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(Circulation. 2000;102:2541.)
© 2000 American Heart Association, Inc.

Basic Science Reports

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

Qinghua Hu, PhD; Roy C. Ziegelstein, MD

From the Department of Medicine, Division of Cardiology, Johns Hopkins Bayview Medical Center, Johns Hopkins University School of Medicine, Baltimore, Md.

Correspondence to Roy C. Ziegelstein, MD, Department of Medicine, Division of Cardiology, Johns Hopkins Bayview Medical Center, 4940 Eastern Ave, Baltimore, MD 21224-2780. E-mail rziegel{at}jhmi.edu

Background—We have previously shown that hydrogen peroxide stimulates endothelial [Ca²⁺]_i oscillations. This study was performed to determine whether posthypoxic reoxygenation stimulates [Ca²⁺]_i oscillations in vascular endothelial cells.

Methods and Results—Hypoxia (glucose-free 95% N₂/5% CO₂ bicarbonate buffer for 60 minutes) stimulated an increase in [Ca²⁺]_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% CO₂ bicarbonate buffer), 13 of 16 cells responded with repetitive [Ca²⁺]_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. [Ca²⁺]_i oscillations were still observed in 4 of 7 cells studied in Ca²⁺-free buffer but did not occur when the intracellular Ca²⁺ store was first depleted during hypoxia by either 1 µmol/L thapsigargin or by 10 mmol/L caffeine (n=6 for each). Reoxygenation-induced [Ca²⁺]_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 [Ca²⁺]_i oscillations that are dependent on Ca²⁺ release from an intracellular pool and require extracellular Ca²⁺ 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.

Key Words: calcium • endothelium • hypoxia

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