Biomedical subjects

T Bisogno

Publications and source records attributed to T Bisogno.

At least 55 records · Page 3Linked to original sources

PubMed2000

Endocannabinoids: new targets for drug development.

The possible therapeutic use of marijuana s active principles, the cannabinoids, is currently being debated. It is now known that these substances exert several of their pharmacological actions by activating specific cell membrane receptors, the CB1 and CB2 cannabinoid receptor subtypes. This knowledge led to the design of synthetic cannabinoid agonists and antagonists with high therapeutic potential. The recent discovery of the endocannabinoids, i.e. endogenous metabolites capable of activating the cannabinoid receptors, and the understanding of the molecular mechanisms leading to their biosynthesis and inactivation, opened a new era in research on the pharmaceutical applications of cannabinoids. Ongoing studies on the pathological and physiological conditions regulating the tissue levels of endocannabinoids, and on the pharmacological activity of these compounds and their derivatives, may provide a lead for the development of new drugs for the treatment of nervous and immune disorders, cardiovascular diseases, pain, inflammation and cancer. These studies are reviewed in this article with special emphasis on the chemical features that determine the interaction of endocannabinoids with the proteins mediating their activity and degradation.

Animals↗

PubMed1999

Biosynthesis and inactivation of N-arachidonoylethanolamine (anandamide) and N-docosahexaenoylethanolamine in bovine retina.

N-Arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), the two proposed endogenous agonists of cannabinoid receptors, and the putative AEA biosynthetic precursor, N-arachidonoylphosphatidylethanolamine (NArPE), were identified in bovine retina by means of gas chromatography-electron impact mass spectrometry (GC-EIMS). This technique also allowed us to identify N-docosahexanoylethanolamine (DHEA) and 2-docosahexanoylglycerol (2-DHG), two derivatives of docosahexaenoic acid (DHA), one of the most abundant fatty acids esterified in retina phospholipids and necessary for optimal retinal function. N-Docosahexaenoylphosphatidylethanolamine (NDHPE), the potential biosynthetic precursor for DHEA, was also found. The fatty acid composition of the sn-1 and sn-2 positions of bovine retina's most abundant phospholipid classes, also determined here, were in agreement with a phospholipid-dependent mechanism for 2-AG, 2-DHG, AEA, and DHEA biosynthesis, as very high levels of polyunsaturated fatty acids, including DHA, were found on the sn-2 position of phosphatidylcholine (PC) and -ethanolamine (PE), and measurable amounts of di-docosahexanoyl-PC and -PE, two potential biosynthetic precursors of NDHPE, were detected. Accordingly, we found that isolated particulate fractions from bovine retina could release AEA and DHEA in a time-dependent fashion. Finally, a fatty acid amide hydrolase (FAAH)-like activity with subcellular distribution and pH dependency similar to those reported for the brain enzyme was also detected in bovine retina. This activity was inhibited by FAAH inhibitors, phenylmethylsulfonyl fluoride and arachidonoyltrifluoromethylketone, and appeared to recognize DHEA with a lower efficiency than AEA. These data indicate that AEA and its congeners may play a physiological role in the mammalian eye.

Amides↗

PubMed1999

Unsaturated long-chain N-acyl-vanillyl-amides (N-AVAMs): vanilloid receptor ligands that inhibit anandamide-facilitated transport and bind to CB1 cannabinoid receptors.

We investigated the effect of changing the length and degree of unsaturation of the fatty acyl chain of N-(3-methoxy-4-hydroxy)-benzyl-cis-9-octadecenoamide (olvanil), a ligand of vanilloid receptors, on its capability to: (i) inhibit anandamide-facilitated transport into cells and enzymatic hydrolysis, (ii) bind to CB1 and CB2 cannabinoid receptors, and (iii) activate the VR1 vanilloid receptor. Potent inhibition of [(14)C]anandamide accumulation into cells was achieved with C20:4 n-6, C18:3 n-6 and n-3, and C18:2 n-6 N-acyl-vanillyl-amides (N-AVAMs). The saturated analogues and Delta(9)-trans-olvanil were inactive. Activity in CB1 binding assays increased when increasing the number of cis-double bonds in a n-6 fatty acyl chain and, in saturated N-AVAMs, was not greatly sensitive to decreasing the chain length. The C20:4 n-6 analogue (arvanil) was a potent inhibitor of anandamide accumulation (IC(50) = 3.6 microM) and was 4-fold more potent than anandamide on CB1 receptors (Ki = 0.25-0.52 microM), whereas the C18:3 n-3 N-AVAM was more selective than arvanil for the uptake (IC(50) = 8.0 microM) vs CB1 receptors (Ki = 3.4 microM). None of the compounds efficiently inhibited [(14)C]anandamide hydrolysis or bound to CB2 receptors. All N-AVAMs activated the cation currents coupled to VR1 receptors overexpressed in Xenopus oocytes. In a simple, intact cell model of both vanilloid- and anandamide-like activity, i.e., the inhibition of human breast cancer cell (HBCC) proliferation, arvanil was shown to behave as a "hybrid" activator of cannabinoid and vanilloid receptors.

Amidohydrolases↗

PubMed1999

Brain regional distribution of endocannabinoids: implications for their biosynthesis and biological function.

The amounts, in nine different rat brain regions, of the two endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoylglycerol (2-AG), and of the putative AEA precursor N-arachidonoyl-phosphatidylethanolamine (NArPE), were determined by isotope-dilution gas chromatography-mass spectrometry and compared to the number of cannabinoid binding sites in each region. The distribution of NArPE, reported here for the first time, exhibited a good correlation with that of AEA, the former metabolite being 3-13 times more abundant than the endocannabinoid in all regions. The highest amounts of both metabolites (up to 358.5 and 87 pmol/g wet weight tissue, respectively) were found in the brainstem and striatum, and the lowest in the diencephalon, cortex, and cerebellum. These data support the hypothesis that, in the brain, AEA is a metabolic product of NArPE and may reach levels compatible with its proposed neuromodulatory function. The brain distribution of 2-AG, also described in this study for the first time, was found to correlate with that of AEA with levels ranging from 2.0 to 14.0 nmol/g (in the diencephalon and brainstem, respectively). The distribution of the endocannabinoids did not match exactly with that of cannabinoid binding sites, suggesting either that these compounds are not necessarily produced near their molecular targets, or that they play functional roles additional to the activation of cannabinoid receptors. Regional differences in the ligand/receptor ratios may also lead to predict corresponding differences in the efficiency of receptor activation, as shown by previous studies.

Aging↗

PubMed1999

Metabolism of anandamide and 2-arachidonoylglycerol: an historical overview and some recent developments.

Anandamide (N-arachidonoylethanolamine) and 2-arachidonoylglycerol are the two endogenous agonists of cannabinoid receptors discovered to date. Like other eicosanoids, and unlike classical neuromodulators, these two compounds are synthesized by neurons on demand, i.e., their biosynthesis, rather than release, is stimulated by Ca2+ influx and cell membrane depolarization. Both endocannabinoids can be produced from membrane phosphoglycerides through the action of phospholipases, although de novo pathways have also been suggested. Once released by cells, the action of both anandamide and 2-arachidonoylglycerol is terminated--after their diffusion through the cell membrane--by the hydrolysis of the amide or ester bonds to yield arachidonic acid, which is then immediately reincorporated into phospholipids. One enzyme, fatty acid amide hydrolase, catalyzes the hydrolysis of both endocannabinoids in nervous and nonnervous cells. This enzyme also recognizes N-palmitoylethanolamine, an antiinflammatory congener of anandamide, with a catalytic efficiency that depends on the cell type under study. However, the existence of different isozymes with different affinity for anandamide and N-palmitoylethanolamine has not been investigated. Moreover, little work has been performed on the regulation of anandamide formation and breakdown, and several open questions remain as to the possible biosynthetic and degradative mechanisms of cannabimimetic 2-arachidonoylglycerol in nucleated blood cells such as macrophages. Finally, the co-existence of both endocannabinoids in invertebrates has not been fully established. Here we briefly review the state of the art, and present new data from our laboratory, on these four largely unexplored aspects of endocannabinoid metabolism.

Animals↗

PubMed1999

Finding of the endocannabinoid signalling system in Hydra, a very primitive organism: possible role in the feeding response.

Hydra (Cnidaria) is the first animal organism to have developed a neural network, which has been proposed to control, inter alia, the "feeding response", i.e. a mechanism through which the coelenterate opens and then closes its mouth in the presence of prey and/or glutathione. Here, we report that Hydra contains: (i) selective cannabinoid binding sites; (ii) the endogenous cannabinoid receptor ligand, anandamide (arachidonoylethanolamide); (iii) a fatty acid amide hydrolase-like activity catalysing anandamide hydrolysis; and (iv) the putative biosynthetic precursor of anandamide, N-arachidonoylphosphatidylethanolamine. We suggest that this "endogenous cannabinoid system" is involved in the modulation of the "feeding response". Anandamide (1 nM-1 microM) potently inhibited (up to 45%) the glutathione-induced "feeding response" by accelerating Hydra vulgaris mouth closure. The effect was maximal at 100 nM anandamide and was reversed by the selective antagonist of the CB1 subtype of mammalian cannabinoid receptors, SR 141716A (50-100 nM). Specific cannabinoid binding sites were detected in membranes from Hydra polyps by using [3H]SR 141716A (Kd= 1.87 nM, Bmax = 26.7 fmol/mg protein), and increasing anandamide concentrations were found to displace the binding of [3H]SR 141716A to these membranes (Ki = .505 nM). Hydra polyps were also found to contain amounts of anandamide (15.6 pmol/g) and N-arachidonoylphosphatidylethanolamine (32.4 pmol/g), as well as the other "endocannabinoid" 2-arachidonoylglycerol (11.2 nmol/g), comparable to those described previously for mammalian brain. Finally, a fatty acid amide hydrolase activity (Vmax = 3.4 nmol/min/mg protein), with subcellular distribution, pH dependency and sensitivity to inhibitors similar to those reported for the mammalian enzyme, but with a lower affinity for anandamide (Km = 400 microM), was also detected in Hydra polyps. These data suggest that the endocannabinoid signalling system plays a physiological role in Hydra that is to control the feeding response. Hydra is the simplest living organism described so far to use this recently discovered regulatory system.

Amidohydrolases↗

PubMed1999

Biosynthesis and inactivation of the endocannabinoid 2-arachidonoylglycerol in circulating and tumoral macrophages.

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 microM) and lipopolysaccharide (LPS, 200 microg x mL-1) 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 [3H]2-AG was cleared from the medium of intact J774 macrophages (t1/2 = 19-28 min) and esterified to phospholipids, diacylglycerols and triglycerides or hydrolyzed to [3H]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 Km = 110 microM and Vmax = 7.9 nmol x min-1 x (mg protein)-1 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.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

Identification of endocannabinoids and cannabinoid CB(1) receptor mRNA in the pituitary gland.

Most data on effects of natural and synthetic cannabinoids on anterior pituitary hormone secretion point out to a primary impact on the hypothalamus. There is also some evidence, however, of possible direct actions of these compounds on the anterior pituitary, although the presence of cannabinoid receptors in the pituitary has not been documented as yet. In the present study, we evaluated the presence of cannabinoid CB(1) receptor-mRNA transcripts in the pituitary gland by in situ hybridization. We observed CB(1) receptor-mRNA transcripts in the anterior pituitary and to a lesser extent in the intermediate lobe whereas they were absent in the neural lobe. We then examined whether CB(1) receptor-mRNA levels in both pituitary lobes responded to chronic activation by a specific agonist, as did receptors located in adjacent hypothalamic nuclei and in other brain regions. Daily administration of CP-55,940 for 18 days produced a small, but statistically significant paradoxical increase in CB(1) receptor-mRNA levels in the anterior pituitary, with no changes in the intermediate lobe, in contrast to reduced CB(1) receptor-mRNA levels observed in the ventromedial hypothalamic nucleus (VMN), and to decreased CB(1) receptor binding in the VMN and the arcuate nucleus. The time-course of up-regulation of CB(1) receptor-mRNA transcripts in the anterior lobe was biphasic; daily administration of Delta(9)-tetrahydrocannabinol produced an early and marked decrease in CB(1) receptor-mRNA levels after 1 and 3 days, followed by normalization after 7 days and by a small increase after 14 days. We also checked whether endogenous cannabinoid ligands are present in the anterior pituitary and the hypothalamus. Although anandamide itself was detected only in trace amounts, concentrations of its precursor N-arachidonoyl-phosphatidyl-ethanolamine and of 2-arachidonoyl-glycerol were found in both tissues, suggesting that endocannabinoids may be synthetized in the anterior pituitary. In summary, CB(1) receptors and corresponding ligands seem to be expressed in cells of the anterior and intermediate lobes of the pituitary, but the response of CB(1) receptor-mRNA transcripts in the anterior lobe to chronic agonist activation is different than the desensitization observed in hypothalamic nuclei.

Animals↗

PubMed1999

Cannabimimetic fatty acid derivatives: the anandamide family and other endocannabinoids.

In agreement with the highly lipophilic nature of (-)-delta9-tetrahydrocannabinol, all the endogenous ligands of cannabinoid receptors identified so far are derivatives of long chain fatty acids. N- Arachidonoylethanolamine (anandamide) and some of its polyunsaturated congeners have been found in mammalian brain and shown to activate the CB1 and, with a lower efficacy, CB2 cannabinoid receptor subtypes. More recently, 2-arachidonoylglycerol (2-AG), a widespread intermediate in the metabolism of phosphoglycerides, diacylglycerols and triglycerides, was also found to activate the cannabinoid receptors. The capability of palmitoylethanolamide, an anti-inflammatory metabolite, to activate CB2-like receptors is still being debated. Here we review: 1) the metabolic pathways suggested so far to underlie the biosynthesis and inactivation of anandamide and 2-AG, and 2) the current knowledge of the chemical bases for the interactions of anandamide and 2-AG with proteins of the endogenous cannabinoid system characterized so far, i.e. the CB1 and CB2 receptor subtypes, the membrane anandamide carrier , which facilitates anandamide diffusion into cells, and the enzyme fatty acid amide hydrolase , which catalyzes anandamide and, to a certain extent, 2-AG hydrolysis in vivo.

Animals↗

PubMed1998

Interactions between synthetic vanilloids and the endogenous cannabinoid system.

The chemical similarity between some synthetic agonists of vanilloid receptors, such as olvanil (N-vanillyl-cis-9-octadecenoamide), and the 'endocannabinoid' anandamide (arachidonoyl-ethanolamide, AEA), suggests possible interactions between the cannabinoid and vanilloid signalling systems. Here we report that olvanil is a stable and potent inhibitor of AEA facilitated transport into rat basophilic leukemia (RBL-2H3) cells. Olvanil blocked both the uptake and the hydrolysis of [14C]AEA by intact RBL-2H3 cells (IC50 = 9 microM), while capsaicin and pseudocapsaicin (N-vanillyl-nonanamide) were much less active. Olvanil was more potent than previously reported inhibitors of AEA facilitated transport, i.e. phloretin (IC50 = 80 microM), AM404 (12.9% inhibition at 10 microM) or oleoylethanolamide (27.5% inhibition at 10 microM). Olvanil was a poor inhibitor of [14C]AEA hydrolysis by RBL-2H3 and N18TG2 cell membranes, suggesting that the inhibitory effect on [14C]AEA breakdown observed in intact cells was due to inhibition of [14C]AEA uptake. Olvanil was stable to enzymatic hydrolysis, and (i) displaced the binding of high affinity cannabinoid receptor ligands to membrane preparations from N18TG2 cells and guinea pig forebrain (Ki = 1.64-7.08 microM), but not from cells expressing the CB2 cannabinoid receptor subtype; (ii) inhibited forskolin-induced cAMP formation in intact N18TG2 cells (IC50 = 1.60 microM), this effect being reversed by the selective CB1 antagonist SR141716A. Pseudocapsaicin, but not capsaicin, also selectively bound to CB1 receptor-containing membranes. These data suggest that some of the analgesic actions of olvanil may be due to its interactions with the endogenous cannabinoid system, and may lead to the design of a novel class of cannabimimetics with potential therapeutic applications as analgesics.

Animals↗

PubMed1998

Arachidonoylserotonin and other novel inhibitors of fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis of bioactive fatty acid amides and esters such as the endogenous cannabinoid receptor ligands, anandamide (N-arachidonoyl-ethanolamine) and 2-arachidonoylglycerol, and the putative sleep inducing factor cis-9-octadecenoamide (oleamide). Most FAAH blockers developed to date also inhibit cytosolic phospholipase A2 (cPLA2) and/or bind to the CB1 cannabinoid receptor subtype. Here we report the finding of four novel FAAH inhibitors, two of which, malhamensilipin A and grenadadiene, were screened out of a series of thirty-two different algal natural products, and two others, arachidonoylethylene glycol (AEG) and arachidonoyl-serotonin (AA-5-HT) were selected out of five artificially functionalized polyunsaturated fatty acids. When using FAAH preparations from mouse neuroblastoma N18TG2 cells and [14C]anandamide as a substrate, the IC50s for these compounds ranged from 12.0 to 26 microM, the most active compound being AA-5-HT. This substance was also active on FAAH from rat basophilic leukaemia (RBL-2H3) cells (IC50 = 5.6 microM), and inhibited [14C]anandamide hydrolysis by both N18TG2 and RBL-2H3 intact cells without affecting [14C]anandamide uptake. While AEG behaved as a competitive inhibitor and was hydrolyzed to arachidonic acid (AA) by FAAH preparations, AA-5-HT was resistant to FAAH-catalyzed hydrolysis and behaved as a tight-binding, albeit non-covalent, mixed inhibitor. AA-5-HT did not interfere with cPLA2-mediated, ionomycin or antigen-induced release of [3H]AA from RBL-2H3 cells, nor with cPLA2 activity in cell-free experiments. Finally, AA-5-HT did not activate CB1 cannabinoid receptors since it acted as a very weak ligand in in vitro binding assays, and, at 10-15 mg/kg body weight, it was not active in the 'open field', 'hot plate' and rectal hypothermia tests carried out in mice. Conversely AEG behaved as a cannabimimetic substance in these tests as well as in the 'ring' immobility test where AA-5-HT was also active. AA-5-HT is the first FAAH inhibitor reported to date which is inactive both against cPLA2 and at CB1 receptors, whereas AEG represents a new type of cannabinoid receptor agonist.

Amidohydrolases↗

PubMed1998

An entourage effect: inactive endogenous fatty acid glycerol esters enhance 2-arachidonoyl-glycerol cannabinoid activity.

2-Arachidonoyl-glycerol (2-Ara-GI) has been isolated from various tissues and identified as an endogenous ligand for both cannabinoid receptors, CB1 and CB2. Here we report that in spleen, as in brain and gut, 2-Ara-GI is accompanied by several 2-acyl-glycerol esters, two major ones being 2-linoleoyl-glycerol (2-Lino-Gl) and 2-palmitoyl-glycerol (2-Palm-Gl). These two esters do not bind to the cannabinoid receptors, nor do they inhibit adenylyl cyclase via either CB1 or CB2; however, they significantly potentiate the apparent binding of 2-Ara-Gl and its apparent capacity to inhibit adenylyl cyclase. Together these esters also significantly potentiate 2-Ara-Gl inhibition of motor behavior, immobility on a ring, analgesia on a hot plate and hypothermia caused by 2-Ara-Gl in mice. 2-Lino-Gl, but not 2-Palm-GI, significantly inhibits the inactivation of 2-Ara-Gl by neuronal and basophilic cells. These data indicate that the biological activity of 2-Ara-Gl can be increased by related, endogenous 2-acyl-glycerols, which alone show no significant activity in any of the tests employed. This effect ('entourage effect') may represent a novel route for molecular regulation of endogenous cannabinoid activity.

Adenylyl Cyclase Inhibitors↗

PubMed1998

The endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation.

Anandamide was the first brain metabolite shown to act as a ligand of "central" CB1 cannabinoid receptors. Here we report that the endogenous cannabinoid potently and selectively inhibits the proliferation of human breast cancer cells in vitro. Anandamide dose-dependently inhibited the proliferation of MCF-7 and EFM-19 cells with IC50 values between 0.5 and 1.5 microM and 83-92% maximal inhibition at 5-10 microM. The proliferation of several other nonmammary tumoral cell lines was not affected by 10 microM anandamide. The anti-proliferative effect of anandamide was not due to toxicity or to apoptosis of cells but was accompanied by a reduction of cells in the S phase of the cell cycle. A stable analogue of anandamide (R)-methanandamide, another endogenous cannabinoid, 2-arachidonoylglycerol, and the synthetic cannabinoid HU-210 also inhibited EFM-19 cell proliferation, whereas arachidonic acid was much less effective. These cannabimimetic substances displaced the binding of the selective cannabinoid agonist [3H]CP 55, 940 to EFM-19 membranes with an order of potency identical to that observed for the inhibition of EFM-19 cell proliferation. Moreover, anandamide cytostatic effect was inhibited by the selective CB1 receptor antagonist SR 141716A. Cell proliferation was arrested by a prolactin mAb and enhanced by exogenous human prolactin, whose mitogenic action was reverted by very low (0.1-0.5 microM) doses of anandamide. Anandamide suppressed the levels of the long form of the prolactin receptor in both EFM-19 and MCF-7 cells, as well as a typical prolactin-induced response, i.e., the expression of the breast cancer cell susceptibility gene brca1. These data suggest that anandamide blocks human breast cancer cell proliferation through CB1-like receptor-mediated inhibition of endogenous prolactin action at the level of prolactin receptor.

Arachidonic Acids↗

PubMed1998

Biosynthesis and degradation of bioactive fatty acid amides in human breast cancer and rat pheochromocytoma cells--implications for cell proliferation and differentiation.

The endogenous cannabinoid, anandamide (arachidonoylethanolamide), and the sleep-inducing factor, oleamide (cis-9-octadecenoamide), represent two classes of long-chain fatty acid amides with several neuronal actions and metabolic pathways in common. Here we report that these two compounds are present in human breast carcinoma EFM-19 cells and rat adrenal pheochromocytoma PC-12 cells, together with the enzyme responsible for their degradation, fatty acid amide hydrolase, and the proposed biosynthetic precursors for arachidonoylethanolamide and related acylethanolamides, the N-acyl-phosphatidylethanolamines. Lipids extracted from cells labelled with [14C]ethanolamine contained radioactive compounds with the same chromatographic behaviour as arachidonoylethanolamide and acyl-PtdEtns. The levels of these compounds were not influenced by either stimulation with ionomycin in EFM-19 cells or two-week treatment with the nerve growth factor in PC-12 cells. The chemical nature of arachidonoylethanolamide, related acylethanolamides and the corresponding acyl-PtdEtns was confirmed by gas chromatographic/mass spectrometric analyses of the purified compounds, which also showed the presence of higher levels of oleamide. The latter compound, which does not activate the central CB1 cannabinoid receptor, exhibited an anti-proliferative action on EFM-19 cells at higher concentrations than arachidonoylethanolamide (IC50 = 11.3 microM for oleamide and 2.1 microM for arachidonoylethanolamide), while at a low, inactive dose it potentiated an arachidonoylethanolamide cytostatic effect. The CB1 receptor selective antagonist SR 141716A (0.5 microM) reversed the effect of both arachidonoylethanolamide and oleamide. EFM-19 cells and PC-12 cells were found to contain a membrane-bound [14C]arachidonoylethanolamide-hydrolysing activity with pH dependency and sensitivity to inhibitors similar to those previously reported for fatty acid amide hydrolase. This enzyme was inhibited by oleamide in both intact cells and cell-free preparations. The presence of transcripts of fatty acid amide hydrolase in these cells was shown by northern blot analyses of their total RNA. The rate of [14C]arachidonoylethanolamide hydrolysis by intact cells, the kinetic parameters of arachidonoylethanolamide enzymatic hydrolysis and the amounts of the fatty acid amide hydrolase transcript, were not significantly influenced by a two-week treatment with nerve growth factor and subsequent transformation of PC-12 cells into neuron-like cells. These data show for the first time that: (a) induction by nerve growth factor of a sympathetic neuronal phenotype in PC-12 cells has no effect on arachidonoylethanolamide/oleamide metabolism, (b) arachidonoylethanolamide and oleamide are autacoid suppressors of human breast cancer cell proliferation. Moreover these data lend conclusive support to the previous hypothesis that oleamide may act as an enhancer of arachidonoylethanolamide actions through competitive inhibition of its degradation.

Amidohydrolases↗

PubMed1998

The novel endogenous cannabinoid 2-arachidonoylglycerol is inactivated by neuronal- and basophil-like cells: connections with anandamide.

The novel endogenous cannabinoid 2-arachidonoylglycerol (2-AG) was rapidly inactivated by intact rat basophilic leukaemia (RBL-2H3) and mouse neuroblastoma (N18TG2) cells through diffusion/hydrolysis/reacylation processes. The hydrolysis of 2-AG was inhibited by typical esterase inhibitors and by more specific blockers of 'fatty acid amide hydrolase' (FAAH), the enzyme catalysing the hydrolysis of the other 'endocannabinoid', anandamide (AEA). No evidence for a facilitated-diffusion process was found. A 2-AG-hydrolysing activity was detected in homogenates from both cell lines, with the highest levels in membrane fractions. It exhibited an optimal pH at 10, and recognized both 2- and 1(3)- isomers of monoarachidonoylglycerol with similar efficiencies. The apparent Km and Vmax values for -3H-2-AG hydrolysis were 91 microM and 29 microM and 2.4 and 1.8 nmol.min-1.mg of protein-1 respectively in N18TG2 and RBL-2H3 cells. [3H]2-AG hydrolysis was inhibited by Cu2+, Zn2+ and p-hydroxymercuribenzoate, and by 2- or 1(3)-monolinoleoyl- and -linolenoyl-glycerols, but not by the oleoyl, palmitoyl and myristoyl congeners. Purified fractions from solubilized membrane proteins catalysed, at pH 9.5, the hydrolysis of 2-AG as well as AEA. Accordingly, AEA as well as FAAH inhibitors, including arachidonoyltrifluoromethyl ketone (ATFMK), blocked [3H]2-AG hydrolysis by N18TG2 and RBL-2H3 membranes, whereas 2-AG inhibited [14C]AEA hydrolysis. FAAH blockade by ATFMK preserved from inactivation the 2-AG synthesized de novo by intact N18TG2 cells stimulated with ionomycin. These data suggest that FAAH may be one of the enzymes deputed to the physiological inactivation of 2-AG, and create intriguing possibilities for the cross-regulation of 2-AG and AEA levels.

Amidohydrolases↗

PubMed1998

Endocannabinoids: endogenous cannabinoid receptor ligands with neuromodulatory action.

The existence of an endogenous cannabinoid system was demonstrated conclusively with the discovery of endogenous brain constituents capable of activating the cannabinoid receptors functionally. These compounds are synthesized by neuronal cells and inactivated through re-uptake and enzymatic hydrolysis by both neurons and astrocytes. In analogy with the endorphins they can be referred to as endocannabinoids. Apart from the identification of their metabolic pathways, research carried out in the past six years has focused on the possible cellular and molecular targets for the actions of endocannabinoids. These studies have confirmed a similarity between the endocannabinoids and the psychoactive substance in marijuana, delta9(-)-tetrahydrocannabinol, and have suggested a role for endocannabinoids in the modulation of neurotransmitter action and release.

Animals↗

PubMed1997

The sleep inducing factor oleamide is produced by mouse neuroblastoma cells.

Cis-9,10-octadecenoamide (oleamide) was isolated from the cerebrospinal fluid of sleep-deprived mammals and shown to induce sleep in rats. The enzyme catalyzing the hydrolysis of the amide bond of oleamide as well as of anandamide, the putative endogenous ligand of cannabinoid receptors, was purified from rat liver, cloned, shown to be expressed also in brain and named fatty acid amide hydrolase (FAAH). The enzymatic synthesis of oleamide from oleic acid and ammonia by rat brain microsomes has been also described. However, no evidence has been reported so far on the neuronal origin of oleamide, necessary in order to postulate for this compound a role as a neuromodulator. Here we show for the first time that oleamide is produced by a neuronal cell type and that its biosynthesis in intact neurons is not likely to occur through the direct condensation of oleic acid and ammonia. A lipid metabolite was extracted and purified from mouse neuroblastoma N18TG2 cells through a sequence of chromatographic steps and characterized as oleamide by means of gas chromatography/electron impact mass spectrometry (GC/EIMS). The amount of oleamide, as estimated by GC analyses carried out in comparison with known amounts of synthetic oleamide, was 55.0+/-09.5 pmols/10(7) cells, compared to less than 0.7 pmol/10(7) cells for anandamide in the same cells. When N18TG2 cells were prelabeled with [14C]oleic acid and the lipids extracted and purified, a radioactive component with the same chromatographic behavior as oleamide was found whose levels: (1) were not significantly influenced by stimulation with ionomycin; (2) were slightly increased by incubation with FAAH inhibitor phenyl-methyl-sulphonyl-fluoride (PMSF); (3) appeared to correlate with [14C]oleic acid incorporation into phospholipids but not with free [14C]oleic acid levels. N18TG2 cell membranes were shown to contain an enzymatic activity catalyzing the synthesis of oleamide from oleic acid and ammonia. This activity was inhibited by FAAH selective inhibitors arachidonoyltrifluoromethylketone and methylarachidonoylfluorophosphonate, as well as by an excess of anandamide, and by PMSF at the same concentration which increased oleamide formation in intact cells. These data suggest that a FAAH-like enzyme working "in reverse" may be responsible for the formation of oleamide in cell-free preparations but not in whole cells.

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