The human endocannabinoid system regulates a myriad of physiological processes through a complex lipid signaling network involving cannabinoids and their respective receptors, cannabinoid receptor 1 (hCB1 R) and cannabinoid receptor 2 (hCB2 R). Anandamide (AEA) and cannabidiol (CBD) are classical examples of cannabinoids that elicit a variety of effects, both beneficial and detrimental, through these receptors. Mounting evidence suggested the presence of other potential cannabinoid targets that may be responsible for other observable effects. However, prior pharmacological studies on these cannabinoid compounds provided scant evidence of direct engagement to these proposed targets. Moreover, to the best of our knowledge, no chemoproteomic studies have been demonstrated on CBD. Here we showed that, by taking advantage of a recently developed \'label-free\' 2D-TPP (2 Dimensional-Thermal Protein Profiling) approach, we have identified several new putative targets of both AEA and CBD. Comparison of these interaction landscapes with those obtained from well-established affinity-based protein profiling (AfBPP) platforms has led to the discovery of both shared and unique protein targets. Subsequent target validation of selected proteins led us to conclude that this 2D-TPP strategy complements well with AfBPP.

The cannabis derivative marijuana is the most widely used recreational drug in the Western world and is consumed by an estimated 83 million individuals (∼3% of the world population). In recent years, there has been a marked transformation in society regarding the risk perception of cannabis, driven by its legalization and medical use in many states in the United States and worldwide. Compelling research evidence and the Food and Drug Administration cannabis-derived cannabidiol approval for severe childhood epilepsy have confirmed the large therapeutic potential of cannabidiol itself, Δ9-tetrahydrocannabinol and other plant-derived cannabinoids (phytocannabinoids). Of note, our body has a complex endocannabinoid system (ECS)-made of receptors, metabolic enzymes, and transporters-that is also regulated by phytocannabinoids. The first endocannabinoid to be discovered 30 years ago was anandamide (N-arachidonoyl-ethanolamine); since then, distinct elements of the ECS have been the target of drug design programs aimed at curing (or at least slowing down) a number of human diseases, both in the central nervous system and at the periphery. Here a critical review of our knowledge of the goods and bads of the ECS as a therapeutic target is presented to define the benefits of ECS-active phytocannabinoids and ECS-oriented synthetic drugs for human health. SIGNIFICANCE STATEMENT: The endocannabinoid system plays important roles virtually everywhere in our body and is either involved in mediating key processes of central and peripheral diseases or represents a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of the components of this complex system, and in particular of key receptors (like cannabinoid receptors 1 and 2) and metabolic enzymes (like fatty acid amide hydrolase and monoacylglycerol lipase), will advance our understanding of endocannabinoid signaling and activity at molecular, cellular, and system levels, providing new opportunities to treat patients.

Following the publication of this paper, it was drawn to the Editors\' attention by a concerned reader that certain of the scratch-wound data shown in Fig. 3A were strikingly similar to data appearing in different form in another article by different authors. Owing to the fact that the contentious data in the above article had already been published elsewhere prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 13: 1558‑1662, 2016; DOI: 10.3892/mmr.2015.4721].

Transient receptor potential vanilloid 1 (TRPV1) ion channel serves as the detector for noxious temperature above 42 °C, pungent chemicals like capsaicin, and acidic extracellular pH. This channel has also been shown to function as an ionotropic cannabinoid receptor. Despite the solving of high-resolution three-dimensional structures of TRPV1, how endocannabinoids such as anandamide and N-arachidonoyl dopamine bind to and activate this channel remains largely unknown. Here we employed a combination of patch-clamp recording, site-directed mutagenesis, and molecular docking techniques to investigate how the endocannabinoids structurally bind to and open the TRPV1 ion channel. We found that these endocannabinoid ligands bind to the vanilloid-binding pocket of TRPV1 in the \"tail-up, head-down\" configuration, similar to capsaicin; however, there is a unique interaction with TRPV1 Y512 residue critical for endocannabinoid activation of TRPV1 channels. These data suggest that a differential structural mechanism is involved in TRPV1 activation by endocannabinoids compared with the classic agonist capsaicin.

In this study, a taste bud tissue biosensor was prepared by a starch-sodium alginate cross-linking fixation method. Capsaicin was used as a TRPV1 noxious ion channel activator to investigate the antagonism kinetics of six different substances on capsaicin. The results showed that capsazepine, AMG517, loureirin B, and tetrahydropalmatine were all competitive allosteric regulatory ligands for capsaicin, while aconitine and anandamide were mixed allosteric regulatory ligand that combines non-competition and competition effect. Through analyzing the kinetic parameters of capsaicin and its competitive allosteric regulatory ligands, and comparing the structures between spicy substances and endocannabinoids, the importance of amide groups and similar groups in the allosteric regulation of cannabinoids (CB) receptors and analgesic mechanism was elucidated. This indicates that vanilloid activators turn on the TRPV1 ion channel to transmit only pain and other nociceptive signals, while capsaicin and its competitive ligands are capable of activating intracellular G protein/PI3K/PIP2 signaling pathways by binding to endogenous cannabinoid receptors, and then increase intracellular PIP2 levels (the increasing PIP2 can competitively replace capsaicin and other vanilloid activators), thereby closing the TRPV1 channel and exerting the analgesic effect. The elucidation of this mechanism of pain and analgesia will lay the theoretical foundation and new ideas for investigating nociceptive signal and screening potential analgesic drugs.

OBJECTIVE: The purpose of this study was to investigate the change in plasma anandamide (AEA) levels throughout the normal menstrual cycle, and to analyze the relationship among AEA, sex steroids and gonadotrophins.
METHODS: The patients were fertile women with normal menstrual cycle, proposed to get in vitro fertilization (IVF) treatment due to oviduct obstruction or male infertility. Patients were divided into two groups, cross-sectional (n=79) and longitudinal (n=10). The plasma AEA levels were examined by the ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) system. The serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and progesterone (P) were measured by chemiluminescence.
RESULTS: The AEA levels in the late follicular phase were slightly higher than those in the early follicular phase. Subsequently, the AEA levels peaked at the time of ovulation in both two cohorts. Finally, the lowest AEA levels were measured in the luteal phase. Moreover, there were highly significant positive correlations between the plasma AEA concentration and the serum levels of FSH, LH and E2, whereas the AEA level was not correlated with P during the normal menstrual cycle.
CONCLUSIONS: Our observations reveal a dynamic change in the plasma AEA level, which is closely associated with the levels of gonadotrophin and sex steroid hormones, suggesting that the hormones may be involved in the regulation of AEA levels during the menstrual cycle. Our studies help to design new strategies to improve implantation and treatments for reproductive diseases.

OBJECTIVE: We investigated the role of the endocannabinoid system (ECS) in the endometrium of unexplained infertility (UI) patients, and effect of anandamide (AEA) on leukemia inhibitory factor (LIF).
METHODS: Patients were divided into UI and control groups. Endometrium samples were collected at the midluteal phase. Levels of cannabinoid type 1 (CB1), fatty acid amide hydrolase (FAAH), and LIF were examined. LIF productions were measured after AEA, CB1 antagonist AM251, and CB2 antagonist AM630 stimulation.
RESULTS: Rates of available embryo, successful implantation and pregnancy, and the endometrial thickness of UI group were significantly lower than control, suggesting uterine receptivity was decreased in UI group. FAAH and LIF levels were significantly decreased, whereas endometrial CB1 was slightly increased in UI group. LIF production was promoted by low amount of AEA administration (1-10 μM), while the promotion was reduced by higher concentration of AEA (50 μM). LIF levels were decreased by AM251 or AM630, compared with AEA alone. Expressions of FAAH and LIF were closely associated with uterus receptivity and implantation rate of UI patients. Different concentrations of AEA could stimulate dynamic changes in LIF production.
CONCLUSIONS: Our data indicated the important role of the ECS in human fertility, which may promote new strategies for successful implantation and treatments for reproductive diseases.

This study aimed to investigate the correlation between endocannabinoids and polycystic ovary syndrome (PCOS), and the expression levels of endocannabinoids in different phases of menstruation. The expression of cannabinoid receptors (CB1) and fatty acid amide hydrolase (FAAH) in the endometrium were immunohistochemically stained and compared between women with PCOS and the control group. Integrated optical density (IOD) was assessed to analyze their expression levels. The CB1 and FAAH were expressed in endometrial epithelial cytoplasm. No significant difference in CB1 level was observed between PCOS and non-PCOS women. Additionally, the expression of CB1 did not fluctuate with menstrual cycle. However, the FAAH levels were lower in the PCOS group than the non-PCOS group (p < 0.05). FAAH levels in secretory phases were significantly elevated compared to menstrual and proliferative phases (p < 0.05). Our results demonstrate that the endocannabinoid system may play an important role in menstruation, and dysregulation of the system may result in PCOS.

BACKGROUND: It is reported that endogenous cannabinoids can cause vasodilation and bradycardia. They have anti-inflammatory effect and protect endothelial cells from injury, therefore they have potential application prospect in the prevention of cardio-cerebrovascular diseases. However, the mechanisms of the neuroprotection mediated by cannabinoid 1 receptors (CB1Rs) have not been uncovered in detail.
METHODS: Nearly one hundred of new publications relevant to the theme are almost selected from Pubmed. The advanced details associated with the involvement of CB1R in cerebral ischemia as well as cerebral ischemic tolerance are reviewed.
RESULTS: Anandamide system is mainly made up of cannabinoid receptors, their endogenous ligands and some related enzymes. The activation of the system mediates various molecular events so that plays a crucial role in the neuroprotection of cerebral ischemia. Increasing evidences suggest that CB1R is one of key molecules that mediate cerebral ischemia and cerebral ischemia tolerance. It is likely to provide an appropriate antioxidant balance by increasing endogenous free radical scavengers and helpful to exert the neuroprotective effects. Moreover, MAPKs, including ERK1/2, c-Jun Nterminal kinase (JNK) and p38MAPK can be recruited and stimulated through a complex signaling networks mediated by CB1R. Considerable evidences have indicated that CB1R was a crucial regulator for ERK1/2 signaling pathway. It is known that PI3K/Akt is a classical signaling pathway and its activation exerts neuroprotective effect via significant promoting cell survival. Glycogen synthase kinase-3β (GSK-3β) is an important downstream target of p-Akt. The PI3K/Akt/GSK-3β signaling pathway mediated by CB1Rs takes an important part in cerebral ischemic injury. PKC and CB1R are found to be abundantly co-expressed in presynaptic nerve endings of brain. There are considerable reports that different PKC isozymes played vital roles respectively in cerebral ischemic injury and preconditioning. The CB1R -mediated activation of PKCε can effectively stimulate ischemic tolerance.
CONCLUSIONS: CB1R played an important part via several signaling pathways in the protection from ischemic stroke and in ischemic tolerance. The involved molecular signaling pathways include ERK1/2, PI3K/Akt/GSK-3β and the translocation and activation of PKCε. With the intimate association between CB1R and neuron injuries, to target the receptor will exert neuroprotective effects on cerebral ischemia, which provides wide foreground for a novel therapy target.

While activation of cannabinoid CB1 receptor (CB1R) regulates a variety of retinal neuronal functions by modulating ion channels in these cells, effect of activated cannabinoid receptors on Ca(2+) channels in retinal Müller cells is still largely unknown. In the present work we show that three subunits of T-type Ca(2+) channels, CaV3.1, CaV3.2 and CaV3.3, as well as one subunit of L-type Ca(2+) channels, CaV1.2, were expressed in rat Müller cells by immunofluorescent staining. Consistently, nimodipine- and mibefradil-sensitive Na(+) currents through L- and T-type Ca(2+) channels could be recorded electrophysiologically. The cannabinoid receptor agonist WIN55212-2 significantly suppressed Ca(2+) channel currents, mainly the T-type one, in acutely isolated rat Müller cells in a dose-dependent manner, with an IC50 of 3.98μM. The WIN55212-2 effect was not blocked by AM251/SR141716, specific CB1R antagonists. Similar suppression of the currents was observed when anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), endogenous ligands of cannabinoid receptors, were applied. Moreover, even though CB2 receptors (CB2Rs) were expressed in rat Müller cells, the effects of WIN55212-2 and 2-AG on Ca(2+) channel currents were not blocked by AM630, a selective CB2R antagonist. However, the effect of AEA could be partially rescued by AM630. These results suggest that WIN55212-2 and 2-AG receptor-independently suppressed the Ca(2+) channel currents in Müller cells, while AEA suppressed the currents partially through CB2Rs. The existence of receptor-dependent and -independent mechanisms suggests that cannabinoids may modulate Müller cell functions through multiple pathways.