Expression of a functional extracellular calcium-sensing receptor in human aortic endothelial cells

Extracellular Ca²⁺ concentration ([Ca²⁺]_o) regulates the functions of many cell types through a G protein-coupled [Ca ²⁺]_o-sensing receptor (CaR). Whether the receptor is functionally expressed in vascular endothelial cells is largely unknown. In cultured human aortic endothelial cells (HAEC), RT-PCR yielded the expected 555-bp product corresponding to the CaR, and CaR protein was demonstrated by fluorescence immunostaining and Western blot. RT-PCR also demonstrated the expression in HAEC of alternatively spliced variants of the CaR lacking exon 5. Although stimulation of fura 2-loaded HAEC by several CaR agonists (high [Ca²⁺]_o, neomycin, and gadolinium) failed to increase intracellular Ca²⁺ concentration ([Ca²⁺]_i), the CaR agonist spermine stimulated an increase in [Ca²⁺]_i that was diminished in buffer without Ca²⁺ and was abolished after depletion of an intracellular Ca²⁺ pool with thapsigargin or after blocking IP₃- and ryanodine receptor-mediated Ca²⁺ release with xestospongin C and with high concentration ryanodine, respectively. Spermine stimulated an increase in DAF-FM fluorescence in HAEC, consistent with NO production. Both the increase in [Ca²⁺]_i and in NO production were reduced or absent in HAEC transfected with siRNA specifically targeted to the CaR. HAEC express a functional CaR that responds to the endogenous polyamine spermine with an increase in [Ca²⁺]_i, primarily due to release of IP₃- and ryanodine-sensitive intracellular Ca²⁺ stores, leading to the production of NO. Expression of alternatively spliced variants of the CaR may result in the absence of a functional response to other known CaR agonists in HAEC.