蛋白激酶C 参与内源性大麻素花生四烯乙醇胺对L型钙电流的抑制作用

目的：研究内源性大麻素物质花生四烯乙醇胺是否通过改变蛋白激酶C（ PKC）活性从而抑制心肌L型钙电流,并进一步探讨可能改变PKC活性的信号途径。方法应用全细胞膜片钳技术记录单个心肌细胞的L型钙电流（P ＜0 n．05）;应用PepTag非放射性蛋白激酶C检测系统（ Promega）检测PKC活性;Elisa试剂盒测定细胞中二脂酰甘油（ DAG）的含量;western blot 技术测定磷脂酶Cβ（ PLCβ）和磷酸化磷脂酶C β（ p-PLCβ）表达。结果应用花生四烯乙醇胺灌流心肌细胞后显著抑制心肌L型钙电流（ P ＜0．05）,预先应用大麻素1型受体（ CB1）阻断剂AM251或PKC非特异性激动剂佛波醇酯（PMA）可以完全阻断此抑制效应,而大麻素2型受体（CB2）阻断剂AM630没有阻断花生四烯乙醇胺抑制L型钙电流的作用。检测心肌细胞PKC活性发现,花生四烯乙醇胺明显抑制PKC活性（ P ＜0．05）,同样预先应用AM251或PMA完全阻断花生四烯乙醇胺对PKC活性的抑制效应,而AM630无此效应。应用花生四烯乙醇胺没有影响心肌细胞DAG含量和PLCβ的磷酸化。结论本实验首次证明内源性大麻素花生四烯乙醇胺激活心肌细胞CB1受体后抑制细胞PKC的活性,从而抑制L型钙电流,此过程没有PLCβ-DAG途径参与。     

花生四烯乙醇胺对大鼠心功能与心肌一氧化氮水平及一氧化氮合酶活性的影响

目的 观察大麻素类物质花生四烯乙醇胺（anandamide，Ana）对大鼠体外心脏左心室心肌中一氧化氮(NO)含量和一氧化氮合酶(NOS)活性的影响。方法 采用Langendorff方法观察Ana对大鼠体外心脏心率（HR）、冠脉流量（CF）、冠脉灌注压(CPP)、左室压最大上升速率(＋dp/dtmax)、左室压最大下降速率( dp/dtmax)、左室收缩峰压(LVSP)、左室舒张末压(LVEDP)及左室发展压(LVDP)的影响；NO含量和NOS活性采用联苯胺荧光分光光度法测定。结果 Ana可使体外心脏心率、CPP、＋dp/dtmax、 dp/dtmax、LVSP、LVDP降低，使LVEDP升高，CF增加。选择性大麻素CB1受体拮抗药AM251(1 μmol·L 1)可阻断 Ana的部分心脏效应；另一选择性大麻素CB2受体拮抗药AM630(1 μmol·L 1)对Ana的心脏效应无显著影响。NOS抑制药L N 硝基精氨酸甲酯（L NAME）(100 μmol·L 1)对Ana的心脏效应也无显著影响。Ana能增强原生型NOS(cNOS)活性，抑制诱生型NOS(iNOS)活性，促进心肌NO的释放。结论 Ana使离体大鼠心肌收缩力降低，心率减慢，表现出负性肌力和负性频率作用；Ana可舒张冠脉，增加冠脉流量；大麻素CB2受体可能不参与Ana的这些心脏效应，内源性NO也可能不参与调节Ana的心脏效应；可能存在其他新的作用位点调节Ana的心脏效应。Ana调节心肌NOS同工酶活性，增加cNOS活性，降低iNOS活性，促进NO的释放，可能发挥心肌保护作用，在心肌缺血和高血压治疗中有潜在应用前景。     

内源性大麻素—生物合成、信号转导及生物降解

大麻素系统由内源性大麻素、大麻素受体和内源性大麻素失活系统三部分组成,该系统失衡与多种中枢神经系统和免疫系统疾病有关。内源性大麻素水平是衡量大麻素系统活性的主要指标。内源性大麻素代谢途径的深入研究对揭示大麻素系统生理、病理作用以及设计基于该系统的新型治疗药物至关重要。该文综述内源性大麻素的生物合成、信号转导及其降解过程。     

1992—2010年 N-花生四烯酰乙醇胺研究文献计量分析

摘 要：通过文献计量学方法分析内源性大麻素 N-花生四烯酰乙醇胺（anandamide,AEA）的研究现状。以 anandamide 为关键词在 ISI Web of Knowledge 数据库中检索相关文献,对其发表时间、语种、作者、期刊来源、学科类别和被引用次数等进行分析。共检索相关文献3 015篇,主要作者、国家、期刊、学科类别、发表时间依次为：Di Marzo V（220篇）、美国（1 254篇）、Br J Pharmacol（189篇）、生物化学与分子生物学类（2 631篇）、2009年（318篇）。AEA 领域的研究正逐渐受到世界各国研究者的重视,但其产业化、规模化发展还需要很长的发展过程。     

FAAH/MAGL抑制剂在中枢神经系统疾病治疗中的潜在应用

内源性大麻素系统(ECS)因其对大脑的神经保护作用而受到广泛关注,并被视作是神经精神疾病(包括焦虑、抑郁、阿尔茨海默病(AD)和帕金森病(PD)等神经退行性疾病的治疗靶点。内源性大麻素及其配体,以及参与内源性大麻素系统调控和代谢的蛋白可作为治疗神经精神疾病的潜在对象。2种内源性大麻素水解酶脂肪酸酰胺水解酶(FAAH)和单酰甘油脂肪酶(MAGL)的抑制剂可通过维持下丘脑-垂体-肾上腺轴负反馈调节机制的稳定,抑制炎症反应,从而对抑郁/焦虑产生作用。也有报告显示,其可在减少的淀粉样蛋白Aβ沉积和抑制多巴胺神经元的死亡等AD/PD的发病机制中发挥作用。由于FAAH和MAGL抑制剂是减少其水解而间接增加内源性大麻素水平,因此比直接补充外源性大麻素引起的副作用更少。本研究在大麻素的物质基础上总结了大麻素的发展概况,包括3个来源:植物类大麻素,内源性大麻素,合成类大麻类。并介绍了ECS及其参与的相关神经系统疾病。最终落脚于2种内源性大麻素水解酶抑制剂在治疗神经精神疾病方面的最新研究进展,尤其是针对抑郁/焦虑和AD/PD。我们希望完善内源性大麻素系统抗神经精神性疾病的机制网络,为相关疾病的基础及临床研究和治疗提供更多可能性。     

大麻素CB1受体对条件性药物渴求的控制作用

最近研究表明,大麻素CB1受体是用于治疗药物成瘾的一个新靶标。CB1受体存在于大脑激动环路,能调节药物摄取。在模拟人类成瘾的诱导复发动物模型上,阻断CB1受体能减弱暗示诱导性药物渴求的恢复。在多种滥用药物如精神兴奋剂、阿片、尼古丁及酒精等均可观察到防复发的作用。此外,CB1受体在奖赏相关性记忆中也有重要作用,这与内源性大麻素在记忆相关的可塑性中的作用相一致。临床试验证明,CB1受体拮抗剂利莫那班能够抑制复发反应和体重增加。总之,临床及临床前研究均表明,CB1受体拮抗剂为成瘾行为开辟了一种新的治疗途径。     

哺乳动物体内大麻CB1受体分子生物学功能概述

目的探讨大麻CB1受体分子生物学功能。方法回顾大麻CB1受体相关文献。结果大麻受体CB1在CNS表达并结合Gi/o蛋白转换胞内信号,下调一系列广泛的信号机制。结论大麻CB1受体可能与神经类疾病和代谢失衡有关,选择性调节CB1受体是这类疾病首选治疗方法。     

减肥药的作用新靶点及相关新药研发近况

对近年来发现的减肥药作用新靶点，如瘦素及其受体、睫状神经营养因子、大麻素受体、神经肽Y5受体、β3肾上腺素受体、脂肪酸转运蛋白4等进行了介绍，并对以这些新靶点为基础开发的一系列相关新药的研究进展进行了综述。     

大麻素受体与肝病

内源性大麻素系统(the endocannabinoid system,ECS)包括大麻素受体、内源性大麻素及参与生物合成、降解的酶,主要分布于脑部,在外周组织也有分布,包括肝脏。肝脏各种疾病其ECS均可被激活。肝硬化时血管及心脏的CB1受体激活后引起血管舒张及心肌病,CB1受体受阻后,上述作用可受抑制;肝纤维化小鼠模型中,肝星状细胞CB1被激活可引起肝纤维化,CB1受体受阻则可减缓肝纤维化的进展;因高脂饮食或长期饮酒引起的脂肪肝也依赖于外周CB1的激活,包括肝脏CB1受体,同时CB1受体会引起胰岛素抵抗及血脂异常。虽然CB1受体拮抗剂因其精神神经方面的副作用使其临床应用受到限制,但如果能将其副作用限制则有望用于临床。     

大麻素受体及其Ⅰ型抑制剂的研究进展

目前已经确认的大麻素(cannabinoid,CB)受体有两种亚型:CB1和CB2,它们的分布与生理功能各不相同,其选择性抑制剂的研究也是近年来的一个热点。研究表明,CB1受体抑制剂具有良好的抗肥胖活性。本文综述了CB受体及其Ⅰ型抑制剂的研究进展。     

Modulation of sensory neuron potassium conductances by anandamide indicates roles for metabolites

BACKGROUND AND PURPOSE: The endogenous cannabinoid anandamide (AEA) acts at cannabinoid (CB(1)) and vanilloid (TRPV(1)) receptors. AEA also shows antinociceptive properties; although the underlying mechanism for this is not fully understood, both CB(1) and TRPV(1) may be involved. Voltage-activated Ca(2+) channels in rat-cultured dorsal root ganglion (DRG) neurons are modulated by AEA. However, AEA in different populations of neurons enhanced or attenuated KCl-evoked Ca(2+) influx; these effects were linked with soma size. The aim of this study was to determine how AEA or its metabolites might produce these variable responses. EXPERIMENTAL APPROACH: The whole cell patch-clamp technique and fura-2 Ca(2+) imaging were used to characterize the actions of AEA on action potential firing and voltage-activated K(+) currents and to determine whether AEA metabolism plays any role in its effects on cultured DRG neurons. KEY RESULTS: AEA attenuated multiple action potential firing evoked by 300 ms depolarizing current commands in a subpopulation of DRG neurons. Application of 1 microM AEA attenuated voltage-activated K(+) currents and the recovery of KCl-evoked Ca(2+) transients. The insensitivity of these responses to the CB(1) receptor antagonist rimonabant (100 nM) and preincubation of DRG neurons with pertussis toxin suggested that these actions are not CB(1) receptor-mediated. Preincubating DRG neurons with the fatty acid amide hydrolase (FAAH) inhibitor phenylmethylsulphonyl fluoride (PMSF) attenuated the inhibitory actions of AEA on K(+) currents and Ca(2+) influx. CONCLUSION AND IMPLICATIONS: These data suggest that the products of AEA metabolism by FAAH contribute to the attenuation of K(+) conductances and altered excitability of cultured sensory neurons.     

内源性大麻素联用顺铂对人肺腺癌A549细胞的诱导凋亡作用

目的观察内源性大麻素（AEA）、顺铂（DDP）单用或联用对人肺癌A549细胞增殖抑制和诱导凋亡的作用。方法采用四甲基偶氮唑蓝（MTT）法检测AEA和DDP对肺癌A549细胞的增殖抑制作用,以流式细胞仪（FCM）PI单染检测AEA联用DDP对细胞周期的影响,以Annexinv／PI双染法检测AEA联用DDP对细胞凋亡的影响。结果AEA对肺癌细胞增殖有明显的抑制作用,且呈剂量、时间依赖性。10,20μmol／L的AEA和1,2mg／L的DDP联合作用24,48,72h后,细胞增殖抑制率显著高于单用组;两药联用后诱导细胞凋亡的作用显著增强,AEA可使A549细胞阻滞于G2／M期。结论AEA及DDP对肺癌A549细胞的增殖具有显著的抑制作用,并可诱导细胞凋亡,在一定范围内呈量效关系,且两者联合应用具有相加或协同作用。     

CB1 receptors and post-ischemic brain damage: studies on the toxic and neuroprotective effects of cannabinoids in rat organotypic hippocampal slices

Cannabinoids (CBs) are implicated in a number of physiological and pathological mechanisms in the central nervous system, but their exact role in post-ischemic brain injury is unclear. The toxic and neuroprotective effects of synthetic and endogenous CBs were evaluated in rat organotypic hippocampal slices exposed to 20 min oxygen-glucose deprivation (OGD) and in gerbils subjected to bilateral carotid occlusion for 5 min. When present in the incubation medium, the synthetic CB agonists WIN 55212-2 and CP 55940 (1-30 μM) and the CB1 agonist ACEA exacerbated CA1 injury induced by OGD, whereas the CB1 receptor antagonists AM 251 and LY 320135 were neuroprotective with maximal activity at 1 μM. AM 251 (at 3 mg/kg, i.p.) also attenuated CA1 pyramidal cell death in gerbils in vivo. The endocannabinoid 2-arachidonoylglycerol (2-AG) reduced OGD injury in hippocampal slices at 0.1-1 μM, whereas anandamide (AEA) was neurotoxic at the same concentrations. The effects of WIN 55212-2, AEA and 2-AG in slices were all dependent on the activation of CB1 but not CB2 receptors, except for the toxic effects of AEA that were also dependent on vanilloid TRPV1 receptors. Our results suggest that exogenous administration of CB1 agonists and the production of endocannabinoids “on demand” may produce different, if not opposite, effects on the fate of neurons following cerebral ischemia.     

Design, synthesis, and binding studies of new potent ligands of cannabinoid receptors

Despite their different chemical structures, delta9-tetrahydrocannabinol (THC) and anandamide (AEA) have common pharmacological properties. This study was aimed at finding new cannabinoid receptor ligands that overcome the instability of AEA and its analogues. To this end we planned the synthesis of a series of compounds which retained both a rigid structure, like that of plant cannabinoids, and a flexible portion similar to that of anandamide. Binding studies on CB1 and CB2 receptors, anandamide membrane transporter (AMT), and fatty acid amide hydrolase (FAAH) showed that some of the newly developed compounds have high affinity and specificity for cannabinoid CB1 and CB2 receptors. Compound 25 is a potent CB1 and CB2 ligand, with affinity constants significantly lower than AEA and similar to WIN 55-212, compound 52 is a potent CB2 ligand, although not very selective over CB1 receptors, and compound 43 is CB2 ligand, with at least a 26-fold selectivity over CB1 receptors. Compound 25 behaved as a inverse agonist at CB1 receptors as assessed in the cyclic AMP functional assay.     

Binding affinity and agonist activity of putative endogenous cannabinoids at the human neocortical CB1 receptor

We investigated the affinity of putative endocannabinoids (2-arachidonylglycerol, 2-AG; noladin ether, virodhamine) for the human neocortical CB1 receptor. Functional activity of these compounds (including anandamide, AEA) was determined by examining basal and forskolin-stimulated cAMP formation. Assays were performed with synaptosomes, prepared from fresh human neocortical tissue. Receptor affinity was assessed from competition binding experiments with the CB1/2 agonist [3H]-CP55.940 in absence or presence of a protease inhibitor to assess enzymatic stability. Noladin ether and virodhamine inhibited [3H]-CP55.940 binding (Ki: 98, 1740 nM, respectively). Protease inhibition decreased the Ki value of virodhamine (Ki: 912 nM), but left that of noladin ether unchanged. 2-AG almost lacked affinity (Ki lymphoblasic )10 microM). Basal cAMP formation was unaffected by AEA and noladin ether, but strongly enhanced by 2-AG and virodhamine. Forskolin-stimulated cAMP formation was inhibited by AEA and noladin ether (IC50: 69, 427 nM, respectively) to the same extent as by CP55.940 (Imax each approximately 30%). Inhibitions by AEA or noladin ether were blocked by the CB1 receptor antagonist AM251. Virodhamine increased forskolin-stimulated cAMP formation, also in presence of AM251, by approximately 20%. 2-AG had no effect; in presence of AM251, however, 10 microM 2-AG stimulated cAMP formation by approximately 15%. Our results suggest, that AEA and noladin ether are full CB1 receptor agonists in human neocortex, whereas virodhamine may act as a CB1 receptor antagonist/inverse agonist. Particularly the (patho)physiological role of 2-AG should be further investigated, since its CB1 receptor affinity and agonist activity especially in humans might be lower than generally assumed.     

Anandamide and NADA bi-directionally modulate presynaptic Ca2+ levels and transmitter release in the hippocampus

BACKGROUND AND PURPOSE: Inhibitory CB(1) cannabinoid receptors and excitatory TRPV(1) vanilloid receptors are abundant in the hippocampus. We tested if two known hybrid endocannabinoid/endovanilloid substances, N-arachidonoyl-dopamine (NADA) and anandamide (AEA), presynapticaly increased or decreased intracellular calcium level ([Ca(2+)](i)) and GABA and glutamate release in the hippocampus. EXPERIMENTAL APPROACH: Resting and K(+)-evoked levels of [Ca(2+)](i) and the release of [(3)H]GABA and [(3)H]glutamate were measured in rat hippocampal nerve terminals. KEY RESULTS: NADA and AEA per se triggered a rise of [Ca(2+)](i) and the release of both transmitters in a concentration- and external Ca(2+)-dependent fashion, but independently of TRPV(1), CB(1), CB(2), or dopamine receptors, arachidonate-regulated Ca(2+)-currents, intracellular Ca(2+) stores, and fatty acid metabolism. AEA was recently reported to block TASK-3 potassium channels thereby depolarizing membranes. Common inhibitors of TASK-3, Zn(2+), Ruthenium Red, and low pH mimicked the excitatory effects of AEA and NADA, suggesting that their effects on [Ca(2+)](i) and transmitter levels may be attributable to membrane depolarization upon TASK-3 blockade. The K(+)-evoked Ca(2+) entry and Ca(2+)-dependent transmitter release were inhibited by nanomolar concentrations of the CB(1) receptor agonist WIN55212-2; this action was sensitive to the selective CB(1) receptor antagonist AM251. However, in the low micromolar range, WIN55212-2, NADA and AEA inhibited the K(+)-evoked Ca(2+) entry and transmitter release independently of CB(1) receptors, possibly through direct Ca(2+) channel blockade. CONCLUSIONS AND IMPLICATIONS: We report here for hybrid endocannabinoid/endovanilloid ligands novel dual functions which were qualitatively similar to activation of CB(1) or TRPV(1) receptors, but were mediated through interactions with different targets.     

Good news for CB1 receptors: endogenous agonists are in the right place

Endocannabinoids are endogenous ligands of brain-type (CB1) and spleen-type (CB2) cannabinoid receptors. N-Arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) are prototype members of the fatty acid amides and the monoacylglycerols, two groups of endocannabinoids. Unlike CB1, CB2 receptors do not reside within 'caveolae', specialized membrane microdomains that are well-known modulators of the activity of a number of G protein-coupled receptors. In this issue of the British Journal of Pharmacology, Rimmerman and coworkers demonstrate that 2-AG is entirely localized in the caveolae of dorsal root ganglion cells, where also part of AEA (approximately 30%) can be detected. However, most of AEA (approximately 70%) was detected in non-caveolae fractions, that is where CB2 receptors are localized. The different interaction of AEA and 2-AG with membrane microdomains might have significant implications for endocannabinoid-dependent autocrine and/or retrograde-paracrine signalling pathways. It also raises an important question about the structural determinants responsible for a different localization of two apparently similar endocannabinoids within lipid bilayers.     

The endogenous cannabinoid anandamide shares discriminative stimulus effects with ∆(9)-tetrahydrocannabinol in fatty acid amide hydrolase knockout mice

The endogenous cannabinoid system has been noted for its therapeutic potential, as well as the psychoactivity of cannabinoids such as Δ9-tetrahydrocannabinol (THC). However, less is known about the psychoactivity of anandamide (AEA), an endocannabinoid ligand. Thus, the goals of this study were to establish AEA as a discriminative stimulus in transgenic mice lacking fatty acid amide hydrolase (i.e., FAAH -/- mice unable to rapidly metabolize AEA), evaluate whether THC or oleamide, a fatty acid amide, produced AEA-like responding, and assess for CB(1) mediation of AEA's discriminative stimulus. Mice readily discriminated between 6mg/kg AEA and vehicle in a two-lever drug discrimination task. AEA dose-dependently generalized to itself. THC elicited full AEA-like responding, whereas oleamide failed to substitute. The CB(1) antagonist rimonabant attenuated AEA- and THC-induced AEA-appropriate responding, demonstrating CB(1) mediation of AEA's discriminative stimulus. These findings suggest that, in the absence of FAAH, AEA produces intoxication comparable to THC, and consequently to marijuana.     

Biochanin A, a naturally occurring inhibitor of fatty acid amide hydrolase

Background and purpose:

Inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the metabolism of the endogenous cannabinoid (CB) receptor ligand anandamide (AEA), are effective in a number of animal models of pain. Here, we investigated a series of isoflavones with respect to their abilities to inhibit FAAH.

Experimental approach:

In vitro assays of FAAH activity and affinity for CB receptors were used to characterize key compounds. In vivo assays used were biochemical responses to formalin in anaesthetized mice and the ‘tetrad’ test for central CB receptor activation.

Key results:

Of the compounds tested, biochanin A was adjudged to be the most promising. Biochanin A inhibited the hydrolysis of 0.5 µM AEA by mouse, rat and human FAAH with IC₅₀ values of 1.8, 1.4 and 2.4 µM respectively. The compound did not interact to any major extent with CB₁ or CB₂ receptors, nor with FAAH-2. In anaesthetized mice, URB597 (30 µg i.pl.) and biochanin A (100 µg i.pl.) both inhibited the spinal phosphorylation of extracellular signal-regulated kinase produced by the intraplantar injection of formalin. The effects of both compounds were significantly reduced by the CB₁ receptor antagonist/inverse agonist AM251 (30 µg i.pl.). Biochanin A (15 mg·kg⁻¹ i.v.) did not increase brain AEA concentrations, but produced a modest potentiation of the effects of 10 mg·kg⁻¹ i.v. AEA in the tetrad test.

Conclusions and implications:

It is concluded that biochanin A, in addition to its other biochemical properties, inhibits FAAH both in vitro and peripherally in vivo.This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x