Cinzia Rapino

Molecular identification of albumin and Hsp70 as cytosolic anandamide-binding proteins.

The cellular uptake and the intracellular synthesis/degradation of anandamide are crucial steps for controlling its extracellular level and the duration of its activity. Although the biosynthesis and breakdown of anandamide are well understood, little is known about the mechanisms underlying its intracellular transport. Here, we investigated the presence of a potential carrier-mediated trafficking of anandamide within the cytosol, using a biotinylated analog as a tool to catch by affinity chromatography anandamide-interacting proteins. The identity of two of these anandamide-binding proteins, Hsp70 and serum albumin, was determined by mass spectrometry, confirmed by western blotting and confocal microscopy, and further validated through an anandamide-binding assay. These findings suggest that the trafficking of anandamide from the plasma membrane to the internal compartments of a cell occur via a nonvesicular mechanism mediated by cytosolic carriers.

Characterization of the Endocannabinoid System in Human Spermatozoa and Involvement of Transient Receptor Potential Vanilloid 1 Receptor in Their Fertilizing Ability

Human spermatozoa express type-1 cannabinoid receptor (CB1), whose activation by anandamide (AEA) affects motility and acrosome reaction (AR). In this study, we extended the characterization of the AEA-related endocannabinoid system in human spermatozoa, and we focused on the involvement of the AEA-binding vanilloid receptor (TRPV1) in their fertilizing ability. Protein expression was revealed for CB1 ( approximately 56 kDa), TRPV1 ( approximately 95 kDa), AEA-synthesizing phospholipase D (NAPE-PLD) ( approximately 46 kDa), and AEA-hydrolyzing enzyme [fatty acid amide hydrolase (FAAH), approximately 66 kDa]. Both AEA-binding receptors (CB1 and TRPV1) exhibited a functional binding activity; enzymatic activity was demonstrated for NAPE-PLD, FAAH, and the purported endocannabinoid membrane transporter (EMT). Immunoreactivity for CB1, NAPE-PLD, and FAAH was localized in the postacrosomal region and in the midpiece, whereas for TRPV1, it was restricted to the postacrosomal region. Capsazepine (CPZ), a selective antagonist of TRPV1, inhibited progesterone (P)-enhanced sperm/oocyte fusion, as evaluated by the hamster egg penetration test. This inhibition was due to a reduction of the P-induced AR rate above the spontaneous AR rate, which was instead increased. The sperm exposure to OMDM-1, a specific inhibitor of EMT, prevented the promoting effect of CPZ on spontaneous AR rate and restored the sperm responsiveness to P. No significant effects could be observed on sperm motility. In conclusion, this study provides unprecedented evidence that human spermatozoa exhibit a completely functional endocannabinoid system related to AEA and that the AEA-binding TRPV1 receptor could be involved in the sperm fertilizing ability.

Abnormal mGlu 5 Receptor/Endocannabinoid Coupling in Mice Lacking FMRP and BC1 RNA

Transcriptional silencing of the gene encoding the fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS). FMRP acts as a translational repressor at central synapses, and molecular and synaptic plasticity studies have shown that the absence of this protein alters metabotropic glutamate 5 receptors (mGlu5Rs)-mediated signaling. In the striatum of mice lacking FMRP, we found enhanced activity of diacylglycerol lipase (DAGL), the enzyme limiting 2-arachidonoylglicerol (2-AG) synthesis, associated with altered sensitivity of GABA synapses to the mobilization of this endocannabinoid by mGlu5R stimulation with DHPG. Mice lacking another repressor of synaptic protein synthesis, BC1 RNA, also showed potentiated mGlu5R-driven 2-AG responses, indicating that both FMRP and BC1 RNA act as physiological constraints of mGlu5R/endocannabinoid coupling at central synapses. The effects of FMRP ablation on DAGL activity and on DHPG-mediated inhibition of GABA synapses were enhanced by simultaneous genetic inactivation of FMRP and BC1 RNA. In double FMRP and BC1 RNA lacking mice, striatal levels of 2-AG were also enhanced compared with control animals and to single mutants. Our data indicate for the first time that mGlu5R-driven endocannabinoid signaling in the striatum is under the control of both FMRP and BC1 RNA. The abnormal mGlu5R/2-AG coupling found in FMRP-KO mice emphasizes the involvement of mGlu5Rs in the synaptic defects of FXS, and identifies the modulation of the endocannabinoid system as a novel target for the treatment of this severe neuropsychiatric disorder.

Evidence for the intracellular accumulation of anandamide in adiposomes

Abstract.Anandamide is a lipid messenger that carries out a wide variety of biological functions. It has been suggested that anandamide accumulation involves binding to a saturable cellular component. To identify the structure(s) involved in this process, we analyzed the intracellular distribution of both biotinylated and radiolabeled anandamide, providing direct evidence that lipid droplets, also known as adiposomes, constitute a dynamic reservoir for the sequestration of anandamide. In addition, confocal microscopy and biochemical studies revealed that the anandamide-hydrolase is also spatially associated with lipid droplets, and that cells with a larger adiposome compartment have an enhanced catabolism of anandamide. Overall, these findings suggest that adiposomes may have a critical role in accumulating anandamide, possibly by connecting plasma membrane to internal organelles along the metabolic route of this endocannabinoid.

The Novel Reversible Fatty Acid Amide Hydrolase Inhibitor ST4070 Increases Endocannabinoid Brain Levels and Counteracts Neuropathic Pain in Different Animal Models

The effect of the enol carbamate 1-biphenyl-4-ylethenyl piperidine-1-carboxylate (ST4070), a novel reversible inhibitor of fatty acid amide hydrolase (FAAH), was investigated for acute pain sensitivity and neuropathic pain in rats and mice. Brain enzymatic activity of FAAH and the endogenous levels of its substrates, anandamide (AEA; N-arachidonoylethanolamine), 2-arachidonoylglycerol (2-AG), and N-palmitoylethanolamine (PEA), were measured in control and ST4070-treated mice. ST4070 (10, 30, and 100 mg/kg) was orally administered to assess mechanical nociceptive thresholds and allodynia by using the Randall-Selitto and von Frey tests, respectively. Neuropathy was induced in rats by either the chemotherapeutic agent vincristine or streptozotocin-induced diabetes, whereas the chronic constriction injury (CCI) model was chosen to evaluate neuropathy in mice. ST4070 produced a significant increase of nociceptive threshold in rats and counteracted the decrease of nociceptive threshold in the three distinct models of neuropathic pain. In diabetic mice, ST4070 inhibited FAAH activity and increased the brain levels of AEA and PEA, without affecting that of 2-AG. The administration of ST4070 generated long-lasting pain relief compared with pregabalin and the FAAH inhibitors 1-oxo-1[5-(2-pyridyl)-2-yl]-7-phenylheptane (OL135) and cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-ylester (URB597) in CCI neuropathic mice. The antiallodynic effects of ST4070 were prevented by pretreatment with cannabinoid type 1 and cannabinoid type 2 receptor antagonists and by the selective peroxisome proliferator-activated receptor α antagonist [(2S)-2-[[(1Z)-1-methyl-3-oxo-3-[4-(trifluoromethyl)phenyl]-1-propenyl]amino]-3-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]phenyl]propyl]-carbamic acid ethyl ester (GW6471). The administration of ST4070 generated long-lasting neuropathic pain relief compared with pregabalin and the FAAH inhibitors OL135 and URB597. Taken together, the reversible FAAH inhibitor ST4070 seems to be a promising novel therapeutic agent for the management of neuropathic pain.

The endogenous cannabinoid system in the gut of patients with inflammatory bowel disease

Activation of cannabinoid receptors (CBs) by endocannabinoids impacts on a number of gastrointestinal functions. Recent data indicate that CB1 agonists improve 2,4-dinitrobenzene sulfonic acid-induced colitis in mice, thus suggesting a role for the endocannabinoid agonist anandamide (AEA) in protecting the gut against inflammation. We here examined the gut endocannabinoid system in inflammatory bowel disease (IBD) patients, and investigated the ex vivo and in vitro effects of the non-hydrolysable AEA analog methanandamide (MAEA) on the mucosal proinflammatory response. The content of AEA, but not of 2-arachidonoyl-glycerol and N-palmitoylethanolamine, was significantly lower in inflamed than uninflamed IBD mucosa, and this was paralleled by lower activity of the AEA-synthesizing enzyme N-acyl-phosphatidylethanolamine-specific phospholipase D and higher activity of the AEA-degrading enzyme fatty acid amide hydrolase. MAEA significantly downregulated interferon-γ and tumor necrosis factor-α secretion by both organ culture biopsies and lamina propria mononuclear cells. Although these results are promising, further studies are needed to determine the role of cannabinoid pathways in gut inflammation.

Differences in the Endocannabinoid System of Sperm from Fertile and Infertile Men

Male infertility is a major cause of problems for many couples in conceiving a child. Recently, lifestyle pastimes such as alcohol, tobacco and marijuana have been shown to have further negative effects on male reproduction. The endocannabinoid system (ECS), mainly through the action of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) at cannabinoid (CB1, CB2) and vanilloid (TRPV1) receptors, plays a crucial role in controlling functionality of sperm, with a clear impact on male reproductive potential. Here, sperm from fertile and infertile men were used to investigate content (through LC-ESI-MS), mRNA (through quantitative RT-PCR), protein (through Western Blotting and ELISA) expression, and functionality (through activity and binding assays) of the main metabolic enzymes of AEA and 2-AG (NAPE-PLD and FAAH, for AEA; DAGL and MAGL for 2-AG), as well as of their binding receptors CB1, CB2 and TRPV1. Our findings show a marked reduction of AEA and 2-AG content in infertile seminal plasma, paralleled by increased degradation: biosynthesis ratios of both substances in sperm from infertile versus fertile men. In addition, TRPV1 binding was detected in fertile sperm but was undetectable in infertile sperm, whereas that of CB1 and CB2 receptors was not statistically different in the two groups. In conclusion, this study identified unprecedented alterations of the ECS in infertile sperm, that might impact on capacitation and acrosome reaction, and hence fertilization outcomes. These alterations might also point to new biomarkers to determine male reproductive defects, and identify distinct ECS elements as novel targets for therapeutic exploitation of ECS-oriented drugs to treat male fertility problems.

Regulation of male fertility by the endocannabinoid system

Mammalian conception is a complex process regulated by both sexual behavior and reproductive performance. Alcohol, marijuana and tobacco are among the main factors which affect negatively fertility in women and men. Several studies have demonstrated that marijuana impairs the male copulatory activity, and that smokers of this illegal drug show reduced fertility due, for instance, to decrease in sperm concentration, defective sperm function or alteration of sperm morphology. The discovery of endocannabinoids and all components responsible for their metabolism has allowed to collect valuable information on the effects of these endogenous lipids, able to mimic the actions of delta-9-tetrahydrocannabinol (THC), in reproductive functions. The purpose of this review is to describe the actions of cannabinoids and endocannabinoids on the control of procreation and hormonal release during the fertilization process in males.

Detailed characterization of the endocannabinoid system in human macrophages and foam cells, and anti-inflammatory role of type-2 cannabinoid receptor

OBJECTIVE Cannabinoid receptors are activated in murine macrophages upon exposure to oxidized low-density lipoproteins (oxLDL), and type-1 cannabinoid receptor (CB1R) is considered as a risk factor in atherosclerosis, because it promotes cholesterol accumulation and release of inflammatory mediators. Conversely, accumulated evidence suggests a protective role for type-2 cannabinoid receptor (CB2R). Here, we sought to ascertain whether different elements of the endocannabinoid system (ECS) were activated in human lipid-laden macrophages, and whether CB2R played any role in atherogenesis and inflammation of these cells. METHODS AND RESULTS Human macrophages were exposed to oxLDL in order to obtain lipid-laden foam cells. Liquid chromatography/mass spectrometry (LC/MS) was used to measure the production of the endocannabinoids in both macrophages and foam cells, and radiometric assays were performed to measure cannabinoid receptor binding and activity of endocannabinoid metabolizing enzymes. OxLDL accumulation was investigated by confocal imaging, and cytokine production and release were measured by means of flow cytometry and ELISA. The results showed that human macrophages possess a fully functional ECS, which was modulated by oxLDL. Selective CB2R activation reduced cellular oxLDL accumulation, which was associated with decreased expression of CD36 scavenger receptor, and decreased production of TNFα, IL-12 and IL-10. These anti-atherogenic and anti-inflammatory effects were reverted by the selective CB2R antagonist SR144528. CONCLUSIONS A fully active ECS is present in human macrophages and macrophage-derived foam cells. Selective activation of CB2R reduces CD36-dependent oxLDL accumulation and modulates production of inflammatory cytokines, thus representing a potential therapeutic strategy to combat atherosclerosis.

5-Lipoxygenase-dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment

The functional adaptation of the immune system to the surrounding environment is also a fundamental issue in space. It has been suggested that a decreased number of lymphocytes might be a cause of immunosuppression, possibly due to the induction of apoptosis. Early activation of 5‐lipoxygenase (5‐LOX) might play a central role in the initiation of the apoptotic program. The goal of the role of apoptosis in lymphocyte depression (ROALD) experiment, flown on the International Space Station as part of the BIO‐4 mission of the European Space Agency, was to ascertain the induction of apoptosis in human lymphocytes under authentic microgravity, and to elucidate the possible involvement of 5‐LOX. Our results demonstrate that exposure of human lymphocytes to microgravity for 48 h onboard the ISS remarkably increased apoptotic hallmarks such as DNA fragmentation (~3‐fold compared to ground‐based controls) and cleaved‐poly (ADP‐ribose) polymerase (PARP) protein expression (~3‐fold), as well as mRNA levels of apoptosis‐related markers such as p53 (~3‐fold) and calpain (~4‐fold); these changes were paralleled by an early increase of 5‐LOX activity (~2‐fold). Our findings provide a molecular background for the immune dysfunction observed in astronauts during space missions, and reveal potential new markers to monitor health status of ISS crew members.—Battista, N., Meloni, M. A., Bari, M., Mastrangelo, N., Galleri, G., Rapino, C., Dainese, E., Finazzi Agrò, A., Pippia, P., Maccarrone, M. 5‐Lipoxygenase‐dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment. FASEB J. 26, 1791‐1798 (2012). www.fasebj.org