Actions of cannabinoids on amoebae

Al-Hammadi, Israa and Parry, Jackie and Wright, Karen (2020) Actions of cannabinoids on amoebae. PhD thesis, Lancaster University.

Text (2020Israa Al-hammadi PhD)
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Abstract

Endocannabinoids, such as Anandamide (AEA), are lipid compounds which, together with the receptors they bind to, form the endocannabinoid system (ECS) which, in animals, modulates mood, cognition, appetite etc. The ECS can be activated by phytocannabinoids such as cannabidiol (CBD) and as such, interest in its therapeutic use has grown substantially over recent years. However, not all of CBD’s effects can be explained by its binding to cannabinoid receptors and many other targets have been proposed. A better understanding of these alternative targets would increase the therapeutic potential of this phytocannabinoid in the future. Single-celled protists, such as amoebae, do not possess cannabinoid receptors yet they respond negatively to endo- and phyto-cannabinoids, suggesting they possess alternative targets only. This study therefore examined the involvement of three alternative targets, i.e., the Peroxisome-Proliferator Activated Receptor (PPAR), Dopamine Receptor and Serotonin Receptor, in the action of AEA and CBD on amoebae. Of the 20-amoeba species tested, only 6 showed a reduction in population growth in the presence of CBD (IC50, 0.98-7.31μM), i.e., Hartmannella cantabrigiensis, Naegleria gruberi, Vahlkampfia avara, Vermamoeba vermiformis, Acanthamoeba castellanii and Flamella arnhemensis. All but the latter two species also showed reduced population growth in the presence of AEA (IC50, 0.96-9.89μM). The negative effect of AEA could not be alleviated by blocking receptors with antagonists against the three PPAR isoforms (PPARs α, β and γ), the dopamine receptor or the serotonin receptor, suggesting that none were involved in the mode of action of AEA. However, the negative effect of CBD was alleviated with the antagonist for the PPARα receptor in V. vermiformis and that for the serotonin receptor (specifically 5-HT1A) in N. gruberi. Interestingly, CBD significantly affected the feeding behaviour of these two amoebae (but AEA did not), by stopping amoebic feeding completely (and causing a lag in the ingestion of prey), which was then followed by a reduced ingestion rate; both of which were dose-dependent. Further work with V. vermiformis showed that this CBD-induced cessation in feeding was not due to the halting of phagosome processing and defecation however, there was an interaction between CBD and C-type lectins recognising mannose, N-Acetyl-D-glucosamine (GlcNAc) and 2 N-Acetyl-D-galactosamine (GalNAc). Receptor-mediated phagocytosis in V. vermiformis involved all three C-type lectins and although CBD did not appear to directly interfere (bind) with any of them, its presence (together with sugars to block these receptors) led to a synergistic reduction in ingestion rate with mannose and GalNac (but no effect on lag) and an extension of the lag with GlcNAc (but no effect on ingestion rate). The ‘CBD-receptor’ (putative PPARα) and GlcNAc receptor were therefore considered to be involved with phagosome formation and not prey capture and phagosome filling. It is therefore hypothesised that, because only the three PPAR isoforms exist in vertebrates, V. vermiformis possesses a promiscuous PPAR-like molecule that can bind CBD at the same site it binds the PPARα antagonist. The instantaneous nature of the feeding lag suggests the presence of a non-genomic PPAR response whereby in its ligated state PPAR cannot bind with a necessary protein (possibly Syk, LAT or PKC) to initiate a downstream signaling cascade, which would normally culminate in actin polymerisation and phagocytic cup formation. In conclusion, amoebae provide an ideal model organism to evaluate the significance of alternative targets in the functioning of the ECS. And, as is the case with animals, multiple alternative targets appear to be present in amoebae; PPARα (for CBD) in one species, Serotonin Receptor (for CBD) in another species, with the other alternative targets for CBD and indeed AEA currently unknown. And, considering multicellularity originated from free-living single-celled protists, amoebae have also provided an opportunity to investigate the historical functioning of the ECS which appears to be in the main, involved with the feeding response.