Pierangelo Orlando

Endocannabinoid signalling in the blood of patients with schizophrenia

AimTo test the hypothesis that schizophrenia might be associated with alterations of the endogenous cannabinoid system in human blood.ResultsBlood from 20 healthy volunteers and 12 patients with schizophrenia, 5 of which both before and after a successful antipsychotic treatment, was analysed for: 1) the amounts of the endocannabinoid anandamide; 2) the levels of cannabinoid CB1 and CB2 receptor mRNAs, and 3) the levels of the mRNA encoding the enzyme fatty acid amide hydrolase (FAAH), responsible for anandamide degradation.The amounts of anandamide were significantly higher in the blood of patients with acute schizophrenia than in healthy volunteers (7.79 ± 0.50 vs. 2.58 ± 0.28 pmol/ml). Clinical remission was accompanied by a significant decrease of the levels of anandamide (3.88 ± 0.72 pmol/ml) and of the mRNA transcripts for CB2 receptors and FAAH.ConclusionThese findings indicate that endocannabinoid signalling might be altered during the acute phase of schizophrenia not only in the central nervous system but also in the blood. These changes might be related to the several immunological alterations described in schizophrenia.

Plant-Derived Cannabinoids Modulate the Activity of Transient Receptor Potential Channels of Ankyrin Type-1 and Melastatin Type-8

The plant cannabinoids (phytocannabinoids), cannabidiol (CBD), and Δ9-tetrahydrocannabinol (THC) were previously shown to activate transient receptor potential channels of both vanilloid type 1 (TRPV1) and ankyrin type 1 (TRPA1), respectively. Furthermore, the endocannabinoid anandamide is known to activate TRPV1 and was recently found to antagonize the menthol- and icilin-sensitive transient receptor potential channels of melastatin type 8 (TRPM8). In this study, we investigated the effects of six phytocannabinoids [i.e., CBD, THC, CBD acid, THC acid, cannabichromene (CBC), and cannabigerol (CBG)] on TRPA1- and TRPM8-mediated increase in intracellular Ca2+ in either HEK-293 cells overexpressing the two channels or rat dorsal root ganglia (DRG) sensory neurons. All of the compounds tested induced TRPA1-mediated Ca2+ elevation in HEK-293 cells with efficacy comparable with that of mustard oil isothiocyanates (MO), the most potent being CBC (EC50 = 60 nM) and the least potent being CBG and CBD acid (EC50 = 3.4–12.0 μM). CBC also activated MO-sensitive DRG neurons, although with lower potency (EC50 = 34.3 μM). Furthermore, although none of the compounds tested activated TRPM8-mediated Ca2+ elevation in HEK-293 cells, they all, with the exception of CBC, antagonized this response when it was induced by either menthol or icilin. CBD, CBG, THC, and THC acid were equipotent (IC50 = 70–160 nM), whereas CBD acid was the least potent compound (IC50 = 0.9–1.6 μM). CBG inhibited Ca2+ elevation also in icilin-sensitive DRG neurons with potency (IC50 = 4.5 μM) similar to that of anandamide (IC50 = 10 μM). Our findings suggest that phytocannabinoids and cannabis extracts exert some of their pharmacological actions also by interacting with TRPA1 and TRPM8 channels, with potential implications for the treatment of pain and cancer.

Control by the endogenous cannabinoid system of ras oncogene-dependent tumor growth

We investigated the effect of 2‐methyl‐arachidonyl‐2′‐fluoro‐ethylamide (Met‐F‐AEA), a stable analog of the endocannabinoid anandamide, on a rat thyroid epithelial cell line (FRTL‐5) transformed by the K‐ras oncogene, and on epithelial tumors derived from these cells. Met‐F‐AEA effect in vivo was evaluated in a nude mouse xenograft model, where K‐ras‐transformed (KiMol) cells were implanted subcutaneously. Met‐F‐AEA (0.5 mg/kg/dose) induced a drastic reduction in tumor volume. This effect was inhibited by the CBi receptor antagonist SR141716A (0.7 mg/kg/dose) and was accompanied by a strong reduction of K‐ras activity. Accordingly, KiMol cells and tumors express CB1 receptors. Met‐F‐AEA inhibited (IC50 ~5 μM) the proliferation in vitro and the transition to the S phase of KiMol cells and it reduced K‐ras activity; these effects were antagonized by SR141716A. Met‐F‐AEA cytostatic action was significantly smaller in nontransformed FRTL‐5 cells than in KiMol cells. Met‐F‐AEA treatment exerted opposite effects on the expression of CB1 receptors in KiMol and FRTL‐5 cells, with a strong up‐regulation in the former case and a suppression in nontransformed cells. The data suggest that: 1) Met‐F‐AEA inhibits ras oncogene‐dependent tumor growth in vivo through CB1 cannabinoid receptors; and 2) responsiveness of FRTL‐5 cells to endocannabinoids depends on whether or not they are transformed by K‐ras.

Microencapsulation in food science and biotechnology

Microencapsulation can represent an excellent example of microtechnologies applied to food science and biotechnology. Microencapsulation can be successfully applied to entrap natural compounds, like essential oils or vegetal extracts containing polyphenols with well known antimicrobial properties to be used in food packaging. Microencapsulation preserves lactic acid bacteria, both starters and probiotics, in food and during the passage through the gastrointestinal tract, and may contribute to the development of new functional foods.

Increased endocannabinoid levels reduce the development of precancerous lesions in the mouse colon.

Colorectal cancer is an increasingly important cause of death in Western countries. Endocannabinoids inhibit colorectal carcinoma cell proliferation in vitro. In this paper, we investigated the involvement of endocannabinoids on the formation of aberrant crypt foci (ACF, earliest preneoplastic lesions) in the colon mouse in vivo. ACF were induced by azoxymethane (AOM); fatty acid amide hydrolase (FAAH) and cannabinoid receptor messenger ribonucleic acid (mRNA) levels were analyzed by the quantitative reverse transcription polymerase chain reaction (RT-PCR); endocannabinoid levels were measured by liquid chromatography–mass spectrometry; caspase-3 and caspase-9 expressions were measured by Western blot analysis. Colonic ACF formation after AOM administration was associated with increased levels of 2-arachidonoylglycerol (with no changes in FAAH and cannabinoid receptor mRNA levels) and reduction in cleaved caspase-3 and caspase-9 expression. The FAAH inhibitor N-arachidonoylserotonin increased colon endocannabinoid levels, reduced ACF formation, and partially normalized cleaved caspase-3 (but not caspase-9) expression. Notably, N-arachidonoylserotonin completely prevented the formation of ACF with four or more crypts, which have been show to be best correlated with final tumor incidence. The effect of N-arachidonoylserotonin on ACF formation was mimicked by the cannabinoid receptor agonist HU-210. No differences in ACF formation were observed between CB1 receptor-deficient and wild-type mice. It is concluded that pharmacological enhancement of endocannabinoid levels (through inhibition of endocannabinoid hydrolysis) reduces the development of precancerous lesions in the mouse colon. The protective effect appears to involve caspase-3 (but not caspase-9) activation.

The role of endocannabinoids in the regulation of gastric emptying : alterations in mice fed a high-fat diet

Endocannabinoids (via cannabinoid CB1 receptor activation) are physiological regulators of intestinal motility and food intake. However, their role in the regulation of gastric emptying is largely unexplored. The purpose of the present study was to investigate the involvement of the endocannabinoid system in the regulation of gastric emptying in mice fed either a standard diet (STD) or a high‐fat diet (HFD) for 14 weeks.

Cannabidiol, a safe and non-psychotropic ingredient of the marijuana plant Cannabis sativa, is protective in a murine model of colitis

Inflammatory bowel disease affects millions of individuals; nevertheless, pharmacological treatment is disappointingly unsatisfactory. Cannabidiol, a safe and non-psychotropic ingredient of marijuana, exerts pharmacological effects (e.g., antioxidant) and mechanisms (e.g., inhibition of endocannabinoids enzymatic degradation) potentially beneficial for the inflamed gut. Thus, we investigated the effect of cannabidiol in a murine model of colitis. Colitis was induced in mice by intracolonic administration of dinitrobenzene sulfonic acid. Inflammation was assessed both macroscopically and histologically. In the inflamed colon, cyclooxygenase-2 and inducible nitric oxide synthase (iNOS) were evaluated by Western blot, interleukin-1β and interleukin-10 by ELISA, and endocannabinoids by isotope dilution liquid chromatography–mass spectrometry. Human colon adenocarcinoma (Caco-2) cells were used to evaluate the effect of cannabidiol on oxidative stress. Cannabidiol reduced colon injury, inducible iNOS (but not cyclooxygenase-2) expression, and interleukin-1β, interleukin-10, and endocannabinoid changes associated with 2,4,6-dinitrobenzene sulfonic acid administration. In Caco-2 cells, cannabidiol reduced reactive oxygen species production and lipid peroxidation. In conclusion, cannabidiol, a likely safe compound, prevents experimental colitis in mice.

Obesity-driven synaptic remodeling affects endocannabinoid control of orexinergic neurons.

Significance Endocannabinoids act retrogradely at presynaptic sites to activate cannabinoid receptor type 1 (CB1) receptors, thereby inhibiting neurotransmitter release and fine-tuning synaptic transmission. In murine models of obesity with leptin deficiency, we report that orexin-A neurons undergo a shift from predominant control by CB1-expressing excitatory to CB1-expressing inhibitory inputs. In addition, endocannabinoid biosynthesis is increased in these neurons. CB1 activation by endocannabinoids reduces the inhibition of orexinergic neurons in obese mice, thereby enhancing orexin-A release in target brain areas and contributing to hyperphagia and increased body weight, as well as to alterations of hormone levels typical of obesity. Acute or chronic alterations in energy status alter the balance between excitatory and inhibitory synaptic transmission and associated synaptic plasticity to allow for the adaptation of energy metabolism to new homeostatic requirements. The impact of such changes on endocannabinoid and cannabinoid receptor type 1 (CB1)-mediated modulation of synaptic transmission and strength is not known, despite the fact that this signaling system is an important target for the development of new drugs against obesity. We investigated whether CB1-expressing excitatory vs. inhibitory inputs to orexin-A–containing neurons in the lateral hypothalamus are altered in obesity and how this modifies endocannabinoid control of these neurons. In lean mice, these inputs are mostly excitatory. By confocal and ultrastructural microscopic analyses, we observed that in leptin-knockout (ob/ob) obese mice, and in mice with diet-induced obesity, orexinergic neurons receive predominantly inhibitory CB1-expressing inputs and overexpress the biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol, which retrogradely inhibits synaptic transmission at CB1-expressing axon terminals. Patch-clamp recordings also showed increased CB1-sensitive inhibitory innervation of orexinergic neurons in ob/ob mice. These alterations are reversed by leptin administration, partly through activation of the mammalian target of rapamycin pathway in neuropeptide-Y-ergic neurons of the arcuate nucleus, and are accompanied by CB1-mediated enhancement of orexinergic innervation of target brain areas. We propose that enhanced inhibitory control of orexin-A neurons, and their CB1-mediated disinhibition, are a consequence of leptin signaling impairment in the arcuate nucleus. We also provide initial evidence of the participation of this phenomenon in hyperphagia and hormonal dysregulation in obesity.

Beneficial effect of the non-psychotropic plant cannabinoid cannabigerol on experimental inflammatory bowel disease

Inflammatory bowel disease (IBD) is an incurable disease which affects millions of people in industrialized countries. Anecdotal and scientific evidence suggests that Cannabis use may have a positive impact in IBD patients. Here, we investigated the effect of cannabigerol (CBG), a non-psychotropic Cannabis-derived cannabinoid, in a murine model of colitis. Colitis was induced in mice by intracolonic administration of dinitrobenzene sulphonic acid (DNBS). Inflammation was assessed by evaluating inflammatory markers/parameters (colon weight/colon length ratio and myeloperoxidase activity), by histological analysis and immunohistochemistry; interleukin-1β, interleukin-10 and interferon-γ levels by ELISA, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by western blot and RT-PCR; CuZn-superoxide dismutase (SOD) activity by a colorimetric assay. Murine macrophages and intestinal epithelial cells were used to evaluate the effect of CBG on nitric oxide production and oxidative stress, respectively. CBG reduced colon weight/colon length ratio, myeloperoxidase activity, and iNOS expression, increased SOD activity and normalized interleukin-1β, interleukin-10 and interferon-γ changes associated to DNBS administration. In macrophages, CBG reduced nitric oxide production and iNOS protein (but not mRNA) expression. Rimonabant (a CB1 receptor antagonist) did not change the effect of CBG on nitric oxide production, while SR144528 (a CB2 receptor antagonist) further increased the inhibitory effect of CBG on nitric oxide production. In conclusion, CBG attenuated murine colitis, reduced nitric oxide production in macrophages (effect being modulated by the CB2 receptor) and reduced ROS formation in intestinal epithelial cells. CBG could be considered for clinical experimentation in IBD patients.

The endocannabinoid system and pivotal role of the CB2 receptor in mouse spermatogenesis

The exact role of the endocannabinoid system (ECS) during spermatogenesis has not been clarified. We used purified germ cell fractions representative of all phases of spermatogenesis and primary cultures of spermatogonia. This approach allowed the precise quantification of the cannabinoid receptor ligands, anandamide and 2-arachidonoylglycerol, and of the expression at transcriptional and transductional levels of their metabolic enzymes and receptors. Our data indicate that male mouse germ cells possess an active and complete ECS, which is modulated during meiosis, and suggest the presence of an autocrine endocannabinoid signal during spermatogenesis. Mitotic cells possess higher levels of 2-arachidonoylglycerol, which decrease in spermatocytes and spermatids. Accordingly, spermatogonia express higher and lower levels of 2-arachidonoylglycerol biosynthetic and degrading enzymes, respectively, as compared to meiotic and postmeiotic cells. This endocannabinoid likely plays a pivotal role in promoting the meiotic progression of germ cells by activating CB2 receptors. In fact, we found that the selective CB2 receptor agonist, JWH133, induced the Erk 1/2 MAPK phosphorylation cascade in spermatogonia and their progression toward meiosis, because it increased the number of cells positive for SCP3, a marker of meiotic prophase, and the expression of early meiotic prophase genes.