Hydrogen peroxide induces intracellular calcium oscillations in human aortic endothelial cells

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 κ1 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.