Histamine‐induced Ca 2+ oscillations in a human endothelial cell line depend on transmembrane ion flux, ryanodine receptors and endoplasmic reticulum Ca 2+ ‐ATPase

1 Using single cell microfluorometry to monitor changes in bulk Ca 2+ concentration ([Ca 2+ ] bulk ) and the whole‐cell configuration of the patch clamp technique to measure K + currents (voltage clamp) and membrane potential (current clamp), the mechanisms of histamine‐induced Ca 2+ oscillations in the umbilical vein endothelial cell‐derived cell line EA.hy926 were studied. 2 In single cells, histamine (10 μM) evoked sinusoidal Ca 2+ oscillations in low extracellular Ca 2+ concentrations ([Ca 2+ ] o = 10–30 μM). In contrast, histamine did not initiate Ca 2+ oscillations either in the absence of extracellular Ca 2+ (10 μM EGTA) or in the presence of 2.5 mM extracellular Ca 2+ . 3 Ca 2+ oscillations were accompanied by rhythmic activation of Ca 2+ ‐activated K + (K Ca ) channels and membrane hyperpolarization of 18.1 ± 3.9 mV. Hence, cell depolarization with 70 mM extracellular K + or the inhibition of non‐selective cation channels (NSCCs) and K Ca channels by 10 μM Loe 908 and 10 mM tetrabutylammonium prevented histamine‐evoked Ca 2+ oscillations. 4 Preventing Na + ‐Ca 2+ exchange (NCX) by 10 μM 2′,4′‐dichlorobenzamil, or removal of extracellular Na + , abolished histamine‐induced Ca 2+ oscillations. Lowering the extracellular Na + concentration and thus promoting the reversed mode of NCX (3Na + out and 1Ca 2+ in) increased the amplitude and frequency of histamine‐induced Ca 2+ oscillations by 25 and 13 %, respectively. Hence, in the absence of extracellular Ca 2+ , 10 μM histamine induced an elevation of intracellular Na + concentration in certain subplasmalemmal domains. 5 The inhibitor of sarco/endoplasmic reticulum Ca 2+ ‐ATPase (SERCA) 2,5‐di‐tert‐butyl‐1,4‐benzo‐hydroquinone (15 μM) prevented histamine‐induced Ca 2+ oscillations. In addition, blockage of ryanodine‐sensitive Ca 2+ release (RsCR) by 25 μM ryanodine blunted Ca 2+ oscillations. 6 In endothelial cells that were treated for 16 h with 10 μM nocodazole to collapse the superficial endoplasmic reticulum (sER), no histamine‐induced Ca 2+ oscillations were found. 7 We conclude that in low [Ca 2+ ] o conditions histamine‐induced Ca 2+ oscillations depend on transmembrane Na + loading through NSCCs that leads to Ca 2+ entry via NCX. Cation influx is controlled by K Ca channel activity that triggers membrane hyperpolarization and, thus, provides the driving force for cation influx. Hence, the Ca 2+ entering needs to be sequestrated via SERCA into sER to become released by RsCR to evoke Ca 2+ spiking. These data further support our previous work on localized Ca 2+ signalling as a key phenomenon in endothelial Ca 2+ homeostasis.