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

Roy C. Ziegelstein, Yali Xiong, Chaoxia He, Qinghua Hu

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)153-163
Number of pages11
JournalBiochemical and Biophysical Research Communications
Volume342
Issue number1
DOIs
StatePublished - Mar 31 2006

Keywords

  • Endothelial cells
  • Extracellular Ca -sensing receptor
  • Extracellular Ca concentration
  • Intracellular Ca concentration
  • Nitric oxide
  • Spermine

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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