Monica Bari

Anandamide Induces Apoptosis in Human Cells via Vanilloid Receptors EVIDENCE FOR A PROTECTIVE ROLE OF CANNABINOID RECEPTORS

The endocannabinoid anandamide (AEA) is shown to induce apoptotic bodies formation and DNA fragmentation, hallmarks of programmed cell death, in human neuroblastoma CHP100 and lymphoma U937 cells. RNA and protein synthesis inhibitors like actinomycin D and cycloheximide reduced to one-fifth the number of apoptotic bodies induced by AEA, whereas the AEA transporter inhibitor AM404 or the AEA hydrolase inhibitor ATFMK significantly increased the number of dying cells. Furthermore, specific antagonists of cannabinoid or vanilloid receptors potentiated or inhibited cell death induced by AEA, respectively. Other endocannabinoids such as 2-arachidonoylglycerol, linoleoylethanolamide, oleoylethanolamide, and palmitoylethanolamide did not promote cell death under the same experimental conditions. The formation of apoptotic bodies induced by AEA was paralleled by increases in intracellular calcium (3-fold over the controls), mitochondrial uncoupling (6-fold), and cytochrome c release (3-fold). The intracellular calcium chelator EGTA-AM reduced the number of apoptotic bodies to 40% of the controls, and electrotransferred anti-cytochrome c monoclonal antibodies fully prevented apoptosis induced by AEA. Moreover, 5-lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid and MK886, cyclooxygenase inhibitor indomethacin, caspase-3 and caspase-9 inhibitors Z-DEVD-FMK and Z-LEHD-FMK, but not nitric oxide synthase inhibitorNω-nitro-l-arginine methyl ester, significantly reduced the cell death-inducing effect of AEA. The data presented indicate a protective role of cannabinoid receptors against apoptosis induced by AEA via vanilloid receptors.

Experimental Parkinsonism Alters Endocannabinoid Degradation: Implications for Striatal Glutamatergic Transmission

Cannabinoid receptors and their endogenous ligands have been recently identified in the brain as potent inhibitors of neurotransmitter release. Here we show that, in a rat model of Parkinsons disease induced by unilateral nigral lesion with 6-hydroxydopamine (6-OHDA), the striatal levels of anandamide, but not that of the other endocannabinoid 2-arachidonoylglycerol, were increased. Moreover, we observed a decreased activity of the anandamide membrane transporter (AMT) and of the anandamide hydrolase [fatty acid amide hydrolase (FAAH)], whereas the binding of anandamide to cannabinoid receptors was unaffected. Spontaneous glutamatergic activity recorded from striatal spiny neurons was higher in 6-OHDA-lesioned rats. Inhibition of AMT byN-(4-hydroxyphenyl)-arachidonoylamide (AM-404) or by VDM11, or stimulation of the cannabinoid CB1 receptor by HU-210 reduced glutamatergic spontaneous activity in both naı̈ve and 6-OHDA-lesioned animals to a similar extent. Conversely, the FAAH inhibitors phenylmethylsulfonyl fluoride and methyl-arachidonoyl fluorophosphonate were much more effective in 6-OHDA-lesioned animals. The present study shows that inhibition of anandamide hydrolysis might represent a possible target to decrease the abnormal cortical glutamatergic drive in Parkinsons disease.

Anandamide inhibits metabolism and physiological actions of 2-arachidonoylglycerol in the striatum

Of the endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have received the most study. A functional interaction between these molecules has never been described. Using mouse brain slices, we found that stimulation of metabotropic glutamate 5 receptors by 3,5-dihydroxyphenylglycine (DHPG) depressed inhibitory transmission in the striatum through selective involvement of 2-AG metabolism and stimulation of presynaptic CB1 receptors. Elevation of AEA concentrations by pharmacological or genetic inhibition of AEA degradation reduced the levels, metabolism and physiological effects of 2-AG. Exogenous AEA and the stable AEA analog methanandamide inhibited basal and DHPG-stimulated 2-AG production, confirming that AEA is responsible for the downregulation of the other eCB. AEA is an endovanilloid substance, and the stimulation of transient receptor potential vanilloid 1 (TRPV1) channels mimicked the effects of endogenous AEA on 2-AG metabolism through a previously unknown glutathione-dependent pathway. Consistently, the interaction between AEA and 2-AG was lost after pharmacological and genetic inactivation of TRPV1 channels.

Relation between decreased anandamide hydrolase concentrations in human lymphocytes and miscarriage

BACKGROUND Endocannabinoids such as anandamide are thought to have adverse effects on pregnancy and embryonic development. The activity of the degradative enzyme anandamide hydrolase may therefore be crucial for prevention of excessive concentrations of anandamide in the uterus, and thus prevention of pregnancy failure or female infertility. We tested this hypothesis in a preliminary study, and then used the results to find out whether anandamide hydrolase activity could predict miscarriage in a group of pregnant women. METHODS We assessed anandamide hydrolase activity in peripheral lymphocytes from 50 healthy, pregnant women at weeks 6-11 of gestation by a specific radiochromatographic method. The expression of the enzyme at the protein level was measured by ELISA with specific polyclonal antibodies. In a further study, we measured anandamide hydrolase concentration in 120 women who were 7-8 weeks pregnant and compared these findings with subsequent pregnancy outcome. FINDINGS In the first study, seven of the 50 women had a miscarriage. Anandamide hydrolase activity was lower in the seven women who miscarried than in the 43 who did not (60.43 pmol/min per mg protein [SD 29.34] vs 169.60 pmol/min per mg protein [30.20]; difference 109.17 pmol/min per mg protein [95% CI 26.64-191.70]; p<0.0001 by the Mann-Whitney test). Enzyme activity correlated with enzyme concentration, and a threshold concentration represented by an optical density (after ELISA) of 0.15 absorbance units at 450 nm separated the women who had miscarriages from those who did not. In the second study, 15 women had anandamide hydrolase concentrations below the threshold, and 105 had concentrations at or above the threshold. All 15 women in the low anandamide hydrolase group had miscarriages, compared with one of the 105 women with high concentrations (p<0.0001 by Fishers exact test). INTERPRETATION Decreased anandamide hydrolase activity and expression in peripheral lymphocytes is an early (<8 weeks of gestation) marker of spontaneous abortion, and may prove useful as a diagnostic tool for large-scale, routine monitoring of gestation. Our results also suggest that endocannabinoids might be critical in regulating the lymphocyte-dependent cytokine network associated with human fertility and successful pregnancy.

Lipid rafts control signaling of type-1 cannabinoid receptors in neuronal cells: Implications for anandamide-induced apoptosis

Several G protein-coupled receptors function within lipid rafts plasma membrane microdomains, which may be important in limiting signal transduction. Here we show that treatment of rat C6 glioma cells with the raft disruptor methyl-β-cyclodextrin (MCD) doubles the binding efficiency (i.e. the ratio between maximum binding and dissociation constant) of type-1 cannabinoid receptors (CB1R), which belong to the rhodopsin family of G protein-coupled receptors. In parallel, activation of CB1R by the endogenous agonist anandamide (AEA) leads to ∼3-fold higher [35S]GTPγS binding in MCD-treated cells than in controls, and CB1R-dependent signaling via adenylate cyclase, and p42/p44 MAPK is almost doubled by MCD. Unlike CB1R, the other AEA-binding receptor TRPV1, the AEA synthetase NAPE-PLD, and the AEA hydrolase FAAH are not modulated by MCD, whereas the activity of the AEA membrane transporter (AMT) is reduced to ∼50% of the controls. We also show that MCD reduces dose-dependently AEA-induced apoptosis in C6 cells but not in human CHP100 neuroblastoma cells, which mirror the endocannabinoid system of C6 cells but are devoid of CB1R. MCD reduces also cytochrome c release from mitochondria of C6 cells, and this effect is CB1R-dependent and partly mediated by activation of p42/p44 MAPK. Altogether, the present data suggest that lipid rafts control CB1R binding and signaling, and that CB1R activation underlies the protective effect of MCD against apoptosis.

Progesterone Up-Regulates Anandamide Hydrolase in Human Lymphocytes: Role of Cytokines and Implications for Fertility

Physiological concentrations of progesterone stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human lymphocytes. At the same concentrations, the membrane-impermeant conjugate of progesterone with BSA was ineffective, suggesting that binding to an intracellular receptor was needed for progesterone activity. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational level, and was partly mediated by the Th2 cytokines. In fact, lymphocyte treatment with IL-4 or with IL-10 had a stimulating effect on FAAH, whereas the Th1 cytokines IL-12 and IFN-γ reduced the activity and the protein expression of FAAH. Human chorionic gonadotropin or cortisol had no effect on FAAH activity. At variance with FAAH, the lymphocyte anandamide transporter and cannabinoid receptors were not affected by treatment with progesterone or cytokines. Good FAAH substrates such as anandamide and 2-arachidonoylglycerol inhibited the release of leukemia-inhibitory factor from human lymphocytes, but N-palmitoylethanolamine, a poor substrate, did not. A clinical study performed on 100 healthy women showed that a low FAAH activity in lymphocytes correlates with spontaneous abortion, whereas anandamide transporter and cannabinoid receptors in these cells remain unchanged. These results add the endocannabinoids to the hormone-cytokine array involved in the control of human pregnancy.

Levodopa treatment reverses endocannabinoid system abnormalities in experimental parkinsonism

Cannabinoid receptors and their endogenous ligands are potent inhibitors of neurotransmitter release in the brain. Here, we show that in a rat model of Parkinsons disease induced by unilateral nigral lesion with 6‐hydroxydopamine (6‐OHDA), the striatal levels of the endocannabinoid anandamide (AEA) were increased, while the activity of its membrane transporter and hydrolase (fatty‐acid amide hydrolase, FAAH) were decreased. These changes were not observed in the cerebellum of the same animals. Moreover, the frequency and amplitude of glutamate‐mediated spontaneous excitatory post‐synaptic currents were augmented in striatal spiny neurones recorded from parkinsonian rats. Remarkably, the anomalies in the endocannabinoid system, as well as those in glutamatergic activity, were completely reversed by chronic treatment of parkinsonian rats with levodopa, and the pharmacological inhibition of FAAH restored a normal glutamatergic activity in 6‐OHDA‐lesioned animals. Thus, the increased striatal levels of AEA may reflect a compensatory mechanism trying to counteract the abnormal corticostriatal glutamatergic drive in parkinsonian rats. However, this mechanism seems to be unsuccessful, since spontaneous excitatory activity is still higher in these animals. Taken together, these data show that anomalies in the endocannabinoid system induced by experimental parkinsonism are restricted to the striatum and can be reversed by chronic levodopa treatment, and suggest that inhibition of FAAH might represent a possible target to decrease the abnormal cortical glutamatergic drive in Parkinsons disease.

Endocannabinoids in adipocytes during differentiation and their role in glucose uptake.

Abstract.The molecular basis for the control of energy balance by the endocannabinoid anandamide (AEA) is still unclear. Here, we show that murine 3T3-L1 fibroblasts have the machinery to bind, synthesize and degrade AEA, and that their differentiation into adipocytes increases by approximately twofold the binding efficiency of cannabinoid receptors (CBR), and by approximately twofold and approximately threefold, respectively, the catalytic efficiency of the AEA transporter and AEA hydrolase. In contrast, the activity of the AEA synthetase and the binding efficiency of vanilloid receptor were not affected by the differentiation process. In addition, we demonstrate that AEA increases by approximately twofold insulin-stimulated glucose uptake in differentiated adipocytes, according to a CB1R-dependent mechanism that involves nitric oxide synthase, but not lipoxygenase or cyclooxygenase. We also show that AEA binding to peroxisome proliferator-activated receptor-γ, known to induce differentiation of 3T3-L1 fibroblasts into adipocytes, is not involved in the stimulation of glucose uptake.

Excitability of prefrontal cortical pyramidal neurons is modulated by activation of intracellular type-2 cannabinoid receptors.

The endocannabinoid (eCB) system is widely expressed throughout the central nervous system (CNS) and the functionality of type-1 cannabinoid receptors in neurons is well documented. In contrast, there is little knowledge about type-2 cannabinoid receptors (CB2Rs) in the CNS. Here, we show that CB2Rs are located intracellularly in layer II/III pyramidal cells of the rodent medial prefrontal cortex (mPFC) and that their activation results in IP3R-dependent opening of Ca2+-activated Cl− channels. To investigate the functional role of CB2R activation, we induced neuronal firing and observed a CB2R-mediated reduction in firing frequency. The description of this unique CB2R-mediated signaling pathway, controlling neuronal excitability, broadens our knowledge of the influence of the eCB system on brain function.

New Insights into Endocannabinoid Degradation and its Therapeutic Potential

Endocannabinoids are amides, esters and ethers of long chain polyunsaturated fatty acids, which act as new lipidic mediators. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the main endogenous agonists of cannabinoid receptors, able to mimic several pharmacological effects of (-)-Delta9-tetrahydrocannabinol (THC), the active principle of Cannabis sativa preparations like hashish and marijuana. The activity of AEA and 2-AG at their receptors is limited by cellular uptake through an anandamide membrane transporter (AMT), followed by intracellular degradation. A fatty acid amide hydrolase (FAAH) is the main AEA hydrolase, whereas a monoacylglycerol lipase (MAGL) is critical in degrading 2-AG. Here, we will review growing evidence that demonstrates that these hydrolases are pivotal regulators of the endogenous levels of AEA and 2-AG in vivo, overall suggesting that specific inhibitors of AMT, FAAH or MAGL may serve as attractive therapeutic targets for the treatment of human disorders. Recently, the N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), which synthesizes AEA from N-arachidonoylphosphatidylethanolamine (NArPE), and the diacylglycerol lipase (DAGL), which generates 2-AG from diacylglycerol (DAG) substrates, have been characterized. The role of these synthetic routes in maintaining the endocannabinoid tone in vivo will be discussed. Finally, the effects of inhibitors of endocannabinoid degradation in animal models of human disease will be reviewed, with an emphasis on their ongoing applications in anxiety, cancer and neurodegenerative disorders.