Identification of adenosine receptor subtypes expressed in the human endothelial-like ECV304 cells

Adenosine, acting through its receptors, is a potent endogenous regulator of endothelial cells. The cultured endothelial cells expressing adenosine receptors are thus important for elucidation of molecular mechanism of adenosine functions in these cell systems. Therefore, identification of adenosine receptors in the human ECV304 cell line derived from a human umbilical vein endothelial cell culture was performed. RT-PCR experiments revealed that ECV304 cells express mRNAs for A1 and A2B adenosine receptors. The expression of mRNA for A2A adenosine receptor was not in a significant level and that for A3 adenosine receptor was not detected. The binding study of ECV304 cell membrane fractions using various radiolabeled ligands for adenosine receptors indicated the presence of A1 adenosine receptors 245 fmol/mg of membrane proteins, but the specific binding for A2A and for A3 adenosine receptors were found to be negligible. The functional expression of A1 and A2B adenosine receptors in ECV304 cells was detected by assays for adenosine-3',5'-cyclic monophosphate and for extracellular signal-regulated kinase, but that of A2A adenosine receptors was not confirmed under the assay conditions employed. In conclusion, this study presented evidence for functional A1 and A2B adenosine receptors in human endothelial-like ECV304 cells, indicating that ECV304 cells can be a good model for the study of adenosine receptors, especially for A2B adenosine receptor, in endothelial cells.