Characterization of novel N-Acyl neurotransmitter/amino acid conjugates (NAAN) on CB1 and TRP family member receptors

Abstract

"A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy""26th of June 2014"Includes bibliographical references.The endocannabinoid family has been extended in recent years from its two first well described members, anandamide and 2-arachidonoyl glycerol, to include a new family of related endogenous lipid-derived compounds composed of a neurotransmitter or amino acid head and a fatty acid tail termed N-acyl amino acid/neurotransmitter conjugate (NAAN). This family of molecules have important effects on G protein-coupled receptors, including the cannabinoid receptors CB1 and CB2; but also have diverse effects on members of the transient receptor potential (TRP) family and other ion channels, thus making them important candidates for the modulation of pain. The aim of this thesis was to investigate the effect of some of these NAAN on heterologously expressed CB1, CB2 and TRPA1 receptors. The effects of NAAN on the signalling pathways and desensitization mechanisms of the CB1 and CB2 receptors were investigated. For the TRPA1 receptor, we investigated how these chemically unreactive compounds might activate a receptor commonly seen as activated by electrophilic agonists, temperature, pressure and pH. Studies were conducted using a 96-wells micro plate reader measuring in real time changes in membrane potential and calcium levels in cell lines expressing recombinant receptors and ion channels.N-arachidonoyl dopamine (NADA) coupled to Gq-mediated elevations ofcalcium in CB1 and CB2 expressing cells, but did not act through these receptors to modulate adenylyl cyclase, K channels or ERK activation. NADA did not change the dynamics of CB1 receptor desensitization or affect the allosteric modulators ORG 27569 and PSNCAM-1. Thus, NADA is a highlyxiiibiased agonist of CB1 and CB2 toward Gq coupling. Various NAANs activated the TRPA1 receptor, but were less efficacious than the parent molecule, arachidonic acid. Activation of TRPA1 was reduced in a mutant TRPA1 receptor lacking the cysteines necessary for the activation of the receptor by reactive compounds, hinting at possibly another binding site for the receptor. The activation of TRPA1 by arachidonic acid was not blocked by inhibiting enzymes which metabolize it. On the contrary, NDGA, a lipoxygenase inhibitor, activates the TRPA1 receptor and caused cold hyperalgesia in an acetone test in mice .The findings of this thesis give new insight in the intricacies of the action of NAANs and related compounds on CB1, CB2 as well as the TRPA1 receptor.Mode of access: World wide web1 online resource (xvi, 228 pages) illustrations, graphs, chart