Hypoxia/Reoxygenation Stimulates Intracellular Calcium Oscillations in Human Aortic Endothelial Cells

Abstract

Background —We have previously shown that hydrogen peroxide stimulates endothelial [Ca 2+ ] i oscillations. This study was performed to determine whether posthypoxic reoxygenation stimulates [Ca 2+ ] i oscillations in vascular endothelial cells. Methods and Results —Hypoxia (glucose-free 95% N 2 /5% CO 2 bicarbonate buffer for 60 minutes) stimulated an increase in [Ca 2+ ] i from 111.9±7.9 to 161.7±17.7 nmol/L (n=12, P <0.01) in indo 1–loaded human aortic endothelial cells. On reoxygenation (glucose-containing 95% air/5% CO 2 bicarbonate buffer), 13 of 16 cells responded with repetitive [Ca 2+ ] i oscillations with an average amplitude of 570.6±59.3 nmol/L, occurring at a mean interval of 0.28±0.04/min and persisting for ≥60 minutes. [Ca 2+ ] i oscillations were still observed in 4 of 7 cells studied in Ca 2+ -free buffer but did not occur when the intracellular Ca 2+ store was first depleted during hypoxia by either 1 μmol/L thapsigargin or by 10 mmol/L caffeine (n=6 for each). Reoxygenation-induced [Ca 2+ ] i oscillations were abolished by 10 μmol/L diphenyleneiodonium, an inhibitor of NAD(P)H oxidase (n=7), and by polyethylene glycol (PEG)–catalase (5000 U/mL, n=4) but were not prevented by inhibitors of xanthine oxidase (n=5), cyclooxygenase (n=4), nitric oxide synthase (n=5), the mitochondrial electron transport chain (n=4), or by PEG–superoxide dismutase (n=5). Conclusions —Posthypoxic reoxygenation stimulates repetitive [Ca 2+ ] i oscillations that are dependent on Ca 2+ release from an intracellular pool and require extracellular Ca 2+ to be maintained. These oscillations may be initiated by NAD(P)H oxidase–derived hydrogen peroxide and may play a role in signal transduction during ischemia/reperfusion in vivo.