An Investigation into the Molecular Targets Mediating Cannabidiol Action in the Free-living Ciliate Tetrahymena pyriformis

Tims, Nathan and Parry, Jackie and Wright, Karen (2021) An Investigation into the Molecular Targets Mediating Cannabidiol Action in the Free-living Ciliate Tetrahymena pyriformis. Masters thesis, Lancaster University.

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Abstract

This study aimed to characterised the effect of cannabidiol (CBD) on the feeding behaviour of Tetrahymena pyriformis and to determine its molecular target(s). Experiments involved feeding T. pyriformis with a Synechococcus sp. prey (live or dead) or latex beads, in the presence/absence of CBD with/without the pre-blocking of specific receptors or components of the MAPK pathway. CBD caused a reduction in ingestion rate (IR) at ca. 2 µM until it was zero at ≥2.5 µM and this gave rise to a feeding lag. The lag was temporary, with its duration being positively correlated to CBD concentration. Post-lag, the ciliate began to feed again, and the resulting IR correlated positively with the duration of the lag; reflecting the satiation level of the ciliate, as food vacuoles continued to be defecated during this feeding lag. A feeding lag of >32 min, with the defecation of ca. 3 food vacuoles, induced ‘starvation’ in T. pyriformis and this resulted in the ciliate exhibiting hyperphagia. The role of feeding receptors (C-type lectins [CLRs] and Scavenger Receptors [SRs]) in this CBD response was evaluated. T. pyriformis utilised SRs and CLRs for mannose, N-acetylglucosamine (GlcNAc), and N- acetylgalactosamine (GalNAc) for the ingestion of live Synechococcus, which were ingested at higher rates, and gave rise to more food vacuoles, than dead cells/beads. Heat-treatment destroyed the mannose, GlcNAc and SR ligands on Synechococcus cells, leaving only GalNAc residues to facilitate the uptake of dead cells. None of the receptors were involved in the uptake of beads. The blocking of these receptors, prior to adding CBD, did not abolish the CBD-induced feeding lag but post-lag IRs were not hyperphagic; being equivalent to that expected when blocking these receptors in the absence of CBD. This suggests that these receptors do not directly interact with CBD. The blocking of other receptors (GCPRs and TRPV, PPAR, Dopamine, Serotonin and Adenosine), which are known to interact with CBD in other eukaryotic cells, was also tested but none abolished the CBD-induced feeding lag, suggesting that T. pyriformis has few, if any, CBD-interacting receptors that are associated with its feeding mechanism. The ciliate also did not perceive CBD as an external stress as no evidence of activation of the MAPK pathway was recorded. It was therefore proposed that the target(s) of CBD, for reducing IR, might be vacuole formation/membrane recycling (which would prevent the development of nascent food vacuoles) and/or ciliary function (which would reduce swimming speed and the formation of feeding currents in the oral cavity); both of which involve actin, microtubules, and dynein. The mechanism behind the action of CBD on T. pyriformis feeding is more complicated than first thought, but with its considerably reduced repertoire of potential targets, compared to human cells, elucidating its targets, and whether these are evolutionary conserved, might be achievable. It is hoped that the knowledge gleaned will aid in the future implementation of CBD as an effective therapeutic agent for a number of conditions.