Histamine-induced Ca2+ oscillations in a human endothelial cell line depend on transmembrane ion flux, ryanodine receptors and endoplasmic reticulum Ca2+-ATPase.
Using single cell microfluorometry to monitor changes in bulk Ca2+ concentration ([Ca2+]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 Ca2+ oscillations in the umbilical vein endothelial cell-derived cell line EA.hy926 were studied. In single cells, histamine (10 microM) evoked sinusoidal Ca2+ oscillations in low extracellular Ca2+ concentrations ([Ca2+]o = 10-30 microM). In contrast, histamine did not initiate Ca2+ oscillations either in the absence of extracellular Ca2+ (10 microM EGTA) or in the presence of 2.5 mM extracellular Ca2+. Ca2+ oscillations were accompanied by rhythmic activation of Ca2+-activated K+ (KCa) 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 KCa channels by 10 microM Loe 908 and 10 mM tetrabutylammonium prevented histamine-evoked Ca2+ oscillations. Preventing Na+-Ca2+ exchange (NCX) by 10 microM 2', 4'-dichlorobenzamil, or removal of extracellular Na+, abolished histamine-induced Ca2+ oscillations. Lowering the extracellular Na+ concentration and thus promoting the reversed mode of NCX (3Na+ out and 1Ca2+ in) increased the amplitude and frequency of histamine-induced Ca2+ oscillations by 25 and 13 %, respectively. Hence, in the absence of extracellular Ca2+, 10 microM histamine induced an elevation of intracellular Na+ concentration in certain subplasmalemmal domains. The inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2,5-di-tert-butyl-1, 4-benzo-hydroquinone (15 microM) prevented histamine-induced Ca2+ oscillations. In addition, blockage of ryanodine-sensitive Ca2+ release (RsCR) by 25 microM ryanodine blunted Ca2+ oscillations. In endothelial cells that were treated for 16 h with 10 microM nocodazole to collapse the superficial endoplasmic reticulum (sER), no histamine-induced Ca2+ oscillations were found. We conclude that in low [Ca2+]o conditions histamine-induced Ca2+ oscillations depend on transmembrane Na+ loading through NSCCs that leads to Ca2+ entry via NCX. Cation influx is controlled by KCa channel activity that triggers membrane hyperpolarization and, thus, provides the driving force for cation influx. Hence, the Ca2+ entering needs to be sequestrated via SERCA 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 phenomenon in endothelial Ca2+ homeostasis.
Paltauf-Doburzynska
J
J
Department of Medical Biochemistry and Medical Molecular Biology, Karl-Franzens University of Graz, Harrachgasse 21/III, A-8010 Graz, Austria.
Frieden
M
M
Spitaler
M
M
Graier
W F
WF
D000970:Antineoplastic Agents / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002478:Cells, Cultured; D004558:Electric Stimulation; D004594:Electrophysiology; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D015739:Nocodazole / Q000494:pharmacology; D010507:Periodicity; D015221:Potassium Channels / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000494:pharmacology*; D012508:Sarcolemma / Q000737:chemistry / Q000201:enzymology; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology
D000970:Antineoplastic Agents; D015221:Potassium Channels; D019837:Ryanodine Receptor Calcium Release Channel; D019831:Sodium-Calcium Exchanger; D006632:Histamine; D000252:Calcium-Transporting ATPases; D015739:Nocodazole; D002118:Calcium