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(Circulation. 1998;97:268-275.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Hydrogen Peroxide Induces Intracellular Calcium Oscillations in Human Aortic Endothelial Cells

Qinghua Hu, PhD; Stefano Corda, MD; Jay L. Zweier, MD; Maurizio C. Capogrossi, MD; ; Roy C. Ziegelstein, MD

From the Department of Medicine (Q.H., J.L.Z., M.C.C., R.C.Z.), Division of Cardiology, Johns Hopkins Bayview Medical Center, Johns Hopkins University School of Medicine, Baltimore, Md; Laboratory of Cardiovascular Science (S.C., M.C.C.), Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Md; and Laboratorio di Patologia Vascolare (M.C.C.), Istituto Dermopatico dell' Immacolata, Rome, Italy.

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 rziegels{at}welchlink.welch.jhu.edu

Background—Because the vascular endothelium is exposed to oxidant stress resulting from ischemia/reperfusion and from the products of polymorphonuclear leukocytes or monocytes, studies were performed to examine the effect of hydrogen peroxide (1 µmol/L to 10 mmol/L) on endothelial Ca²⁺ signaling.

Methods and Results—At low concentrations (1 to 10 µmol/L), hydrogen peroxide did not affect intracellular Ca²⁺ concentration in subconfluent, indo 1–loaded human aortic endothelial monolayers. At a concentration of 100 µmol/L hydrogen peroxide, intracellular free Ca²⁺ gradually increased from 125.3±6.8 to 286.3±19.9 nmol/L over 4.2±0.9 minutes before repetitive Ca²⁺ oscillations were observed, consisting of an initial large, transient spike of 1 µmol/L followed by several spikes of decreasing amplitudes at a frequency of 0.7±0.1 min^-1 over 12.0±1.1 minutes. After these oscillations, intracellular Ca²⁺ reached a plateau of 543.4±64.0 nmol/L, which was maintained above baseline levels for >5 minutes and then partially reversible on washout of hydrogen peroxide in most monolayers. Intracellular Ca²⁺ oscillations were typically observed when monolayers were exposed to 100 to 500 µmol/L hydrogen peroxide. Higher concentrations of hydrogen peroxide (1 and 10 mmol/L) increased intracellular Ca²⁺ but only rarely (2 of 6 monolayers at 1 mmol/L) or never (at 10 mmol/L) stimulated intracellular Ca²⁺ oscillations. Removal of Ca²⁺ from the buffer either before hydrogen peroxide stimulation or during an established response did not block intracellular Ca²⁺ oscillations in response to 100 µmol/L hydrogen peroxide, but prior depletion of an intracellular Ca²⁺ store with either caffeine, histamine, or thapsigargin abolished Ca²⁺ oscillations.

Conclusions—Hydrogen peroxide induces concentration-dependent intracellular Ca²⁺ oscillations in human endothelial cells, which results from release of an endoplasmic reticulum Ca²⁺ store. Because oxidant production appears to occur in the micromolar range in the postischemic/anoxic endothelium and is associated with impaired endothelium-dependent relaxation, the effects of micromolar concentrations of hydrogen peroxide on endothelial Ca²⁺ signaling described in the present study may be important in the pathogenesis of postischemic endothelial dysfunction.

Key Words: calcium • endothelium • free radicals

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