Effects of cocaine in 5-lipoxygenase-deficient mice

Summary. 5-Lipoxygenase (5-LOX), along with 12-lipoxygenase and cyclooxygenases, metabolizes arachidonic acid into eicosanoids. In rodents, 12-lipoxygenase deficiency alters behavioral responses to cocaine. We used 5-LOX-deficient mice and their controls to investigate cocaine’s actions. After repeated cocaine injections, the increase in locomotor activity was greater in 5-LOX-deficient mice. Since the 5-LOX pathway may regulate the levels/metabolism of arachidonoylethanolamide (AEA) we assayed the AEA levels in the striatum, the binding of the endogenous AEA to the cannabinoid receptor CB1R, and anandamide hydrolase (FAAH) activity in the striatum, hippocampus, and cortex. Striatal AEA levels decreased after repeated cocaine injections. Cocaine also decreased CB1R binding in all brain regions studied and the only significant differences between 5-LOX-deficient and control mice was the greater hippocampal FAAH activity in 5-LOX-deficient mice. Our results demonstrated that a 5-LOX deficiency alters sensitivity to repeated cocaine. It should be investigated whether a human 5-LOX gene polymorphism affects cocaine’s actions. M. Maccarrone, H. Manev: These authors contributed equally as the senior authors. Correspondence: Hari Manev, Department of Psychiatry, The Psychiatric Institute, University of Illinois, 1601 West Taylor Street, MC912 Chicago, IL 60612, USA     

Localization of N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) expression in mouse brain: A new perspective on N-acylethanolamines as neural signaling molecules

N-acylethanolamines (NAEs) are membrane-derived lipids that are utilized as signaling molecules in the nervous system (e.g., the endocannabinoid anandamide). An N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) that catalyzes formation of NAEs was recently identified as a member of the zinc metallohydrolase family of enzymes. NAPE-PLD(-/-) mice have greatly reduced brain levels of long-chain saturated NAEs but wild-type levels of polyunsaturated NAEs (e.g., anandamide), suggesting an important role for NAPE-PLD in the biosynthesis of at least a subset of endogenous NAEs in the mammalian nervous system. To provide a neuroanatomical basis for investigation of NAPE-PLD function, here we have analyzed expression of NAPE-PLD in the mouse brain using mRNA in situ hybridization and immunocytochemistry. NAPE-PLD(-/-) mice were utilized to establish the specificity of probes/antibodies used. The most striking feature of NAPE-PLD expression in the brain was in the dentate gyrus, where a strong mRNA signal was detected in granule cells. Accordingly, immunocytochemical analysis revealed intense NAPE-PLD immunoreactivity in the axons of granule cells (mossy fibers). Intense NAPE-PLD immunoreactivity was also detected in axons of the vomeronasal nerve that project to the accessory olfactory bulb. NAPE-PLD expression was detected in other brain regions (e.g., hippocampus, cortex, thalamus, hypothalamus), but the intensity of immunostaining was weaker than in mossy fibers. Collectively, the data obtained indicate that NAPE-PLD is expressed by specific populations of neurons in the brain and targeted to axonal processes. We suggest that NAEs generated by NAPE-PLD in axons may act as anterograde synaptic signaling molecules that regulate the activity of postsynaptic neurons.     

Inhibition of fatty acid amide hydrolase suppresses referred hyperalgesia induced by bladder inflammation

Study Type – Aetiology (case series) Level of Evidence 4

OBJECTIVE

? To determine (i) the presence of fatty acid amide hydrolase (FAAH) in the urinary bladder; (ii) whether or not endogenous fatty acid ethanolamides are synthesized by the bladder; (iii) the effects of FAAH inhibition on referred hyperalgesia associated with acute bladder inflammation in rats.

MATERIALS AND METHODS

? Immunohistochemistry and immunoblotting were performed to detect FAAH in the bladder. Acrolein (1 mM, 400 µL) was instilled into bladders of female Wistar rats to induce cystitis. Referred mechanical hyperalgesia was assessed by application of Von Frey monofilaments to the hind paws. ? Animals were killed 4, 24, 48 and 72 h after acrolein instillation, and the fatty acid ethanolamide content of bladders was measured using isotope‐dilution liquid chromatography/mass spectrometry. ? Other rats were treated with the FAAH inhibitor URB597 (0.3 mg/kg, i.p.) after the induction of cystitis, and the mechanical sensitivity of the hind paws was determined.

RESULTS

? Immunohistochemistry and immunoblotting showed the presence of FAAH in the bladder, with greatest abundance in the urothelium. ? Acrolein‐induced cystitis increased fatty acid ethanolamide content (including anandamide) in the bladder in a time‐dependent manner. Inhibition of FAAH diminished referred hyperalgesia associated with acute bladder inflammation.

CONCLUSIONS

? The results obtained in the present study indicate that (i) FAAH is present in the urinary bladder; (ii) fatty acid ethanolamides are increased during bladder inflammation; (iii) inhibition of FAAH could be an effective therapeutic approach for the treatment of bladder pain. ? These results raise the possibility that inhibitors of enzymes responsible for metabolism of fatty acid ethanolamides could inhibit pain associated with bladder inflammation.     

微粒体环氧化酶与谷胱甘肽S转移酶的遗传多态性对新生儿出生体重的影响

「目的」探讨母亲微粒体环氧化酶（ＥＰＨＸ１）与谷胱甘肽Ｓ转移酶（ＧＳＴＭ１，ＧＳＴＴ１）的遗传多态笥对新生儿出生体重的影响。「方法」采用回顾性流行病学调查方法，使用统一调查表，由经过培训的调查员在北京燕山某医院对入院分娩的６３０个母亲婴儿对进行调查。「结果」单因素分析发现：ＥＰＨＸ１和ＧＳＴＴ１、ＧＳＴＭ１基因多态对新生儿出生体重的影响无显著意义。经母亲年龄、文化程度、生育史、芳香烃溶剂暴露、在家     

Detoxication Strategy of Epoxide Hydrolase��The Basis for a Novel Threshold for Definable Genotoxic Carcinogens

From our recent work on the three-dimensional structure of epoxide hydrolases we theoretically deduced the likelihood of a two-step catalytic mechanism that we and others have subsequently experimentally confirmed. Analysis of the rate of the two steps by us and by others show that the first step—responsible for removal of the reactive epoxide from the system—works extraordinarily fast (typically three orders of magnitude faster than the second step), sucking up the epoxide like a sponge. Regeneration of the free enzyme (the second step of the catalytic mechanism) is slow. This becomes a toxicological problem only at doses of the epoxide that titrate the enzyme out. Our genotoxicity work shows that indeed this generates a practical threshold below which no genotoxicity is observed. This shows that—contrary to old dogma—practical thresholds exist for definable genotoxic carcinogens.     

泛素羧基末端水解酶-1抑制剂对多巴胺能神经元的神经毒性作用

Objective To investigate the neurotoxic effects ofLDN-57444, a specific ubiquitin C-termiual hydrolase L1 (UCH-L1) inhibitor, on dopaminergic neurons and the possible mechanism. Methods The viability of SK-N-SH cells exposed to 5, 10, 25, 50, 75 or 100 μmol/L LDN-57444 for 24 h was assessed using MTT assay, and the cell apoptosis was detected with Hoechst staining. Western blot was performed to identify the expressions of UCH-L1 protein, ubiquitin monomer and polyubiquitinated proteins, and the activity of the ubiquitin-proteasome system (UPS) was evaluated with fluorometry. Results After exposure to UCH-LI inhibitor for 24 h, the cell process-like structures of SK-N-SH cells diminished, and the cell body shrank and became spherical. Exposure to LDN-57444 resulted in concentration-dependent reduction of the cell viability, and the reduction became statistically significant following the exposure to 50 μmol/L LDN-57444, as compared with that in the control group (P<0.05). The exposure also resulted in obvious cell apoptosis as shown by nuclear fragmentation and presence of the apoptotie bodies. Western blot detected no obvious changes in UCH-L1 protein expression but identified reduced ubiquitin monomer and increased polyubiquitinated protein expression in the cells. Fluorometry showed reduced activity of UPS in the exposed cells. Conclusion UCH-L1 inhibitor produces neurotoxicity to dopaminergie neurons and induces cell apoptosis possibly as the result of impaired UPS activity and intracellular accumulation of polyubiquitinated proteins following the exposure.     

ACT and UCH-L1 polymorphisms in Parkinson's disease and age of onset.

alpha1-Antichymotrypsin (ACT) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) have been suggested as susceptibility factors for Parkinson's disease (PD). We replicated these findings in a Chinese case-control sample consisting of 160 PD cases and 160 carefully matched control subjects. Genotypes were determined using polymerase chain reaction and BstN1 or Rsa1 restriction enzyme assay. Analysis showed no significant difference between PD patients and controls for genotype or allele frequencies of the ACT and UCH-L1 S18Y polymorphisms. UCH-L1 S18Y polymorphism carriers, however, were found to be significantly less frequent in early-onset PD patients with a reduced risk of 0.557 (95% C.I. = 0.314-0.985; P = 0.043). These data suggest that ACT polymorphism does not influence the risk for developing PD. UCH-L1 S18Y polymorphism, however, may be a weak protective factor against early-onset PD.     

室旁核内脂肪酸酰胺水解酶上调介导慢性心衰大鼠交感神经兴奋亢进

目的:探讨慢性心衰大鼠室旁核(PVN)内脂肪酸酰胺水解酶(FAAH)表达失衡导致中枢交感传出增加的机制。方法:雄性Wistar大鼠左冠状动脉结扎8周后,用超声心动图检测心功能,并通过组织病理学测定心肌梗死面积,鉴定大鼠心衰模型构建成功;酶联免疫吸附法测血浆去甲肾上腺素(NE)水平;Western blot测定PVN内FAAH蛋白表达量;高效液相色谱法测定PVN内N-花生四烯酰乙醇胺(AEA)的生成量;PVN微量注射AEA、FAAH抑制剂PF3845或r AAV2-FAAH sh RNA病毒,记录交感驱动和心功能指标的变化。结果:与假手术组相比,心衰组心功能显著降低,血浆NE显著增加,PVN内FAAH表达量显著上调,AEA生成量显著减少(P0.05);PVN微量灌注PF3845、AEA或r AAV2-FAAH sh RNA病毒后,心衰组交感驱动指标显著降低,心功能明显改善。结论:心衰状态下PVN内FAAH蛋白表达上调可能引起AEA生成量的减少,从而增强交感神经兴奋性,恶化心衰。     

Association between polymorphisms of microsomal epoxide hydrolase and COPD: results from meta-analyses

Background and objective: COPD is a complex polygenic disease in which gene–environment interactions are very important. The gene encoding microsomal epoxide hydrolase (EPHX1) is one of several candidate loci for COPD pathogenesis and is highly polymorphic. Based χ on the polymorphisms of EPHX1 gene (tyrosine/histidine 113, histidine/arginine 139), the population can be classified into four groups of putative EPHX1 phenotypes (fast, normal, slow and very slow). A number of studies have investigated the association between the genotypes and phenotypes of EPHX1 and COPD susceptibility in different populations, with inconsistent results. A systematic review and meta‐analysis of the published data was performed to gain a clearer understanding of this association. Methods: The MEDLINE database was searched for case–control studies published from 1966 to August 2007. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. Results: Sixteen eligible studies, comprising 1847 patients with COPD and 2455 controls, were included in the meta‐analysis. The pooled result showed that the EPHX1 113 mutant homozygote was significantly associated with an increased risk of COPD (OR 1.59, 95% CI: 1.14–2.21). Subgroup analysis supported the result in the Asian population, but not in the Caucasian population. When the analysis was limited to only the larger‐sample‐size studies, studies in which controls were in Hardy–Weinberg equilibrium and studies in which controls were smokers/ex‐smokers, the pooled results supported the conclusion. The EPHX1 139 heterozygote protected against the development of COPD in the Asian population, but not in the Caucasian population. The other gene types of EPHX1 113 and EPHX1 139 were not associated with an increased risk of COPD. The slow activity phenotype of EPHX1 was associated with an increased risk of COPD. The fast activity phenotype of EPHX1 was a protective factor for developing COPD in the Asian population, but not in the Caucasian population. However, the very slow activity phenotype of EPHX1 was a risk for developing COPD in the Caucasian population, but not in the Asian population. Conclusions: The polymorphisms of EPHX1 113 and EPHX1 139 are genetic contributors to COPD susceptibility in Asian populations. The phenotypes of EPHX1 were contributors to overall COPD susceptibility.