Abstract
The CB1 cannabinoid receptor (CB1R) and extracellular calcium (eCa2+)-stimulated Calcium Sensing receptor (CaSR) can exert cellular signaling by modulating levels of intracellular calcium ([Ca2+]i). We investigated the mechanisms involved in the ([Ca2+]i) increase in N18TG2 neuroblastoma cells, which endogenously express both receptors. Changes in [Ca2+]i were measured in cells exposed to 0.25 or 2.5 mM eCa2+ by a ratiometric method (Fura-2 fluorescence) and expressed as the difference between baseline and peak responses (ΔF340/380). The increased ([Ca2+]i) in cells exposed to 2.5 mM eCa2+ was blocked by the CaSR antagonist, NPS2143, this inhibition was abrogated upon stimulation with WIN55212-2. WIN55212-2 increased [Ca2+]i at 0.25 and 2.5 mM eCa2+ by 700% and 350%, respectively, but this increase was not replicated by CP55940 or methyl-anandamide. The store-operated calcium entry (SOCE) blocker, MRS1845, attenuated the WIN55212-2-stimulated increase in [Ca2+]i at both levels of eCa2+. Simultaneous perfusion with the CB1 antagonist, SR141716 or NPS2143 decreased the response to WIN55212-2 at 0.25 mM but not 2.5 mM eCa2+. Co-perfusion with the non-CB1/CB2 antagonist O-1918 attenuated the WIN55212-2-stimulated [Ca2+]i increase at both eCa2+ levels. These results are consistent with WIN55212-2-mediated intracellular Ca2+ mobilization from store-operated calcium channel-filled sources that could occur via either the CB1R or an O-1918-sensitive non-CB1R in coordination with the CaSR. Intracellular pathway crosstalk or signaling protein complexes may explain the observed effects.