Histamine-induced Ca2+ oscillations in a human endothelial cell line depend on transmembrane ion flux, ryanodine receptors and endoplasmic reticulum Ca2+-ATPase

1. Using single cell microfluorometry to monitor changes in bulk Ca2+ conce ntration ([Ca2+](bulk)) and the whole-cell configuration of the patch clamp technique to measure K+ currents (voltage clamp) and membrane potential (c urrent clamp), the mechanisms of histamine-induced Ca2+ oscillations in the umbilical vein endothelial cell-derived cell line EA.hy926 were studied. 2. In single cells, histamine (10 mu M) evoked sinusoidal Ca2+ oscillations in low extracellular Ca2+ concentrations ([Ca2+](o) = 10-30 mu M). In cont rast, histamine did not initiate Ca2+ oscillations either in the absence of extracellular Ca2+ (10 mu M EGTA) or in the presence of 2.5 mM extracellul ar Ca2+. 3. Ca2+ oscillations were accompanied by rhythmic activation of Ca2+-activa ted K+ (K-Ca) channels and membrane hyperpolarization of 18.1 +/- 3.9 mV. H ence, cell, depolarization with 70 mM extracellular K+ or the inhibition of nea-selective cation channels (NSCCs) and K-Ca channels by 10 mu M Loe 908 and 10 mM tetrabutylammonium presented histamine-evoked Ca2+ oscillations. 4. Preventing Na+-Ca2+ exchange (NCX) by 10 mu M 2',4'-dichlorobenzamil, or removal of extracellular Na+, abolished histamine-induced Ca2+ oscillation s. Lowering the extracellular Na+ concentration and thus promoting the reve rsed mode of NCX (3Na(+) out and 1Ca(2+) in) increased the amplitude and fr equency of histamine-induced Ca2+ oscillations by 25 and 13%, respectively. Hence, in the absence of extracellular Ca2+, 10 mu M histamine induced an elevation of intracellular Na+ concentration in certain subplasmalemmal dom ains. 5. The inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)2,5-di-t ert-butyl-1,4-benzo-hydroquinone (15 mu M) pre-vented histamine-induced Ca2 + oscillations. In addition, blockage of ryanodine-sensitive Ca2+ release ( RsCR) by 25 mu M ryanodine blunted Ca2+ oscillations. 6. In endothelial cells that were treated for 16 h with 10 mu M nocodazole to collapse the superficial endoplasmic reticulum (sER), no histamine-induc ed Ca2+ oscillations were found. 7. We conclude that; in low [Ca2+](o) conditions histamine-induced Ca2+ osc illations depend on transmembrane Na+ loading through NSCCs that leads to C a2+ entry via NCX. Cation influx is controlled by K-Ca channel activity tha t triggers membrane hyperpolarization and, thus, provides the driving force for cation influx. Hence, the Ca2+ entering needs to be sequestrated via S ERCA into sER to become released by RsCR to evoke Ca2+ spiking. These data further support our previous work on localized Ca2+ signalling as a key phe nomenon in endothelial Ca2+ homeostasis.