TY - JOUR
T1 - Expression of a functional extracellular calcium-sensing receptor in human aortic endothelial cells
AU - Ziegelstein, Roy C.
AU - Xiong, Yali
AU - He, Chaoxia
AU - Hu, Qinghua
N1 - Funding Information:
This work was supported in part by American Heart Association Scientist Development Grant 0335020N to Q. Hu.
Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2006/3/31
Y1 - 2006/3/31
N2 - 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.
AB - 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.
KW - Endothelial cells
KW - Extracellular Ca -sensing receptor
KW - Extracellular Ca concentration
KW - Intracellular Ca concentration
KW - Nitric oxide
KW - Spermine
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U2 - 10.1016/j.bbrc.2006.01.135
DO - 10.1016/j.bbrc.2006.01.135
M3 - Article
C2 - 16472767
AN - SCOPUS:32644474035
VL - 342
SP - 153
EP - 163
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 1
ER -