P2Y-and P2U-Mediated Increases in Internal Calcium in Single Bovine Aortic Endothelial Cells in Primary Culture

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Abstract
Increases in intracellular calcium ([Ca2+]|) to ATP, ADP, AMP, adenosine, UTP, 2-methylthio ATP (2-MeSATP), 2-methylthio ADP (2-MeSADP) and α,β-methylene ATP (α,β-meATP) were investigated in single bovine aortic endothelial cells (BAEC) in primary culture using Indo-1. Evidence was obtained for the presence of P2Y and P2U, but not P2X receptors. Normalized concentration-effect curves for ATP, UTP and 2-MeSATP were biphasic in shape. At 10 nM, the agonist rank order was UTP ATP ≥ 2-MeSATP, while above 1 μM, it was ATP ≥ UTP ≥ 2-MeSATP. No cross-desensitization between responses to P2U and P2Y receptors was observed in normal external solution. However, when internal Ca2+ stores were depleted by exposure to 2-MeSATP or UTP in Ca2+-free solution and agonists then re-applied in presence of external Ca2+, homologous but not heterologous desensitization was seen. In the same conditions, heterologous desensitization was observed for UTP after ATP but not for ATP after UTP. Taken together, the results are consistent with the coexistence of P2Y and P2U receptors in primary-cultured BAEC and suggest that upon activation, different intracellular signaling pathways could be involved in increasing [Ca2+]i.