Effect of β-naphthoflavone on AhR-regulated genes (CYP1A1, 1A2, 1B1, 2S1, Nrf2, and GST) and antioxidant enzymes in various brain regions of pig

The constitutive and inducible expression of aryl hydrocarbon receptor (AhR) and of the AhR-regulated genes coding for CYP1A1, CYP1A2, CYP1B1, CYP2S1, and Nrf2 was investigated by real-time or traditional PCR in cerebral areas (cortex, cerebellum, midbrain, and hippocampus), blood–brain interfaces (meninges and brain microvessels) and liver obtained from control pigs and from pigs treated with β-naphthoflavone (βNF), a potent AhR agonist. The enzymatic activities of ethoxyresorufin-O-deethylase (EROD), and methoxyresorufin-O-deethylase (MEROD), marker for CYP1A1 and CYP1A2, the GST and various antioxidant enzymes (catalase, superoxide dismutase, GSSG-reductase, and GSH-peroxidase) were also determined in the same CNS regions. The AhR, CYP1A1, CYP1A2, CYP1B1, Nrf2 mRNAs were detected, although at different extent, in all the CNS regions, while CYP2S1 mRNA was detected only in midbrain. In the blood–brain interfaces, the constitutive basal expression of AhR and CYP1A1 was comparable to the hepatic one and even higher for CYP1B1 and Nrf2. The treatment with βNF determined the induction of CYP1A1 and 1B1 (but not of AhR, CYP1A2, and Nrf2) mRNA levels in various CNS areas; notably, CYP1A1 mRNA was increased to about 300-fold in the microvessels. The analysis of enzymatic activities revealed that EROD, but not MEROD, was induced in microsomes but not in mitochondria of all the CNS areas. However, the mitochondrial EROD activities were comparable (in midbrain, meninges) or higher (in cortex, cerebellum, hippocampus) than the microsomal ones, suggesting an important metabolic function of CYP1A1 in this subcellular localization. The activities of GST and antioxidant enzymes were detected in all CNS tissues, with levels lower than the hepatic ones, but found quite evenly distributed and marginally affected by βNF treatment. The high expression of metabolic enzymes found in blood–brain interfaces could represent a very important defence toward toxins of CNS.     

A comparison of the central actions of prostaglandins A1, E1, E2, F1α, and F2α in the rat

The effect of prostaglandins (PGs) A₁, E₁, E₂, F₁, and F₂ administered intraventricularly at doses of 0.02–4.0 g/rat were studied in some behavioral, antinociceptive and anticonvulsant tests in rats. Exploratory and locomotor activity were decreased by all PGs except A₁ and F₂ which had no effect on locomotor activity. All PGs studied, except A₁, induced hyperthermia and afforded protection in the hot-plate analgesic test and against maximal electroshock seizures.     

A comparison of the central actions of prostaglandins A1, E1, E2, F1α, and F2α in the rat

Prostaglandins (PGs) injected into the right lateral brain ventricle (i.v.c.) of the rat increased the sleeping time induced by hexobarbital, chloral hydrate, and ethanol. PGE₁ and PGE₂ intensified chlorpromazine-induced catalepsy, inhibited amphetamine hyperactivity, and significantly depressed the amphetamine-induced stereotypy. NA concentrations were decreased by PGE₁ and PGE₂ and were increased by PGF₂. PGF₂ increased both 5-HT and 5-HIAA levels in rat brain. Total ACh concentrations were increased by PGF₁ and PGF₂. PGE₁, PGE₂, and PGF₂ enhanced the turnover of NA, DA, and 5-HT. PGE₂ counteracted the decreased activity induced by -MT and abolished the hypothermic action of -MT. PGF₂ had little effect on the activity of PCPA pretreated rats, whereas the higher doses of PGF₂ increased body temperature in these animals.     

Proteomic analysis of trichloroethylene-induced alterations in expression, distribution, and interactions of SET/TAF-Iα and two SET/TAF-Iα-binding proteins, eEF1A1 and eEF1A2, in hepatic L-02 cells

Emerging evidence indicates that trichloroethylene (TCE) exposure causes severe hepatotoxicity. However, the mechanisms of TCE hepatotoxicity remain unclear. Recently, we reported that TCE exposure up-regulated the expression of the oncoprotein SET/TAF-Iα and SET knockdown attenuated TCE-induced cytotoxicity in hepatic L-02 cells. To decipher the function of SET/TAF-Iα and its contributions to TCE-induced hepatotoxicity, we employed a proteomic analysis of SET/TAF-Iα with tandem affinity purification to identify SET/TAF-Iα-binding proteins. We identified 42 novel Gene Ontology co-annotated SET/TAF-Iα-binding proteins. The identifications of two of these proteins (eEF1A1, elongation factor 1-alpha 1; eEF1A2, elongation factor 1-alpha 2) were confirmed by Western blot analysis and co-immunoprecipitation (Co-IP). Furthermore, we analyzed the effects of TCE on the expression, distribution and interactions of eEF1A1, eEF1A2 and SET in L-02 cells. Western blot analysis reveals a significant up-regulation of eEF1A1, eEF1A2 and two isoforms of SET, and immunocytochemical analysis reveals that eEF1A1 and SET is redistributed by TCE. SET is redistributed from the nucleus to the cytoplasm, while eFE1A1 is translocated from the cytoplasm to the nucleus. Moreover, we find by Co-IP that TCE exposure significantly increases the interaction of SET with eEF1A2. Our data not only provide insights into the physiological functions of SET/TAF-Iα and complement the SET interaction networks, but also demonstrate that TCE exposure induces alterations in the expression, distribution and interactions of SET and its binding partners. These alterations may constitute the mechanisms of TCE cytotoxicity.     

A study on expression of hMLH1, hMSH2 and PCNA in colorectal adenomas and malignant change type in adenomas

object To explore the effect of hMLHland hMSH2 in carcinogenesis and progression of colorectal carcinoma,and their influences on proliferation in colorectal adenoma and malignant change in adenomas, Methods we investigated the expression of mismatch repair (MMR） gene hMLH1, hMSH2 and proliferation cell nuclear antigen (PCNA)by immunohistochemistry in 63 cases colorectal adenomas,20cases malignant change in adenomas and 20ceses colorectal carcinomas (CBCs). Results The positive rate of hMLH1 and hMSH2 in colorectal adenomas,malignant change in adenomas and CBCs were significantly lower than that in normal mucosa. With increasing dysplasia in adenomas, the expression rate of hMLH1 and hMSH2 protein decreased gradually; The PCNA label index of tumors with overexpression hMSH2 were significantly higher than that of tumors with negative hMSH2 in colorectal malignant change type in adenomas and CRCs. PCNA lable index of tumors with overexpression hMSH2 were significant higher than that of tumors with negative hMSH2 in adenomas with grade Ⅱ, Ⅲ dysplasia. The changes of PCNA label index in colorectal adenomas, malignant change type in adenomas and CBCs were not association with the expression of hMLH1. Conclusion It indicated that the MMB genes mutation and abnormal function of DNA mismatch repair maybe participate in carcinogenesis of CBCs. It is might be an early event in carcinogenesis of CBCs. It suggested that the reduction of proliferating activities in colorectal neoplasms might be related to mutation and defect of MMB gene.     

A comparative double-blind study of loprazolam, 1 mg and 2 mg,versus placebo in anxiety-induced insomnia

Summary

A double-blind trial was carried out in 40 anxious in-patients with insomnia to compare the hypnotic effectiveness and tolerance of loprazolam, 1?mg and 2?mg, versus placebo. Groups of 10 patients received one or other dose of loprazolam or placebo for 7 nights and then placebo for the following 3 nights. Sleep quality and morning residual efiects were assessed each morning during the 10-day trial period by means of a sleep questionnaire. The results showed that I?mg loprazolam was superior to placebo but less effective than 2?mg loprazolam. No side-effects were reported at either dose level.     

Influence of polymorphisms in genes SLC1A1, GRIN2B, and GRIK2 on clozapine-induced obsessive–compulsive symptoms

Rationale

Clinical observations indicate that atypical antipsychotics, especially clozapine, induce obsessive–compulsive (OC) symptoms in schizophrenia patients. Recent data from neuroimaging and clinical trials suggest a role for altered glutamate neurotransmission in the etiology of OC disorder (OCD), and SLC1A1, GRIN2B, and GRIK2 have all been reported to regulate glutamate transmission and affect OCD pathophysiology.

Objectives

This study aimed to determine whether SLC1A1, GRIN2B, and GRIK2 are associated with clozapine-induced OC symptoms.

Methods

A total of 250 clinically stable schizophrenia patients receiving clozapine treatment were recruited. The Yale-Brown Obsessive Compulsive Scale (Y-BOCS) was used to evaluate the severity of OC symptoms. Based on their Y-BOCS scores, 250 patients were divided into the OC and non-OC groups (patients with or without OC symptoms, respectively). Additionally, three reported OCD susceptibility polymorphisms, SLC1A1 (rs2228622), GRIN2B (rs890), and GRIK2 (rs1556995), were genotyped.

Results

Trends of association with OC symptoms were observed in rs2228622A and rs890T alleles. SLC1A1 and GRIN2B interaction was found in the significant two-locus gene–gene interaction model (p?=?0.0021), using the multifactor dimensionality reduction method. Further analysis showed a significant interaction between SLC1A1 and GRIN2B on the Y-BOCS score (F _{6, 137}?=?7.650, p?<?0.001), and individuals with AA/TT genotypes had a significantly higher mean Y-BOCS score than those with other genotypes, except AG/TT.

Conclusions

These results suggest that SLC1A1, GRIN2B, and interactions between the two may potentially confer a susceptibility to OC symptoms in schizophrenia patients receiving clozapine.     

Assessment of CYP1A2 Activity in Clinical Practice: Why,How, and When?

The cytochrome P450 enzyme CYP1A2 mediates the rate-limiting step in the metabolism of many drugs including theophylline, clozapine, and tacrine as well as in the bioactivation of procarcinogens. CYP1A2 activity shows both pronounced intra- and interindividual variability, which is, among other factors, related to smoking causing enzyme induction, to drug intake and to dietary factors which may result in induction or inhibition. In contrast to these exogenous factors, genetic influences on enzyme activity seem to be less pronounced. Therefore, phenotyping of CYP1A2, i.e. the determination of the actual activity of the enzyme in vivo, represents a useful approach both for scientific and clinical applications. CYP1A2 is almost exclusively expressed in the liver. Since liver tissue cannot be obtained for direct phenotyping, a probe drug which is metabolized by CYP1A2 has to be given. Proposed probe drugs include caffeine, theophylline, and melatonin. Caffeine is most often used because of the predominating role of CYP1A2 in its overall metabolism and the excellent tolerability. Various urinary, plasma, saliva, and breath based CYP1A2 caffeine metrics have been applied. While caffeine clearance is considered as the gold standard, the salivary or plasma ratio of paraxanthine to caffeine in a sample taken approximately 6 hr after a defined dose of caffeine is a more convenient, less expensive but also fully validated CYP1A2 phenotyping metric. CYP1A2 phenotyping is applied frequently in epidemiologic and drug-drug interaction studies, but its clinical use and usefulness remains to be established.     

Determination of caffeine and five metabolites simultaneously by HPLC and application on evaluating the activity of CYP1A2, CYP2A6, NAT2 and XO in Chinese healthy volunteers

HPLC同时检测咖啡因及其代谢产物并在健康中国人群中CYP1A2,CYP2A6,NATR和XO酶活性评价中的应用@陈尧$Pharmacogenetics Research Institute, Central South University!Changsha 410078, Hunan, China @欧阳冬生$Pharmacogenetics Research Institute, Central South Univer     

Ochratoxin A and β2-microglobulin in BEN patients and controls

Ochratoxin A (OTA) is a mycotoxin naturally occurring in different foods. OTA is arguably a risk factor for Balkan endemic nephropathy (BEN). The aims of this study are to (1) test the OTA-BEN association in BEN-groups and controls and (2) determine whether urine β2-microglobulin, a marker of impaired ability of the kidneys to re-absorb, is related to OTA. BEN patients had significantly higher OTA serum levels. Within the offspring, OTA was significantly related to higher β2-microglobulin excretion. OTA (2005/2006) was related to a higher incidence of BEN after 2008, providing further evidence that OTA is a risk factor for BEN.     

Induction and decline of hepatic cytochromes P4501A1 and 1A2 in rats exposed to hyperoxia are not paralleled by changes in glutathione S-transferase-α

We investigated the effects of hyperoxia on the activities of hepatic ethoxyresorufin 0-deethylase (EROD) (CYP1A1), methoxyresorufin 0-demethylase (MROD) (CYP1A2), and glutathione transferase-α (GST-α), and the status of protein thiols (PSH) in male Sprague-Dawley rats. Twenty-four h of hyperoxia more than doubled EROD and MROD activities, which were increased 7.6- and 3.3-fold, respectively, after 48 h of hyperoxia. The increases in EROD and MROD activities were paralleled by enhanced CYP1A1/1A2 apoproteins contents, as detected by Western analysis. At 60 h of hyperoxia, by which time hyperoxic Sprague-Dawley rats display marked respiratory distress, pulmonary edema, and other markers of pulmonary dysfunction, the activities and levels of hepatic CYP1A1 and 1A2 had declined dramatically and returned to levels observed in air-breathing control animals. Hepatic activities of GST-α, as well as PSH status, were not altered significantly in the hyperoxic animals at any time point. The marked induction and subsequent decline of hepatic CYP1A1/1A2 activities in rats exposed to hyperoxia suggest that these enzymes may contribute to the mechanisms of injury and/or to adaptive responses to hyperoxic exposures in vivo.     

Gene cloning, expression, purification and characterization of lipoprotein- associated phospholipase A2 in Pichia pastoris

AIM To express and purify Lipoprotein -associated phospholipase A2 (Lp-PLA2), and to establish a screening model for Lp-PLA2 inhibitors through the recombinant Lp-PLA2. METHODS The full-length gene of Lp-PLA2 was cloned from the differentiated THP-1 cells by RT-PCR and PCR. The Lp-PLA2 gene was subcloned into the Pichia expression vector pPIC9 and introduced a sequence encoding a C-terminal stretch of six histidine residues at the same time. The recombinant plasmid was transformed into Pichia pastoris GS115 by spheroplasting and the gene was then integrated into the GS115 genome. Lp- PLA2 was expressed in the yeast strain GS115 by inducing with 0.5% methanol.     

Occurrence, pharmacology and function of presynaptic α2-autoreceptors in α2 A/D-adrenoceptor-deficient mice

The occurrence, pharmacological properties and function of alpha2-autoreceptors were studied in hippocampal slices, occipito-parietal cortex slices, segments of heart atria and segments of the vas deferens of wildtype (WT) mice and mice in which the alpha2 A/D-adrenoceptor gene had been disrupted (alpha2 A/D(KO)). Tissues were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically. Stimulation periods for brain slices consisted either of 1 pulse or of 2-64 pulses delivered at 1-s intervals; stimulation periods for peripheral tissues consisted either of 1 POP (pseudo-one-pulse; brief burst of 20 pulses/50 Hz) or of 2-4 POPs delivered at 1-s intervals. Single pulses or POPs were used to study the effect of medetomidine and its interaction with antagonists. One or more pulses or POPs per stimulation period were used to study alpha2-autoinhibition. Medetomidine decreased the evoked overflow of tritium in WT tissues. In alpha2 A/D(KO) tissues, the inhibition was slightly (peripheral tissues) or greatly (brain slices) attenuated but not abolished. Phentolamine, rauwolscine, spiroxatrine, 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), tolazoline and prazosin antagonized the effect of medetomidine in all tissues. Their pKd values against medetomidine were compared with pKd values at prototypical alpha2 binding sites by means of a correlation analysis. For WT brain and atrial autoreceptors, the correlations indicated an alpha2D pharmacology, whereas for WT vas deferens autoreceptors they favoured an alpha2B pharmacology. In the KO tissues, any correlation with alpha2D was lost, and the non-alpha2 A/D-autoreceptors displayed alpha2B or alpha2C pharmacology. When 2 or more pulses or POPs were applied to WT tissues per stimulation period, the pulse number-overflow curve (POP number-overflow curve) was flat, indicating that overflow elicited by p pulses (POPs) was much smaller than p times the overflow elicited by a single pulse (POP); moreover, rauwolscine caused a pulse (POP) number-dependent and, at high pulse (POP) numbers, large increase in evoked tritium overflow. In alpha2 A/D(KO) tissues, the pulse (POP) number-overflow curve was much steeper, indicating that overflow elicited by p pulses (POPs) was closer to p times the overflow elicited by a single pulse (POP); moreover, rauwolscine caused no (atria) or only a small increase in overflow, and did so in brain slices only at high pulse numbers (16 and 64). In conclusion, the predominant alpha2D pharmacology of the autoreceptors in WT tissues supports the idea that the main mammalian presynaptic alpha2-autoreceptors belong to the alpha2 A/D subtype. However, alpha2 A/D-deficient animals also possess autoreceptors. As expected, these non-alpha2 A/D-autoreceptors display alpha2B or alpha2C pharmacology. In WT animals, alpha2B- or alpha2C-autoreceptors or both may coexist with alpha2 A/D-autoreceptors, at least in peripheral tissues. Little autoinhibition by released noradrenaline in trains of pulses remains when the alpha2 A/D-adrenoceptor is lacking, again in accord with a predominance of alpha2 A/D-autoreceptors.     

Sodium arsenite-induced abnormalities in expressions of Caveolin-1, eNOS,IKKβ, and COX-2 in SV-40 immortalized human uroepithelial cells and in urothelial carcinomas

Arsenic, a known human carcinogen, is found throughout the crust of the earth. Prolonged arsenic exposure is a known cause of urothelial carcinoma (UC) and blackfoot disease (BFD). The aim of this study was to determine the effect of sodium arsenite on Caveolin-1 and downstream signaling molecules (eNOS, IKKβ and COX-2) expression in human urothelial cells (SV-HUC-1). Immunohistochemical (IHC) staining of Caveolin-1, eNOS, IKKβ, and COX-2 was also compared between UC patients from endemic and non-endemic areas of BFD in Taiwan. Immunocytochemical staining and Western blotting results revealed increased expression of Caveolin-1, IKKβ, and COX-2 but decreased eNOS in SV-HUC-1 cells treated with low concentration of arsenite. Additionally, MEK inhibitor (U0126) significantly attenuated arsenite-induced expression of Caveolin-1, IKKβ and COX-2 while reducing eNOS expression. The IHC staining of UCs revealed that expressions of Caveolin-1, IKKβ, and COX-2 were significantly higher in patients from endemic areas of BFD compared to patients from non-endemic areas (p = 0.011, p = 0.002, p = 0.0001) whereas eNOS was significantly lower (p = 0.0001). The correlation observed between Caveolin-1 and downstream signaling molecule expression may be an important mechanism of arsenic-induced urothelial carcinogenesis.     

Involvement of central β2-adrenergic, NMDA and thromboxane A2 receptors in the pressor effect of anandamide in rats

Intravenous (i.v.) injection of the endocannabinoid anandamide induces triphasic cardiovascular responses, including a pressor effect mediated via unknown central and peripheral mechanism(s). The aim of the present study was to determine the central mechanism(s) responsible for the pressor response to anandamide. For this purpose, the influence of antagonists at thromboxane A₂ TP (sulotroban, daltroban, SQ 29548), NMDA (MK-801) and β₂-adrenergic receptors (ICI 118551) on the pressor effect induced by i.v. and intracerebroventricularly (i.c.v.) administered anandamide was examined in urethane-anaesthetized rats. Anandamide (1.5–3 µmol/kg, i.v.) or its stable analogue methanandamide (0.75 µmol/kg, i.v.) increased blood pressure by 25%. Anandamide (0.03 μmol per animal i.c.v.) caused a pure pressor effect (by 20%) but only in the presence of antagonists of CB₁ and TRPV1 receptors. The effects of cannabinoids (i.v. or i.c.v.) were diminished by i.v. daltroban, sulotroban (10 μmol/kg each), and/or SQ 29548 (1 μmol/kg). The effect of anandamide i.v. was reduced by SQ 29548 (0.02 μmol per animal i.c.v.) and by the thromboxane A₂ synthesis inhibitor furegrelate i.c.v. (1.8 µmol per animal). ICI 118551, MK-801 (1 µmol/kg i.v. each), and bilateral adrenalectomy diminished the effect of anandamide i.c.v. Sulotroban (i.v.) failed to affect the response to anandamide (i.v.) in pithed rats, and anandamide and methanandamide did not bind to TP receptors in rat platelets. The present study suggests that central β₂-adrenergic, NMDA and thromboxane A₂ receptors are involved in the anandamide-induced adrenal secretion of catecholamines and their pressor effect in urethane-anaesthetized rats.     

Metabolism of sanguinarine in human and in rat: Characterization of oxidative metabolites produced by human CYP1A1 and CYP1A2 and rat liver microsomes using liquid chromatography–tandem mass spectrometry

The quaternary benzo[c]phenanthridine alkaloid, sanguinarine (SA), has been detected in the mustard oil contaminated with Argemone mexicana, which produced severe human intoxications during epidemic dropsy in India. Today, SA metabolism in human and in rat has not yet been fully elucidated. The goal of this study is to investigate the oxidative metabolites of SA formed during incubations with rat liver microsomes (RLM) and recombinant human cytochrome P450 (CYP) and to tentatively identify the CYP isoforms involved in SA detoxification. Metabolites were analyzed by liquid chromatography combined with electrospray ionization-tandem mass spectrometry. Up to six metabolites were formed by RLM and their modified structure has been proposed using their mass spectra and mass shifts from SA (m/z 332). The main metabolite M2 (m/z 320) resulted from ring-cleavage of SA followed by demethylation, whereas M4 (m/z 348) is oxidized by CYP in the presence of NADPH. The diol-sanguinarine metabolite M6 (m/z 366) formed by RLM might derive from a putative epoxy-sanguinarine metabolite M5 (m/z 348). M4 and M6 could be detected in rat urine as their respective glucuronides. 5,6-Dihydrosanguinarine is the prominent derivative formed from SA in cells expressing no CYP. Oxidative biotransformation of SA was investigated using eight human CYPs: only CYP1A1 and CYP1A2 displayed activity.     

Presynaptic α2-adrenoceptor,opioid κ-receptor and adenosine A1-receptor interactions on noradrenaline release in rabbit brain cortex

Summary The interaction of presynaptic, release-inhibiting ₂-adrenoceptors, opioid -receptors and adenosine A₁-receptors was studied in slices of the occipito-parietal cortex of the rabbit. The slices were preincubated with ³H-noradrenaline and then superfused and stimulated electrically twice for 2 min each (S₁, S₂). The stimulation-evoked overflow of tritium was taken to reflect action potential-evoked release of noradrenaline. One of two release-modulating compounds to be examined for interaction was kept in the medium throughout superfusion, the other one was added before S2. In many experiments, the stimulation parameters were adjusted (frequency 0.5–7 Hz; voltage drop 2–5 V/cm) in order to obtain similar reference release (S₁) values despite the presence of the first release-modulating compound.The selective -receptor agonist ethylketocyclazocine (EK) attenuated markedly the release-inhibiting effects of the ₂-adrenoceptor-selective agonists clonidine and -methylnoradrenaline as well as the release-facilitating effect of the ₂-adrenoceptor-selective antagonist yohimbine. The attenuation occurred both when the parameters of electrical stimulation were kept constant and when they were adjusted to obtain similar S₁ release values. The selective A₁-receptor agonist R-N⁶-phenylisopropyladenosine (PIA) also attenuated the effects of clonidine and yohimbine. Conversely, clonidine attenuated and yohimbine enhanced the release-inhibiting effect of PIA. Yohimbine also enhanced the release-facilitating effect of the adenosine receptor antagonist 8-phenyltheophylline. Again, these changes occurred both at constant stimulation parameters and when stimulation parameters were adjusted. EK attenuated the release-inhibiting effect of PIA, and conversely PIA attenuated the effect of EK, both at constant and at adjusted parameters of electrical stimulation. The release-inhibiting effects of tetrodotoxin and Cd²⁺ remained unchanged in the presence of clonidine or EK.These results demonstrate mutual interactions between presynaptic a2-, opioid K- and adenosine A₁-receptors. As soon as any one of the three systems is activated, the inhibition due to activation of either of the two remaining systems is blunted. The interactions are not a consequence of the change in release per se that the first receptor ligand inevitably produces. -Adrenoceptors interact with opioid -receptors in a similar manner, independently of the chemical nature (imidazoline or phenylethylamine derivative) of the -agonist used. The interaction is specific for release-modulating receptors and does not extend to Na⁺ ar Ca²⁺ channel blockers. It may occur at the level of the receptors themselves or at the post-receptor transduction mechanisms.Send offprint requests to N. Limberger at the above address     

Anticholinesterase effects of huperzine A,E2020,and tacrine in rats

目的：比较石杉碱甲、Ｅ２０２０和他克林对大鼠胆碱酯酶的抑制作用．方法：比色法测定大鼠各脑区的ＡＣｈＥ及血清中ＢｕＣｈＥ活力．结果：三药灌胃后对大鼠脑中ＡＣｈＥ均产生剂量依赖性抑制．以石杉碱甲作用最强．他克林对ＢｕＣｈＥ的抑制显著强于Ｅ２０２０和石杉碱甲，其副反应最为明显．单次经口给药后，石杉碱甲对脑内ＡＣｈＥ的抑制作用长于Ｅ２０２０和他克林．多次给药后，对胆碱酯酶的抑制作用三药均未有耐受性产生．结论：石杉碱甲是一种高选择性ＡＣｈＥ抑制剂，具有口服活性高，作用时程长，副反应小的优点，适于临床应用．     

Presynaptic α2-autoreceptors in brain cortex: α2D in the rat and α2A in the rabbit

Summary Presynaptic ₂-autoreceptors in rat and rabbit brain cortex were compared by means of antagonists and agonists. Brain cortex slices were preincubated with [³H]-noradrenaline and then superfused and stimulated by 3 (rat) or 4 (rabbit) pulses at a frequency of 100 Hz.The ₂-adrenoceptor agonist bromoxidine (UK 14 304) reduced the electrically evoked overflow of tritium with EC₅₀ values of 4.5 nmol/l in the rat and 0.7 nmol/l in the rabbit. The antagonists phentolamine, 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole (BRL 44408), rauwolscine, 1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo(c,f)imidazo(1,5-a)azepine (BRL 41992), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4, 5-tetrahydro-3-benzazepine (SKF 104078), imiloxan, prazosin and corynanthine did not per se increase the evoked overflow of tritium but shifted the concentration-inhibition curve of bromoxidine to the right in a manner compatible with competitive antagonism. Up to 4 concentrations of each antagonist were used to determine its dissociation constant K_D. The K_D values correlated only weakly between the rat and the rabbit. Dissociation constants K_A of bromoxidine were calculated from equieffective concentrations in unpretreated brain slices and slices in which part of the ₂-adrenoceptors had been irreversibly blocked by phenoxybenzamine. The K_A value was 123 nmol/l in the rat and 7.2 nmol/l in the rabbit.The results confirm the species difference between rat and rabbit brain presynaptic ₂-autoreceptors. Comparison with data from the literature indicates that the rat brain autoreceptors can be equated with the _2D subtype as defined by radioligand binding, whereas the rabbit brain autoreceptors conform to the _2A subtype. For example, the antagonist affinities for the rat autoreceptors correlate with their binding affinities for the gene product of ₂-RG20, the putative rat _2D-adrenoceptor gene (r = 0.97; P<0.01), but not with their binding affinities for the gene product of ₂-C10, the putative human _2A-adrenoceptor gene. Conversely, the rabbit autoreceptors correlate with the ₂-C10 (r = 0.98; P<0.001) but not with the ₂-RG20 gene product. Since presynaptic ₂-autoreceptors are also _2D in rat submaxillary gland and perhaps vas deferens and _2A in rabbit pulmonary artery, the possibility arises that the majority of ₂-autoreceptors generally are _2D in the rat and _2A in the rabbit. Moreover, receptors of the _2A/D group generally may be the main mammalian ₂-autoreceptors.Correspondence to: N. Limberger at the above address