(Endo)cannabinoids mediate different Ca2+ entry mechanisms in human bronchial epithelial cells

Abstract

In human bronchial epithelial (16HBE14o-) cells, CB1 and CB2 cannabinoid receptors are present, and their activation by the endocannabinoid virodhamine and the synthetic non-selective receptor agonist CP55,940 inhibits adenylyl cyclase and cellular interleukin-8 release. Here, we analyzed changes in intracellular calcium ([Ca2+]i) evoked by Δ9-tetrahydrocannabinol (Δ9-THC), CP55,940, and virodhamine in 16HBE14o- cells. Δ9-THC induced [Ca2+]i increase and a large transient [Ca 2+]i mobilization, the latter probably reflecting store-depletion-driven capacitative Ca2+ entry (CCE). In contrast, CP55,940 induced a rather moderate Ca2+ influx and a sustained [Ca2+]i mobilization. CP55,940-induced Ca2+ influx was inhibited by Ni2+, indicating CCE, possibly mediated by transient receptor potential channel TRPC1, the mRNA of which is expressed in 16HBE14o- cells. CP55,940-induced calcium alterations were mimicked by virodhamine concentrations below 30∈μM. Interestingly, higher virodhamine induced an additional Ca2+ entry, insensitive to Ni 2+, but sensitive to the TRPV1 antagonist capsazepine, the TRPV1-TRPV4 inhibitor ruthenium red, and the non-CCE (NCCE) inhibitors La 3+ and Gd3+. Such pharmacological profile is supported by the presence of TRPV1, TRPV4, and TRPC6 mRNAs as well as TRPV1 and TRPC6 proteins in 16HBE14o- cells. Cannabinoid receptor antagonists increased virodhamine-induced Ca2+ entry. Virodhamine also enhanced arachidonic acid release, which was insensitive to cannabinoid receptor antagonism, but sensitive to the phospholipase A2 inhibitor quinacrine, and to capsazepine. Arachidonic acid induced [Ca2+] i increase similar to virodhamine. Collectively, these observations suggest that [Ca2+]i alterations induced by Δ9-THC, CP55,940 and by low concentrations of virodhamine involve mobilization and subsequent CCE mechanisms, whereas such responses by high virodhamine concentrations involve NCCE pathways.