脂筏介导的内源性大麻素对HepG2细胞的作用及其机制

目的 研究内源性大麻素(AEA)以脂质为基础的信号途径对肝癌细胞株HepG2的作用机制,探讨AEA在肝癌发生和发展中的作用.方法 免疫荧光检测脂肪酸水解酶、大麻素受体(CB)1和CB2在胎肝细胞株L02和肝癌细胞株HepG2中的定位.以不同浓度的AEA及膜胆固醇耗竭剂甲基-β-环糊精(MCD)处理,分为AEA 10 μmol/L组、AEA 20 μmol/L组、AEA40 μmol/L组,MCD 10 mmol/L+AEA 10 μmol/L组、MCD 10 mmol/L+AEA 20 μmol/L组和MCD 10 mmol/L+AEA 40 μmol/L组,分别孵育L02细胞和HepG2细胞,流式细胞术碘化丙啶单染法检测细胞的坏死率.Western Blot检测L02和HepG2细胞中脂肪酸水解酶、CB1和CB2的蛋白表达及其下游信号通路磷酸化P38促分裂原活化蛋白激酶(p-P38MAPK)和磷酸化c-Jun氨基端激酶(P-JNK)的表达变化.组间均数的比较采用独立样本t检验或单因素方差分析.结果 AEA可有效地导致肝癌细胞坏死,以浓度为AEA 40 μmol/L组达到最大效应,F=108.594,P<0.05,差异有统计学意义.MCD 10mmol/L预孵育后的HepG2细胞坏死率在AEA 10 μmol/L组、AEA 20 μmol/L组和AEA 40 μmol/L组分别为7.83%±2.13%,16.30%±0.94%,43.09%±5.10%,MCD处理前AEA 10 μmol/L组、AEA 20 μmol/L组、AEA 40 μmol/L组分别为13.64%±1.69%、20.28%±0.91%,52.71%±4.29%,处理前后比较,t值分别为3.702,5.274和3.503,P值均<0.05,差异均有统计学意义.同时,AEA能激活HepG2细胞中P38 MAPK和JNK,以AEA 40 μmol/L组作用明显,F值分别为11.908和26.054,P值均<0.05,差异均有统计学意义,MCD作用前p-P38 MAPK和p-JNK/β-肌动蛋白灰度值分别为1.63±0.06,1.60±0.31,MCD作用后p-P38 MAPK/β-肌动蛋白、p-JNK灰度值分别为1.14±0.01、1.17±0.29,作用前后相比,t值分别为2.801和12.829,P值均<0.05,差异均有统计学意义.结论 AEA可以有效地导致HepG2细胞坏死而对L02细胞无影响,AEA激活了HepG2细胞p-P38 MAPK和P-JNK相关信号传导途径,且此过程与脂筏有关.     

Anti-dyskinetic effects of cannabinoids in a rat model of Parkinson's disease: role of CB(1) and TRPV1 receptors

Levodopa is the most commonly prescribed drug for Parkinson's disease (PD). Although levodopa improves PD symptoms in the initial stages of the disease, its long-term use is limited by the development of side effects, including abnormal involuntary movements (dyskinesias) and psychiatric complications. The endocannabinoid system is emerging as an important modulator of basal ganglia functions and its pharmacologic manipulation represents a promising therapy to alleviate levodopa-induced dyskinesias. Rats with 6-OHDA lesions that are chronically treated with levodopa develop increasingly severe axial, limb, locomotor and oro-facial abnormal involuntary movements (AIMs). Administration of the cannabinoid agonist WIN 55,212-2 attenuated levodopa-induced axial, limb and oral AIMs dose-dependently via a CB(1)-mediated mechanism, whereas it had no effect on locomotive AIMs. By contrast, systemic administration of URB597, a potent FAAH inhibitor, did not affect AIMs scoring despite its ability to increase anandamide concentration throughout the basal ganglia. Unlike WIN, anandamide can also bind and activate transient receptor potential vanilloid type-1 (TRPV1) receptors, which have been implicated in the modulation of dopamine transmission in the basal ganglia. Interestingly, URB597 significantly decreased all AIMs subtypes only if co-administered with the TRPV1 antagonist capsazepine. Our data indicate that pharmacological blockade of TRPV1 receptors unmasks the anti-dyskinetic effects of FAAH inhibitors and that CB(1) and TRPV1 receptors play opposite roles in levodopa-induced dyskinesias.     

The differential contractile responses to capsaicin and anandamide in muscle strips isolated from the rat urinary bladder

The contractile responses to capsaicin and anandamide, exogenous and endogenous agonists for transient receptor potential vanilloid receptor 1 (TRPV1), respectively, were investigated in muscle strips isolated from the rat urinary bladder. Capsaicin and anandamide produced concentration-dependent contractions of the muscle strips. The contractile response induced by capsaicin disappeared within approximately 20 min. In contrast, anandamide produced contractile responses lasting at least for 30 min. Capsaicin produced additive contractile responses in anandamide-treated muscle strips. The contractile response to anandamide was attenuated, but not abolished in strips desensitized by capsaicin. The response to capsaicin was abolished in the presence of a TRPV1 antagonist, N-(4-tertiarybutylphenyl)-4-(3-chlorphyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC), but not altered in the presence of either tetrodotoxin, atropine or indomethacin. In the presence of SR140333, a tachykinin NK₁ receptor antagonist or SR48968, an NK₂ receptor antagonist, the response to capsaicin was attenuated. The response to anandamide was partially attenuated in the presence of ONO8130, a prostanoid EP₁ receptor antagonist, URB597, a fatty-acid amide hydrolase inhibitor, BCTC, SR140333 or SR48968, and almost completely abolished by indomethacin. Neither tetrodotoxin, atropine, a cannabinoid CB₁ receptor antagonist, AM251, nor a cannabinoid CB₂ receptor antagonist, AM630, had any effect on the response to anandamide. These results indicate that capsaicin produces muscle contractions by stimulating the TRPV1 receptor, followed by release of neuropeptides that can activate tachykinin NK₁ and/or NK₂ receptors in the bladder and that the contractile response to anandamide is mediated at least in part by activation of prostanoid EP₁ receptors due to production of prostaglandins in addition to TRPV1 receptor activation.     

Anxiolytic-like effect of cannabinoids injected into the rat dorsolateral periaqueductal gray

Contradictory results exist concerning the effects of systemic injections of CB(1) cannabinoid receptor agonists on anxiety-related behaviors. Direct drug administration into brain structures related to aversive responses can potentially help to clarify the role of cannabinoids on anxiety. One such structure is the midbrain dorsolateral periaqueductal gray (dlPAG). Therefore, the aim of this study was to test the hypothesis that the activation of the CB(1) receptor in the dlPAG would attenuate anxiety-related behaviors. Male Wistar rats with cannula aimed at the dlPAG received injections of the endogenous cannabinoid anandamide, the anandamide transport inhibitor AM404, the anandamide analogue ACEA or the CB(1) receptor antagonist AM251, and were submitted to the elevated plus maze (EPM), an animal model of anxiety. Anandamide (0.5-50pmol) and ACEA (0.05-5pmol) induced anxiolytic-like effects with bell-shaped dose-response curves, the higher doses being ineffective. The anandamide anxiolytic effect was potentiated by AM404 (50pmol) and prevented by AM251 (100pmol). Neither AM404 (0.5-50pmol) nor AM251 (1-100pmol) alone modified the animal behavior in the EPM. The present study suggests that the dlPAG is a possible neuroanatomical site for anxiolytic-like effects mediated by CB(1) agonists. Furthermore, this work supports the importance of neuronal uptake as a mechanism that limits the in vivo actions of anandamide.     

The endocannabinoid anandamide inhibits kinin B1 receptor sensitization through cannabinoid CB1 receptor stimulation in human umbilical vein

The possible inhibition of kinin B₁ receptor up-regulation by arachidonoylethanolamide (anandamide) was evaluated in isolated human umbilical vein. Anandamide and its metabolically stable analogue, R-N-(2-Hydroxy-1-methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (R-(+)-methanandamide), produced a selective and dose-dependent inhibition of kinin B₁ receptor-sensitized contractile responses. The inhibitory effect of anandamide on B₁ receptor-sensitized responses failed to be modified either by 5-biphenyl-4-ylmethyl-tetrazole-1-carboxylic acid dimethylamide (LY2183240), a selective anandamide uptake inhibitor, or 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-y l](4-methoxyphenyl) methanone (AM630), selective cannabinoid CB₂ receptor antagonist. However, the cannabinoid CB₁ receptor antagonist, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophen yl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), abolished anandamide effects on kinin B₁ receptor sensitization. The present results provide strong pharmacological evidence indicating that endocannabinoid anandamide inhibits kinin B₁ receptor up-regulation through cannabinoid CB₁ receptor stimulation in human umbilical vein.     

Discriminative stimulus functions in rats of AM1346, a high-affinity CB1R selective anandamide analog

Objective  To characterize in vivo the high-affinity CB₁ cannabinoid receptor (CB₁R) selective anandamide analog AM1346 [alkoxyacid amide of N-eicosa-tetraenylamine] using drug discrimination. Substitution tests involved Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and R-(+)-methanandamide (mAEA), a metabolically stable analog of anandamide (AEA), as well as the CB₁R antagonist/inverse agonist rimonabant; d-amphetamine and morphine were also examined to assess pharmacological specificity. Materials and methods  Rats were initially trained to discriminate between i.p.-injected vehicle and 3 mg/kg AM1346 (group 3 mg/kg; t′ = 20 min); subsequently, the rats were retrained with 5.6 mg/kg AM1346 (group 5.6 mg/kg; t′ = 20 min). Results  Dose-generalization curves of AM1346, Δ⁹-THC, and mAEA suggested the following order of potency: Δ⁹-THC > AM1346 > mAEA both for rats discriminating between 3 and 5.6 mg/kg AM1346 from vehicle. In group 3 mg/kg, challenge by 1 mg/kg rimonabant resulted in parallel shifts to the right of the dose-generalization curves for Δ⁹-THC and AM1346, suggesting surmountable antagonism. Surmountable antagonism was not demonstrated with rimonabant–mAEA combinations. A long duration of effect was indicated when 3 mg/kg AM1346 was examined after different time intervals following i.p. administration (group 3 mg/kg). The in vivo half-life was close to 5 h. Neither d-amphetamine nor morphine generalized in either of groups 3 mg/kg and 5.6 mg/kg, suggesting pharmacological specificity. Conclusion  Unlike mAEA, the surmountable antagonism between rimonabant and AM1346 showed that the structural features of AEA can be modified to produce novel ligands that reduce the dissociation between the discriminative stimulus and rate decreasing effects of CB₁R agonists derived from an AEA template.     

Falcarinol is a covalent cannabinoid CB1 receptor antagonist and induces pro-allergic effects in skin

The skin irritant polyyne falcarinol (panaxynol, carotatoxin) is found in carrots, parsley, celery, and in the medicinal plant Panax ginseng. In our ongoing search for new cannabinoid (CB) receptor ligands we have isolated falcarinol from the endemic Sardinian plant Seseli praecox.We show that falcarinol exhibits binding affinity to both human CB receptors but selectively alkylates the anandamide binding site in the CB₁ receptor (K_i = 594 nM), acting as covalent inverse agonist in CB₁ receptor-transfected CHO cells. Given the inherent instability of purified falcarinol we repeatedly isolated this compound for biological characterization and one new polyyne was characterized. In human HaCaT keratinocytes falcarinol increased the expression of the pro-allergic chemokines IL-8 and CCL2/MCP-1 in a CB₁ receptor-dependent manner. Moreover, falcarinol inhibited the effects of anandamide on TNF-alpha stimulated keratinocytes. In vivo, falcarinol strongly aggravated histamine-induced oedema reactions in skin prick tests. Both effects were also obtained with the CB₁ receptor inverse agonist rimonabant, thus indicating the potential role of the CB₁ receptor in skin immunopharmacology. Our data suggest anti-allergic effects of anandamide and that falcarinol-associated dermatitis is due to antagonism of the CB₁ receptor in keratinocytes, leading to increased chemokine expression and aggravation of histamine action.     

Effects of intrathecal anandamide on somatosensory evoked responses in rats

Anandamide, endogenous ligand of cannabinoid receptors produces similar effects of cannabinoids via CB1 receptors in the central nervous system. Its effect on ascending pathways of somatosensory conduction and somatosensory cortex is not known. The aim of this study was to determine the effects of anandamide on somatosensory evoked potentials (SEP). In this study, 24 Wistar male rats were used. The rats were divided into 4 groups. At the beginning, sciatic nerve stimulated scalp SEP traces were obtained from all of the rats. Later, 0.02 cm(3) anhydrous ethanol, 100 microg/kg, 200 microg/kg and 400 microg/kg anandamide dissolved in anhydrous ethanol were injected intrathecally to the first (control), second, third and fourth groups, respectively. Five minutes later, second SEP traces were started. In every SEP trace, two negative waves (N1, N2) following positive deflections were obtained. The latency and amplitudes of these waves assessed were compared in each group. In control and second groups, the parameters of these waves before and after the injections were not significantly different. However, in the third and fourth groups, latencies of N1 and N2 after injections were found significantly longer. This effect was dose dependent. In any of the groups, no significant changes were detected in the amplitudes after injections. In conclusion, anandamide, when injected intrathecally in pharmacological doses caused an induction of moderate conduction delay in SEP systems.     

内源性大麻素对小鼠肝突触小体摄取去甲肾上腺素递质的影响

目的探讨内源性大麻素（anandamide,AEA）对肝组织神经末梢去甲肾上腺素（NE）的影响及可能的神经生物学机制。方法制备小鼠肝突触小体,用高效液相色谱-电化学（HPLC-ECD）方法检测AEA在体外对肝突触小体NE递质的影响。结果小鼠肝突触小体分离成功,体外实验中随着AEA浓度逐渐升高至10μmol／L,肝突触小体内NE的含量减少27．81％～70．79％,存在明显的剂量效应关系,差异有统计学意义（P〈0．01）;AEA浓度继续升高时,去甲肾上腺素含量不再继续减少。结论AEA能通过干扰小鼠肝突触部位NE递质的再摄取,产生抑制性作用。     

Endocannabinoids and cannabinoid analogues block human cardiac Kv4.3 channels in a receptor-independent manner

Endocannabinoids are amides and esters of long chain fatty acids that can modulate ion channels through both receptor-dependent and receptor-independent effects. Nowadays, their effects on cardiac K⁺ channels are unknown even when they can be synthesized within the heart. We have analyzed the direct effects of endocannabinoids, such as anandamide (AEA), 2-arachidonoylglycerol (2-AG), the endogenous lipid lysophosphatidylinositol, and cannabinoid analogues such as palmitoylethanolamide (PEA), and oleoylethanolamide, as well as the fatty acids from which they are endogenously synthesized, on human cardiac Kv4.3 channels, which generate the transient outward K⁺ current (I_to1). Currents were recorded in Chinese hamster ovary cells, which do not express cannabinoid receptors, by using the whole-cell patch-clamp. All these compounds inhibited I_Kv4.3 in a concentration-dependent manner, AEA and 2-AG being the most potent (IC₅₀ ∼ 0.3-0.4 µM), while PEA was the least potent. The potency of block increased as the complexity and the number of C atoms in the fatty acyl chain increased. The effects were not mediated by modifications in the lipid order and microviscosity of the membrane and were independent of the presence of MiRP2 or DPP6 subunits in the channel complex. Indeed, effects produced by AEA were reproduced in human atrial I_to1 recorded in isolated myocytes. Moreover, AEA effects were exclusively apparent when it was applied to the external surface of the cell membrane. These results indicate that at low micromolar concentrations the endocannabinoids AEA and 2-AG directly block human cardiac Kv4.3 channels, which represent a novel molecular target for these compounds.