Visualization of 2-arachidonoylglycerol accumulation and cannabinoid CB1 receptor activity in rat brain cryosections by functional autoradiography

In neuronal signalling mediated by the endocannabinoid 2-arachidonoylglycerol, both synthetic and inactivating enzymes operate within close proximity to the G(i/o)-coupled pre-synaptic CB(1) receptors, thus allowing for rapid onset and transient duration of this lipid modulator. In rat brain, 2-arachidonoylglycerol is inactivated mainly via hydrolysis by serine hydrolase inhibitor-sensitive monoacylglycerol lipase activity. We show in this study that comprehensive pharmacological elimination of this activity in brain cryosections by methyl arachidonylfluorophosphonate or hexadecylsulphonyl fluoride results in endocannabinoid-mediated CB(1) receptor activity, which can be visualized by functional autoradiography. URB597, a specific inhibitor of anandamide hydrolysis proved ineffective. TLC indicated that the bioactivity resided in 2-arachidonoylglycerol-containing fraction and gas chromatography-mass spectroscopy detected elevated levels of monoacylglycerols, including 2-arachidonoylglycerol in this fraction. Although two diacylglycerol lipase inhibitors, tetrahydrolipstatin (THL) and RHC80267, blocked the bulk of 2-arachidonoylglycerol accumulation in methyl arachidonylfluorophosphonate-treated sections, only THL reversed the endocannabinoid-dependent CB(1) receptor activity. Further studies indicated that at the used concentrations, THL rather specifically antagonized the CB(1) receptor. These findings confirm that in brain sections there is preservation of enzymatic pathways regulating the production of endogenous receptor ligands. Furthermore, the presently described methodology may serve as an elegant and intuitive approach to identify novel membrane-derived lipid modulators operating in the CNS.     

Phosphatidic acid as the biosynthetic precursor of the endocannabinoid 2-arachidonoylglycerol in intact mouse neuroblastoma cells stimulated with ionomycin

In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2-arachidonoylglycerol (2-AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2-AG. [3H]2-AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N-ethylmaleimide, two inhibitors of the Mg2+/Ca2(+)-dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin-induced [3H]2-AG formation. These data indicate that DAG precursors for 2-AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl-CoA acylase inhibitor thimerosal significantly reduced [3H]2-AG biosynthesis. The implications of these latter findings, and of the PA-dependent pathways of 2-AG formation described here, are discussed.     

The inhibition of arachidonic acid metabolism in human platelets by RHC 80267, a diacylglycerol lipase inhibitor

The diacylglycerol lipase inhibitor, RHC 80267, 1,6-di(O-(carbamoyl)cyclohexanone oxime)hexane, was tested for its ability to block the release of arachidonic acid from human platelets. At a concentration (10 microM) reported to completely inhibit diacylglycerol lipase in fractions of broken platelets, RHC 80267 had no effect on diacylglycerol lipase activity or the release of arachidonic acid from washed human platelets stimulated with collagen. At a high concentration (250 microM), the compound inhibited the formation of arachidonyl-monoacylglycerol by 70% and the release of arachidonate by 60%. However, at this concentration RHC 80267 was found to inhibit cyclooxygenase activity, phospholipase C activity and the hydrolysis of phosphatidylcholine (PC) (presumably by inhibiting phospholipase A2). The phospholipase C inhibition was attributed to the inhibition of prostaglandin H2 formation, as it was alleviated by the addition of the endoperoxide analog, U-46619. PC hydrolysis was only partially restored with U-46619, suggesting that RHC 80267 directly alters phospholipase A2 activity. The inhibition of arachidonate release observed was accounted for by the inhibition of PC hydrolysis. We conclude that RHC 80267, because of its lack of specificity at concentrations needed to inhibit diacylglycerol lipase, is an unsuitable inhibitor for studying the release of arachidonic acid in intact human platelets.     

Effects of RHC 80267, a diglyceride lipase inhibitor, on prolactin secretion and calcium uptake in GH3 pituitary cells

The effect of the diglyceride lipase inhibitor RHC 80267 on the prolactin secretory process was examined in clonal anterior pituitary GH3 cells. This compound reduced basal prolactin secretion as well as secretion induced by TRH and phospholipase C but not that induced by phorbol myristate acetate. Although exogenous phospholipase C increased diglyceride, no increase in the products of diglyceride lipase was detected. Moreover, low doses of RHC 80267 were observed to effectively block potassium-stimulated 45calcium influx. It is unlikely that RHC 80267 inhibits prolactin release solely by inhibiting diglyceride lipase. These data suggest blockade of plasma membrane calcium channels as an alternate mechanism for the inhibitory actions of RHC 80267 on intact GH3 cells. These observations may have implications for RHC 80267 action in other cell types.     

Inhibitors of diacylglycerol lipase and diacylglycerol kinase inhibit carbamylcholine-stimulated responses in guinea pig pancreatic minilobules

We earlier showed that the diacylglycerol (DG) lipase inhibitor, RHC 80267, increased the steady-state level of DG and inhibited the release of arachidonic acid (AA) in carbamylcholine (CCh)-stimulated pancreatic minilobules (J. F. Dixon and L. E. Hokin, (1984) J. Biol. Chem. 259, 14418-14425). There was no effect on phospholipid metabolism. We have now investigated the effect of RHC 80267 on CCh-stimulated formation of inositol monophosphate formation, cGMP formation, and amylase release. CCh (10 microM) increased cGMP formation by approximately 20-fold, and this response was inhibited 55-75% by RHC 80267 (75-100 microM). RHC 80267 had no effect on either nitroprusside- or calcium ionophore-stimulated cGMP formation, arguing against a direct inhibition of guanylate cyclase by RHC 80267. Arachidonic acid, the release of which is inhibited by RHC 80267, neither stimulated cGMP formation nor reversed the effect of RHC 80267 on CCh-stimulated cGMP formation. This suggests, but does not prove, that the rise in cGMP in response to CCh is not due to an increase in AA as has been suggested. Both phorbol myristate acetate (25 nM) and the DG kinase inhibitor R 59022 (10 microM) inhibited CCh-stimulated cGMP formation by 40%. RHC 80267 also inhibited CCh-stimulated inositol phosphate accumulation and amylase release by 60 and 40%, respectively. The data suggest that the inhibition of CCh-stimulated cGMP formation and other muscarinic responses by RHC 80267 is probably the result of feedback inhibition of the cholinergic receptor via activation of protein kinase C by the elevated DG.     

RHC 80267 does not inhibit the diglyceride lipase pathway in intact platelets

RHC 80267 inhibits diglyceride lipase activity in microsomes from canine platelets (1). Chau and Tai (2) reported that RHC 80267 prevents the transient accumulation of monoglyceride in thrombin-stimulated human platelets, while leaving arachidonate release unimpaired. In contrast, we find that while the drug inhibits both diglyceride lipase (I₅₀=15 μM) and monoglyceride lipase (I₅₀=11 μM) activities in platelet microsomes, it is ineffective when added to intact platelets. The transient intermediates in the diglyceride lipase pathway, 1,2-diglyceride and 2-monoglyceride, both accumulated after thrombin stimulation of intact platelets treated with RHC 80267, and arachidonate release was not inhibited. We conclude that RHC 80267 cannot be used to evaluate the diglyceride lipase pathway in intact platelets.     

Diacylglycerol Enrichment of Endoplasmic Reticulum or Lipid Droplets Recruits Perilipin 3/TIP47 during Lipid Storage and Mobilization

Fatty acid-induced triacylglycerol synthesis produces triacylglycerol droplets with a protein coat that includes perilipin 3/TIP47 and perilipin 4/S3-12. This study addresses the following two questions. Where do lipid droplets emerge, and how are their coat proteins recruited? We show that perilipin 3- and perilipin 4-coated lipid droplets emerge along the endoplasmic reticulum (ER). Blocking membrane trafficking with AlF₄⁻ during fatty acid-induced triacylglycerol synthesis drove perilipin 3 to the tubular ER. Forskolin, which like AlF₄⁻ activates adenylate cyclase, did not redistribute perilipin 3, but when added together with AlF₄⁻ perilipin 3 was recruited to lipid droplets rather than the ER. Thus inhibiting trafficking with AlF₄⁻ redistributed perilipin 3 differently under conditions of triacylglycerol synthesis (fatty acid addition) versus hydrolysis (forskolin) suggesting a shared acylglycerol-mediated mechanism. We tested whether diacylglycerol (DG), the immediate precursor of triacylglycerol and its first hydrolytic product, affects the distribution of perilipin 3. Stabilizing DG with the DG lipase inhibitor {"type":"entrez-protein","attrs":{"text":"RHC80267","term_id":"1470879788","term_text":"RHC80267"}}RHC80267 enhanced the perilipin 3 recruited to lipid droplets and raised DG levels in this fraction. Treating cells with a membrane-permeable DG recruited perilipin 3 to the ER. Stabilizing DG, by blocking its hydrolysis with {"type":"entrez-protein","attrs":{"text":"RHC80267","term_id":"1470879788","term_text":"RHC80267"}}RHC80267 or its acylation with triacsin C, enhanced recruitment of perilipin 3 to the ER. Expressing the ER enzyme DGAT1, which removes DG by converting it to triacylglycerol, attenuated perilipin 3 DG-induced ER recruitment. Membrane-permeable DG also drove perilipin 4 and 5 onto the ER. Together the data suggest that these lipid droplet proteins are recruited to DG-enriched membranes thereby linking lipid coat proteins to the metabolic state of the cell.     

Melittin exerts multiple effects on the release of free fatty acids from L1210 cells: lack of selective activation of phospholipase A2 by melittin

Melittin is known as a phospholipase A2 (PLA2) activator, but the selectivity of its effect on PLA2 is uncertain. We examined the selectivity of melittin effect on the release of free fatty acids (FFAs) from L1210 cells using various inhibitors. A systemic lipid analysis by HPLC and GLC revealed that melittin induced release of various FFAs including saturated, monounsaturated, and polyunsaturated FFAs. Various PLA2 inhibitors examined exerted only minimal effects on the melittin-induced arachidonic acid (AA) and palmitic acid (PAL) releases. Specific inhibitors of phosphatidylinositol-phospholipase C (U73122) and diacylglycerol lipase (RHC80267) exerted significant inhibitory effects on both AA and PAL releases. These results suggest that melittin-induced FFA release is most likely due to multiple participations of various types of lipases. Since BAPTA/AM, an intracellular Ca2+ chelator, did not influence the FFA release, the Ca2+ influxed by melittin appeared not to be a key factor for the FFA release. The mimicking of the melittin-induced FFA release by digitonin, a membrane-permeabilizing agent, implies that the membrane-perturbing action of melittin is likely the cause of the FFA release. Melittin also induced release of multiple FFAs from other cell lines including P388D1 and HL60. The rapid melittin-stimulated phospholipase D (PLD) observed in L1210 cells appeared not directly related to the steady release of FFA, as indicated by the fact that the PLD was not blocked by RHC80267. In view of melittin's multiple effects on the composition of cellular lipids, we conclude that melittin does neither exclusively release any single FFA nor selectively activate PLA2 in L1210 cells. The problem of using melittin as a PLA2 activator is discussed.     

Stimulation of inositol phosphate and diacylglycerol production by RHC 80267, a diacylglycerol-lipase inhibitor, in rat gastric parietal cells: effects on hydrogen ion secretion

RHC 80267, on inhibitor of diacylglycerol lipase, was used to investigate the role of diacylglycerol in acid secretion by isolated rat gastric parietal cells. Unexpectedly, RHC 80267 stimulated the production of inositol phosphates in [3H]inositol-prelabeled cells and increased levels of 32P-labeled phosphatidic acid to the same degree as did carbachol. RHC 80267 increased diacylglycerol to a greater extent than did carbachol, and additionally decreased levels of [3H]arachidonic acid. This suggests that RHC 80267 stimulated phospholipase C and inhibited diacylglycerol lipase in parietal cells. RHC inhibited [14C]aminopyrine uptake, a measure of acid secretion, stimulated by carbachol or by simultaneous addition of carbachol and dibutyryl-cAMP. These data support the model that the diacylglycerol/protein kinase C branch of the phosphoinositide system is inhibitory to acid secretion.     

Endothelium-derived 2-arachidonylglycerol: an intermediate in vasodilatory eicosanoid release in bovine coronary arteries

Acetylcholine stimulates the release of endothelium-derived arachidonic acid (AA) metabolites including prostacyclin and epoxyeicosatrienoic acids (EETs), which relax coronary arteries. However, mechanisms of endothelial cell (EC) AA activation remain undefined. We propose that 2-arachidonylglycerol (2-AG) plays an important role in this pathway. An AA metabolite isolated from bovine coronary ECs was identified as 2-AG by mass spectrometry. In ECs pretreated with the fatty acid amidohydrolase inhibitor diazomethylarachidonyl ketone (DAK; 20 micromol/l), methacholine (10 micromol/l)-stimulated 2-AG release was blocked by the phospholipase C inhibitor U-73122 (10 micromol/l) or the diacylglycerol lipase inhibitor RHC-80267 (40 micromol/l). In U-46619-preconstricted bovine coronary arterial rings, 2-AG relaxations averaging 100% at 10 micromol/l were inhibited by endothelium removal, by DAK, by the hydrolase inhibitor methyl arachidonylfluorophosphate (10 micromol/l), by the cyclooxygenase inhibitor indomethacin (10 micromol/l), but not by the CB1 cannabinoid receptor antagonist SR-141716 (1 micromol/l). The cytochrome P-450 inhibitor SKF-525a (10 micromol/l) and the 14,15-epoxyeicosa-5Z-enoic acid EET antagonist (14,15-EEZE; 10 micromol/l) further attenuated the indomethacin-resistant relaxations. The nonhydrolyzable 2-AG analogs noladin ether, 2-AG amide, and 14,15-EET glycerol amide did not induce relaxation. N-nitro-L-arginine-resistant relaxations to methacholine were also inhibited by U-73122, RHC-80267, and DAK. 14,15-EET glycerol ester increased opening of large-conductance K(+) channels 12-fold in cell-attached patches of isolated smooth muscle cells and induced relaxations averaging 95%. These results suggest that methacholine stimulates EC 2-AG production through phospholipase C and diacylglycerol lipase activation. 2-AG is further hydrolyzed to AA, which is metabolized to vasoactive eicosanoids. These studies reveal a role for 2-AG in EC AA release and the regulation of coronary tone.     

Activation of the endocannabinoid system by organophosphorus nerve agents

Delta(9)-tetrahydrocannabinol (THC), the psychoactive ingredient of marijuana, has useful medicinal properties but also undesirable side effects. The brain receptor for THC, CB(1), is also activated by the endogenous cannabinoids anandamide and 2-arachidonylglycerol (2-AG). Augmentation of endocannabinoid signaling by blockade of their metabolism may offer a more selective pharmacological approach compared with CB(1) agonists. Consistent with this premise, inhibitors of the anandamide-degrading enzyme fatty acid amide hydrolase (FAAH) produce analgesic and anxiolytic effects without cognitive defects. In contrast, we show that dual blockade of the endocannabinoid-degrading enzymes monoacylglycerol lipase (MAGL) and FAAH by selected organophosphorus agents leads to greater than ten-fold elevations in brain levels of both 2-AG and anandamide and to robust CB(1)-dependent behavioral effects that mirror those observed with CB(1) agonists. Arachidonic acid levels are decreased by the organophosphorus agents in amounts equivalent to elevations in 2-AG, which indicates that endocannabinoid and eicosanoid signaling pathways may be coordinately regulated in the brain.     

RHC 80267 inhibits thyrothopin-stimulated prostaglandin release from rat thyroid lobes

In the present report, we studied the effect of the diglyceride (DG) lipase inhibitor, RHC 80267 on basal and thyrotropin (TSH) - stimulated prostaglandin (PG) release from rat thyroid lobes Further, we tested the effect of RHC 80267 on phosphatidylinositol phospholipase C (PIPLC), DG lipase, and arachidonate cyclo-oxygenase acdtivities in rat thyroid cytosol, plasma membrane, and whole homogenate preparations, r espectively. Whereas RHC 80267 inhibited DG lipase activity in a dose - re;ated manner from 0.5 – 10 μM (17 – 80% inhibition), it failed either PIPLC or arachidonate cyclo-oxygenase activities by more than 9% when tested at 5 and 10 μM (n = 3). RHC 80267 reduced TSH-stimulated 6-keto-PGF_1α and PGE_2α relase by 100 ± 14% and 57 ± 12%, respectively 9$\text{x}\text{+}\text{S.E.}\text{;}\text{p}\text{< 0.01}$ for both; n = 10 – 12; the diglyceride lipase inhibitor did not reduce basal release of either PG. These data provide additional evidence which implicate a PIPLC - DG lipase pathway in TSH-stimulated PG synthesis in thyroid.     

Cytochrome P-450 metabolites of 2-arachidonoylglycerol play a role in Ca2+-induced relaxation of rat mesenteric arteries

The perivascular sensory nerve (PvN) Ca(2+)-sensing receptor (CaR) is implicated in Ca(2+)-induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries, which involves the vascular endogenous cannabinoid system. We determined the effect of inhibition of diacylglycerol (DAG) lipase (DAGL), phospholipase A(2) (PLA(2)), and cytochrome P-450 (CYP) on Ca(2+)-induced relaxation of PE-contracted rat mesenteric arteries. Our findings indicate that Ca(2+)-induced vasorelaxation is not dependent on the endothelium. The DAGL inhibitor RHC 802675 (1 microM) and the CYP and PLA(2) inhibitors quinacrine (5 microM) (EC(50): RHC 802675 2.8 +/- 0.4 mM vs. control 1.4 +/- 0.3 mM; quinacrine 4.8 +/- 0.4 mM vs. control 2.0 +/- 0.3 mM; n = 5) and arachidonyltrifluoromethyl ketone (AACOCF(3), 1 microM) reduced Ca(2+)-induced relaxation of mesenteric arteries. Synthetic 2-arachidonoylglycerol (2-AG) and glycerated epoxyeicosatrienoic acids (GEETs) induced concentration-dependent relaxation of isolated arteries. 2-AG relaxations were blocked by iberiotoxin (IBTX) (EC(50): control 0.96 +/- 0.14 nM, IBTX 1.3 +/- 0.5 microM) and miconazole (48 +/- 3%), and 11,12-GEET responses were blocked by IBTX (EC(50): control 55 +/- 9 nM, IBTX 690 +/- 96 nM) and SR-141716A. The data suggest that activation of the CaR in the PvN network by Ca(2+) leads to synthesis and/or release of metabolites of the CYP epoxygenase pathway and metabolism of DAG to 2-AG and subsequently to GEETs. The findings indicate a role for 2-AG and its metabolites in Ca(2+)-induced relaxation of resistance arteries; therefore this receptor may be a potential target for the development of new vasodilator compounds for antihypertensive therapy.     

Anandamide and 2-arachidonoylglycerol: Pharmacological Properties, Functional Features, and Emerging Specificities of the Two Major Endocannabinoids

Since the discovery of endocannabinoids and their receptors, two major members of the endocannabinoid family, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), have been regarded almost as twin brothers. Pharmacological properties were initially considered to be similar, as these molecules were believed mutually exchangeable and almost indistinguishable in the regulation of synaptic functions, such as long- and short-term synaptic plasticity, and in behavioral aspects, such as learning and memory, reward and addiction, antinociception, and anxiety. In recent years, however, endocannabinoid signaling specificity began to emerge, in particular, due to the production of genetically engineered mice lacking key enzymes in endocannabinoid synthesis or degradation, together with the development of selective inhibitors of AEA or 2-AG catabolic enzymes. Evidence now suggests that AEA and 2-AG possess specific pharmacological properties, are engaged in different forms of synaptic plasticity, and take part in different behavioral functions. In this review, we provide an overview on similarities and specificities of the two endocannabinoids in the CNS and on the unresolved questions concerning their role in synaptic signaling.     

Diacylglycerol lipase and pituitary prolactin release in vitro: studies employing RHC 80267

We studied the possible involvement of diacylglycerol lipase in the regulatory mechanisms governing the release of prolactin by primary cultures of anterior pituitary cells. This was accomplished by studying the effect of a selective inhibitor of diacylglycerol lipase activity, RHC 80267, on basal prolactin release and that stimulated by TRH and elevated potassium concentrations. RHC 80267 produced a concentration-dependent reduction in basal prolactin release and abolished its increase produced by TRH and potassium. These results are consistent with the hypothesis that the production of arachidonate from lipids via the diacylglycerol lipase pathway is an important event in the governance of prolactin release.     

The effect of inhibition of both diacylglycerol metabolism and phospholipase A2 activity on superoxide generation by human neutrophils

A 'cocktail' consisting of an inhibitor of diacylglycerol kinase (R59022, 10 microM), an inhibitor of diacylglycerol lipase (RHC80267, 10 microM), and an inhibitor of phospholipase A2 (either 100 microM indomethacin, or 100 microM sodium meclofenamate) markedly enhanced superoxide production by human neutrophils stimulated with post-receptor stimuli, fluoride and gamma-hexachlorocyclohexane. On the other hand, the response to the C3b/Fc receptor stimulus, opsonized zymosan, was marginally decreased whilst that to the Fc receptor stimulus, aggregated IgG, was virtually unaffected. Since the inhibitors used are deemed to inhibit the main routes of arachidonate production, these results call into question the role of arachidonate in the transduction of O2- generation by post-receptor stimuli, but support a role for arachidonate in receptor-mediated transduction.     

13-Methylarachidonic Acid Is a Positive Allosteric Modulator of Endocannabinoid Oxygenation by Cyclooxygenase

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing k_cat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism.     

Development of the first potent and specific inhibitors of endocannabinoid biosynthesis

Enzymes for the biosynthesis and degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG) have been cloned and are the sn-1-selective-diacylglycerol lipases alpha and beta (DAGLalpha and beta) and the monoacylglycerol lipase (MAGL), respectively. Here, we used membranes from COS cells over-expressing recombinant human DAGLalpha to screen new synthetic substances as DAGLalpha inhibitors, and cytosolic fractions from wild-type COS cells to look for MAGL inhibitors. DAGLalpha and MAGL activities were assessed by using sn-1-[14C]-oleoyl-2-arachidonoyl-glycerol and 2-[3H]-arachidonoylglycerol as substrates, respectively. We screened known compounds as well as new phosphonate derivatives of oleic acid and fluoro-phosphinoyl esters of different length. Apart from the general lipase inhibitor tetrahydrolipstatin (orlistat) (IC50 approximately 60 nM), the most potent inhibitors of DAGLalpha were O-3640 [octadec-9-enoic acid-1-(fluoro-methyl-phosphoryloxymethyl)-propylester] (IC50 = 500 nM), and O-3841 [octadec-9-enoic acid 1-methoxymethyl-2-(fluoro-methyl-phosphinoyloxy)-ethyl ester] (IC50 = 160 nM). Apart from being almost inactive on MAGL, these two compounds showed high selectivity over rat liver triacylglycerol lipase, rat N-acylphosphatidyl-ethanolamine-selective phospholipase D (involved in anandamide biosynthesis), rat fatty acid amide hydrolase and human recombinant cannabinoid CB1 and CB2 receptors. Methylarachidonoyl-fluorophosphonate and the novel compound UP-101 [O-ethyl-O-p-nitro-phenyl oleylphosphonate] inhibited both DAGLalpha and MAGL with similar potencies (IC50 = 0.8-0.1 and 3.7-3.2 microM, respectively). Thus, we report the first potent and specific inhibitors of the biosynthesis of 2-AG that may be used as pharmacological tools to investigate the biological role of this endocannabinoid.     

Impact of tetrahydrocannabinol on the endocannabinoid 2-arachidonoylglycerol metabolism: ABHD6 and ABHD12 as novel players in human placenta

Cannabis use has been increasing worldwide for recreational and medical purposes. Consumption by pregnant women is associated with disturbances in pregnancy outcome, such as low birth weight, prematurity and intrauterine growth retardation, though the underlying biochemical mechanisms are unknown. The endocannabinoid system is involved in several reproductive events and the disruption of its homeostasis by ∆⁹-tetrahydrocannabinol (THC), the main psychoactive cannabinoid, may lead to a negative gestational outcome. In human placenta, THC impairs the levels of the endocannabinoid anandamide (AEA). The other major endocannabinoid, 2-arachidonoylglycerol (2-AG) also plays an important role on proper placentation and pregnancy success. However, THC impact on 2-AG homeostasis has never been addressed. Hence, the effects of THC in 2-AG levels and metabolic enzymes expression were explored. Long-term treatment impairs the expression of the main 2-AG synthetic and degradative enzymes. Curiously, with the highest concentration, despite the maintenance of diacylglycerol lipase alpha (DAGLα) and the decrease in monoacylglycerol lipase (MAGL) expression, 2-AG levels remain constant. Given the endocannabinoid signalling local tight regulation, we hypothesize the involvement of other 2-AG degradative enzymes. Indeed, THC increases the expression of the hydrolyzing enzymes alpha beta hydrolase domain-6 (ABHD6) and -12 (ABHD12), that we firstly describe in human placental tissues. The results show that THC, depending on time of exposure, induces alterations in 2-AG metabolic enzymes expression in placental explants, highlighting the importance of 2-AG regulation and endocannabinoid signalling in placental development. Alterations in this homeostasis may explain the negative pregnancy outcome related to cannabis consumption.     

Characterization of the lipolytic pathways that mediate free fatty acid release during Fas/CD95-induced apoptosis

We have undertaken a study to characterize the lipolytic pathway responsible for the generation of free fatty acids (FFA) during Fas/CD95-induced apoptosis in Jurkat cells. It was initially shown that the cellular lipid fraction that suffered the major quantitative decrease during Fas-induced apoptosis was that of phosphatidylcholine (PC). In addition, the secretion of palmitic acid-derived FFA was largely prevented by D609, an inhibitor of PC-specific phospholipase C (PC-PLC) and also by the diacylglycerol lipase (DAGL) inhibitor RHC-80267, suggesting that the secretion of these FFA during Fas-induced apoptosis is mediated by the generation of DAG by a PC-PLC activity and, sequentially, by a 1-DAGL activity which generates the FFA from its sn-1 position. The endocannabinoid 2-arachidonoyl glycerol (2-AG) should be generated as a sub-product of this pathway, but it did not accumulate inside the cells nor was secreted into the supernatant. Interestingly, the complete inhibition of free AA secretion during Fas-induced apoptosis was only achieved by using the AA trifluoromethylketone, which not only inhibits all types of phospholipase-A₂ (PLA₂) activities, but also the described lytic activities on 2-AG. Using a combination of RHC-80267 and the iPLA₂-specific inhibitor bromoenol lactone, it was shown that the DAGL pathway also cooperates with iPLA₂ in the generation of free arachidonate.