Cannabinoid receptors, the molecular targets of the cannabis constituent Delta9-tetrahydrocannabinol, are present throughout the body and are normally bound by a family of endogenous lipids - the endocannabinoids. Release of endocannabinoids is stimulated in a receptor-dependent manner by neurotransmitters and requires the enzymatic cleavage of phospholipid precursors present in the membranes of neurons and other cells. Once released, the endocannabinoids activate cannabinoid receptors on nearby cells and are rapidly inactivated by transport and subsequent enzymatic hydrolysis. These compounds might act near their site of synthesis to serve a variety of regulatory functions, some of which are now beginning to be understood. Recent advances in the biochemistry and pharmacology of the endocannabinoid system in relation to the opportunities that this system offers for the development of novel therapeutic agents will be discussed.

The endogenous fatty acid ethanolamide, palmitylethanolamide, alleviated, in a dose-dependent manner, pain behaviors elicited in mice by injections of formalin (5%, intraplantar), acetic acid (0.6%, 0.5 ml per animal, intraperitoneal, i.p.), kaolin (2.5 mg per animal, i.p.), and magnesium sulfate (120 mg per kg, i.p.). The antinociceptive effects of palmitylethanolamide were prevented by the cannabinoid CB2 receptor antagonist SR144528 [N-([1s]-endo-1.3.3-trimethylbicyclo[2.3.1]heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide], not by the cannabinoid CB1 receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide x HCl]. By contrast, palmitylethanolamide had no effect on capsaicin-evoked pain behavior or thermal nociception. The endogenous cannabinoid, anandamide (arachidonylethanolamide), alleviated nociception in all tests (formalin, acetic acid, kaolin, magnesium sulfate, capsaicin and hot plate). These effects were prevented by the cannabinoid CB1 receptor antagonist SR141716A, not the cannabinoid CB2 receptor antagonist SR141716A. Additional fatty acid ethanolamides (oleylethanolamide, myristylethanolamide, palmitoleylethanolamide, palmitelaidylethanolamide) had little or no effect on formalin-evoked pain behavior, and were not investigated in other pain models. These results support the hypothesis that endogenous palmitylethanolamide participates in the intrinsic control of pain initiation. They also suggest that the putative receptor site activated by palmitylethanolamide may provide a novel target for peripherally acting analgesic drugs.

We describe some properties on an Mr 30,000 thermolabile and trypsin-sensitive protein that activates phospholipase A2 (PLA2) and which was isolated from nervous tissue of the marine mollusk, Aplysia californica. A similar protein is present in rat cerebral cortex. This protein was partially purified from crude homogenates of nervous tissue by ion exchange chromatography on DEAE-Sephadex followed by size-exclusion high performance liquid chromatography (HPLC). It is loosely associated with membrane fractions, and is extracted by 0.05% Tween 20. Although similar in size to several previously described PLA2-stimulating proteins from non-neural mammalian cells and tissues, it differs from them in some aspects of biological activity. The protein promotes the release of eicosanoids from the membranes of intact Aplysia neurons prelabeled with [3H]arachidonic acid and appears to be an in vitro substrate for protein kinase C (PKC). PLA2-stimulating activity is greatly enhanced after exposing isolated ganglia to phorbol dibutyrate (PDBu) and is reduced by treatment with immobilized E. coli alkaline phosphatase. These observations suggest that phosphorylation of this stimulatory protein by PKC regulates PLA2 in neurons.

The potent analgesic effects of cannabis-like drugs and the presence of CB1-type cannabinoid receptors in pain-processing areas of the brain and spinal cord indicate that endogenous cannabinoids such as anandamide may contribute to the control of pain transmission within the central nervous system (CNS). Here we show that anandamide attenuates the pain behaviour produced by chemical damage to cutaneous tissue by interacting with CB1-like cannabinoid receptors located outside the CNS. Palmitylethanolamide (PEA), which is released together with anandamide from a common phospholipid precursor, exerts a similar effect by activating peripheral CB2-like receptors. When administered together, the two compounds act synergistically, reducing pain responses 100-fold more potently than does each compound alone. Gas-chromatography/mass-spectrometry measurements indicate that the levels of anandamide and PEA in the skin are enough to cause a tonic activation of local cannabinoid receptors. In agreement with this possibility, the CB1 antagonist SR141716A and the CB2 antagonist SR144528 prolong and enhance the pain behaviour produced by tissue damage. These results indicate that peripheral CB1-like and CB2-like receptors participate in the intrinsic control of pain initiation and that locally generated anandamide and PEA may mediate this effect.

The putative endogenous cannabinoid, anandamide (0.2-2 mg/kg i.v.), decreased systemic blood pressure dose-dependently in anesthesized guinea pigs. These effects were prevented by the CB1 cannabinoid receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide x HCl] at the dose of 0.2 mg/kg i.v. The vasodepressor responses to anandamide were significantly potentiated and prolonged by a novel inhibitor of carrier-mediated anandamide transport, N-(4-hydroxyphenyl) arachidonylethanolamide (AM404) (10 mg/kg, i.v.). These results suggest that anandamide transport participates in terminating the vascular actions of anandamide.

Palmitoylethanolamide (PEA), the naturally occurring amide of palmitic acid and ethanolamine, reduces pain and inflammation through an as-yet-uncharacterized mechanism. Here, we identify the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPAR-alpha) as the molecular target responsible for the anti-inflammatory properties of PEA. PEA selectively activates PPAR-alpha in vitro with an EC(50) value of 3.1 +/- 0.4 microM and induces the expression of PPAR-alpha mRNA when applied topically to mouse skin. In two animal models, carrageenan-induced paw edema and phorbol ester-induced ear edema, PEA attenuates inflammation in wild-type mice but has no effect in mice deficient in PPAR-alpha. The natural PPAR-alpha agonist oleoylethanolamide (OEA) and the synthetic PPAR-alpha agonists GW7647 and Wy-14643 mimic these effects in a PPAR-alpha-dependent manner. These findings indicate that PPAR-alpha mediates the anti-inflammatory effects of PEA and suggest that this fatty-acid ethanolamide may serve, like its analog OEA, as an endogenous ligand of PPAR-alpha.

Palmitoylethanolamide (PEA), the naturally occurring amide of ethanolamine and palmitic acid, is an endogenous lipid that modulates pain and inflammation. Although the anti-inflammatory effects of PEA were first characterized nearly 50 years ago, the identity of the receptor mediating these actions has long remained elusive. We recently identified the ligand-activated transcription factor, peroxisome proliferator-activated receptor-alpha (PPAR-alpha), as the receptor mediating the anti-inflammatory actions of this lipid amide. Here we outline the history of PEA, starting with its initial discovery in the 1950s, and discuss the pharmacological properties of this compound, particularly in regards to its ability to activate PPAR-alpha.

The analgesic properties of cannabinoid receptor agonists are well characterized. However, numerous side effects limit the therapeutic potential of these agents. Here we report a synergistic antinociceptive interaction between the endogenous cannabinoid receptor agonist anandamide and the synthetic peroxisome proliferator-activated receptor-alpha (PPAR-alpha) agonist 2-(4-(2-(1-Cyclohexanebutyl)-3-cyclohexylureido)ethyl)phenylthio)-2-methylpropionic acid (GW7647) in a model of acute chemical-induced pain. Moreover, we show that anandamide synergistically interacts with the large-conductance potassium channel (KCa1.1, BK) activator isopimaric acid. These findings reveal a synergistic interaction between the endocannabinoid and PPAR-alpha systems that might be exploited clinically and identify a new pharmacological effect of the BK channel activator isopimaric acid.

Activation of peroxisome proliferator-activated receptor (PPAR)-alpha, a member of the nuclear receptor superfamily, modulates inflammation and tissue injury events associated with spinal cord trauma in mice. Palmitoylethanolamide (PEA), the naturally occurring amide of palmitic acid and ethanolamine, reduces pain and inflammation through a mechanism dependent on PPAR-alpha activation. The aim of the present study was to evaluate the effect of the PEA on secondary damage induced by experimental spinal cord injury (SCI) in mice. SCI was induced by application of vascular clips to the dura mater via a four-level T(5)-T(8) laminectomy. This resulted in severe trauma characterized by edema, neutrophil infiltration, and production of inflammatory mediators, tissue damage, and apoptosis. Repeated PEA administration (10 mg/kg i.p.; 30 min before and 1 and 6 h after SCI) significantly reduced: 1) the degree of spinal cord inflammation and tissue injury, 2) neutrophil infiltration, 3) nitrotyrosine formation, 4) proinflammatory cytokine expression, 5) nuclear transcription factor activation-kappaB activation, 6) inducible nitric-oxide synthase expression, and 6) apoptosis. Moreover, PEA treatment significantly ameliorated the recovery of motor limb function. Together, the results indicate that PEA reduces inflammation and tissue injury associated with SCI and suggest a regulatory role for endogenous PPAR-alpha signaling in the inflammatory response associated with spinal cord trauma.

The endocannabinoids anandamide and 2-arachidonoyglycerol (2-AG) may contribute to the regulation of mood and emotion. In this study, we investigated the impact of the endocannabinoid transport inhibitor AM404 on three rat models of anxiety: elevated plus maze, defensive withdrawal and separation-induced ultrasonic vocalizations. AM404 (1-5 mg kg(-1), intraperitoneal (i.p.)) exerted dose-dependent anxiolytic-like effects in the three models. These behavioral effects were associated with increased levels of anandamide, but not 2-AG, in the prefrontal cortex and were prevented by the CB(1) cannabinoid antagonist rimonabant (SR141716A), suggesting that they were dependent on anandamide-mediated activation of CB(1) cannabinoid receptors. We also evaluated whether AM404 might influence motivation (in the conditioned place preference (CPP) test), sensory reactivity (acoustic startle reflex) and sensorimotor gating (prepulse inhibition (PPI) of the startle reflex). In the CPP test, AM404 (1.25-10 mg kg(-1), i.p.) elicited rewarding effects in rats housed under enriched conditions, but not in rats kept in standard cages. Moreover, AM404 did not alter reactivity to sensory stimuli or cause overt perceptual distortion, as suggested by its lack of effect on startle or PPI of startle. These results support a role of anandamide in the regulation of emotion and point to the anandamide transport system as a potential target for anxiolytic drugs.