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

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Abstract

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-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.

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Materials and methods

Cell culture and [Ca2+]imeasurements. HAEC were purchased from Clonetics (Walkersville, MD 21793). The maintenance of HAEC in culture and [Ca2+]i measurements were performed as previously described in detail [8], [9], [10]. Briefly, HAEC were grown to passage 5–9 at ∼70% confluence on gelatin-coated, 25-mm diameter circular glass coverslips (VWR Scientific, West Chester, PA 19380). HAEC [Ca2+]i was measured after loading with 10 μM of the acetoxymethyl ester form of fura 2 (Invitrogen-Molecular

RT-PCR analysis of CaR mRNA expression in HAEC

To determine whether the CaR is present in HAEC, RT-PCR was performed using specific primers for the CaR. As shown in Fig. 1A, reverse transcription and PCR amplification of HAEC RNA with CaR-specific primers yielded the expected 555-bp product corresponding to the CaR gene detected in RT-PCR product agarose gel. As a positive control, under the identical conditions of reverse transcription and amplification, a RT-PCR product from a human medullary C cell thyroid carcinoma cell line (TT) [13]

Discussion

The present study shows that CaR mRNA and protein are expressed in HAEC and that when the CaR is stimulated by spermine, [Ca2+]i increases primarily due to intracellular Ca2+ release and leads to the synthesis and release of nitric oxide. Spermine was the only known CaR agonist examined in this study that stimulated an increase in [Ca2+]i in this cell type. Of the polyamines, spermine contains the highest number of free amino groups and is therefore the most potent agonist [22]. In the presence

Acknowledgment

This work was supported in part by American Heart Association Scientist Development Grant 0335020N to Q. Hu.

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    Abbreviations: [Ca2+]o, extracellular Ca2+ concentration; CaR, extracellular Ca2+-sensing receptor; [Ca2+]i, intracellular Ca2+ concentration; HAEC, human aortic endothelial cells; IP3, inositol 1,4,5-triphosphate; NO, nitric oxide; SOCE, store-operated calcium entry; TRPC, transient receptor potential canonical; HBS, Hepes-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin; siRNA, small interference RNA; XeC, xestospongin C.

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