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