Effects of P 2 Purinoceptor Agonists on Membrane Potential and Intracellular Ca 2+ of Human Cardiac Endothelial Cells

Vasoactive agonists like adenosine‐5′‐triphosphate (ATP) increase intracellular Ca 2+ ([Ca 2+ ] i ) in vascular endothelial cells with an initial peak due to inositol 1,4,5‐triphosphate‐mediated Ca 2+ release from intracellular stores followed by a sustained plateau that is dependent on the presence of extracellular Ca 2+ , thus leading to an increased synthesis and release of prostacyclin and nitric oxide. We studied the effects of nucleotides on membrane potential and [Ca 2+ ] i in confluent human microvascular cardiac endothelial cells obtained from patients with dilated cardiomyopathy. The whole‐cell configuration of the patch‐clamp technique and a confocal laser scanning microscope employing fluo‐3 as a Ca 2+ indicator were used. Both uridine‐5′‐triphosphate (UTP) and 2‐methylthioadenosine‐5′‐triphosphate (2MeSATP) induced depolarizations in human microvascular cardiac endothelial cells and increased [Ca 2+ ] i with a rank order of potency 2MeSATP>ATP=UPP (EC 50 values (in μM) were 0.084 2MeSATP, 0.67 ATP and 1.1 UTP). This suggests that both P 2u and P 2y purinoceptors are present on human microvascular cardiac endothelial cells. Maximal [Ca 2+ ] i responses of confluent human microvascular cardiac endothelial cell monolayers to UTP were lower when compared to 2MeSATP. Nucleotide‐induced increases in [Ca 2+ ] i consisted of a transient peak, which was also observed in the absence of extracellular Ca 2+ , and a sustained [Ca 2+ ] i plateau. This plateau, which was not observed in all monolayers studied, was not markedly influenced by increasing extracellular [K + ]. Previous incubation with thapsigargin abolished ATP‐induced increases of [Ca 2+ ] i . It is concluded that human microvascular cardiac endothelial cells express both P 2y and P 2u purinoceptors. P 2 purinoceptor agonists release Ca 2+ from intracellular thapsigargin‐sensitive stores and stimulate capacitative Ca 2+ influx pathways. K + efflux through Ca 2+ ‐dependent K + (K ca ) channels does not play a major role in the regulation of nucleotide‐induced Ca 2+ influx in human microvascular cardiac endothelial cells, which might be related to an impaired function of the cells.