Peripheral cannabinoid receptors exert a powerful inhibitory control over pain initiation, but the endocannabinoid signal that normally engages this intrinsic analgesic mechanism is unknown. To address this question, we developed a peripherally restricted inhibitor of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the endocannabinoid anandamide. The compound, called URB937, suppresses FAAH activity and increases anandamide levels outside the central nervous system (CNS). Despite its inability to access brain and spinal cord, URB937 attenuates behavioral responses indicative of persistent pain in rodent models of peripheral nerve injury and inflammation, and prevents noxious stimulus-evoked neuronal activation in spinal cord regions implicated in nociceptive processing. CB1 cannabinoid receptor blockade prevents these effects. The results suggest that anandamide-mediated signaling at peripheral CB1 receptors controls the access of pain-related inputs to the CNS. Brain-impenetrant FAAH inhibitors, which strengthen this gating mechanism, might offer a new approach to pain therapy.
The endocannabinoid anandamide is removed from the synaptic space by a selective transport system, expressed in neurons and astrocytes, which remains molecularly uncharacterized. Here we describe a partly cytosolic variant of the intracellular anandamide-degrading enzyme, fatty acid amide hydrolase-1 (FAAH-1), termed FAAH-like anandamide transporter (FLAT), which lacks amidase activity but binds anandamide with low micromolar affinity and facilitates its translocation into cells. Known anandamide transport inhibitors, such as AM404 and OMDM-1, block these effects. Additionally, we identify a competitive antagonist of the interaction of anandamide with FLAT, the phthalazine derivative ARN272, which prevents anandamide internalization in vitro, interrupts anandamide deactivation in vivo, and exerts profound analgesic effects in rodent models of nociceptive and inflammatory pain, which are mediated by CB1 cannabinoid receptors. The results identify FLAT as a critical molecular component of anandamide transport in neural cells and a potential target for therapeutic drugs.
The purpose of the present study was to characterize daily variations in haematology and blood biochemistry in healthy male and female tench (Tinca tinca) in the four seasons of the year. Red blood cells, haematocrit and haemoglobin presented a similar daily pro-¢le in spring and summer, with high values during the scotophase. Total and di¡erential types of leucocytes showed signi¢cant daily £uctuations, with a di¡erent pattern depending on the season and/or the type of leucocytes studied. A daily rhythm in plasma glucose and triglycerides was detected in winter, but signi¢cant changes in cholesterol concentrations were only detected in females during summer. The lowest mean level for triglycerides and cholesterol was observed in winter. The daily pro¢le of plasma total proteins was similar in the four seasons, without a signi¢cant daily oscillation. All these daily and seasonal changes in haematology and blood biochemistry indicate that the time of the day and the season must be considered when such parameters are used for assessing the physiological status of the ¢sh, and as biomarkers for disease and/or pollutant exposure.
The endocannabinoid 2-arachidonoylglycerol (2-AG) is degraded primarily by monoacylglycerol lipase (MGL). We compared peripheral antinociceptive effects of JZL184, a novel irreversible MGL inhibitor, with the reversible MGL-preferring inhibitor URB602 and exogenous 2-AG in rats. EXPERIMENTAL APPROACHNociception in the formalin test was assessed in groups receiving dorsal paw injections of vehicle, JZL184 (0.001-300 mg), URB602 (0.001-600 mg), 2-AG (ED50), 2-AG + JZL184 (at their ED50), 2-AG + URB602 (at their ED50), AM251 (80 mg), AM251 + JZL184 (10 mg), AM630 (25 mg) or AM630 + JZL184 (10 mg). Effects of MGL inhibitors on endocannabinoid accumulation and on activities of endocannabinoid-metabolizing enzymes were assessed. KEY RESULTSIntra-paw administration of JZL184, URB602 and 2-AG suppressed early and late phases of formalin pain. JZL184 and URB602 acted through a common mechanism. JZL184 (ED50 Phase 1: 0.06 Ϯ 0.028; Phase 2: 0.03 Ϯ 0.011 mg) produced greater antinociception than URB602 (ED50 Phase 1: 120 Ϯ 51.3; Phase 2: 66 Ϯ 23.9 mg) or 2-AG. Both MGL inhibitors produced additive antinociceptive effects when combined with 2-AG. Antinociceptive effects of JZL184, like those of URB602, were blocked by cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) antagonists. JZL184 suppressed MGL but not fatty-acid amide hydrolase or N-arachidonoyl-phosphatidylethanolamine phospholipase D activities ex vivo. URB602 increased hind paw 2-AG without altering anandamide levels. CONCLUSIONS AND IMPLICATIONSMGL inhibitors suppressed formalin-induced pain through peripheral CB1 and CB2 receptor mechanisms. MGL inhibition increased paw skin 2-AG accumulation to mediate these effects. MGL represents a target for the treatment of inflammatory pain.
SUMMARY The endocannabinoid system plays a critical role in the control of energy homeostasis, but the identity and localization of the endocannabinoid signal involved remain unknown. In the present study we developed transgenic mice that over-express in forebrain neurons the presynaptic hydrolase, monoacylglycerol lipase (MGL), which deactivates the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). MGL-overexpressing mice show a 50 percent decrease in forebrain 2-AG levels, but no overt compensation in other endocannabinoid components. This biochemical abnormality is accompanied by a series of metabolic changes that include leanness, elevated energy cost of activity and hypersensitivity to β3-adrenergic-stimulated thermogenesis, which is corrected by reinstating 2-AG activity at CB1-cannabinoid receptors. Additionally, the mutant mice are resistant to diet-induced obesity and express high levels of thermogenic proteins, such as uncoupling protein-1, in their brown adipose tissue. The results suggest that 2-AG signaling through CB1 regulates the activity of forebrain neural circuits involved in the control of energy dissipation.
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