WIN55212-2 Modulates Intracellular Calcium via CB₁ Receptor-Dependent and Independent Mechanisms in Neuroblastoma...

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 (ΔF_340/380). 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.