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Detection of cyclooxygenase-2-derived oxygenation products of the endogenous cannabinoid 2-arachidonoylglycerol in mouse brain Amanda Morgan, Philip J. Kingsley, Michelle M Mitchener, Megan Altemus, Toni Patrick, Andrew Gaulden, Lawrence J. Marnett, and Sachin Patel ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00499 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 4, 2018
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ACS Chemical Neuroscience
Detection of cyclooxygenase-2-derived oxygenation products of the endogenous cannabinoid 2-arachidonoylglycerol in mouse brain Amanda Morgan1,3, Phillip J. Kingsley2, Michelle M. Mitchener2, Megan Altemus1, Toni Patrick1, Andrew Gaulden 1, Lawrence J. Marnett2, and Sachin Patel1,3,4* Department of 1Psychiatry and Behavioral Sciences, 2A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry, and Pharmacology, Vanderbilt Institute of Chemical Biology, 3Department of Molecular Physiology & Biophysics and 4The Vanderbilt Brain Institute Vanderbilt University Medical Center, Nashville, TN 37232
*Correspondence: Sachin Patel, MD, PhD Associate Professor Departments of Psychiatry and Behavioral Sciences, Molecular Physiology & Biophysics, and Pharmacology 2213 Garland Avenue Medical Research Building IV, Rm 8425B Vanderbilt University Medical Center Nashville, TN 37232 Email:
[email protected] Phone: (615) 936-7768 Fax: (615) 322-1462
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Abstract Cyclooxygenase-2 (COX-2) catalyzes the formation of prostaglandins, which are involved in immune regulation, vascular function, and synaptic signaling. COX-2 also inactivates the endogenous cannabinoid (eCB) 2-arachidonoylglycerol (2-AG) via oxygenation of its arachidonic acid backbone to form a variety of prostaglandin glyceryl esters (PG-Gs). Although this oxygenation reaction is readily observed in vitro and in intact cells, detection of COX-2derived 2-AG oxygenation products has not been previously reported in neuronal tissue. Here we show that 2-AG is metabolized in the brain of transgenic COX-2-overexpressing mice and mice treated with the lipopolysaccharide to form multiple species of PG-Gs that are detectable only when monoacylglycerol lipase is concomitantly blocked. Formation of these PG-Gs is prevented by acute pharmacological inhibition of COX-2. These data provide evidence that neuronal COX2 is capable of oxygenating 2-AG to form a variety PG-Gs in vivo and support further investigation of the physiological functions of PG-Gs.
Keywords Endocannabinoid, prostaglandin glyceryl esters, cannabinoid, monoacylglycerol lipase, Lumiracoxib, lipopolysaccharide, prostamide
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Introduction Endogenous cannabinoids (eCBs) are a class of bioactive lipid signaling molecules implicated in a variety of physiological processes including appetite regulation, energy homeostasis, cardiovascular function, motivation, anxiety, and sleep1-5. Within neurons, the eCBs 2-AG and anandamide (AEA) are generated in an activity-dependent manner and serve primarily as retrograde signaling molecules that reduce presynaptic neurotransmitter release via activation of type-1 cannabinoid receptors6. eCBs may also have direct postsynaptic effects on membrane potential and neuronal excitability7, 8. Despite these well-established roles of eCBs, how these lipids are metabolized via non-canonical routes to generate potentially distinct signaling molecules is an active area of investigation. The exact biosynthesis of AEA is poorly understood, but AEA is primarily metabolized by neuronal fatty-acid amide hydrolase (FAAH) to arachidonic acid (AA)9. However, COX-2 can also metabolize AEA to form prostaglandin ethanolamides (PG-EAs; prostamides)10. The prostamides include PGE2-EA, PGD2-EA, and PGF2α-EA and have been previously detected in vivo, supporting the notion that COX-2 serves as a non-canonical eCB metabolic enzyme. For example, PGF2α-EA is detected upon COX-2 upregulation in both rodent dorsal spinal neurons after carrageenan-induced knee inflammation11 and in preadipocyte cells where PGF2α-EA acts to prevent adiopogenesis12. It has been suggested that the ability to detect prostamides in vivo may be facilitated by their relative metabolic stability and lack of metabolism by FAAH13. 2-AG is a highly abundant eCB that mediates retrograde inhibition at central glutamatergic and GABAergic synapses14. Synaptic 2-AG is generated via the sequential actions of phospholipase C and diacylglycerol lipase-α, in response to a variety of activity-dependent mechanisms. In addition to its signaling functions, 2-AG is thought to be the most common
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source of AA for PG production in the CNS15. The synaptic actions of 2-AG are terminated predominantly via monoacylglycerol lipase (MAGL), but also by α/β-hydrolase domain 6 and 12, as well as COX-216. Indeed, 2-AG is efficiently oxygenated by COX-2 in vitro to form prostaglandin glyceryl esters (PG-Gs), and 2-AG-mediated retrograde synaptic inhibition can be enhanced by pharmacological inhibition of COX-210,
17-20
. Despite being detected in
macrophages following lipopolysaccharide (LPS) administration21-23 and in inflamed rat hindpaw24, COX-2-derived oxidative metabolites of 2-AG in brain have remained elusive. It is possible that PG-Gs are hydrolytically unstable in vivo or rapidly further metabolized, and thus have eluded detection. Given that PG-Gs have been recently identified as endogenous agonists of P2Y6 receptors25 and implicated as neuronal signaling molecules16 and pain modulators in the periphery24, a clearer understanding of the conditions under which PG-Gs are formed is needed. Here we use an LPS model of neuroinflammation, a transgenic COX-2 overexpression model, and pharmacological approaches combined with stable isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and quantify PG-G formation in mouse brain for the first time. These data provide evidence that neuronal COX-2 can produce PG-Gs in the brain when 2-AG levels are elevated via MAGL inhibition and provide direct support for the contention that neuronal COX-2 metabolizes 2-AG in vivo.
Results and Discussion We first optimized our analytical detection of PG-Gs by LC/MS/MS using deuterated PGE2-G and PGD2-G as authentic standards. PGE2-G, PGD2-G and PGF2α-G were identified and quantified by LC-MS/MS analysis. These three analytes and the internal standards (PGE2-G-d5 and PGF2α-G-d5) were ionized by complexation with the ammonium cation, resulting in an
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[M+NH4]+ complex. Multiple product ions were observed when this complex was subjected to collision induced dissociation (CID). For example, the CID spectrum of the [PGE2-G+NH4]+ complex (m/z = 444.3) is shown in Fig. 1a, where the product ions at m/z 391.2, 317.3, and 299.0 are observed. Fragmentation of the [PGD2-G+NH4]+ complex gives a similar spectrum (not shown). For both PGE2-G and PGD2-G, the selected reaction monitoring (SRM) transition m/z 444.2 – 391.1 was used for quantitative analysis as this transition gave the greatest signal:noise ratio. Table S1 gives the SRM transitions for all analytes reported here. The chromatographic system we employed here was able to resolve the sn-1 and sn-2 isomers of PGE2-G, PGD2-G and PGF2α-G (Fig. S1). PG-Gs are monoacylglycerols and thus will undergo isomerization from the presumed original sn-2 conformation to the thermodynamically favored sn-1 conformation. Importantly, LC-MS/MS analysis using alternative SRM transitions (m/z 444.2 – 317.1 & 444.2 – 299.0) generated nearly identical chromatograms to that generated by m/z 444.2 – 391.1 transition, providing compelling support for our accurate and reliable detection of authentic PGGs using our analytical approach (Fig. S2 a-b).
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a 100%
391. 2 -74 (C3 O2 H 6) OH
O O
80% O
Rel. Int. (%)
HO
OH
H
OH
NH 3
60% -18 (H 2 O)
444. 2
m /z [PGE2-G + NH4]+ = 444.3
299.0
373. 3
40%
409. 3
281. 1
20%
200
240
427.2
317.3
280
320
360
400
440
m/z (Da)
****
250
c
***
d
600
AA - nmol/g
2-AG - nmol/g
200
800
150 100
AEA - pmol/g
b
400
30
20
10
200 50 0
Ve h
40
20
0.6 0.4 0.2 0.0
84
JZ L1 84
1.0 0.8 0.6 0.4 0.2
4 18 JZ L
JZ L1 84
0.0
Ve hi cl e
4 18 JZ L
Ve h
ic le
0.0
Ve hi cl e
4 18
0.8
icl e
0.2
** 20
Ve h
0.4
j
1.0
PGF2α-G- pmol/g
0.6
40
JZ L
Ve h
PGD2-G - pmol/g
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60
0 ic le
84 JZ L1
ic le Ve h
i
1.0
JZ L1
icl e
84
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g
80
PGF2α - pmol/g
5
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ic le
*
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PGD2 - pmol/g
PGE2 - pmol/g
e
JZ L
Ve h
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0
PGE2-G - pmol/g
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Figure. 1. PG-Gs are not detected in C57/Bl6J mouse brain after MAGL inhibition. (a) CID spectra of PGE2-G. Upon CID, the ammonium complex of PGE2-G (m/z 444) undergoes sequential loss of NH3 and two molecules of H2O to generate the product ions at m/z 427, 409 and 391, respectively. Subsequently, m/z 391 species will undergo a loss of H2O and C3H6 O2 (74 Da) – in either order – to generate the product ions at m/z 373, 317 and 299. Circled peak 391 was used for quantitative analysis while 299 and 317 were used for confirmatory analysis (see Fig. S1). (b-d) Effects of JZL184 on 2-AG, AA, and AEA levels in mouse brain. (e-g) Effects of JZL184 on prostaglandin levels in mouse brain. (h-j) Effects of JZL184 on PG-G levels in mouse brain, relative to vehicle-treated mice. Each point (circle) represents a biological replicate (one mouse). Veh n=10, JZL184 n=9. Error bars represent S.E.M. * p
