Biochemical and Biophysical Research Communications
Expression of a functional extracellular calcium-sensing receptor in human aortic endothelial cells☆
Section snippets
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.