No abstract

Anandamide (arachidonoylethanolamide, AnNH) and palmitoylethanolamide (PEA) have been proposed as the physiological ligands, respectively, of central and peripheral cannabinoid receptors. Both of these receptors are expressed in immune cells, including macrophages and mast cells/basophils, where immunomodulatory and/or anti-inflammatory actions of AnNH and PEA have been recently reported. We now provide biochemical grounds to these actions by showing that the biosynthesis, uptake, and degradation of AnNH and PEA occur in leukocytes. On stimulation with ionomycin, J774 macrophages and RBL-2H3 basophils produced AnNH and PEA, probably through the hydrolysis of the corresponding N-acylphosphatidylethanolamines, also found among endogenous phospholipids. Immunological challenge of RBL-2H3 cells also caused AnNH and PEA release. The chemical structure and the amounts of AnNH and PEA produced upon ionomycin stimulation were determined by means of double radiolabeling experiments and isotope dilution gas chromatography/ electron impact mass spectrometry. Both cell lines rapidly sequestered the two amides from the culture medium through temperature-dependent, saturable and chemically inactivable mechanisms. Once uptaken by basophils, AnNH and PEA compete for the same inactivating enzyme which catalyzes their hydrolysis to ethanolamine. This enzyme was found in both microsomal and 10,000 ؋ g fractions of RBL cell homogenates, and exhibited similar inhibition and temperature/pH dependence profiles but a significantly higher affinity for PEA with respect to neuronal "anandamide amidohydrolase." The finding of biosynthetic and inactivating mechanisms for AnNH and PEA in macrophages and basophils supports the previously proposed role as local modulators of immune/inflammatory reactions for these two long chain acylethanolamides.In the past 15 years evidence has been accumulating to support the existence of a neuroimmune axis. The presence of neuropeptide receptors in immune cells allows them to respond to peptidergic stimulation with decreased or augmented proliferation, chemotaxis, phagocytosis, degranulation, lymphokine and cytokine release, superoxide radical formation, and leukotriene biosynthesis (see Refs. 1 and 2, for reviews). The concomitant action on vascular permeability of neuropeptides like substance P, calcitonin gene-related peptide, and neurokinins, concurs to the onset of immune/inflammatory reactions, as in the "axon-reflex" model for neurogenic inflammation. These reactions can be modulated through feed-back actions on autonomic and sensorial fibers by mediators and neurotransmitters produced by immune cells (3, 4). Some circulating leukocytes can, in fact, synthesize, store, and release neuropeptides such as vasoactive intestinal peptide, endorphins, and substance P (1, 4), and the recent finding of nerve growth factor in mast cells (5) widens the spectrum of potential responses that backfed peripheral neurons can produce during neuroimmune interactions. Among the receptor classes whose expression and fun...

ABSTRACT

The stimulus-induced biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in intact mouse J774 macrophages and the inactivation of 2-AG by the same cells or by rat circulating macrophages was studied. By using gas chromatography-mass spectrometry, we found that ionomycin (5 mm) and lipopolysaccharide (LPS, 200 mg´mL 21 ) cause a 24-fold and 2.5-fold stimulation of 2-AG levels in J774 cells, respectively, thus providing unprecedented evidence that this cannabimimetic metabolite can be synthesized by macrophages. In J774 cells, LPS also induced a 7.8-fold increase of the levels of the other endocannabinoid, anandamide, and, in rat circulating macrophages, an almost twofold increase of 2-AG levels. Extracellular [ 3 H]2-AG was cleared from the medium of intact J774 macrophages (t 1/2 = 19±28 min) and esterified to phospholipids, diacylglycerols and triglycerides or hydrolyzed to [ 3 H]arachidonic acid and glycerol. These catabolic processes were attenuated differentially by various enzyme inhibitors. Rat circulating macrophages were shown to contain enzymatic activities for the hydrolysis of 2-AG, including: (a) fatty acid amide hydrolase (FAAH), the enzyme responsible for anandamide breakdown and previously shown to catalyse also 2-AG hydrolysis, and (b) a 2-AG hydrolase activity different from FAAH and down-regulated by LPS. High levels of FAAH mRNA were found in circulating macrophages but not platelets, which, however, contain a 2-AG hydrolase. Both platelets and macrophages were shown to express the mRNA for the CB1 cannabinoid receptor. A macrophage 2-AG hydrolase with apparent K m = 110 mm and V max = 7.9 nmol´min 21´( mg protein) 21 was partially characterized in J774 cells and found to exhibit an optimal pH of 6±7 and little or no sensitivity to typical FAAH inhibitors. These findings demonstrate for the first time that macrophages participate in the homeostasis of the hypotensive and immunomodulatory endocannabinoid 2-AG through metabolic mechanisms that are subject to regulation.Keywords: cannabinoids; lipopolysaccharide; anandamide; monoacylglycerol lipase; FAAH.The discovery in 1992 [1] of anandamide (N-arachidonoylethanolamine), the first`endocannabinoid' (i.e. an endogenous ligand of cannabinoid receptors) was followed three years later by the finding that a well known intermediate in phosphoglyceride metabolism, 2-arachidonoylglycerol (2-AG), could also act as a potential agonist at cannabinoid receptors [2±4]. Pharmacological studies carried out on synthetic 2-AG revealed for this compound a series of cannabimimetic properties both in vivo [2] and in vitro [3]. In particular, it was found that 2-AG, like (±)-D 9 -tetrahydrocannabinol, induces inhibition of locomotor activity, catalepsy, hypothermia and analgesia in mice [2], inhibits the forskolin-induced stimulation of cAMP formation [3], and modulates Ca 2+ currents in NG108±15 hybrid cells [5]. Recently, cannabinoid receptor-mediated biological activities were found for 2-AG also in peripheral tissues. These are the inhibition of mo...

The monoacylglycerol 2-arachidonoylglycerol (2-AG) has been recently suggested as a possible endogenous agonist at cannabinoid receptors both in brain and peripheral tissues. Here we report that a widely used model for neuronal cells, mouse N18TG2 neuroblastoma cells, which contain the CB1 cannabinoid receptor, also biosynthesize, release and degrade 2-AG. Stimulation with ionomycin (1-5 microM) of intact cells prelabelled with [3H]arachidonic acid ([3H]AA) led to the formation of high levels of a radioactive component with the same chromatographic behaviour as synthetic standards of 2-AG in TLC and HPLC analyses. The amounts of this metabolite were negligible in unstimulated cells, and greatly decreased in cells stimulated in the presence of the Ca2+-chelating agent EGTA. The purified component was further characterized as 2-AG by: (1) digestion with Rhizopus arrhizus lipase, which yielded radiolabelled AA; (2) gas chromatographic-MS analyses; and (3) TLC analyses on borate-impregnated plates. Approx. 20% of the 2-AG produced by stimulated cells was found to be released into the incubation medium when this contained 0.1% BSA. Subcellular fractions of N18TG2 cells were shown to contain enzymic activity or activities catalysing the hydrolysis of synthetic [3H]2-AG to [3H]AA. Cell homogenates were also found to convert synthetic [3H]sn-1-acyl-2-arachidonoylglycerols (AcAGs) into [3H]2-AG, suggesting that 2-AG might be derived from AcAG hydrolysis. When compared with ionomycin stimulation, treatment of cells with exogenous phospholipase C, but not with phospholipase D or A2, led to a much higher formation of 2-AG and AcAGs. However, treatment of cells with phospholipase A2 10 min before ionomycin stimulation caused a 2.5-3-fold potentiation of 2-AG and AcAG levels with respect to ionomycin alone, whereas preincubation with the phospholipase C inhibitor neomycin sulphate did not inhibit the effect of ionomycin on 2-AG and AcAG levels. These results suggest that the Ca2+-induced formation of 2-AG proceeds through the intermediacy of AcAGs but not necessarily through phospholipase C activation. By showing for the first time the existence of molecular mechanisms for the inactivation and the Ca2+-dependent biosynthesis and release of 2-AG in neuronal cells, the present paper supports the hypothesis that this cannabimimetic monoacylglycerol might be a physiological neuromodulator.