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

Abstract

The stimulus-induced biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in intact mouse J774 macrophages and the inactivation of 2-AG by the same cells or by rat circulating macrophages was studied. By using gas chromatography-mass spectrometry, we found that ionomycin (5 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 (t_1/2 = 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 K_m = 110 µm and V_max = 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.

Abbreviations

2-AG

2-arachidonoylglycerol

AA

arachidonic acid

LPS

lipopolysaccharide

FAAH

fatty acid amide hydrolase

Δ₄AchCoCH₂F₃

arachidonoyltrifluoromethane

CH₂Δ₄AchPOF

methylarachidonoylfluorophosphonate

NP-HPLC

normal phase high-pressure liquid chromatography

PhCH₂SO₂F

phenylmethanesulphonyl fluoride

GC-EIMS

gas chromatography-electron impact mass spectrometry

RT-PCR

reverse transcriptase-PCR

HO-BzHgOH

p-hydroxy-mercuribenzoate.

The discovery in 1992 [1] 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[2] and in vitro[3]. In particular, it was found that 2-AG, like (–)-Δ⁹-tetrahydrocannabinol, induces inhibition of locomotor activity, catalepsy, hypothermia and analgesia in mice [2], inhibits the forskolin-induced stimulation of cAMP formation [3], and modulates Ca²⁺ currents in NG108–15 hybrid cells [5]. Recently, cannabinoid receptor-mediated biological activities were found for 2-AG also in peripheral tissues. These are the inhibition of mouse embryo development and implantation [6], the modulation of lymphocyte proliferation [3] with suppression of interleukin-2 secretion from activated T cells [7], and the arrest of human breast cancer cell proliferation [8]. 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 [9]. The production of 2-AG was also found to be enhanced in normal, but not in endothelium denuded, rat aorta on stimulation with carbachol [10]. In a previous study [11], 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 [12]. 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 [11], 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 [11]. 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) [17]. 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 [19], and a 2-AG hydrolase that we have preliminarily characterized in J774 cells.

Materials and methods

Materials

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 [20] 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 [13]. 2-AG was kindly donated by Prof. R. Mechoulam, Hebrew University, Jerusalem, Israel, whereas synthetic anandamide was synthesized as described previously [13]. D₈-anandamide and d₈–2-AG were synthesized from d₈-AA (Cayman Chemicals, Ann Arbor, MI) and ethanolamine or glycerol as described in [13] and [20], 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 [13]. 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 [11]. 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 [11].

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 [13] 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 [11].

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 [21].

Enzyme assays

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 [13]) 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 [21].

Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA was prepared by the acid guanidinium thiocyanate-phenol-chloroform method [23] and then treated with RNase-free DNase as described [24]. 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 [19] 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 [25] 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 [11], 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 [14]. 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 [26], leads to 2-AG formation in these cells through the same mechanism underlying 2-AG biosynthesis in ionomycin-stimulated N18TG2 cells [27], i.e. formation of PA, followed by its hydrolysis to diacylglycerol and by the conversion of diacylglycerol into 2-AG.

Figure 1. Biosynthesis of 2-AG in intact J774 macrophages. (A) GC-EIMS fragmentograms obtained in the selected ion monitoring mode of purified and derivatized 2-AG fractions from vehicle-treated (upper panel) or ionomycin-stimulated cells (lower panel); (B) GC-EIMS fragmentograms obtained in the selected ion monitoring mode of purified and derivatized 2-AG fractions from vehicle-treated (upper panel) or LPS-stimulated cells (lower panel). Ions were monitored at m/z = 507 and 522, for undeuterated 2-AG, and m/z = 515 and 530 for d₈–2-AG (not shown). The two analyses shown in (A) and (B) were performed in two different days and are representative of two separate experiments each. Peaks at m/z = 515 and 530 were eluted after 19 and 18.8 min in the two separate analyses, and their areas were used to calculate the amounts of endogenous 2-AG using the isotope-dilution criterion.

Figure 2. Biosynthesis of 2-AG in rat circulatingmacrophages. GC-EIMS chromatograms (upper panels) and selected ion spectra (lower panels) obtained from purified and derivatized 2-AG fractions from (A) vehicle-treated (B) LPS-stimulated cells and (C) LPS-stimulated cells ‘spiked’ with synthetic 2-AG (10 ng). The chromatograms in the upper panels reflect combined currents obtained by selected ion monitoring at m/z-values of 507, 451, 432, 147, 129 and 103, corresponding to the major EIMS fragmentation peaks of synthetic 2-AG, as described elsewhere [11]. An expanded view of the GC-EIMS peak is also shown in the panels on the right. Chromatographic conditions are different from those of Fig. 1 and are described in [11]. Areas of the peaks obtained with a series of known amounts of synthetic 2-AG were used to calculate the amounts of endogenous 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 [9] or carbachol [10]. 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 [28], 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 ([21] 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 [22]. 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 [29]. 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 [30]. 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 [13] as well as U937 macrophages [18], 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 [21] 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.

Figure 3. Inactivation of [³H]2-AG by intact J744 macrophages. (A) Incorporation of radioactivity into phospholipids (PL), diacylglycerols (DAG), triacylglycerols (TAG) and arachidonic acid (AA) after incubation of J744 cells (1.5 × 10⁶ cells·well⁻¹) with [³H]2-AG (2 µm, 10 000 cpm·0.5 mL⁻¹) for increasing periods of time at 37 °C or 4 °C. The amounts of unprocessed [³H]2-AG found inside cells are also shown. The effect of lowering the temperature to 4 °C is only shown for [³H]2-AG and [³H]phospholipid for the sake of clarity. This picture is representative of three separate experiments carried out in duplicate. (B) Effect of various inhibitors on the incorporation of radioactivity into phospholipids and AA extracted from cells after 30 min incubation with 4 µm[³H]2-AG. Data are means ± S.D. of three separate experiments and were compared by means of anova followed by Fisher’s test. *, P < 0.05; **, P < 0.01 vs vehicle treated cells. p-HMB, HO-BeHgOH; PMSF, PhCH₂SO₂F; MAFP, CH₂Δ₄AchPOF; ATFMK; Δ₄AchCOCH₂F₃.

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 [33]. We found that, as previously reported in N18TG2 cells [20] and rat brain [34], 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 [35], 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 [11]) 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 [19], is also able to recognize 2-AG as a substrate [36], and contributes significantly to the inactivation of the monoglyceride in RBL-2H3 and N18TG2 cells [21]. 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 [21]). 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 [33] and porcine brain [34] 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.

Table 1. Subcellular distribution of [³H]2-AG-and [¹⁴C]anandamide-hydrolyzing activities in rat circulating macrophages and platelets. The enzyme activity is expressed as nmol·min⁻¹·(mg protein⁻¹) of [³H]arachidonic acid or [¹⁴C]ethanolamine (in parentheses) produced from [³H]2-AG or [¹⁴C]anandamide hydrolysis, assayed, respectively, at pH 7.0 and 9.0. The activities in cells treated with 200 µg·mL⁻¹ LPS are shown. Data are means ± SD of three experiments, except for [¹⁴C]anandamide hydrolysis in platelets (n = 2).

Fraction	Platelets	Platelets + LPS	Macrophages	Macrophages + LPS
*, P < 0.05 vs vehicle treated cells, as determined by anova followed by Fisher’s test.
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)

Table 2. Effect of pH and enzyme inhibitors on [³H]2-AG-hydrolyzing activities from rat circulating macrophage and platelet membranes. The enzyme activities were expressed as percentage of the activities without any inhibitor added or as % of maximal activity. Pooled 10 000 and 100 000 g pellets from cell homogenates were used. Inhibitors were tested at pH 7.0. Data are means ± SD of three experiments.

Parameter	Platelets	Macrophages.
*, P < 0.05 for the effect of inhibitor versus vehicle treated cells as determined by anova followed by Fisher’s test.
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 [37], but while anandamide has been shown to affect macrophage function (reviewed in [38]), there is no report as yet on the effects of 2-AG on these cells.

Figure 4. Gel electrophoresis analyses of cDNA sequences obtained from RT-PCR amplification of RNAs from rat circulating macrophages and platelets. The primers for rat FAAH used are described in Materials and methods. Rat liver RNA was used as a positive control for FAAH (lane 1). In rat platelets (lane 2) a very faint band corresponding to FAAH could be seen only after prolonged over-exposure of film. cDNA from rat macrophages is shown in lane 3. Base pair markers are shown (lane 4). Representative of three experiments. Primers for rat CB1 were also used to confirm the integrity of the RNA used in these experiments, and the intense CB1 signal found for rat liver is not meant to serve as positive control for CB1 but rather as evidence for the integrity of rat liver RNA. An intense RT-PCR signal for CB1 in rat liver has been found also by others (David Shire, Sanofi Recherche, France; personal communication). As this signal was here more intense than the CB1 signals found for macrophages and platelets, it is unlikely that it was due to contaminations of rat liver with 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 [21]. 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 K_m and V_max values of 110 ± 15 µm and 7.9 ± 0.9 nmol·min⁻¹·(mg protein)⁻¹ when using [³H]2-AG as the substrate (V_max/K_m ratio = 0.072), and 170 ± 12 µm and 4.8 ± 0.6 nmol·min⁻¹· (mg protein)⁻¹ when using [³H]2-palmitoyl-glycerol (V_max/K_m ratio = 0.028) (n = 2). The presence of an uncharacterized monoacylglycerol lipase in cytosolic fractions from rat alveolar macrophages had been suggested previously [39]. 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].

Figure 5. Preliminary characterization of ‘2-AG hydrolase’ from J774 macrophages. (A) Subcellular distribution of the enzyme activity at pH 7.0, expressed in nmol·min⁻¹·(mg protein⁻¹) of [³H]arachidonic acid produced from [³H]2-AG hydrolysis. The distribution of the [¹⁴C]anandamide-hydrolyzing activity at pH 9.0 is also shown. (B) Dependency on pH of the enzyme activity. Data are expressed as percentage of maximal activity. (C) Effect of the FAAH inhibitor CH₂Δ₄AchPOF on the activity of the enzyme from J774 macrophages and, for a comparison, RBL-2H3 cells. Activity is expressed as percentage of the activity at pH 7.0 with no inhibitor added. (D) Effect of esterase inhibitors (100 µm), Δ₄AchCoCH₂F₃ (50 µm) and 1(3)-monoacylglycerols (100 µm) on the enzyme activity, expressed as percentage of the activity at pH 7.0 with no substance added. In (B-D), pooled 10 000 and 100 000 g pellets from J774 cell homogenates were used. In (A) and (D) data are means ± SD of 3 experiments. In (B) and (C) data are representative of two separate experiments with each data point carried out in triplicate, and error bars are not shown for the sake of clarity. *, P < 0.05; **, P < 0.01 vs vehicle treated cells, as determined by anova followed by Fisher’s test. 1(3)-16 : 0, 1(3)-palmitoylglycerol; 1(3)-18 : 2, 1(3)-linoleoyl-glycerol; 1(3)-18 : 3, 1(3)-γ-linolenoyl-glycerol. For other abbreviations, please see legend to Fig. 3.

Conclusions

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 [42] 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 [29]) 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 [27], 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.

Acknowledgements

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.

Footnotes

1. * Permanent address: Department of Medicine, University of Wurzburg, Germany.