Augmentation of endogenous cannabinoid (eCB) signaling represents an emerging approach to the treatment of affective disorders. Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid to form prostaglandins, but also inactivates eCBs in vitro. However, the viability of COX-2 as a therapeutic target for in vivo eCB augmentation has not been explored. Here we utilized medicinal chemistry and in vivo analytical and behavioral pharmacological approaches to demonstrate a key role for COX-2 in the regulation of endocannabinoid (eCB) levels in vivo. A novel pharmacological strategy involving “substrate-selective” inhibition of COX-2 was used to augment eCB signaling without affecting related non-eCB lipids or prostaglandin synthesis. Behaviorally, substrate-selective inhibition of COX-2reducedanxiety-like behaviors in mice via increasede CB signaling. These data elucidate a key role for COX-2 in the regulation of eCB signaling and suggest substrate-selective pharmacology represents a viable approach for eCB augmentation with broad therapeutic potential.

Comprehensive studies of prostaglandin (PG) synthesis in murine resident peritoneal macrophages (RPM) responding to bacterial lipopolysaccharide (LPS) revealed that the primary PGs produced by RPM were prostacyclin and PGE 2 . Detectable increases in net PG formation occurred within the first hour, and maximal PG formation had occurred by 6 -10 h after LPS addition. Free arachidonic acid levels rose and peaked at 1-2 h after LPS addition and then returned to baseline. Cyclooxygenase-2 (COX-2) and microsomal PGE synthase levels markedly increased upon exposure of RPM to LPS, with the most rapid increases in protein expression occurring 2-6 h after addition of the stimulus. RPM constitutively expressed high levels of COX-1. Studies using isoform-selective inhibitors and RPM from mice bearing targeted deletions of ptgs-1 and ptgs-2 demonstrated that COX-1 contributes significantly to PG synthesis in RPM, especially during the initial 1-2 h after LPS addition. Selective inhibition of either COX isoform resulted in increased secretion of tumor necrosis factor-␣ (TNF-␣); however, this effect was much greater with the COX-1 than with the COX-2 inhibitor. These results demonstrate autocrine regulation of TNF-␣ secretion by endogenous PGs synthesized primarily by COX-1 in RPM and suggest that COX-1 may play a significant role in the regulation of the early response to endotoxemia.

This article is available online at http://www.jlr.org ture, shape, and fluidity of cell membranes (1)(2)(3). Each LysoPL comprises one nonpolar acyl chain, varying in length and degree of unsaturation, and a polar glycerophosphate headgroup. Based on the structure of the headgroup, LysoPLs belong primarily to one of six classes, including lysophosphatidic acids (LPAs), lysophosphatidylcholines (LPCs), lysophosphatidylethanolamines (LPEs), lysophosphatidylglycerols (LPGs), lysophosphatidylinositols (LPIs), and lysophosphatidylserines (LPSs), each with distinct biological functions dependent on physiological location and availability of their respective cellular receptors.LysoPLs have been shown to elicit a wide range of biological effects, including cell proliferation, intracellular calcium mobilization, metabolic activity, inflammatory and antiinflammatory processes, and neuritogenesis (4-20). These effects are generally evoked through ligand-receptor interactions, but this is not always the case. For example, high physiological concentrations of LPCs (200 µM in human plasma) call into question a receptor agonist role in some circumstances, and accruing evidence supports the hypothesis that they can elicit effects by altering membrane properties or interacting directly with proteins in ways other than saturable binding to a specific site (21-24). Abstract Lysophospholipids (LysoPLs) are bioactive lipid species involved in cellular signaling processes and the regulation of cell membrane structure. LysoPLs are metabolized through the action of lysophospholipases, including lysophospholipase A1 (LYPLA1) and lysophospholipase A2 (LYPLA2).A new X-ray crystal structure of LYPLA2 compared with a previously published structure of LYPLA1 demonstrated nearidentical folding of the two enzymes; however, LYPLA1 and LYPLA2 have displayed distinct substrate specificities in recombinant enzyme assays. To determine how these in vitro substrate preferences translate into a relevant cellular setting and better understand the enzymes' role in LysoPL metabolism, CRISPR-Cas9 technology was utilized to generate stable KOs of Lypla1 and/or Lypla2 in Neuro2a cells. Using these cellular models in combination with a targeted lipidomics approach, LysoPL levels were quantified and compared between cell lines to determine the effect of losing lysophospholipase activity on lipid metabolism. This work suggests that LYPLA1 and LYPLA2 are each able to account for the loss of the other to maintain lipid homeostasis in cells; however, when both are deleted, LysoPL levels are dramatically increased, causing phenotypic and morphological changes to the cells.-Wepy, pholipases cooperate to mediate lipid homeostasis and lysophospholipid signaling. J. Lipid Res. 2019. 60: 360-374.Supplementary key words brain lipids • eicosanoids • prostaglandins • fatty acid • protein kinases/MAP kinase • lipidomics • mass spectrometry • fluorescence microscopy • neurons • X-ray crystallography Lysophospholipids (LysoPLs) are detergent-like lipid species that play a c...

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