The discovery in 1992  of anandamide (N-arachidonoyl-ethanolamine), 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 and in vitro. In particular, it was found that 2-AG, like (–)-Δ9-tetrahydrocannabinol, induces inhibition of locomotor activity, catalepsy, hypothermia and analgesia in mice , inhibits the forskolin-induced stimulation of cAMP formation , and modulates Ca2+ currents in NG108–15 hybrid cells . Recently, cannabinoid receptor-mediated biological activities were found for 2-AG also in peripheral tissues. These are the inhibition of mouse embryo development and implantation , the modulation of lymphocyte proliferation  with suppression of interleukin-2 secretion from activated T cells , and the arrest of human breast cancer cell proliferation . More particularly, a role for 2-AG as an endogenous hypotensive factor now seems to emerge from several recent sets of experimental data. First, 2-AG formation and release from vascular endothelial cells in culture can be induced by thrombin and a Ca2+ ionophore . The production of 2-AG was also found to be enhanced in normal, but not in endothelium denuded, rat aorta on stimulation with carbachol . In a previous study , it was found that 2-AG induces a reduction in rat mean arterial pressure which could be mimicked by preparations of circulating macrophages and platelets from rat donors treated with lipopolysaccharide (LPS). In both cases, the hypotensive effect was attenuated by a selective antagonist of the CB1 subtype of cannabinoid receptors. Blood circulating macrophages were also shown to participate in haemorrhage-induced and CB1 receptor-mediated hypotension in rats . It was proposed that endocannabinoids derived from these two blood cell types could contribute to both haemorrhage-and endotoxin-induced hypotension [11,12]. While it was clear, however, that platelets synthesize only 2-AG , and that stimulated macrophages from various sources can produce and inactivate anandamide [12–15], the capability of these latter cells to biosynthesize 2-AG could not be demonstrated due to the presence of platelets in preparations of circulating macrophages . Furthermore, no study to date has addressed the question of whether macrophages can inactivate 2-AG produced by these or neighbouring cells (e.g. endothelial cells) and, if so, by which mechanism. These two issues are extremely important in order to understand to what extent macrophages participate in the homeostasis of a powerful hypotensive substance such as 2-AG. In the present study we have circumvented the problem of platelet contamination of blood macrophage preparations by investigating the metabolism of 2-AG in a tumoral macrophage cell line. Immortalized monocytes/macrophages have been successfully used in recent years to study the biosynthesis, action and inactivation of anandamide [13,14,16–18]. In particular, mouse J774 macrophages were shown to: (a) respond to ionomycin stimulation by producing anandamide, very probably through the hydrolysis of a preformed phospholipid precursor [13,16], (b) take up exogenous anandamide through a temperature-dependent and saturable mechanism [13,17], and (c) respond to anandamide by releasing arachidonic acid (AA) . We present here data indicating that stimulation of J774 macrophages with either ionomycin or LPS leads to the formation of 2-AG, and that exogenous 2-AG, once taken up by these cells, can be inactivated through parallel re-esterification into phosphoglycerides and hydrolysis to AA. Moreover, we show that 2-AG can also be hydrolyzed by subcellular fractions of rat circulating macrophages through at least two enzymes, including fatty acid amide hydrolase (FAAH), previously found to catalyze the hydrolysis of anandamide , and a 2-AG hydrolase that we have preliminarily characterized in J774 cells.
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 µm) and lipopolysaccharide (LPS, 200 µg·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 µm and Vmax = 7.9 nmol·min−1·(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.
fatty acid amide hydrolase
normal phase high-pressure liquid chromatography
gas chromatography-electron impact mass spectrometry
Materials and methods
Mouse J774 A1 cells were purchased from DSMG (Braunschweig, Germany) and cultured according to the instructions of the manufacturer. Circulating macrophages and platelet were prepared from whole rat blood as described below. The number of platelets in macrophage preparations was estimated to be 20–25 per macrophage. In experiments with macrophage preparations 5 × 106 cells were used, whereas at least 5 × 109 cells had to be used in experiments with platelet preparations in order to observe 2-AG biosynthesis or hydrolysis. Therefore, the data obtained in the former type of experiments were biased by the presence of platelets only for a minor part. [3H]2-AG (5 mCi·mmol−1) was either synthesized as described elsewhere  or kindly donated, together with [3H]2-palmitoyl-glycerol (5 mCi mmol−1) by Dr T. Sugiura (Teikyo University, Kanagawa, Japan). [3H]Anandamide (220 Ci mmol−1) was purchased from NEN Dupont (Boston, MA). [14C]Anandamide was synthesized as described previously . 2-AG was kindly donated by Prof. R. Mechoulam, Hebrew University, Jerusalem, Israel, whereas synthetic anandamide was synthesized as described previously . D8-anandamide and d8–2-AG were synthesized from d8-AA (Cayman Chemicals, Ann Arbor, MI) and ethanolamine or glycerol as described in  and , respectively. Ionomycin and LPS (0127:B8) as well as synthetic AA, di-oleoyl-glycerol, tri-oleoyl-glycerol, 1(3)-linoleoyl-glycerol, 1(3)-palmitoyl-glycerol, 1(3)-γ-linolenoyl-glycerol and sn-1-palmitoyl-2-oleoyl-phosphatidylethanolamine and the esterase inhibitors were purchased from Sigma, UK. Arachidonoyltrifluoromethane (Δ4AchCoCH2F3) and methylarachidonoylfluorophosphonate (CH2Δ4AchPOF) were purchased from Biomol (Plymouth Meeting, PA).
Cell stimulation and lipid extraction and purification
Experiments with J774 macrophages were performed in the Naples laboratory. Confluent intact J774 cells from 20 100 mm Petri dishes were washed three times with serum-free medium and stimulated with either 5 µm ionomycin or 200 µg·mL−1 LPS in serum-free medium, respectively, for 20 or 90 min at 37 °C. After the stimulation, ice-cold methanol containing 2 nmol each of d8-anandamide and d8-2-AG was added to the media, cells were scraped from the dishes, transferred to 50 mL Falcon tubes and an equal volume of chloroform added prior to sonication for 2 min at 4 °C. The organic phase was then dried under nitrogen and purified by means of a series of chromatographic steps consisting of open bed chromatography on silica gel and normal phase high-pressure liquid chromatography (NP-HPLC) carried out as previously described . In the case of rat circulating macrophage preparations, biosynthetic experiments were performed in the Richmond laboratory. Mononuclear cells were isolated from a total of 60 mL of heparinized whole blood from adult, male Sprague-Dawley rats by the method described in detail elsewhere . The mononuclear cell fraction was resuspended in phosphate buffered saline in siliconized glass tubes. The cells were divided into two equal aliquots and plated onto plastic culture dishes. After the adhesion of macrophages and contaminating platelets, the nonadherent cells (mostly lymphocytes) were removed by aspiration and the medium was replaced by serum-free RPMI 1640. The cells were incubated for 90 min at 37 °C with vehicle or 200 µg·mL−1 LPS, respectively. The medium was then replaced with fresh RPMI 1640 medium containing 200 µm phenylmethanesulphonyl fluoride (PhCH2SO2F) and 30 min later, the cells plus medium were extracted three times with 2 volumes of ethyl acethate and the organic phase dried under nitrogen, to be submitted to purification by reverse phase HPLC as described previously .
Gas chromatography-mass spectrometric analysis of anandamide and 2-AG. NP-HPLC fractions of J774 cell extracts with the same retention time as 2-AG (21 min) and anandamide (35 min) were liophylized and derivatized with 20 µL N-methyl-N-trimethylsilyl-trifluoroacetamide +1% trimethylchlorosylane (Pierce, Rockford, IL) for 2 h at room temperature, prior to analysis by gas chromatography-electron impact mass spectrometry (GC-EIMS), carried out as described previously  in the selected ion monitoring mode. In the case of 2-AG NP-HPLC fractions, selected ions were monitored at m/z = 530 and 522 (molecular ion peaks) and m/z = 515 and 507 (−15, loss of a methyl group) for d8–2-AG and 2-AG, respectively. In the case of anandamide NP-HPLC fractions, selected ions were monitored at m/z = 427 and 419 (molecular ion peaks) and m/z = 412 and 404 (−15, loss of a methyl group) for d8-anandamide and anandamide, respectively. The amounts of 2-AG and anandamide were calculated from the peak area ratios of the −15 fragments for the nondeuterated versus the deuterated compounds. In the case of samples from rat circulating macrophages, 2-AG was analysed in the Richmond laboratory as described in .
Inactivation of [3H]2-AG by intact J774 cells
Experiments on the inactivation of [3H]2-AG, [3H]- or [14C]anandamide by intact J774 cells were performed as described previously [13,21]. Confluent or subconfluent cells in six-well dishes (respectively 1.5 or 0.5 × 106 cells·well−1) were washed with serum-free medium and incubated with [3H]2-AG compounds at different concentrations (2, 4, 8 µm) or for increasing periods of time. Incubations were terminated by separating the media from the cells and by addition of ice-cold methanol. In some experiments, the incubations were carried out on ice at 0–4 °C, or in the presence of various inhibitors. Media and cell extracts were analyzed for the presence of radiolabelled 2-AG, anandamide, AA, triglicerides, diglycerides and phospholipids as described previously .
J774 cells and rat circulating macrophages and platelets were homogenized and subcellular fractions prepared as described previously [13,21]. In some experiments macrophages and platelets were stimulated with 200 µg·mL−1 LPS as described above. Aliquots of membrane and cytosolic fractions (0.02–0.05 mg) were assayed for their capability to hydrolyze [3H]2-AG or [14C]anandamide to [3H]AA or [14C]ethanolamine, respectively. These assays have been widely described in previous reports [13,20–22]. In the case of assays of [3H] 2-AG-hydrolyzing activity, incubations were performed at pH 7 with increasing concentrations of the substrate, or in buffers at various pH (prepared as described in ) or at pH 7 in the presence of various inhibitors, with a substrate concentration of 50 µm. [3H]AA produced from [3H]2-AG hydrolysis was purified and quantitated as described previously .
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was prepared by the acid guanidinium thiocyanate-phenol-chloroform method  and then treated with RNase-free DNase as described . The integrity of RNAs was assessed by analysing 18S and 28S rRNA contents on a denaturating 1% agarose gel. 1 µg of RNA in a single tube was reverse transcribed for 60 min and subsequently amplified by the PCR process using a single thermostable DNA polymerase for both reactions. Each mixture (50 µL) comprised 0.45 µm primers specific for the rat CB1 receptor or for the rat FAAH, 300 µm dNTPs, 115 mm potassium acetate, 8% (w/v) glycerol, 50 mm bicine pH 8.2, 5 units of rTth DNA polymerase (Perkin-Elmer, Branchburg, NJ) and 2.5 mm manganese acetate. The primer sets used to amplify the coding regions of the rat CB1 receptor gene and the rat FAAH gene  were as follows: CB1 upper primer 5′-CCACAGAAATTCCCTCTAACTTCC-3′; CB1 lower primer 5′-GAAAATGTCCGAGCAAACAGATTG-3′; FAAH upper primer 5′-GCCTGAAAGCTCTACTGTGTGAGC-3′; FAAH lower primer 5′-GAAGGTCCAGACTTGGTTGTGGCT-3′. Amplification of the CB1 receptor gene (675 bp) and FAAH gene (1027 bp) were performed through 30 cycles of denaturation of 95 °C for 1 min, annealing at 55 °C for 1 min and elongation at 72 °C for 1 min in a PE Gene Amp PCR System 9600 (Perkin Elmer). After the reaction, the PCR products were electrophoresed on a 2% agarose gel. Photographs were taken under UV light. No PCR product was detected when the reverse-transcriptase step was omitted (data not shown), indicating that there was no contamination by genomic DNA of each gene in the RT-PCR templates. Northern blotting for FAAH mRNA was carried out as described in  on 25 µg of total RNA from circulating macrophages.
Results and discussion
Stimulus-induced formation of 2-AG in macrophages
The stimulatory effect of LPS and ionomycin on anandamide formation from rat circulating macrophages was reported previously [11,12]. Here we found that lipids extracted from J774 macrophages contain amounts of 2-AG that were augmented from 5.0 ± 5.0 to 120.0 ± 29.0 pmol per 107 cells by stimulation with 5 µm ionomycin, and from 7.5 ± 2.1 to 19.5 ± 1.2 pmol per 107 cells by stimulation with 200 µg·mL−1 LPS (Fig. 1a,b, means ± SD, n = 2). Likewise, the amount of 2-AG in control and LPS-treated rat circulating macrophages was 2.3 and 4.2 ng (about 10.0 and 18.1 pmol per 107 cells), respectively (Fig. 2). Similar amounts of 2-AG were earlier detected in pure platelet preparations containing about 50 times more platelets than the platelets contaminating the present macrophage preparation , suggesting that the 2-AG detected here must have come predominantly from macrophages. As to anandamide formation in J774 macrophages, LPS elevated the amounts of this compound from almost undetectable levels (0.8 ± 0.2 pmol per 107 cells) to 6.9 ± 1.0 pmol per 107 cells (means ± SD, n = 2). This latter finding provides mass spectrometric confirmation to the very recent report of LPS-induced stimulation of anandamide formation in the mouse RAW264.7 macrophage line, based on chromatographic and double-isotope labelling evidence . In summary, our data indicate that a several-fold enhancement of 2-AG (and anandamide) levels can be induced in macrophages by endotoxin-treatment and/or Ca2+ influx, and lend biochemical support to the hypothesis that these cells contribute to endocannabinoid-mediated hypotension arizing during certain pathological conditions [11,12]. Macrophage-derived 2-AG might also control the activity and function of lymphocytes during immunological responses [3,7]. Future studies should investigate whether LPS, which enhances the levels of polyunsaturated phosphatidic acid (PA) and diacylglycerols in macrophages , leads to 2-AG formation in these cells through the same mechanism underlying 2-AG biosynthesis in ionomycin-stimulated N18TG2 cells , i.e. formation of PA, followed by its hydrolysis to diacylglycerol and by the conversion of diacylglycerol into 2-AG.
Inactivation of 2-AG by intact J774 macrophages
Vascular endothelial and smooth muscle cells were shown to produce 2-AG on stimulation with either thrombin, the Ca2+ ionophore A23187  or carbachol . Apart from contributing to the formation of hypotensive 2-AG, blood macrophages (and platelets), if present near the vascular wall and capable of inactivating the monoglyceride, might also limit or terminate 2-AG action. For example, enzymatic hydrolysis is a limiting factor for the potency of the vasodilatory effect of anandamide in the mesenteric artery , and is likely to be important also for 2-AG. Therefore, we carried out a series of experiments aimed at assessing whether intact, living J774 macrophages can inactivate exogenous [3H]2-AG. We found that the amounts of [3H]2-AG incubated with cells decreased with an half-life varying between 19 and 28 min depending on the cell density and on the initial concentration of the compound (Fig. 3a and data not shown). In cell extracts, increasing amounts of radioactivity with increasing incubation times were found associated with AA and phospholipids in a temperature-sensitive manner. Unprocessed [3H]2-AG was also found whose levels were only slightly decreased at 0–4 °C. These findings are in agreement with analogous data reported previously for rat basophilic leukaemia RBL-2H3 cells ( and Di Marzo, unpublished data). In addition, we found that some radioactivity was also associated with diacylglycerol and triacylglycerols. It is possible that [3H]AA produced from [3H]2-AG hydrolysis is partly esterified into phosphoglycerides, as previously shown for anandamide in rat central neurons . However, when we tested several enzyme inhibitors, we observed that the incorporation of radioactivity into phospholipids and AA was inhibited to a different extent by some of these substances (Fig. 3b). In particular, [3H]AA, but not [3H]phospholipid, formation was inhibited by 100 µmp-hydroxy-mercuribenzoate (HO-BzHgOH). Conversely, 100 µm thimerosal significantly inhibited the formation of [3H]phospholipid (as well as [3H]diacylglycerols and [3H]triacylglycerols, data not shown) without increasing the amount of [3H]AA, and so did 100 µm PhCH2SO2F. 1-linoleoyl-glycerol was more potent as inhibitor of [3H]phospholipid than [3H]AA formation. The FAAH inhibitors CH2Δ4AchPOF and Δ4AchCoCH2F3 were, respectively, ineffective or weakly active against both processes. Interestingly, of the compounds tested, none significantly affected the levels of [3H]2-AG found in cell extracts (data not shown), including 1-linoleoyl-glycerol, which was previously found to inhibit 2-AG diffusion into RBL-2H3 and N18TG2 cells . On the basis of these findings, it is possible to hypothesize that 2-AG, once taken up by macrophages, is catabolized through several parallel enzymatic reactions, i.e. hydrolysis to AA and esterification into phospholipids, diacylglycerols and triacylglycerols. Clearly, our data do not allow to assess to what extent radiolabelled phosphoglycerides are produced from the direct esterification of [3H]2-AG or of [3H]AA produced from [3H]2-AG hydrolysis. As to whether the monoglyceride is taken up through passive or facilitated diffusion, we analysed the possibility that the same mechanism previously shown to facilitate anandamide transport inside RBL-2H3 and J774 cells [13,17] also recognizes 2-AG as substrate. This possibility was recently suggested by a study carried out in human astrocytoma cells . However, three sets of experimental data argue against 2-AG being a ligand for the anandamide ‘carrier’ in J774 macrophages. Firstly, the effect of decreasing the temperature of incubation to 4 °C on the amounts of [3H]2-AG found in J774 cell extracts was not comparable in its extent to that observed for [3H]anandamide uptake by the same or other cells [13,18,22,31]. In fact, the little effect observed (Fig. 3a) may have been due to the strong inhibition of [3H]2-AG hydrolysis or esterification to phospholipids which may result in augmented amounts of unprocessed [3H]2-AG, thus preventing the gradient-dependent diffusion of the monoglyceride into the cells. Secondly, 100 µm PhCH2SO2F, which inhibits anandamide re-uptake from RBL-2H3 and J774 cells  as well as U937 macrophages , did not affect the levels of [3H]2-AG found inside J774 cells (89.1 ± 13.5% of control). Finally, also 100 µm anandamide did not significantly reduce the levels of [3H]2-AG inside cells (88.0 ± 10% of control), nor did 50 µm 2-AG reduce the uptake of 3 nm[3H]anandamide from J774 cells (104.7 ± 2.2% of control, means ± SD, n = 3). In RBL-2H3 cells, where the facilitated transport mechanism for anandamide has been well characterized and is counteracted by several inhibitors [13,32], 50 µm 2-AG did not affect the uptake of 4 µm[14C]anandamide  but it did reduce the uptake of 3 nm[3H]anandamide, although only to a little extent (67.6 ± 5.5% of control, n = 3). These data suggest that, in J774 (and RBL-2H3) cells, 2-AG and anandamide are transported into cells through distinct mechanisms.
Enzymatic hydrolysis of 2-AG in macrophages
We next investigated the presence of hydrolytic enzymes for 2-AG in rat circulating macrophages and, for a comparison, in platelets, where a monoacylglycerol lipase has been partially characterized . We found that, as previously reported in N18TG2 cells  and rat brain , both membrane and soluble fractions from rat blood macrophages and platelets could efficiently catalyse the hydrolysis of [3H]2-AG (Table 1). Interestingly, analogous to macrophage lipoprotein lipase activity , the monoacylglycerol lipase activity of the membrane fractions was reduced by treatment of cells with LPS. Thus, the endotoxin may raise the levels of 2-AG in platelets and macrophages (Fig. 2 and ) by stimulating its synthesis and/or inhibiting its inactivation. Particulate fractions from macrophages, and, to a very little extent, platelets, catalysed also the hydrolysis of [14C]anandamide (in a manner that was not sensitive to LPS) (Table 1). This latter observation is important in consideration of the fact that FAAH, the membrane-bound enzyme responsible for anandamide hydrolysis , is also able to recognize 2-AG as a substrate , and contributes significantly to the inactivation of the monoglyceride in RBL-2H3 and N18TG2 cells . In fact, here we found that 100 µm anandamide and two specific FAAH inhibitors, CH2Δ4AchPOF and Δ4AchCoCH2F3, inhibited [3H]2-AG hydrolysis by macrophage membranes, in agreement with a possible contribution to this enzymatic activity by FAAH (Table 2; both inhibitors blocked [3H]2-AG hydrolysis by RBL-2H3 cells ). A slight effect of high doses of Δ4AchCoCH2F3, but not CH2Δ4AchPOF or anandamide, was also observed in platelet membranes. However, the [3H]2-AG-hydrolysing activities from both macrophage and platelet membranes displayed an optimal pH around 7, which is sensibly different from FAAH optimal pH 9–10 [13,15,18,36] (and similar to what was reported for rat platelet  and porcine brain  monoacylglycerol lipases), even though high activity was retained at pH = 9. Furthermore, PhCH2SO2F and HO-BzHgOH inhibited these activities to a different extent and in a manner dissimilar to that reported for FAAH (Table 2). These findings, taken together, suggest that, in circulating macrophages, [3H]2-AG may be hydrolysed by both FAAH and another hydrolase which is not sensitive to some FAAH inhibitors. In platelets the latter enzyme seems to be predominantly expressed.
|Fraction||Platelets||Platelets + LPS||Macrophages||Macrophages + LPS|
|Debris||0.08 ± 0.01||0.08 ± 0.02||0.05 ± 0.006||0.02 ± 0.003*|
|(800 g pellet)||(0.03 ± 0.01)||(0.07 ± 0.04)||(0.21 ± 0.10)||(0.27 ± 0.09)|
|Mitochondria||0.23 ± 0.01||0.14 ± 0.02*||0.17 ± 0.002||0.04 ± 0.002*|
|(10 000 g pellet)||(0.07 ± 0.026)||(0.06 ± 0.012)||(0.33 ± 0.07)||(0.27 ± 0.06)|
|Microsomes||0.87 ± 0.03||0.71 ± 0.06*||0.36 ± 0.03||0.26 ± 0.01*|
|(100 000 g pellet)||(0.09 ± 0.04)||(0.10 ± 0.04)||(0.37 ± 0.07)||(0.36 ± 0.14)|
|Cytosol||0.67 ± 0.04||0.74 ± 0.02||0.62 ± 0.02||0.61 ± 0.03|
|(100 000 g supernantant)||(0.08 ± 0.04)||(0.07 ± 0.02)||(0.13 ± 0.06)||(0.20 ± 0.05)|
|pH 4||35.6 ± 0.4||36.6 ± 4.4|
|pH 5||69.3 ± 4.3||51.3 ± 1.7|
|pH 6||100||87.1 ± 0.5|
|pH 7||99.4 ± 2.9||100|
|pH 8||94.8 ± 3.2||97.1 ± 1.5|
|pH 9||85.4 ± 2.7||77.5 ± 7.9|
|pH 10||55.9 ± 5.1||36.0 ± 4.8|
|pH 11||3.2 ± 0.5||16.9 ± 1.7|
|Anandamide (100 µm)||81.7 ± 9.6||22.7 ± 2.2*|
|PhCH2SO2F (100 µm)||98.2 ± 9.2||79.4 ± 2.4*|
|HO-BzHgOH (100 µm)||121.0 ± 18.9||33.8 ± 3.7*|
|Δ4AchCoCH2F3 (1 µm)||106.6 ± 14.0||65.1 ± 2.6*|
|Δ4AchCoCH2F3 (10 µm)||92.5 ± 4.2||11.2 ± 0.2*|
|Δ4AchCoCH2F3 (50 µm)||47.5 ± 6.5*||0*|
|CH2Δ4AchPOF (0.5 nm)||85.4 ± 11.0||90.7 ± 2.7|
|CH2Δ4AchPOF (5 nm)||109.5 ± 6.0||104.2 ± 2.3|
|CH2Δ4AchPOF (50 nm)||111.0 ± 4.7||79.3 ± 0.9*|
By analysing the cDNA fragments obtained from macrophage and platelet RNA by RT-PCR, we confirmed that rat macrophages, but much less so platelets, express FAAH (Fig. 4). Northern analyses carried out on total RNA from macrophages also confirmed the presence of FAAH mRNA (data not shown). As a further control for the integrity of RNA preparations, we also investigated, again by using RT-PCR, if circulating platelets and macrophages express the mRNA for the CB1 cannabinoid receptor subtype. We found that both cell preparations contain transcripts for this protein (Fig. 4), thus raising the possibility that macrophage- and platelet-derived endogenous cannabinoids may also act as autacoid mediators on these cells and control their activity. CB1 receptors in macrophages have been already described , but while anandamide has been shown to affect macrophage function (reviewed in ), there is no report as yet on the effects of 2-AG on these cells.
A 2-AG hydrolase distinct from FAAH in macrophages
We wanted to partially characterize the 2-AG hydrolase(s) from macrophages. In order to avoid interferences from FAAH or platelet 2-AG hydrolase, we utilized again the J774 cells, which do not express FAAH [13,25] and hydrolyse 2-AG enzymatically (see above). As for circulating macrophages and platelets, the hydrolysis of [3H]2-AG was catalysed very efficiently by both particulate and soluble fractions prepared from J774 cells (Fig. 5a). Very little [14C]anandamide hydrolysis was observed, in agreement with previous data [13,17]. The [3H]2-AG-hydrolysing activity in J774 cell membranes was optimally active at pH 6–7 (Fig. 5b) but, unlike the enzyme(s) from rat circulating macrophages, exhibited little residual activity at pH = 9. Furthermore, the enzyme was inhibited by HO-BzHgOH, CH2Δ4AchPOF and Δ4AchCoCH2F3 (Fig. 5c,d) to a lesser extent than that observed with membranes from either circulating macrophages (Table 2) or, particularly, RBL-2H3 cells . These findings confirm that macrophages do express ‘2-AG hydrolase(s)’ distinct from FAAH. Apart from 2-AG, 1(3)-monoacylglycerols also inhibited [3H]2-AG hydrolysis by J774 cell membranes to a degree depending on unsaturation, the most potent being 1(3)-AG and 1(3)-γ-linolenoyl-glycerol (Fig. 5d), thus suggesting a certain preference of the enzyme for polyunsaturated 1(3)- and 2-monoacylglycerols. In fact, the enzyme, which followed typical Michaelis–Menten kinetics, displayed apparent Km and Vmax values of 110 ± 15 µm and 7.9 ± 0.9 nmol·min−1·(mg protein)−1 when using [3H]2-AG as the substrate (Vmax/Km ratio = 0.072), and 170 ± 12 µm and 4.8 ± 0.6 nmol·min−1· (mg protein)−1 when using [3H]2-palmitoyl-glycerol (Vmax/Km ratio = 0.028) (n = 2). The presence of an uncharacterized monoacylglycerol lipase in cytosolic fractions from rat alveolar macrophages had been suggested previously . Further studies are now needed in order to purify and fully characterize this enzyme in order to assess whether it is similar to the other monoacylglycerol lipases characterized from platelets, neutrophils and erithrocytes [33,40,41].
The data reported here have shown that macrophages: (a) biosynthesize 2-AG in response to ionomycin and LPS, and (b) possess several rapid mechanisms for the catabolism of 2-AG and its recycling into cell (phospho)glycerides. One of these mechanisms is based on the presence of hydrolytic enzymes, including: (a) a 2-AG lipase subject to down-regulation by LPS, and (b) in the case of rat circulating, but not mouse J774, macrophages, the FAAH which can inactivate also the other endocannabinoid anandamide. Therefore, it is now possible to propose that macrophages play an important role in the homeostasis of endocannabinoids, previously shown to participate in shock-induced hypotension ([11,12] and  for a review). Our study indicates also that the use, in biochemical and pharmacological studies on 2-AG, of FAAH inhibitors such as PhCH2SO2F, HO-BzHgOH, CH2Δ4AchPOF and Δ4AchCoCH2F3, previously shown to block 2-AG hydrolysis in some models [20,21,34,36], may not be always sufficient to inhibit 2-AG inactivation. Substances that minimize the acylation of 2-AG, such as 1-linoleoyl-glycerol (see also ) and thimerosal, should also be utilized for these purposes. It will be interesting to assess whether these substances, as well as inhibitors of 2-AG biosynthesis , can be used: (a) pharmacologically, to modulate the hypotensive state arising from haemorrhage and septic shock in rodents, or (b) as biochemical tools to investigate the controversial issue (reviewed in [42–44]) of whether an endocannabinoid, and 2-AG in particular, is an endothelium-derived hyperpolarizing factor.
The authors are grateful to Dr T. Sugiura, Teikyo University, Kanagawa, Japan, for the gift of part of the tritiated substrates used in this study, to Prof. R. Mechoulam, Hebrew University, Jerusalem, Israel, for the gift of 2-AG, and to Dr M. Maccarrone, Università‘Tor Vergata’, Roma, Italy, for fruitful discussions. We also thank Mr S. Piantedosi and R. Turco for some of the artwork. This work was partly supported by grants from the INTAS (grant 97/1297 to VDM) and the Human Frontier Science Program. JAW was supported by the Deutsche Forschungsgemeinschaft.
* Permanent address: Department of Medicine, University of Wurzburg, Germany.