Prostaglandin E2 glycerol ester, an endogenous COX-2 metabolite of 2-arachidonoylglycerol, induces hyperalgesia and modulates NFkappaB activity

Abstract

Background and purpose: Recombinant cyclooxygenase-2 (COX-2) oxygenates 2-arachidonoylglycerol (2-AG) in vitro. We examined whether prostaglandin E2 glycerol ester (PGE2-G), a COX-2 metabolite of 2-AG, occurs endogenously and affects nociception and immune responses.

Experimental approach: Using mass spectrometric techniques, we examined whether PGE2-G occurs in vivo and if its levels are altered by inhibition of COX-2, monoacylglycerol (MAG) lipase or inflammation induced by carrageenan. We also examined the effects of PGE2-G on nociception in rats and NFkappaB activity in RAW264.7 cells.

Key results: PGE2-G occurs endogenously in rat. Its levels were decreased by inhibition of COX-2 and MAG lipase but were unaffected by carrageenan. Intraplantar administration of PGE2-G induced mechanical allodynia and thermal hyperalgesia. In RAW264.7 cells, PGE2-G and PGE2 produced similar, dose-related changes in NFkappaB activity. PGE2-G was quickly metabolized into PGE2. While the effects of PGE2 on thermal hyperalgesia and NFkappaB activity were completely blocked by a cocktail of antagonists for prostanoid receptors, the same cocktail of antagonists only partially antagonized the actions of PGE2-G.

Conclusions and implications: Thermal hyperalgesia and immunomodulation induced by PGE2-G were only partially mediated by PGE2, which is formed by metabolism of PGE2-G. PGE2-G may function through a unique receptor previously postulated to mediate its effects. Taken together, these findings demonstrate that 2-AG is oxygenated in vivo by COX-2 producing PGE2-G, which plays a role in pain and immunomodulation. COX-2 could act as an enzymatic switch by converting 2-AG from an antinociceptive mediator to a pro-nociceptive prostanoid.

Figure 1

Identification of PGE₂-G in the rat hind paw. (a) Identical mass spectra of material in rat hindpaw extract and PGE₂-G. The mass spectrum of the material in rat hindpaw extract (top plot) and synthetic PGE₂-G (bottom plot) via QqTOF LC/MS/MS were virtually identical. The prominent parent ion at m/z 444.5 [M+ NH₃]⁺ of PGE₂-G was observed for the material in the paw extract and synthetic PGE₂-G. The inset shows the structure of ammonium adduct of PGE₂-G. (b) Multiple-reaction monitoring on a triple-quadrupole mass spectrometer revealed co-eluting peaks with molecular/fragment ions at m/z 444.5/391.2 from PGE₂-G and the material in rat hindpaw extract. LC, liquid chromatography; MS, mass spectrometry; PGE₂-G, prostaglandin E₂ glycerol ester; QqTOF, quadrupole time-of-flight.

Figure 2

COX inhibitors modulate the endogenous levels of PGE₂ and PGE₂-G in the rat hind paw. (a) Effects of COX inhibitors on the in vitro production of PGE₂ (left) and PGE₂-G (right). AA or 2-AG (6 μM) were incubated with COX-2 (5.9 μg protein=69.4 U) at 37 °C for 3 min. As shown, 42% of AA and 58% of 2-AG were oxygenated into PGE₂ and PGE₂-G, respectively (shown as 100% yield). The production of PGE₂ and PGE₂-G was significantly decreased in the presence of the nonselective COX inhibitor ibuprofen (1 mM) or the selective COX-2 inhibitor nimesulide (500 μM) (^***P<0.0001). (b) Effects of COX inhibitors on the in vivo production of PGE₂ (left) and PGE₂-G (right). Ibuprofen (100 mg kg⁻¹, i.p.) or nimesulide (50 mg kg⁻¹, i.p.) was injected, and 2 h later the rat hind paws were removed, homogenized, extracted, and partially purified, and subjected to LC/MS/MS to measure the endogenous level of PGE₂ and PGE₂-G. Both ibuprofen and nimesulide significantly decreased the levels of PGE₂ and PGE₂-G in the rat hind paw (^***P<0.0001). AA, arachidonic acid; 2-AG, 2-arachidonoylglycerol; COX-2, cyclooxygenase-2; i.p., intraperitoneally; LC, liquid chromatography; MS, mass spectrometry; PGE₂, prostaglandin E₂; PGE₂-G, prostaglandin E₂ glycerol ester. ^#P<0.05 (ibuprofen vs nimesulide).

Figure 3

Inhibition of MAG lipase modulates the endogenous levels of 2-AG and PGE₂-G in the rat hind paw. The MAG lipase inhibitor, URB602, was injected into rats (1 μmol 50 μl⁻¹, i.pl.) alone or in combination with the selective COX-2 inhibitor nimesulide (50 mg kg⁻¹, i.p.), and 2 h later rat hind paws were removed, homogenized, extracted, partially purified and subjected to LC/MS/MS to measure endogenous level of PGE₂ and PGE₂-G. (a) Both URB602 and URB602+nimesulide significantly increased the level of 2-AG in the rat hind paw (^*P<0.05, ^***P<0.0001, ^#P<0.05). (b) Both URB602 and URB602+nimesulide significantly decreased the level of PGE₂-G in the rat hind paw (^***P<0.0001). (c) URB602 alone did not change the level of PGE₂ in the rat hind paw (P>0.05). URB602+nimesulide significantly decreased the level of PGE₂ in the rat hind paw (^***P<0.0001, ^###P<0.05). 2-AG, 2-arachidonoylglycerol; COX-2, cyclooxygenase-2; i.p., intraperitoneally; i.pl., intraplantar; LC, liquid chromatography; MAG, monoacylglycerol; MS, mass spectrometry; PGE₂, prostaglandin E₂; PGE₂-G, prostaglandin E₂ glycerol ester; URB602, [1,1′-biphenyl]-3-yl-carbamic acid cyclohexyl ester.

Figure 4

PGE₂-G was hydrolysed into PGE₂ and other unknown metabolites after it was injected into the rat hind paw. (a) Levels of PGE₂-G following its injection into the rat hind paw. Hind paws were removed at 0, 15, 30, 60 or 120 min following PGE₂-G injection (50 μg 50 μl⁻¹, i.pl.), extracted, partially purified and subjected to LC/MS/MS to measure the level of PGE₂-G. At 0 and 15 min, only 6.8 and 1%, respectively, of the injected amount of PGE₂-G were recovered (^***P<0.0001, ^**P<0.001). The amount of PGE₂-G fell to the baseline level 30 min after injection. (b) Rat hind paws were removed at 0, 15, 30, 60 or 120 min after PGE₂-G injection (50 μg 50 μl⁻¹, i.pl) and the levels of PGE₂ were assessed by LC/MS/MS. Immediately after injection (0 min), approximately 10% of the injected amount of PGE₂-G was hydrolysed into PGE₂ and the level of PGE₂ then decreased with time (^***P<0.0001, ^*P<0.05). i.pl., intraplantar; LC, liquid chromatography; MS, mass spectrometry; PGE₂, prostaglandin E₂; PGE₂-G, prostaglandin E₂ glycerol ester.

Figure 5

PGE₂-G produced thermal hyperalgesia and mechanical allodynia. (a) After determination of baseline withdrawal latencies from a radiant heat source, PGE₂-G (0.1, 1 or 10 μg 50 μl⁻¹, i.pl.) was administered and withdrawal latencies were measured at 20, 40, 60, 80, 100, 120, 160, 200 and 240 min after injection. PGE₂-G produced a dose- and time-dependent decrease in withdrawal latency (F_3,41=17.7, P<0.0001). (b) Following determination of mechanical withdrawal thresholds with von Frey filaments, PGE₂-G (0.1 or 10 μg 50 μl⁻¹, i.pl.) was injected. Withdrawal thresholds were recorded at 20, 40, 60, 80, 100, 120, 160 and 200 min after injection. PGE₂-G produced a dose- and time-dependent decrease in withdrawal thresholds (F_2,19=19.3, P<0.0001). i.pl., intraplantar; PGE₂-G, prostaglandin E₂ glycerol ester.

Figure 6

PGE₂-G-induced thermal hyperalgesia is partially mediated by prostanoid receptors. (a) After determination of baseline withdrawal latencies from a radiant heat source, PGE₂ (0.1 μg 50 μl⁻¹, i.pl.) or PGE₂ (0.1 μg 50 μl⁻¹, i.pl.) combined with a cocktail of EP₁, EP₂, EP₃ and EP₄ antagonists (L-335677, AH6809, L-826266 and L-161982) (20 nmol 50 μl⁻¹, i.pl.) was administered and withdrawal latencies were recorded for the following 160 min. PGE₂ (0.1 μg) caused a decrease in withdrawal latency (P<0.0001), which was completely blocked by a cocktail of EP₁, EP₂, EP₃ and EP₄ antagonists. (b) Using the same treatment protocol as in (a), PGE₂-G (1 μg 50 μl⁻¹, i.pl.) caused a decrease in withdrawal latency, but the cocktail of prostanoid receptor antagonists (20 nmol 50 μl⁻¹, i.pl.) only partially blocked its effects. The degree of reversal was significantly less than that produced by PGE₂ (P<0.0001). EP, PGE₂ receptor; i.pl., intraplantar; PGE₂-G, prostaglandin E₂ glycerol ester.

Figure 7

The immunomodulatory effects of PGE₂-G on NFκB activity in RAW264.7 cells was partially mediated by prostanoid receptors. RAW264.7 cells were transiently transfected with the NFκB-Luc plasmid and 24 h later the cells were treated with lipopolysaccharide (100 ng ml⁻¹) for 2 h, followed by various concentrations of PGE₂, PGE₂-G or vehicle (dimethylsulphoxide), or a combination with a cocktail of EP₁, EP₂, EP₃ and EP₄ antagonists (L-335677, AH6809, L-826266 and L-161982, 50 μM preincubated for 30 min). (a) PGE₂ produced a bell-shaped dose–response curve of NFκB activity, which was completely blocked by a cocktail of prostanoid receptor antagonists. (b) PGE₂-G produced a bell-shaped dose–response curve of NFκB activity, which was only partially blocked by a cocktail of prostanoid receptor antagonists. The degree of inhibition by the cocktail of antagonists was significantly greater for cells treated with PGE₂ compared with those treated with PGE₂-G (P<0.04). EP, PGE₂ receptor; NFκB; nuclear factor-κB; PGE₂, prostaglandin E₂; PGE₂-G, prostaglandin E₂ glycerol ester.