WIN55212-2 Modulates Intracellular Calcium via CB(1) Receptor-Dependent and Independent Mechanisms in Neuroblastoma Cells

The CB(1) cannabinoid receptor (CB(1)R) and extracellular calcium (eCa(2+))-stimulated Calcium Sensing receptor (CaSR) can exert cellular signaling by modulating levels of intracellular calcium ([Ca(2+)](i)). We investigated the mechanisms involved in the ([Ca(2+)](i)) increase in N18TG2 neuroblastoma cells, which endogenously express both receptors. Changes in [Ca(2+)](i) were measured in cells exposed to 0.25 or 2.5 mM eCa(2+) by a ratiometric method (Fura-2 fluorescence) and expressed as the difference between baseline and peak responses (ΔF(340/380)). The increased ([Ca(2+)](i)) in cells exposed to 2.5 mM eCa(2+) was blocked by the CaSR antagonist, NPS2143, this inhibition was abrogated upon stimulation with WIN55212-2. WIN55212-2 increased [Ca(2+)](i) at 0.25 and 2.5 mM eCa(2+) 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 [Ca(2+)](i) at both levels of eCa(2+). Simultaneous perfusion with the CB(1) antagonist, SR141716 or NPS2143 decreased the response to WIN55212-2 at 0.25 mM but not 2.5 mM eCa(2+). Co-perfusion with the non-CB(1)/CB(2) antagonist O-1918 attenuated the WIN55212-2-stimulated [Ca(2+)](i) increase at both eCa(2+) levels. These results are consistent with WIN55212-2-mediated intracellular Ca(2+) mobilization from store-operated calcium channel-filled sources that could occur via either the CB(1)R or an O-1918-sensitive non-CB(1)R in coordination with the CaSR. Intracellular pathway crosstalk or signaling protein complexes may explain the observed effects.