内源性大麻素系统与老年性痴呆

老年性痴呆即阿尔茨海默病（Alzheimer’Sdisease,AD）是一种慢性进行性的神经退行性病变,其特征性病理改变为β-淀粉样蛋白（β-amy-loidpeptide,Aβ）沉积形成的细胞外老年斑和tau蛋白过度磷酸化形成的神经细胞内神经原纤维缠结,以及神经元丢失伴随胶质细胞增生等。最近的研究发现内源性大麻素系统对AD的病程有预防治疗作用,主要基于其对神经的保护作用和抗炎功效。本文综述了内源性大麻素对AD的保护作用的几个方面,旨在为AD的治疗开辟新的策略和思路。     

内源性大麻素系统对心血管功能的调节作用

内源性大麻素通过受体依赖性和非依赖性途径经一系列细胞内信号传导通路,可发挥多种重要的心血管效应,如舒张血管、降低血压和抑制心肌收缩等。它还参与抗炎、保护内皮细胞的作用。因此,对其作用机制的深入研究在防治高血压、动脉粥样硬化、缺血/再灌注损伤和心衰等心血管疾病中有重要意义。     

内源性大麻素系统在精神疾病治疗和麻醉觉醒的调控作用

内源性大麻素系统因其在多种行为和脑功能中的重要作用,以及作为包括焦虑、抑郁等神经精神疾病中的治疗靶点而得到广泛关注。内源性大麻素信号失调会导致负性情绪状态和应激反应增多。对于其潜在的神经细胞特异性和神经环路调节的深入研究,有助于神经精神疾病治疗药物的开发,并有助于更好地了解内源性大麻素系统对包括麻醉觉醒在内的神经功能的环路调节。本文聚焦内源性大麻素系统在神经精神疾病治疗以及麻醉觉醒调节中作用的最新研究进展。     

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。     

内源性大麻素系统对中枢神经的调控及临床应用

内源性大麻素系统（ECS）是人体神经内分泌免疫的调控网络,主要分布在中枢神经系统和免疫系统,与认知、情绪调控功能密切相关。文章介绍了ECS在中枢神经边缘系统的分布,揭示ECS与情绪调节结构在分布上的高度重合;简述了ECS的遗传特质,指出ECS的遗传多样性与人类对多种疾病的易感性有关;阐述了ECS对下丘脑一垂体一肾上腺功能轴（HPA）的负反馈机制;SY析了作为多种情绪失调和精神疾病治疗靶点的潜在可能。     

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。     

蛋白激酶C参与内源性大麻素1型受体对心肌L型钙电流的抑制作用

目的研究激活心肌内源性大麻素1型(CB1)受体是否通过改变蛋白激酶C(PKC)活性从而抑制L型钙电流,探讨激活CB1受体导致PKC活性变化的信号途径。方法利用酶解法制备大鼠心室肌细胞,分别单独应用CB1受体特异性激动剂2-氯乙胺花生四烯酸(ACEA)100 nmol·L-1、CB1受体阻断剂AM251 100 nmol·L-1或PKC非特异性激动剂十四烷酸乙酸大戟二萜醇酯(PMA)500 nmol·L-1孵育细胞10 min;此外提前应用AM251或PMA孵育细胞5 min后,再用ACEA孵育细胞10 min。应用全细胞膜片钳技术记录单个心肌细胞的L型钙电流;应用Pep Tag非放射性蛋白激酶C检测系统检测细胞PKC活性;ELISA试剂盒测定细胞中二酰甘油(DAG)的含量;Western蛋白印迹法测定磷脂酶Cβ(PLCβ)和磷酸化磷脂酶Cβ(p-PLCβ)表达。结果给予ACEA激动大鼠心肌细胞的CB1受体,可显著抑制L型钙电流,最大峰值电流密度由11.4±0.8降至(6.4±1.5)p A·p F-1;预先给予AM251或PMA可以完全阻断此抑制效应。检测心肌细胞PKC活性发现,与正常对照组(1.59±0.50)μmol·min-1·L-1相比,ACEA组心肌细胞PKC活性为(0.69±0.48)μmol·min-1·L-1,明显降低;同样预先给予AM251或PMA完全阻断ACEA对PKC活性的抑制效应。而给予ACEA没有影响心肌细胞DAG含量和PLCβ的磷酸化。结论激活心肌细胞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。     

大麻二酚改善福尔马林诱导的口腔颌面部炎性痛作用及中枢机制

目的 研究大麻二酚（CBD）对口腔颌面部炎性痛的镇痛作用及其中枢机制。方法 采用上唇右侧sc给1%福尔马林溶液（37%～40%甲醛）诱发口腔颌面部炎性痛模型。(1)成年雄性C57BL/6J小鼠随机分为正常对照组、模型组和模型+CBD(0.01,0.03和0.06 mg)组（上唇右侧sc给药），给药5 min后，上唇右侧sc给予1%福尔马林溶液。(2)成年雄性C57BL/6J小鼠随机分为正常对照组、模型组和模型+CBD 0.06 mg组，给药方式及部位同分组(1)。观察记录分组(1)小鼠一相痛（0～6 min）和二相痛（15～45 min）小鼠抓脸时间；行为实验结束45 min后（相当于注射福尔马林90 min后），分组(1)中每组随机选取4只，采用免疫荧光方法检测小鼠三叉神经脊束核尾侧亚核（Sp5C）和前扣带回（ACC）脑区c-Fos蛋白表达。采用内源性大麻素探针结合光纤记录方法检测分组(2)中小鼠二相痛时期小鼠中央导水管周围灰质（PAG）、基底外侧杏仁核（BLA）和ACC脑区内源性大麻素水平。结果 (1)与正常对照组相比，模型组小鼠一相痛和二相痛抓脸时间显著延长（P<0.01）...     

内源性大麻素CB2受体拮抗剂AM630对电针镇痛作用的影响

目的：研究内源性大麻素2型受体（CB2受体）拮抗剂AM630对电针镇痛作用的影响。方法：本课题组采用健康成年雄性SD大鼠共50只，随机分为五组：空白对照组（n=10）；炎症组（n=10）；电针治疗组（n=10）；电针＋CB2拮抗剂AM630组（n=10）：电针＋溶媒对照组（n=10）。除空白对照组外其它各组大鼠于左后肢外踝关节皮下注射完全弗式佐剂50μl，制备单发局限性佐剂关节炎模型，     

合成大麻素类新精神活性物质及其管制研究

合成大麻素类物质是种类最多、结构最多样化、滥用最严重的新精神活性物质。本文综述了合成大麻素类物质的危害,分析了我国合成大麻素类物质缴获和检出情况,探讨了国内外合成大麻素类物质列管方法。提出我国合成大麻素类物质整类列管方法尚需进一步完善,建议加强对合成大麻素类物质制毒物品研究和管控。     

Compartmentalization of endocannabinoids into lipid rafts in a microglial cell line devoid of caveolin-1

BACKGROUND AND PURPOSE

N-acyl ethanolamines (NAEs) and 2-arachidonoyl glycerol (2-AG) are endogenous cannabinoids and along with related lipids are synthesized on demand from membrane phospholipids. Here, we have studied the compartmentalization of NAEs and 2-AG into lipid raft fractions isolated from the caveolin-1-lacking microglial cell line BV-2, following vehicle or cannabidiol (CBD) treatment. Results were compared with those from the caveolin-1-positive F-11 cell line.

EXPERIMENTAL APPROACH

BV-2 cells were incubated with CBD or vehicle. Cells were fractionated using a detergent-free continuous OptiPrep density gradient. Lipids in fractions were quantified using HPLC/MS/MS. Proteins were measured using Western blot.

KEY RESULTS

BV-2 cells were devoid of caveolin-1. Lipid rafts were isolated from BV-2 cells as confirmed by co-localization with flotillin-1 and sphingomyelin. Small amounts of cannabinoid CB₁ receptors were found in lipid raft fractions. After incubation with CBD, levels and distribution in lipid rafts of 2-AG, N-arachidonoyl ethanolamine (AEA), and N-oleoyl ethanolamine (OEA) were not changed. Conversely, the levels of the saturated N-stearoyl ethanolamine (SEA) and N-palmitoyl ethanolamine (PEA) were elevated in lipid raft fractions. In whole cells with growth medium, CBD treatment increased AEA and OEA time-dependently, while levels of 2-AG, PEA and SEA did not change.

CONCLUSIONS AND IMPLICATIONS

Whereas levels of 2-AG were not affected by CBD treatment, the distribution and levels of NAEs showed significant changes. Among the NAEs, the degree of acyl chain saturation predicted the compartmentalization after CBD treatment suggesting a shift in cell signalling activity.

LINKED ARTICLES

This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7     

Evidence for both inverse agonism at the cannabinoid CB1 receptor and the lack of an endogenous cannabinoid tone in the rat and guinea-pig isolated ileum myenteric plexus-longitudinal muscle preparation

Background and purpose:

Cannabinoid receptor agonists reduce intestinal propulsion in rodents through the CB₁ receptor. In addition to its antagonistic activity at this receptor, rimonabant (N-(piperidino)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxyamide) alone augments intestinal transit. Using rat and guinea-pig ileum MPLM (myenteric plexus-longitudinal muscle) preparations, we investigated whether the latter effect was through inverse agonism or antagonism of endocannabinoid agonist(s).

Experimental approach:

Inverse agonism was investigated by comparing the maximal enhancement of electrically evoked contractions of the MPLM by two CB₁ receptor antagonists, AM 251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) and O-2050 [(6aR,10aR)-3-(1-methanesulphonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6-H-dibenzo[b,d]pyran], with that produced by rimonabant. To reveal ongoing endocannabinoid activity, effects of inhibiting endocannabinoid hydrolysis by fatty acid amide hydrolase (FAAH) using AA-5HT (arachidonyl-5-hydroxytryptamine), PMSF (phenylmethylsulphonyl fluoride) or URB-597 (3′-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate), or putative uptake using VDM-11 [(5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide] was evaluated.

Key results:

The presence of CB₁ receptors was revealed by antagonism of exogenous anandamide, arachidonylethanolamide (AEA) and WIN 55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate] by rimonabant. The rank order of potentiation of contractions was AM 251 > rimonabant > O-2050. Neither the FAAH inhibitors nor VDM-11 affected electrically evoked contractions. Each FAAH inhibitor increased the potency of AEA but not WIN 55,212-2. VDM-11 did not alter the inhibitory effect of AEA.

Conclusions and implications:

The different levels of maximal potentiation of contractions by the CB₁ receptor antagonists suggest inverse agonism. The potentiation of the action of AEA by the FAAH inhibitors showed that FAAH was present. The lack of effect of FAAH inhibitors and VDM-11 alone on electrically evoked contractions, and on the potency of exogenous AEA suggests that pharmacologically active endocannabinoids were not released and the endocannabinoid transporter was absent. Thus, the CB₁ receptor antagonists behave as inverse agonists.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     

Cannabinoids and bone: endocannabinoids modulate human osteoclast function in vitro

BACKGROUND AND PURPOSE

Both CB₁ and CB₂ cannabinoid receptors have been shown to play a role in bone metabolism. Crucially, previous studies have focussed on the effects of cannabinoid ligands in murine bone cells. This study aimed to investigate the effects of cannabinoids on human bone cells in vitro.

EXPERIMENTAL APPROACH

Quantitative RT-PCR was used to determine expression of cannabinoid receptors and liquid chromatography-electrospray ionization tandem mass spectrometry was used to determine the presence of endocannabinoids in human bone cells. The effect of cannabinoids on human osteoclast formation, polarization and resorption was determined by assessing the number of cells expressing α_vβ₃ or with F-actin rings, or measurement of resorption area.

KEY RESULTS

Human osteoclasts express both CB₁ and CB₂ receptors. CB₂ expression was significantly higher in human monocytes compared to differentiated osteoclasts. Furthermore, the differentiation of human osteoclasts from monocytes was associated with a reduction in 2-AG levels and an increase in anandamide (AEA) levels. Treatment of osteoclasts with LPS significantly increased levels of AEA. Nanomolar concentrations of AEA and the synthetic agonists CP 55 940 and JWH015 stimulated human osteoclast polarization and resorption; these effects were attenuated in the presence of CB₁ and/or CB₂ antagonists.

CONCLUSIONS AND IMPLICATIONS

Low concentrations of cannabinoids activate human osteoclasts in vitro. There is a dynamic regulation of the expression of the CB₂ receptor and the production of the endocannabinoids during the differentiation of human bone cells. These data suggest that small molecules modulating the endocannabinoid system could be important therapeutics in human bone disease.

LINKED ARTICLES

This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7     

Recent advances in the cannabinoids

This article gives an overview of recent advances in the field of cannabinoid research, with an emphasis on patent literature. The review covers the period from January 2000 to July 2002. The period up to the year 2000 was previously reviewed by Goya and Jagerovic in this journal [1]. In addition to compounds acting directly at the cannabinoid receptor, recent advances in regulation of the endocannabinoid system are also discussed.     

Antinociceptive effects of tetrazole inhibitors of endocannabinoid inactivation: cannabinoid and non-cannabinoid receptor-mediated mechanisms

Background and purpose:

Tetrazoles were recently developed as inhibitors of the cellular uptake of the endocannabinoid anandamide or of its hydrolysis by fatty acid amide hydrolase (FAAH), but were proposed to act also on non-endocannabinoid-related serine hydrolases.

Experimental approach:

We tested, in a model of inflammatory pain induced in mice by formalin, five chemically similar inhibitors: (i) OMDM119 and OMDM122, two potent carbamoyl tetrazole FAAH inhibitors with no effect on anandamide uptake; (ii) LY2183240, a carbamoyl tetrazole with activity as both FAAH and uptake inhibitor; (iii) OMDM132, a non-carbamoyl tetrazole with activity only as uptake inhibitor and iv) OMDM133, a non-carbamoyl tetrazole with no activity at either FAAH or uptake.

Results:

All compounds (2.5–10 mg kg⁻¹, i.p.) inhibited the second phase of the nocifensive response induced by intraplantar injection of formalin. The effects of OMDM119, OMDM122 and OMDM133 were not antagonized by pretreatment with cannabinoid CB₁ receptor antagonists, such as rimonabant or AM251 (1–3 mg kg⁻¹, i.p.). The effects of LY2183240 and OMDM132 were fully or partially antagonized by rimonabant, respectively, and the latter compound was also partly antagonized by the CB₂ receptor antagonist, AM630.

Conclusions and implications:

(i) non-FAAH hydrolases might be entirely responsible for the antinociceptive activity of some, but not all, tetrazole FAAH inhibitors, (ii) the presence of a carbamoylating group is neither necessary nor sufficient for such compounds to act through targets other than FAAH and (iii) inhibition of anandamide uptake is responsible for part of this antinociceptive activity, independently of effects on FAAH.     

A comprehensive patents review on cannabinoid 1 receptor antagonists as antiobesity agents

Introduction: Obesity is a rapidly expanding worldwide health problem. Various targets are investigated presently for the treatment of obesity, but there remains an unmet need for an effective drug therapy with acceptable efficacy levels and reduced side effects. Targeting peripherally located cannabinoid 1 (CB1) receptors is an attractive strategy as these receptors play a vital role in energy homeostasis.

Areas covered: CB1 receptor antagonists constitute one of the most important categories of compounds of interest for the control of obesity. In this review, the authors focus on recent advances (since 2007) in diverse chemical classes of patented compounds belonging to the category of CB1 receptor antagonists.

Expert opinion: Safer CB1 receptor antagonists for the treatment of obesity can be discovered by developing such compounds that act peripherally. Increasing the polar service area, decreasing the lipophilicity and designing of neutral antagonists and allosteric inhibitors are some interesting strategies that could offer promising results.     

The biosynthesis, fate and pharmacological properties of endocannabinoids

The finding of endogenous ligands for cannabinoid receptors, the endocannabinoids, opened a new era in cannabinoid research. It meant that the biological role of cannabinoid signalling could be finally studied by investigating not only the pharmacological actions subsequent to stimulation of cannabinoid receptors by their agonists, but also how the activity of these receptors was regulated under physiological and pathological conditions by varying levels of the endocannabinoids. This in turn meant that the enzymes catalysing endocannabinoid biosynthesis and inactivation had to be identified and characterized, and that selective inhibitors of these enzymes had to be developed to be used as (1) probes to confirm endocannabinoid involvement in health and disease, and (2) templates for the design of new therapeutic drugs. This chapter summarizes the progress achieved in this direction during the 12 years following the discovery of the first endocannabinoid.     

Allosteric modulation of glycine receptors

Inhibitory (or strychnine sensitive) glycine receptors (GlyRs) are anion-selective transmitter-gated ion channels of the cys-loop superfamily, which includes among others also the inhibitory γ-aminobutyric acid receptors (GABA(A) receptors). While GABA mediates fast inhibitory neurotransmission throughout the CNS, the action of glycine as a fast inhibitory neurotransmitter is more restricted. This probably explains why GABA(A) receptors constitute a group of extremely successful drug targets in the treatment of a wide variety of CNS diseases, including anxiety, sleep disorders and epilepsy, while drugs specifically targeting GlyRs are virtually lacking. However, the spatially more restricted distribution of glycinergic inhibition may be advantageous in situations when a more localized enhancement of inhibition is sought. Inhibitory GlyRs are particularly relevant for the control of excitability in the mammalian spinal cord, brain stem and a few selected brain areas, such as the cerebellum and the retina. At these sites, GlyRs regulate important physiological functions, including respiratory rhythms, motor control, muscle tone and sensory as well as pain processing. In the hippocampus, RNA-edited high affinity extrasynaptic GlyRs may contribute to the pathology of temporal lobe epilepsy. Although specific modulators have not yet been identified, GlyRs still possess sites for allosteric modulation by a number of structurally diverse molecules, including alcohols, neurosteroids, cannabinoids, tropeines, general anaesthetics, certain neurotransmitters and cations. This review summarizes the present knowledge about this modulation and the molecular bases of the interactions involved.