NADPH氧化酶家族在心血管疾病中作用的研究进展

烟酰胺腺嘌呤二核苷酸磷酸氧化酶是心血管系统中活性氧(reactive oxygen species,ROS)的主要来源,它包 括7种亚型并分别表达在不同的心血管细胞及其细胞器中,参与调节细胞增殖、迁移、分化、凋亡、衰老和炎症反应 等多种活动,其衍生的ROS参与高血压、动脉粥样硬化、糖尿病血管病变、心肌梗死后心室重构等多种心血管疾病 的病理过程。     

别嘌呤醇对离体大鼠缺血再灌注心肌的保护作用

为探讨别嘌呤醇(allo)对心肌的保护作用与机理,采用离体大鼠心脏灌注模型,研究allo对缺血再灌注心肌的保护作用。结果表明心肌缺血再灌注后磷酸肌酸肌酶(CPK)释放增多,超氧化物歧化酶(SOD)活性降低,黄嘌呤氧化酶(XO)活性和丙二醛(MDA)量增加,心肌超微结构破坏明显。allo通过抑制氧自由基的生成对缺血再灌注心肌起保护作用。     

Stoichiometry of the Reaction Catalyzed by Skin Sulfhydryl Oxidase

The stoichiometry of the reaction catalyzed by skin sulfhydryl oxidase was investigated. Dithiothreitol (DTT) was used as the substrate for skin sulfhydryl oxidase. The consumption of DTT, consumption of oxygen, and production of hydrogen peroxide were measured during the enzyme reaction. The molar ratio of DTT:O₂:H₂O₂ in the enzyme reaction was 1:1.02:0.89. Correspondingly, the stoichiometry of the enzyme reaction was calculated to be R(SH)₂ + O₂ → + H₂O₂.     

In vivo comparison of the effects of inhibition of MAO-A versus MAO-B on striatal L-DOPA and dopamine metabolism

Summary Utilizing the cerebral microdialysis technique, we have compared in vivo the effects of selective MAO-A, MAO-B, and nonselective MAO inhibitors on striatal extracellular levels of dopamine (DA) and DA metabolites (DOPAC and HVA). The measurements were made in rats both under basal conditions and following L-DOPA administration. Extracellular levels of dopamine were enhanced and DA metabolite levels strongly inhibited both under basal conditions and following L-DOPA administration by pretreatment with the nonselective MAO inhibitor pargyline and the MAO-A selective inhibitors clorgyline and Ro 41-1049. The MAO-B inhibitor deprenyl had no effect on basal DA, HVA, or DOPAC levels. Nervertheless, deprenyl significantly increased DA and decreased DOPAC levels following exogenous L-DOPA administration, a finding compatible with a significant glial metabolism of DA formed from exogenous L-DOPA. We conclude that DA metabolism underbasal conditions is primarily mediated by MAO-A. In contrast, both MAO-A and MAO-B mediate DA formation when L-DOPA is administered exogenously. The efficacy of newer, reversible agents which lack the cheese effect such as Ro 41-1049 are comparable to the irreversible MAO-A inhibitor clorgyline. The possible relevance of these findings for the treatment of Parkinson's disease is discussed.     

Subtype specificity of γ-aminobutyric acid type A receptor antagonism by clozapine

Clozapine, an atypical neuroleptic, functionally antagonizes the -aminobutyric acid-induced chloride uptake via the main central inhibitory receptor, -aminobutyric acid type A (GABA_A) receptor, in brain vesicles. GABA_A antagonism by micromolar concentrations of clozapine is more efficient in rat cerebrocortical and hippocampal membranes than in cerebellar membranes, as evidenced by clozapine reversal GABA-inhibition of [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding. A typical neuroleptic, haloperidol, failed to antagonize GABA in any of these brain regions, while the specific GABA_A antagonist 2-(3-carboxy-2,3-propyl)-3-amino-6-p-methoxyphenylpyrazinium bromide (SR 95531) was efficient in all three brain regions. Clozapine action on [³⁵S]TBPS binding was unaffected by the benzodiazepine receptor antagonist flumazenil. Clozapine inhibited the binding of [³H]muscimol and [³H]SR 95531 to the GABA recognition site, but this effect only partially correlated with the regional differences in and the potency of clozapine antagonism of GABA-inhibition of [³⁵S]TBPS binding, suggesting that also other than GABA sites may mediate clozapine actions. Autoradiography of [35S]TBPS binding revealed GABA antagonism by clozapine in most brain regions. Main exceptions were cerebellar granule cell and molecular layers, olfactory bulb external plexiform and glomerular layers and primary olfactory cortex, where clozapine antagonized GABA inhibition less than average, and lateral hypothalamic and preoptic areas where its antagonism was greater than average. Recombinant 622 receptors, the predominant 6 subunit-containing receptor subtype in cerebellar granule cells, failed to show GABA antagonism by clozapine up to 100 M. In contrast, recombinant 122 receptors, forming the predominant receptor subtype in the brain, were clozapine sensitive. Recombinant 622 and 632 receptors resulted in clozapine-insensitive receptors, whereas 612 receptors were clozapine sensitive. The efficacy of clozapine to antagonize GABA in 1x2 receptors decreased in the order of 112>122>132. The results indicate that clozapine antagonizes the function of most GABA_A receptor subtypes, and that the interaction is determined by the interaction of the and subunit variants. GABA antagonism is a unique property of clozapine, not shared by haloperidol, which might be involved in the pharmacological mechanism for the increased seizure susceptibility associated with clozapine treatment.     

Xanthine oxidase inhibition by allopurinol affects the reliability of urinary caffeine metabolic ratios as markers for N-acetyltransferase 2 and CYP1A2 activities

Objectives: To evaluate the in vivo effect of xanthine oxidase (XO) inhibition by allopurinol on the determination of polymorphic N-acetyltransferase 2 (NAT2) and cytochrome P450 1A2 (CYP1A2) with urinary caffeine metabolic ratios. Methods: In an open, prospective study involving 21 healthy subjects (eight fast, 13 slow NAT2 acetylators) allopurinol (300 mg perday) was administered orally on trial days 1–8, followed by a wash-out period of 8 days. Urinary caffeine tests (200 mg caffeine p.o.) were performed repetitively. Urine was collected for 8 h and venous blood samples for the determination of allopurinol, oxypurinol and uric acid were drawn. The urinary caffeine metabolites 1-methyluric acid (1MU), 1-methylxanthine (1MX), 1,7-dimethyluric acid (17MU), 1,7-dimethylxanthine (17MX), 5-acetylamino-6-formylamino-3-methyluracil (AFMU), plasma allopurinol and oxypurinol were analysed using high-performance liquid chromatography (HPLC). Results: During XO inhibition by allopurinol, the formation of 1MU from 1MX and therefore the XO ratio 1MU/1MX decreased to 15.9 (1.2)% [mean with (SEM)] of baseline values (P < 0.005). The NAT2 ratio AFMU/1MX decreased likewise to 56.7 (6.3)% (P < 0.005). AFMU/(AFMU + 1MX + 1MU), an alternative NAT2 ratio, remained constant, but the CYP1A2 ratio (AFMU + 1MX + 1MU)/17MU, used to express CYP1A2 activity, transiently increased to 167 (13)% (P < 0.005). The NAT2 phenotype did not influence CYP1A2 and XO ratios or plasma oxypurinol pharmacokinetics. Conclusions: Several caffeine metabolic ratios are commonly used to express the activities of NAT2, CYP1A2 and XO both in healthy volunteers and in polymedicated patients, although their reliability has not been evaluated thoroughly during concurrent drug administration. The findings of this study suggest that NAT2 phenotyping should be performed using the ratio AFMU/(AFMU + 1MX + 1MU) if an XO inhibitor may be present. It also shows that the determination of CYP1A2 activity with caffeine as a metabolic probe is considerably altered under these conditions. Thus, concomitant drug administration may impair the robustness of multiple pathways of the complex caffeine test. This points to the need for alternative probes, designed to assess only the activity of a single enzyme because, in contrast to healthy volunteers, in patients known or unknown drug interactions may often be present. Received: 10 August 1998 / Accepted in revised form: 5 October 1998     

Monoamine oxidase-dependent metabolism of dopamine in the striatum and substantia nigra of

Summary Adult female NMRI mice exposed four times to 100 ppm hydrogen sulfide vapour for 2 h at 4-day intervals showed increasing inhibition of the cerebral cytochrome oxidase activity. Cerebral RNA decreased significantly after the fourth exposure. This change was accompanied by the reduced orotic acid uptake in the RNA fraction. At the same time, 2,3-cyclic nucleotide 3-phosphohydrolase activity used as a marker for glia increased. Acetylcholine esterase activity remained unchanged. The initial exposures also caused an increase in the superoxide dismutase activity and an increase in the glutathione concentration. The latter effects were abolished in the third and fourth exposures. The present data seem to indicate that the biochemical effects of repeated subclinical hydrogen sulfide intoxications are cumulative.     

Cumulative biochemical effects of repeated subclinical hydrogen sulfide intoxication in mouse brain

Summary Adult female NMRI mice exposed four times to 100 ppm hydrogen sulfide vapour for 2 h at 4-day intervals showed increasing inhibition of the cerebral cytochrome oxidase activity. Cerebral RNA decreased significantly after the fourth exposure. This change was accompanied by the reduced orotic acid uptake in the RNA fraction. At the same time, 2,3-cyclic nucleotide 3-phosphohydrolase activity used as a marker for glia increased. Acetylcholine esterase activity remained unchanged. The initial exposures also caused an increase in the superoxide dismutase activity and an increase in the glutathione concentration. The latter effects were abolished in the third and fourth exposures. The present data seem to indicate that the biochemical effects of repeated subclinical hydrogen sulfide intoxications are cumulative.     

Titration of human brain monoamine oxidase-A and-B by clorgyline andl-deprenil

Summary The interaction of clorgyline andl-deprenil with the-A and-B forms of human brain monoamine oxidase (MAO) has been studied. Both compounds inhibit cerebrocortical MAO in a manner consistent with a suicide inactivation of the enzyme. The interaction of clorgyline with the-A form of the enzyme appears to take place almost entirely at specific binding sites, and the conditions required for this inhibitor to titrate the concentrations of MAO-A have been elucidated.l-Deprenil has also been used to titrate the concentration of the-B form of MAO in cerebrocortical homogenates, but there is a considerable degree of non-specific binding of this compound. The two inhibitors have been used to titrate the concentrations of the two enzyme forms in frontal cortex homogenates from different age groups. There was a significantly higher MAO-B activity for the age range 73–95 years than for the age range 2–63 years. No significant differences between the two age groups were found for MAO-A. The activity of MAO-A in the samples correlated very well with the concentration of this enzyme form. Titration of the B-form of the enzyme withl-deprenil indicated an increased enzyme concentration with age, although other factors, such as the non-specific binding of this compound, could contribute to this effect.     

Evolution of changes in neuronal activity in the subthalamic nucleus of rats with unilateral lesion of the substantia nigra assessed by metabolic and electrophysiological measurements

Cellular expression of cytochrome oxidase subunit I (COI) mRNA has recently been used as a metabolic marker for neuronal activity to study the functional changes in the subthalamic nucleus (STN) in parkinsonism. The previous experimental studies have been performed when the pathological state was stabilized at a maximal level. In order to determine the evolution of changes in neuronal activity in the STN after nigrostriatal denervation, we analysed by in situ hybridization the cellular expression of COI mRNA in the subthalamic neurons at different times, from 6 h to 14 days, after unilateral intranigral microinjection of 6-hydroxydopamine (6-OHDA) in rats. In parallel, the time-dependent changes of the unit neuronal activity of subthalamic neurons have been recorded. Levels of COI mRNA increased by 41% in subthalamic neurons from 24 h after 6-OHDA intoxication, to 14 days (+26%). Similarly, electrical activity started to increase slightly 24 h after lesion (+20%) and remained significantly higher at 14 days after the lesion (+189%). Changes in neuronal mean discharge rate were associated with changes in the pattern of spiking activity, from a regular firing pattern to an irregular one with a high bursting activity. These results show that: (i) the hyperactivity of the STN represents a very early phenomenon in the physiopathology of parkinsonian syndromes; and (ii) that changes in COI mRNA expression slightly precede changes in electrical neuronal activity.