Cannabinoids and haemostasis.

Elements of the endocannabinoid system (cannabinoid receptors CB1, CB2, CBPT and CBED, endocannabinoids, enzymes involved in the synthesis and metabolism of endocannabinoids) are located on the structures involved in the process of hemostasis. An increasing level of endocannabinoids was also observed in some pathological conditions, which may occur in disorders of hemostasis. At the same time, disconcertingly, there is an increased number of reports about incidents of cardiovascular events in smokers of marijuana. Experimental and clinical studies demonstrated multidirectional, often contradictory, effects of cannabinoids on hemostasis, including effects of the compounds on platelets, vascular endothelium, fibrinolysis and plasma coagulation systems. The mechanisms of action of cannabinoids on homeostasis depend on the cannabinoid receptors CB1, CB2, CBPT and CBED, receptors of other systems stimulated by endocannabinoids, as well as metabolites of endocannabinoids and nitrogen oxide. The range of biological functions of endo- and plant cannabinoids, expanded to include the process of hemostasis, may constitute a condition for their recognition as a new factor responsible for thromboembolism in smokers of marijuana, in pathological disorders with increased levels of endocannabinoids and in individuals with polymorphisms of FAAH C385A and A385A. On the other hand, there are compelling reasons for anti­hemostatic action of cannabinoids.

Quantification of endocannabinoids in biological systems by chromatography and mass spectrometry: a comprehensive review from an analytical and biological perspective.

The endocannabinoids anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2AG) are physiologically occurring, biologically active compounds on CB(1) and CB(2) receptors with multiple physiological functions. AEA and 2AG have been identified and quantified in many mammalian biological fluids and tissues, such as human plasma, adipocytes, tissues and tissue microdialysates, at concentrations in the picomolar-to-nanomolar range under basal conditions. In this article, recently published chromatographic and mass spectrometric analytical methods, i.e., HPLC with fluorescence or ultraviolet detection, LC-MS, LC-MS/MS, GC-MS and GC-MS/MS, are reviewed and discussed, notably from the quantitative point of view. We focus on and emphasize the particular importance of blood sampling, sample storage and work-up including solvent and solid-phase extraction and derivatization procedures, matrix-effects, and stability of analytes. As 2AG spontaneously isomerizes to its CB(1)/CB(2) receptors biologically inactive 1-arachidonoyl glycerol (1AG) by acyl migration, this phenomenon and its particular importance for accurate quantification of 2AG are discussed in detail. Due to the electrical neutrality of AEA and 2AG their solvent extraction by toluene offers the least matrix-effect and minimum isomerization. LC-MS/MS is the most frequently used analytical technique for AEA and 2AG. At present, the utility of the GC-MS/MS methodology seems to be limited to AEA measurement in human plasma, bronchoalveolar liquid (BAL) and microdialysate samples. Despite great instrumental advances in the LC-MS/MS methodology, sampling and sample treatment remains one of the most crucial analytical steps in 2AG analysis. Extension of the LC-MS/MS methodology, for instance to microdialysate and BAL samples from clinical studies, is a big analytical challenge in endocannabinoid analysis in clinical settings. Currently available LC-MS/MS and GC-MS/MS methods should be useful to investigate the metabolism of AEA and 2AG beyond hydrolysis, i.e., by ß- and ω-oxidation pathways.

A gradient of 2-arachidonoylglycerol regulates mouse epididymal sperm cell start-up.

During transit through the epididymis, spermatozoa are normally kept immotile and do not attain the ability to become motile until they reach the caudal epididymis. This study was undertaken to determine whether endocannabinoids play a role in the epididymis and in particular in suppressing the ability of spermatozoa to become motile. We show that the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are high in mouse spermatozoa isolated from the caput (head) of the epididymis, where these cells do not move (or possess sluggish and irregular motility) and decrease dramatically in spermatozoa isolated from the cauda (tail). The subsequent gradient regulates, via autocrine communication, the activity of cannabinoid receptor CNR1 (previously known as CB1) present on the sperm cell membrane and induces caudal spermatozoa to acquire the potential to become motile ("start-up"). Accordingly, the genetic or pharmacological inactivation of CNR1 increases number of motile spermatozoa in caput. Also, blockers of endocannabinoid cellular uptake inhibit the potential to move of spermatozoa and destroy the 2-AG gradient throughout the epididymis. This gradient-regulated mechanism may encourage further research for future therapies related to male infertility.

Localization of an endocannabinoid system in the hypophysial pars tuberalis and pars distalis of man.

The hypophysial pars tuberalis (PT) acts as an important interface between neuroendocrine brain centers (hypothalamus, pineal organ) and the pars distalis (PD) of the hypophysis. Recently, we have identified an endocannabinoid system in the PT of hamsters and provided evidence that 2-arachidonoylglycerol is a messenger molecule that appears to play an essential role in seasonal reproduction and prolactin release by acting on the cannabinoid receptors in the PD. We now demonstrate the enzymes involved in endocannabinoid synthesis and degradation, namely sn-1-selective diacylglycerol lipase α, N-acylphosphatidylethanolamine-specific phospholipase D, and monoacylglycerol lipase, in the PT of man by means of immunohistochemistry. High-performance liquid chromatography coupled with tandem mass spectrometry revealed 2-arachidonoylglycerol and other endocannabinoids in the human PT. Furthermore, we detected the expression of the cannabinoid receptor 1 (CB1), a primary receptor for endocannabinoids, in the PD. Double-immunofluorescence staining for CB1 and various hypophysial hormones or S-100, a marker for folliculostellate (FS) cells, revealed that CB1 immunoreactivity was mainly localized to corticotrophs and FS-cells. A limited number of lactotrophs and somatotrophs also showed CB1 immunoreactivity, which was however absent from gonadotrophs and thyrotrophs. Our data thus indicate that the human PT comprises an endocannabinoid system, and that corticotrophs and FS-cells are the main target cells for endocannabinoids. The functional significance of this newly discovered pathway remains to be elucidated in man; it might be related to the control of stress responses and/or reflect a remnant seasonal control of hypophysial hormonal secretion.

Cerebrospinal fluid levels of the endocannabinoid anandamide are reduced in patients with untreated newly diagnosed temporal lobe epilepsy.

PURPOSE: The endocannabinoid system is involved in excitatory/inhibitory balance mechanisms within the central nervous system (CNS). Growing evidence shows that its perturbation leads to development of epileptic seizures in experimental models, thus indicating that endocannabinoids play an intrinsic protective role in suppressing pathologic neuronal excitability. Experimental data also demonstrate that the endocannabinoid anandamide (AEA) can antagonize epileptic discharges in hippocampal tissue. The objective of our study was to measure endocannabinoids levels in the cerebrospinal fluid (CSF) of drug-naive patients affected by temporal lobe epilepsy (TLE). METHODS: We measured the levels of both AEA and the other endocannabinoid, 2-arachidonoylglycerol (2-AG), in the CSF of drug-naive patients with TLE. RESULTS: A significant reduction of AEA was found in the CSF of patients with compared with healthy controls (epileptic patients = 2.55 +/- 1.78 pmol/ml; healthy controls = 11.65 +/- 7.53 pmol/ml; n = 9 for both groups, p < 0.01). 2-AG levels, however, were not affected (epileptic patients = 209.5 +/- 146.56; healthy controls = 159.6 +/- 110.2) (n = 6 for both groups, p = 0.48). DISCUSSION: Our findings seem to be consistent with experimental evidence demonstrating a significant prevention of epileptic seizures induced by endocannabinoids in models of epilepsy. Furthermore, they support the hypothesis that AEA may be involved in its pathogenesis, suggesting a hypothetical primary impairment of the endocannabinoid system in untreated TLE. The actual role of this in vivo dysregulation still remains unclear.

Anandamide and AM251, via water, modulate food intake at central and peripheral level in fish.

The endocannabinoid system is a major regulator of food intake in many animal species. Studies conducted so far have mostly focused on mammals, and, therefore, in this study, the role of the endocannabinoid system in food intake in the sea bream Sparus aurata was investigated. The effect of different doses of the endocannabinoid anandamide (AEA), administered via water, was evaluated after different exposure times (30, 60 and 120 min) at both physiological and molecular levels. The results obtained indicate that fish exposed to AEA via water present approximately 1000-fold higher levels of AEA in both the brain and liver, which correlated with a significant increase in food intake and with the elevation of cannabinoid receptor 1 (CB(1)) and neuropeptide Y (NPY) mRNA levels in the brain. A peripheral effect of AEA was also observed, since a time-dependent increase in hepatic CB(1) mRNA and protein levels was detected. These effects were attenuated by the administration, again via water, of a selective cannabinoid CB(1) receptor antagonist (AM251). These findings indicate that the endocannabinoid AEA, at doses that stimulate food intake in fish, concomitantly stimulates the expression of the orexigenic peptide NPY as well that of its own receptor, thereby potentially enhancing its effect on food consumption. In agreement with a role of AEA in food intake in S. aurata, we found increased brain levels of both this and the other endocannabinoid, 2-arachidonoylglycerol (2-AG), following food deprivation.

Differential alterations of the concentrations of endocannabinoids and related lipids in the subcutaneous adipose tissue of obese diabetic patients.

BACKGROUND: The endocannabinoids, anandamide and 2-AG, are produced by adipocytes, where they stimulate lipogenesis via cannabinoid CB1 receptors and are under the negative control of leptin and insulin. Endocannabinoid levels are elevated in the blood of obese individuals and nonobese type 2 diabetes patients. To date, no study has evaluated endocannabinoid levels in subcutaneous adipose tissue (SAT) of subjects with both obesity and type 2 diabetes (OBT2D), characterised by similar adiposity and whole body insulin resistance and lower plasma leptin levels as compared to non-diabetic obese subjects (OB). DESIGN AND METHODS: The levels of anandamide and 2-AG, and of the anandamide-related PPARalpha ligands, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), in the SAT obtained by abdominal needle biopsy in 10 OBT2D, 11 OB, and 8 non-diabetic normal-weight (NW) subjects, were measured by liquid chromatography-mass spectrometry. All subjects underwent a hyperinsulinaemic euglycaemic clamp. RESULTS: As compared to NW, anandamide, OEA and PEA levels in the SAT were 2-4.4-fold elevated (p < 0.05), and 2-AG levels 2.3-fold reduced (p < .05), in OBT2D but not in OB subjects. Anandamide, OEA and PEA correlated positively (p < .05) with SAT leptin mRNA and free fatty acid during hyperinsulinaemic clamp, and negatively with SAT LPL activity and plasma HDL-cholesterol, which were all specifically altered in OBT2D subjects. CONCLUSIONS: The observed alterations emphasize, for the first time in humans, the potential different role and regulation of adipose tissue anandamide (and its congeners) and 2-AG in obesity and type 2 diabetes.

FAAH and anandamide: is 2-AG really the odd one out?

Fatty acid amide hydrolase (FAAH) is a hydrolytic enzyme that recognizes as substrates and inactivates the two most studied endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG). Following the observation that endocannabinoids produced by tissues during pathological conditions often have protective roles, FAAH inhibitors have been proposed as therapeutic drugs. Yet it has been suggested that FAAH functions in vivo only as an anandamide-degrading enzyme because its pharmacological and genetic inactivation is usually accompanied by elevation of anandamide, but not 2-AG, levels. We believe, however, that this concept needs to be revisited in light of reports that, under certain experimental conditions, FAAH inhibitors also elevate 2-AG tissue levels in vivo and, more recently, that FAAH inactivation in the striatum instead reduces 2-AG concentrations through upregulation of anandamide levels, activation of transient receptor potential vanilloid 1 receptors and inhibition of 2-AG biosynthesis.

Minocycline treatment inhibits microglial activation and alters spinal levels of endocannabinoids in a rat model of neuropathic pain.

Activation of spinal microglia contributes to aberrant pain responses associated with neuropathic pain states. Endocannabinoids (ECs) are present in the spinal cord, and inhibit nociceptive processing; levels of ECs may be altered by microglia which modulate the turnover of endocannabinoids in vitro. Here, we investigate the effect of minocycline, an inhibitor of activated microglia, on levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG), and the related compound N-palmitoylethanolamine (PEA), in neuropathic spinal cord. Selective spinal nerve ligation (SNL) in rats resulted in mechanical allodynia and the presence of activated microglia in the ipsilateral spinal cord. Chronic daily treatment with minocycline (30 mg/kg, ip for 14 days) significantly reduced the development of mechanical allodynia at days 5, 10 and 14 post-SNL surgery, compared to vehicle-treated SNL rats (P < 0.001). Minocycline treatment also significantly attenuated OX-42 immunoreactivity, a marker of activated microglia, in the ipsilateral (P < 0.001) and contralateral (P < 0.01) spinal cord of SNL rats, compared to vehicle controls. Minocycline treatment significantly (P < 0.01) decreased levels of 2-AG and significantly (P < 0.01) increased levels of PEA in the ipsilateral spinal cord of SNL rats, compared to the contralateral spinal cord. Thus, activation of microglia affects spinal levels of endocannabinoids and related compounds in neuropathic pain states.

Blockade of endocannabinoid-degrading enzymes attenuates neuropathic pain.

Direct-acting cannabinoid receptor agonists are well known to reduce hyperalgesic responses and allodynia after nerve injury, although their psychoactive side effects have damped enthusiasm for their therapeutic development. Alternatively, inhibiting fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the principal enzymes responsible for the degradation of the respective endogenous cannabinoids, anandamide (AEA) and 2-arachydonylglycerol (2-AG), reduce nociception in a variety of nociceptive assays, with no or minimal behavioral effects. In the present study we tested whether inhibition of these enzymes attenuates mechanical allodynia, and acetone-induced cold allodynia in mice subjected to chronic constriction injury of the sciatic nerve. Acute administration of the irreversible FAAH inhibitor, cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), or the reversible FAAH inhibitor, 1-oxo-1-[5-(2-pyridyl)-2-yl]-7-phenylheptane (OL-135), decreased allodynia in both tests. This attenuation was completely blocked by pretreatment with either CB(1) or CB(2) receptor antagonists, but not by the TRPV1 receptor antagonist, capsazepine, or the opioid receptor antagonist, naltrexone. The novel MAGL inhibitor, 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) also attenuated mechanical and cold allodynia via a CB(1), but not a CB(2), receptor mechanism of action. Whereas URB597 did not elicit antiallodynic effects in FAAH(-/-) mice, the effects of JZL184 were FAAH-independent. Finally, URB597 increased brain and spinal cord AEA levels, whereas JZL184 increased 2-AG levels in these tissues, but no differences in either endo-cannabinoid were found between nerve-injured and control mice. These data indicate that inhibition of FAAH and MAGL reduces neuropathic pain through distinct receptor mechanisms of action and present viable targets for the development of analgesic therapeutics.

Cannabinoid-2 receptor mediates protection against hepatic ischemia/reperfusion injury.

Hepatic ischemia-reperfusion (I/R) injury continues to be a fatal complication that can follow liver surgery or transplantation. We have investigated the involvement of the endocannabinoid system in hepatic I/R injury using an in vivo mouse model. Here we report that I/R triggers several-fold increases in the hepatic levels of the endocannabinoids anandamide and 2-arachidonoylglycerol, which originate from hepatocytes, Kupffer, and endothelial cells. The I/R-induced increased tissue endocannabinoid levels positively correlate with the degree of hepatic damage and serum TNF-alpha, MIP-1alpha, and MIP-2 levels. Furthermore, a brief exposure of hepatocytes to various oxidants (H2O2 and peroxynitrite) or inflammatory stimuli (endotoxin and TNF-alpha) also increases endocannabinoid levels. Activation of CB2 cannabinoid receptors by JWH133 protects against I/R damage by decreasing inflammatory cell infiltration, tissue and serum TNF-alpha, MIP-1alpha and MIP-2 levels, tissue lipid peroxidation, and expression of adhesion molecule ICAM-1 in vivo. JWH133 also attenuates the TNF-alpha-induced ICAM-1 and VCAM-1 expression in human liver sinusoidal endothelial cells (HLSECs) and the adhesion of human neutrophils to HLSECs in vitro. Consistent with the protective role of CB2 receptor activation, CB2-/- mice develop increased I/R-induced tissue damage and proinflammatory phenotype. These findings suggest that oxidative/nitrosative stress and inflammatory stimuli may trigger endocannabinoid production, and indicate that targeting CB2 cannabinoid receptors may represent a novel protective strategy against I/R injury. We also demonstrate that CB2-/- mice have a normal hemodynamic profile.

The rat pineal gland comprises an endocannabinoid system.

In the mammalian pineal gland, the rhythm in melatonin biosynthesis depends on the norepinephrine (NE)-driven regulation of arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme of melatonin biosynthesis. A recent study showed that phytocannabinoids like tetrahydrocannabinol reduce AANAT activity and attenuate NE-induced melatonin biosynthesis in rat pineal glands, raising the possibility that an endocannabinoid system is present in the pineal gland. To test this hypothesis, we analyzed cannabinoid (CB) receptors and specific enzymes for endocannabinoid biosynthesis or catabolism in rat pineal glands and cultured pinealocytes. Immunohistochemical and immunoblot analyses revealed the presence of CB1 and CB2 receptor proteins, of N-acyl phosphatidyl ethanolamine hydrolyzing phospholipase D (NAPE-PLD), an enzyme catalyzing endocannabinoid biosynthesis and of fatty acid amide hydrolase (FAAH), an endocannabinoid catabolizing enzyme, in pinealocytes, and in pineal sympathetic nerve fibers identified by double immunofluorescence with an antibody against tyrosine hydroxylase. The immunosignals for the CB2 receptor, NAPE-PLD, and FAAH found in pinealocytes did not vary under a 12 hr light:12 hr dark cycle. The CB1 receptor immunoreaction in pinealocytes was significantly reduced at the end of the light phase [zeitgeber time (ZT) 12]. The immunosignal for NAPE-PLD found in pineal sympathetic nerve fibers was reduced in the middle of the dark phase (ZT 18). Stimulation of cultured pinealocytes with NE affected neither the subcellular distribution nor the intensity of the immunosignals for the investigated CB receptors and enzymes. In summary, the pineal gland comprises indispensable compounds of the endocannabinoid system indicating that endocannabinoids may be involved in the control of pineal physiology.

Mass-spectrometric identification of anandamide and 2-arachidonoylglycerol in nematodes.

The purpose of the study was to see if nematodes (Caenorhabditis elegans, Caenorhabditis briggsae, and Pelodera strongyloides) produce endocannabinoids; i.e., anandamide (AEA) and 2-arachidonoylglycerol (2-AG). In this study, AEA and 2-AG were identified as endogenous products from nematodes by using electrospray-ionization ion-trap MS/MS (ESI-IT-MS) experiments operated in the positive-ionization mode. Endocannabinoids were identified by product ion scan and concentrations were measured by triple quadrupole mass spectrometry in the multiple reaction monitoring mode (MRM). Both AEA and 2-AG were identified in all of the nematode samples, even though these species lack known cannabinoid receptors. Neither AEA nor 2-AG were detected in the fat-3 mutant of C. elegans, which lacks the necessary enzyme to produce arachidonic acid, the fatty acid precursor of these endocannabinoids.

LC-MS-MS analysis of neutral eicosanoids.

The neutral arachidonic acid derivatives N-arachidonoyl ethanolamine (anandamide or AEA), and 2-arachidonoylglycerol (2-AG) have been identified as endogenous ligands for the cannabinoid receptors. Additionally, these compounds have been identified as substrates of the second isoform of the cyclooxygenase enzyme (COX-2). Through the action of COX-2 and downstream prostaglandin synthases, a diverse family of prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) have been identified. Sensitive and reliable analytical methodology is crucial for the continued research on the biological roles of this family of lipids. In this chapter, we discuss methods for analyzing both the precursor endocannabinoids and their PG-like products by LC-MS-MS. Cation coordination provides the ionization, and selected reaction monitoring is successfully employed to provide a method of analysis that is both sensitive and specific.

Regulation, function, and dysregulation of endocannabinoids in models of adipose and beta-pancreatic cells and in obesity and hyperglycemia.

CONTEXT: Cannabinoid CB(1) receptor blockade decreases weight and hyperinsulinemia in obese animals and humans in a way greatly independent from food intake. OBJECTIVE: The objective of this study was to investigate the regulation and function of the endocannabinoid system in adipocytes and pancreatic beta-cells. DESIGN, SETTING, AND PATIENTS: Mouse 3T3-F442A adipocytes and rat insulinoma RIN-m5F beta-cells, pancreas and fat from mice with diet-induced obesity, visceral and sc fat from patients with body mass index equal to or greater than 30 kg/m(2), and serum from normoglycemic and type 2 diabetes patients were studied. MAIN OUTCOME MEASURE: Endocannabinoid enzyme and adipocyte protein expression, and endocannabinoid and insulin levels were measured. RESULTS: Endocannabinoids are present in adipocytes with levels peaking before differentiation, and in RIN-m5F beta-cells, where they are under the negative control of insulin. Chronic treatment of adipocytes with insulin is accompanied by permanently elevated endocannabinoid signaling, whereas culturing of RIN-m5F beta-cells in high glucose transforms insulin down-regulation of endocannabinoid levels into up-regulation. Epididymal fat and pancreas from mice with diet-induced obesity contain higher endocannabinoid levels than lean mice. Patients with obesity or hyperglycemia caused by type 2 diabetes exhibit higher concentrations of endocannabinoids in visceral fat or serum, respectively, than the corresponding controls. CB(1) receptor stimulation increases lipid droplets and decreases adiponectin expression in adipocytes, and it increases intracellular calcium and insulin release in RIN-m5F beta-cells kept in high glucose. CONCLUSIONS: Peripheral endocannabinoid overactivity might explain why CB(1) blockers cause weight-loss independent reduction of lipogenesis, of hypoadiponectinemia, and of hyperinsulinemia in obese animals and humans.

Endocannabinoid identification in the brain: studies of breakdown lead to breakthrough, and there may be NO hope.

Endocannabinoids are a class of fatty acid derivatives defined by their ability to interact with the specific cannabinoid receptors that were originally identified as the targets of Delta9-tetrahydocannabinol (Delta9-THC), the psychoactive component of cannabis. Endocannabinoids have been implicated in a growing number of important physiological and behavioral events. A full understanding of the functions of endocannabinoids will involve knowing which ones are active, and how they are produced, during any given physical event. However, studying these small lipids in the brain presents many technical challenges. New selective pharmacological tools promise to be very useful in unraveling the complexities of endocannabinoid signaling, but parallel developments from the investigation of the cellular neurophysiology of the endocannabinoid systems highlight the difficulties remaining.

A role for endocannabinoids in the generation of parkinsonism and levodopa-induced dyskinesia in MPTP-lesioned non-human primate models of Parkinson's disease.

Endocannabinoids and cannabinoid CB1 receptors play a role in the control of movement by modulating GABA, glutamate, and other neurotransmitters throughout the basal ganglia. Roles for abnormalities in endocannabinoid signaling in Parkinson's disease (PD) and the major side effect of current treatments, levodopa-induced dyskinesia (LID), have been suggested by rodent studies. Here we show that signaling by endocannabinoids contributes to the pathophysiology of parkinsonism and LID in MPTP-lesioned, non-human primate models of Parkinson's disease. In MPTP-lesioned marmosets previously treated with levodopa to establish LID, attenuation of CB1 signaling by systemic administration of rimonabant (1 and 3 mg/kg) had anti-parkinsonian actions, equivalent to a 71% increase in motor activity at 3 mg/kg. Rimonabant did not elicit dyskinesia. Co-administration of levodopa (8 mg/kg) and rimonabant (1 and 3 mg/kg) resulted in significantly less dyskinesia than levodopa alone, without significantly affecting the anti-parkinsonian action of levodopa. These data suggest that enhanced endocannabinoid signaling may be involved in the pathophysiology of both parkinsonism and LID. To define potential mechanisms by which such a role might be mediated, we determined the levels of the endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG) throughout the basal ganglia in normal and three groups of MPTP-lesioned cynomolgus monkeys (untreated; acutely treated with L-DOPA, non-dyskinetic; long-term treated, with levodopa-induced dyskinesia). In the untreated, MPTP-lesioned primate, parkinsonism was associated with increases in both 2-AG (+88%) and anandamide (+49%) in the striatum, and of 2-AG (+97%) in the substantia nigra, changes that are consistent with the previously suggested role for endocannabinoids in mechanisms attempting to compensate for loss of dopamine in untreated parkinsonism. Increased levels of anandamide (+34%) in the external globus pallidus of MPTP-lesioned animals were normalized by levodopa treatment and may contribute to the generation of parkinsonian symptoms. However, no clear alteration in endocannabinoid levels could be correlated with the expression of LID. These data highlight the potential roles played by endocannabinoids and CB1 in PD and LID and suggest the need for further research to pursue the multiple therapeutic opportunities for manipulating this system in movement disorders.

Changes in endocannabinoid and palmitoylethanolamide levels in eye tissues of patients with diabetic retinopathy and age-related macular degeneration.

Cannabinoid receptors and the endocannabinoids (anandamide (N-arachidonoylethanolamine--AEA) and 2-arachidonoylglycerol (2-AG)), as well as the AEA congener, palmitoylethanolamide (PEA), are involved in ocular physiology. We measured endocannabinoid and PEA levels by isotope-dilution liquid chromatography-mass spectrometric analysis in post-mortem eye tissues of patients with diabetic retinopathy (DR) or age-related macular degeneration (AMD). In eyes with DR, significantly enhanced levels of AEA were found in the retina ( approximately 1.8-fold), ciliary body ( approximately 1.5-fold) and, to a lesser extent, cornea ( approximately 1.3-fold). Surprisingly, 2-AG levels were significantly higher ( approximately 3-fold) only in the iris, whereas PEA levels only slightly increased ( approximately 1.3-fold) in the ciliary body. In eyes with AMD, significantly enhanced levels of AEA were found in the choroid ( approximately 1.3-fold), ciliary body ( approximately 1.4-fold) and cornea ( approximately 1.4-fold), whereas in the retina only a trend towards an increase ( approximately 1.5-fold) was observed. The tissue- and disease-selective nature of the changes observed suggests that the compounds analyzed here may play different roles in the control of eye function under different pathological conditions.

Quantification of endocannabinoids in rat biological samples by GC/MS: technical and theoretical considerations.

In the last several years, interest has increased significantly about the endocannabinoids anandamide and 2-arachidonylglycerol, two lipid messengers that activate cannabinoid receptors. Quantification of these compounds in biological samples presents numerous technical challenges. Because of their low abundance, endocannabinoids are usually quantified by isotope dilution assays using mass spectrometry coupled to either gas chromatography or high-performance liquid chromatography. Although endocannabinoid levels in biological fluids, such as plasma and cerebrospinal fluid, can be directly determined by these techniques, the complex lipid profile of brain tissue samples mandates purification of lipid extracts before GC/MS analysis; this step is not necessary when using HPLC/MS. We have found that when silica gel chromatography is used for endocannabinoid purification, poor recovery and loss of deuterium from the internal standards lead to inaccurate estimation of endocannabinoid levels. By contrast, purification strategies using C(18) solid-phase extraction permits precise and reproducible GC/MS quantification of endocannabinoids in tissue samples.

Cardiovascular risk management by blocking the endocannabinoid system.

The specific blockade of endocannabinoids at the level of the cannabinoid receptor 1 (CB (1) receptor) is a new therapeutic option to reduce body weight and manage cardiovascular risk. Although clinical trials are underway to document the safety and efficacy of this approach, much is still unknown about this endogenous system. Endocannabinoids and their receptors are expressed in the central nervous system as well as in the periphery and regulate the central neural circuits for food uptake and peripheral metabolic circuits. Within the context of food uptake, the stimulation of the CB (1) receptor with Delta (9)-tetrahydrocannabinol (Delta (9)-THC) enhances food consumption, while its blockade with receptor antagonists is an emerging relevant therapeutic means to reduce body weight. Rimonabant is the first of a new class of drugs that interferes with the endocannabinoid system by blocking the CB (1) receptor. In recent clinical studies, a substantial reduction in body weight and waist circumference was associated with an improvement of the cardiovascular risk profile. In particular, increased HDL cholesterol, decreased serum triglycerides and improved insulin sensitivity were observed. Further research will serve to establish the role of these compounds in cardiovascular risk management.