Determinants of cyclooxygenase-2-mediated oxidative metabolism of the endocannabinoid, 2-arachidonoyl glycerol, in vitro and ex vivo

Abstract

DETERMINANTS OF CYCLOOXYGENASE-2-MEDIATED OXIDATIVE METABOLISM OF THE ENDOCANNABINOID, 2-ARACHIDONOYL GLYCEROL, IN VITRO AND EX VIVO

Joel Musee

Dissertation under the direction of Professor Lawrence J. Marnett PGHSs catalyze the oxygenation of fatty acyl substrates (FAH) such as arachidonic acid (AA). This is the committed step in the generation of prostaglandin H2 (PGH2). PGH2 is the substrate for five downstream isomerases that lead to the generation of prostaglandins. Prostaglandins mediate a variety of physiological effects, such as pain, inflammation, fever, vascular homeostasis, and parturition, by their actions at several prostaglandin-specific G-protein coupled receptors (GPCRs). The endogenous ligands for the cannabinoid receptors, arachidonoyl ethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG) are substrates for the second isoform of PGHS, PGHS-2. Their oxygenation leads to the generation of AEA and 2-AG derived prostaglandins (PG-EAs and PG-Gs). PG-Gs have been shown to have unique actions at yet to be identified GPCRs including, mobilizing Ca2+ at the picomolar level in cell culture, and causing a concentration dependent hyperalgesia (exaggerated pain) and allodynia (pain in response to non-pain evoking stimuli). To determine the biochemical determinants of the oxygenation of 2-AG by PGHS-2, I compared the abilities of AA derived hydroperoxides to 2-AG derived peroxides as substrates and activators of the oxygenase function of PGHS. I demonstrated that PGG2 and PGG2-G were both equivalent substrates for PGHS-2. Interestingly, the oxygenation of 2-AG demonstrated and increased need for the concentrations of peroxide required to activate its oxygenation. In the presence of increased peroxide scavenging by glutathione peroxide (GPx), the oxygenation of 2-AG was almost abrogated compared to relatively unaffected AA oxygenation. Specifically, I demonstrated that the depletion of the membrane associated GPx (GPx4), leads to increased oxidant stress and peroxide tone, and significantly increased oxygenation of 2-AG in murine derived cells. These results led us to conclude that the oxygenation of 2-AG is exquisitely sensitive to the concentration of oxygenase activating peroxide. Chemical ligands specifically designed to target the peroxidase active site of PGHS have to date not been reported. I designed a chemical mimic of PGHS-2 substrate PGG2 and developed a chemical ligand that inhibited the turnover of peroxide at the peroxidase active site of PGHS. This lead compound forms a new class of molecule that can specifically and potently inhibit the oxygenation of 2-AG by PGHS-2, while having no impact on AA oxygenation. It could form a useful tool for the dissection of the physiological role of PGHS-2 mediated 2-AG oxygenation.