Phytocannabinoids and endocannabinoids: different in nature

Abstract Cannabis is one of the earliest cultivated plants, of which Cannabis sativa and Cannabis indica are the most widespread and best characterized species. Their extracts contain (phyto)cannabinoids (pCBs) of therapeutic interest, such as Δ9-tetrahydrocannabinol and cannabidiol, along with many other compounds, so that there is no "one cannabis" but several mixtures even from the same plant. This complexity is mirrored, or even exceeded, by the complexity of the molecular targets that pCBs find in our body, most of which belong to the so-called "endocannabinoid (eCB) system". Here, we describe the major pCBs and the main components of the eCB system to appreciate their differences and mutual interactions, as well as the potential of using pCB/eCB-based drugs as novel therapeutics to treat human diseases, both in the central nervous system and at the periphery. Moreover, we address the question of the evolution of pCBs and eCBs, showing that the latter compounds were the first to appear in nature, and that the former substances took a few million years to mimic the three-dimensional structures of the latter, and hence their biological activity in our body. Graphic abstrac

Molecular dynamics study on the Apo- and Holo-forms of 5-lipoxygenase

Lipoxygenases (LOXs) are nonheme iron-containing enzymes catalyzing the dioxygenation of polyunsaturated fatty acids. LOX catalytic activity depends on the presence of iron in the active site and the iron removal is also able to affect the membrane binding properties of the enzyme. Leukotrienes biosynthesis is initiated by the action of 5-LOX at the level of nuclear membrane and the mechanism of enzyme-membrane interaction is thought to involve structural flexibility and conformational changes at the level of the protein tertiary structure. In this study, we have analyzed by molecular dynamics simulations the conformational changes induced by iron removal in 5-LOX. The data indicate that the degree of enzyme flexibility is related to the presence of iron into the active site that is able to stabilize the protein increasing its rigidity. These findings provide further evidence that the conformation and the functional activity of LOXs is tuned by the presence of iron at the active site, suggesting new approaches for the design of enzyme inhibitors.Peer ReviewedPostprint (author's final draft

Quantification of anandamide content in animal cells and tissues: the normalization makes the difference

Anandamide (N-arachidonoylethanolamine, AEA) is an endogenous lipid that binds to cannabinoid receptors in the central nervous system and in peripheral cells. Quantitative analysis of AEA is generally based on the normalization to the fresh weight of the samples. Here, we show that the normalization procedure of AEA content is such a critical factor, that it might introduce per se significant discrepancies in the quantification of AEA even in the same sample. We suggest that a rapid, accurate and most reliable reference to quantify AEA and congeners from different sources is the protein content, a common parameter to cells and tissues

Jekyll and Hyde: Two Faces of Cannabinoid Signaling in Male and Female Fertility

Mammalian reproduction is a complicated process designed to diversify and strengthen the genetic complement of the offspring and to safeguard regulatory systems at various steps for propagating procreation. An emerging concept in mammalian reproduction is the role of endocannabinoids, a group of endogenously produced lipid mediators, that bind to and activate cannabinoid receptors. Although adverse effects of cannabinoids on fertility have been implicated for years, the mechanisms by which they exert these effects were not clearly understood. With the identification of cannabinoid receptors, endocannabinoid ligands, their key synthetic and hydrolytic pathways, and the generation of mouse models missing cannabinoid receptors, a wealth of information on the significance of cannabinoid/endocannabinoid signaling in spermatogenesis, fertilization, preimplantation embryo development, implantation, and postimplantation embryonic growth has been generated. This review focuses on various aspects of the endocannabinoid system in male and female fertility. It is hoped that a deeper insight would lead to potential clinical applications of the endocannabinoid signaling as a target for correcting infertility and improving reproductive health in humans

Anandamide regulates keratinocyte differentiation by inducing DNA methylation in a CB1 receptor-dependent manner.

Anandamide (arachidonoylethanolamide, AEA) belongs to an important class of endogenous lipids including amides and esters of long chain polyunsaturated fatty acids, collectively termed "endocannabinoids." Recently we have shown that AEA inhibits differentiation of human keratinocytes, by binding to type-1 cannabinoid receptors (CB1R). To further characterize the molecular mechanisms responsible for this effect, we investigated the expression of epidermal differentiation-related genes after AEA treatment. We observed that keratin 1 and 10, transglutaminase 5 and involucrin are transcriptionally down-regulated by AEA. Most importantly, we found that AEA is able to decrease differentiating gene expression by increasing DNA methylation in human keratinocytes, through a p38, and to a lesser extent p42/44, mitogen-activated protein kinase-dependent pathway triggered by CB1R. An effect of AEA on DNA methylation because of CB1R-mediated increase of methyltransferase activity is described here for the first time, and we believe that the importance of this effect clearly extends beyond the regulation of skin differentiation. In fact, the modulation of DNA methylation by endocannabinoids may affect the expression of a number of genes that regulate many cell functions in response to these substances

Anandamide uptake by synaptosomes from human, mouse and rat brain: inhibition by glutamine and glutamate

Anandamide (N-arachidonoylethanolamine, AEA) belongs to an emerging class of endogenous lipids, called "endocannabinoids". A specific AEA membrane transporter (AMT) allows the import of this lipid and its degradation by the intracellular enzyme AEA hydrolase. Here, we show that synaptosomes from human, mouse and rat brain might be an ideal ex vivo system for the study of: i) the accumulation of AEA in brain, and ii) the pharmacological properties of AMT inhibitors. Using this ex vivo system, we demonstrate for the first time that glutamine and glutamate act as non-competitive inhibitors of AEA uptake by human, mouse and rat brain AMT

Leptin activates the anandamide hydrolase promoter in human T lymphocytes through STAT3

Physiological concentrations of leptin stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human T lymphocytes up to approximately 300% over the untreated controls. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational levels and involved binding of leptin to its receptor with an apparent dissociation constant (K(d)) of 1.95 +/- 0.14 nm and maximum binding (B(max)) of 392 +/- 8 fmol x mg protein(-1). Leptin binding to the receptor triggered activation of STAT3 but not STAT1 or STAT5 or the mitogen-activated protein kinases p38, p42, and p44. Peripheral lymphocytes of leptin knock-out (ob/ob) mice showed decreased FAAH activity and expression (approximately 25% of the wild-type littermates), which were reversed to control levels by exogenous leptin. Analysis of the FAAH promoter showed a cAMP-response element-like site, which is a transcriptional target of STAT3. Consistently, mutation of this site prevented FAAH activation by leptin in transient expression assays. Electrophoretic mobility shift and supershift assays further corroborated the promoter activity data. Taken together, these results suggest that leptin, by up-regulating the FAAH promoter through STAT3, enhances FAAH expression, thus tuning the immunomodulatory effects of anandamide. These findings might also have critical implications for human fertility

The Endocannabinoid System in the Mediterranean Mussel Mytilus galloprovincialis: Possible Mediators of the Immune Activity?

Anandamide (AEA) is one of the best characterized members of the endocannabinoid family and its involvement in many pathophysiological processes has been well documented in vertebrates and invertebrates. Here, we report the biochemical and functional characterization of key elements of the endocannabinoid system in hemocytes isolated from the Mediterranean mussel Mytilus galloprovincialis. We also show the effects of exogenous AEA, as well as of capsaicin, on the cell ability to migrate and to activate the respiratory burst, upon in vitro stimulation of phagocytosis. Interestingly, our findings show that both AEA and capsaicin suppress the hemocyte response and that the use of selective antagonists of CB2 and TRPV1 receptors revert their inhibitory effects. Overall, present data support previous evidence on the presence of endocannabinoid signaling in mollusks and advance our knowledge about the evolutionary origins of this endogenous system and its role in the innate response of mollusks

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 inhibitor Nomega-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