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

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