Mechanism of cannabinoid-mediated elevation of intracellular calcium T cells

Cannabinoids are a class of over sixty structurally-related plant-derived compounds present in the marijuana plant, Cannabis sativa. Apart from their psychoactive and behavioral effects, cannabinoids are widely known to possess immunomodulatory properties. Generally, it is believed that cannabinoids exert their effects on physiology through the G protein-coupled cannabinoid receptors, CB1 and CB2, expressed primarily in the brain and immune system, respectively. In T cells, cannabinoid treatment has been demonstrated extensively to modulate T cell activation-related events, especially the expression of interleukin (IL)-2. The regulation of IL-2 transcription is critically dependent on intracellular calcium ([Ca2+]i), and the Ca2+-dependent transcription factor, nuclear factor of activated T cells (NFAT). Prior results from this laboratory have found a strong correlation between the modulation of IL-2 expression by cannabinoids and the modulation of NFAT activation and nuclear translocation. However, little is known about the cellular mechanism by which cannabinoids exert their effects on T cells. The present dissertation project, therefore, first examined the hypothesis that the suppression of IL-2 expression in T cells by cannabinoids was dependent on the cannabinoid receptor-mediated elevation of [Ca 2+]i. The first objective of the present studies was to examine the effect of cannabinoids, Delta9-THC and CP55,940, on IL-2 expression and [Ca2+]i elevation in the human T cell lines, HPB-ALL and Jurkat E6-1. It was found that while both Delta 9-THC and CP inhibited the expression of IL-2, only Delta 9-THC treatment resulted in the elevation of [Ca2+] i. Therefore, it was concluded that the modulation of [Ca2+] i and inhibition of IL-2 production by cannabinoids may be simultaneous, yet unrelated events. Regardless, the mechanism by which Delta9-THC elevated [Ca2+]i was characterized as the second objective of the present studies in the HPB-ALL cells, as [Ca2+] i plays a critical role in many T cell processes including activation of enzymes, triggering of apoptotic pathways, and induction of T cell anergy. Results showed that Delta9-THC elevated [Ca2+] i in a cannabinoid receptor antagonist-sensitive and Ca2+ store-independent manner through the TRPC1 class of receptor-operated cation channels (ROCCs). In addition, it was established that the robust induction of [Ca2+]i influx in resting T cells was a characteristic unique to the tricyclic classical cannabinoid compounds, which was not observed with either bicyclic congeners or the eicosinoid-derived endocannabinoids. A final objective of the current dissertation project was to determine unequivocally whether the CB1 and CB2 receptors were involved in the mechanism by which tricyclic cannabinoid compounds elevated [Ca2+]i. Measurements of [Ca2+]i performed in the wildtype and CB1-/-/CB2-/- murine splenocytes demonstrated that the tricyclic cannabinoids induced a rise in [Ca2+] i independently of both CB1 and CB2 receptors. Altogether, the studies from the present dissertation project suggest that tricyclic cannabinoid compounds elevate [Ca2+]i in a CB1 and CB2 receptor- and intracellular Ca2+ store-independent manner through ROCCs in T cells. The elevation of [Ca2+]i mediated by a novel cannabinoid receptor may partly explain the mechanism by which tricyclic cannabinoids modulate T cell function.