all influence uteroplacental vascular tone. Angiotensin II in a pharmacological dose (62.5 μg/h) had a biphasic effect on UBF in the sheep. Initially, there was a rise in UBF as pressure rose; however, by 16–24 h, UBF had fallen. The AngII-induced fall in UBF caused severe foetal hypoxia and hypercapnia. 3. Prostacyclin may protect the uteroplacental circulation from vasoconstrictors such as AngII, as the vasoconstrictor effect of AngII in the uteroplacental circulation is enhanced followin. indomethacin. 4. Oestrogen-induced uterine artery vasodilation is nitrergic dependent. As well, nitrergic nerves alter the responsiveness of pregnant uterine arteries to noradrenaline. 5. Thus, both systemic and local factors are important in the control of UBF and in promoting foetal health an. growth.     

褪黑素对大脑皮层细胞一氧化氮含量及其神经毒性作用的影响

目的：探讨褪黑素(MT)抗衰老作用与神经细胞NO释放之间的联系。方法：连续给予老年小鼠MT，检测大脑皮层神经细胞NO含量的变化,并用原代培养的大鼠皮层神经元，去血清培养后，观察MT对高钾、谷氨酸（Glu）诱发NO释放及硝普钠（SNP）致神经毒性作用的影响。结果：MT能明显抑制老年小鼠脑内NO含量的增高，并拮抗KCI与Glu诱发的NO释放及SNP引起的神经毒性，对大脑神经元有保护作用。结论：MT抑制大脑皮层NO含量增高，可能是其抗衰老作用的机制之一。     

EXAMINATION OF DEVELOPMENTAL NEUROTOXICITY BY THE USE OF TISSUE CULTURE MODEL SYSTEMS

1. The rotation-mediated three-dimensional reaggregate culture system is uniquely suited for studies on developmental neurotoxicity. In this system, it is possible to reconstruct central neuronal pathways and follow their development. 2. Exposure to drugs of abuse including methamphetamine and methylenedioxyamphetamine or the appetite suppressant, fenfluramine, reduces monoamines in the cultures in a dose-dependent manner and interrupts normal monoaminergic development. 3. While the monoaminergic neurones may attain normal rates of development following drug removal, the affected neurones are not capable of overcoming the drug-induced insults and a deficiency in monoamines persists throughout development. 4. In addition, the production of immortalized monoclonal hybrid cells obtained by fusion of fetal mesencephalic neurones with a neuroblastoma has yielded cell lines expressing a dopaminergic phenotype. 5. Such cells have been useful in establishing the relationship of neurotoxicity to cell lineage and can serve as models for the study of the cellular and molecular mechanisms of neurotoxicity.     

OMEPRAZOLE: EFFECTS ON OXIDATIVE DRUG METABOLISM IN THE RAT

Omeprazole, a substituted benzimidazole and a potent gastric antisecretory drug has been tested for inhibition of microsomal drug oxidative function in the rat. A single dose of 40 mg/kg prolonged pentobarbitone sleeping times from 118 (range 73-168) min to 195 (159-222) min (P less than 0.01), pentobarbitone half-lives from 89 (63-114) to 112 (54-146) min (P less than 0.05) and aminopyrine breath 14CO2 half-lives from 43 (37-51) to 56 (49-79) min (P less than 0.05). Omeprazole in doses of 20 mg/kg or less had no significant effect. In prolonging pentobarbitone sleeping times omeprazole 40 mg/kg and an equimolar (30 mg/kg) dose of cimetidine were approximately equipotent. These results contrast with studies in man in which much smaller doses of omeprazole have been shown to produce clinically significant inhibition of drug metabolism.     

甲基苯丙胺条件性位置偏爱大鼠中枢神经系统中一氧化氮合酶表达增加

目的·· :探讨甲基苯丙胺条件性位置偏爱大鼠中枢神经系统中 ,一氧化氮合酶 (NOS)表达变化。方法·· :建立大鼠甲基苯丙胺条件性位置偏爱模型 ,采用还原型辅酶Ⅱ -黄递酶组织化学染色方法对生理盐水组和甲基苯丙胺组大鼠不同脑区NOS阳性神经元数目进行比较。结果·· :与生理盐水相比 ,0.25 -1mg·kg-1甲基苯丙胺在大鼠中可以产生显著的条件性位置偏爱效应 ,1mg·kg-1 甲基苯丙胺组大鼠的伏核壳区、杏仁核、腹侧被盖区和大脑脚区域中NOS阳性细胞数目明显增加。结论·· :一氧化氮在甲基苯丙胺的条件性位置偏爱效应中发挥重要作用     

宁波地区收缴滥用药物的成分分析

目的·· :确定宁波地区收缴滥用药物的主要成分及其掺杂物的种类并对收缴海洛因的含量进行详细分析。方法··:采用气相色谱 -质谱联用法 ,检测2000年度宁波地区收缴的滥用药物的主要成分及其掺杂物。结果··:2000年度宁波地区收缴的滥用药物共送检136份 ,其中检出海洛因123份、氯胺酮5份、曲马朵4份、甲基苯丙胺1份、艾司唑仑1份、大麻1份、三唑仑1份。收缴的滥用药物海洛因的含量差异明显 ,平均含量为35.13 %±s22.63%(n=123) ;海洛因中的掺杂物主要有咖啡因 ,检出率为70.73% (87例)、对乙酰氨基酚 ,检出率为43.90 %(54例)、普鲁卡因 ,检出率为11.38 %(14例)等。结论··:宁波地区收缴的滥用药物以海洛因为主 ,呈现多元化状态 ,氯胺酮滥用上升速度迅猛。海洛因中掺杂物种类繁多。海洛因中乙酰可待因与单乙酰吗啡之间的比值与海洛因的含量有相关性     

INFLUENCE OF EXTRA-INTESTINAL INFLAMMATION ON THE IN VITRO ABSORPTION OF 14C-GLUCOSE AND THE EFFECTS OF ANTIINFLAMMATORY DRUGS IN THE JEJUNUM OF RATS

Inflammation was induced in the hind legs of rats by formalin injection and the in vitro jejunal absorption of ¹⁴C-glucose was studied. Treatment of rats with formalin caused a reduction in the in vitro absorption of glucose from the jejunum. Oral administration of oxyphenbutazone or a herbal anti-inflammatory drug (Withania somnifera) prior to formalin injection, resulted in no alteration in the jejunal absorption of glucose.     

ROLE OF NITRIC OXIDE IN THE DEVELOPMENT OF CORTICOTROPIN-INDUCED HYPERTENSION IN SHEEP

1. The possibility that altered synthesis of vascular nitric oxide (NO) plays a role in the development of corticotropin-induced hypertension in sheep was examined by determining the effect of concomitant infusion of L-arginine, a precursor of NO, on the development of the hypertension. 2. Corticotropin (5 μg/kg per h) infused over 2 days increased mean arterial pressure (MAP) from 83 ± 4 to 99 ± 4 mmHg in five conscious sheep. Concomitant infusion of L-arginine (60 mg/kg per h) did not alter this response; infusion of L-arginine alone had no effect on blood pressure. 3. The dose of L-arginine (60 mg/kg per h) used blocked the rise in MAP (+16 mmHg) in response to a 5 h infusion of N-nitro-L-arginine (1 mg/kg per h). 4. These findings suggest that disruption of NO synthesis does not play a role in the development of corticotropin hypertension in sheep.     

EVIDENCE OF AN ADENOSINE-DEPENDENT MECHANISM IN THE HYPOTENSIVE EFFECT OF l-ARGININE IN MAN

1. The hypothesis that endogenous adenosine could play a role in the haemodynamic response to l-arginine is investigated. 2. The study has been divided into two parts. The first part was a single blind, randomized, placebo-controlled study in which L-arginine i.v. infusion (0.07 mmol/kg per min) in five healthy volunteers caused a significant fall in systolic (-14.2%, from 129.0 ± 8.2 to 110.6 ± 8.5 mmHg; F= 62.89, P<0.0l), diastolic (-16%, from 80.0 ± 7.9 to 67.2 ± 7.0 mmHg; F= 18.97, P < 0.0l) and mean (-15.5%, from 96.4 ± 6.7 to 81.4 ± 6.5 mmHg; F= 28.78, P< 0.01) arterial blood pressure, with a concomitant increase of plasma adenosine concentration (from 244.0 ± 32.2 to 637.0 ± 43.4 nmol/L; F= 79.3 P<10.01). Maximal effects were obtained at the end of L-arginine infusion: haemodynamic parameters returned to basal values in about 30 min while adenosine concentrations normalized in about 15 min. Saline infusion had no effect on these parameters. 3. In the second study the effect of L-arginine i.v. infusion on arterial blood pressure, lower limb blood flow and plasma adenosine, before and after theophylline treatment (1000 mg/day for 3 days, p.o.) was examined. In 10 healthy volunteers the i.v. infusion of l-arginine (0.07 mmol/kg per min) was followed by the same haemodynamic changes as reported above and by a Significant increase in lower limb blood flow (+ 36.7%, from 2.18 ± 0.40 to 2.98 ± 0.71mL/min/lOOmL;t = 4.61, P< 0.01). Pretreatment with theophylline, an adenosine-receptor antagonist, did not affect basal values of arterial pressure, lower limb blood flow and adenosine concentration. The pretreatment with theophylline reduced maximal decrease in systolic pressure (- 8.2 vs -15%), in mean pressure (- 9.9 vs -13.7%) and maximal increase in lower limb blood flow (+19 vs + 37%) caused by i.v. infusion of l-arginine (0.07 mmol/kg per rnin). Such a treatment allowed a progressive restoration of basal blood pressure values and of blood flow, during the second half of l-arginine infusion. This observation was confirmed by the analysis of the area under the curves (AUC). A significant difference in AUC values before and after treatment was obtained for systolic pressure (t = 8.25, P< O.Ol), mean pressure (t= 6.67, P<0.0l) and blood flow (t= 2.31, P<0.05). 4. Theophylline study suggested that the endogenous adenosine increase is sufficient to participate at least in part in the haemodynamic changes caused by l-arginine and that it is involved in a secondary response to l-arginine.