Characterization of Ca²⁺-Sensing Receptor-Mediated Ca²⁺ Influx in Microvascular bEND.3 Endothelial Cells

Ca²⁺-sensing receptors (CaSR), activated by elevated concentrations of extracellular Ca²⁺, have been known to regulate functions of thyroid cells, neurons, and endothelial cells (EC). In this report, we studied CaSR-mediated Ca²⁺ influx in mouse cerebral microvascular EC (bEND.3 cells). Cytosolic free Ca²⁺ concentration and Mn²⁺ influx were measured by fura-2 microfluorometry. High (3 mM) Ca²⁺ (CaSR agonist), 3 mM spermine (CaSR agonist), and 10 μM cinacalcet (positive allosteric modulator of CaSR) all triggered Ca²⁺ influx; however, spermine, unlike high Ca²⁺ and cinacalcet, did not promote Mn²⁺ influx and its response was poorly sensitive to SKF 96365, a TRP channel blocker. Consistently, 2-aminoethoxydiphenyl borate and ruthenium red (two other general TRP channel blockers) suppressed Ca²⁺ influx triggered by cinacalcet and high Ca²⁺ but not by spermine. Ca²⁺ influx triggered by high Ca²⁺, spermine, and cinacalcet was similarly suppressed by A784168, a potent and selective TRPV1 antagonist. Our results suggest that CaSR activation triggered Ca²⁺ influx via TRPV1 channels; intriguingly, pharmacological, and permeability properties of such Ca²⁺ influx depended on the stimulating ligands.