期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 342, 期 1, 页码 153-163出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2006.01.135
关键词
extracellular Ca2+ concentration; extracellular Ca2+-sensing receptor; intracellular Ca2+ concentration; endothelial cells; spermine; nitric oxide
Extracellular Ca2+ concentration ([Ca2+](o)) regulates the functions of many cell types through a G protein-coupled [Ca2+](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-by 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+]), the CaR agonist spermine stimulated an increase in [Ca2+] 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+]; 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. (c) 2006 Elsevier Inc. All rights reserved.
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