Hypoxia/reoxygenation stimulates intracellular calcium oscillations in human aortic endothelial cells

Background-We have previously shown that hydrogen peroxide stimulates endot helial [Ca2+](i) oscillations. This study was performed to determine whethe r posthypoxic reoxygenation stimulates [Ca2+](i) oscillations in vascular e ndothelial cells. Methods and Results-Hypoxia (glucose-free 95% N-2/5% CO2 bicarbonate buffer for 60 minutes) stimulated an increase in [Ca2+](i) from 111.9+/-7.9 to 16 1.7+/-17.7 nmol/L (n=12, P<0.01) in indo 1-loaded human aortic endothelial cells. On reoxygenation (glucose-containing 95% air/5% CO2 bicarbonate buff er), 13 of 16 cells responded with repetitive [Ca2+](i) oscillations with a n average amplitude of 570.6+/-59.3 nmol/L, occurring at a mean interval of 0.28+/-0.04/min and persisting for <greater than or equal to>60 minutes. [ Ca2+](i) oscillations were still observed in 4 of 7 cells studied in Ca2+-f ree buffer but did not occur when the intracellular Ca2+ store was first de pleted during hypoxia by either 1 mu mol/L thapsigargin or by 10 mmol/L caf feine (n=6 for each). Reoxygenation-induced [Ca2+](i) oscillations were abo lished by 10 mu mol/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 [Ca2+](i) oscil lations that are dependent on Ca2+ release from an intracellular pool and r equire extracellular Ca2+ to be maintained. These oscillations may be initi ated by NAD(P)H oxidase-derived hydrogen peroxide and may play a role in si gnal transduction during ischemia/reperfusion in vivo.