Mechanisms of oxygen free radical-induced calcium overload in endothelial cells.
Unregulated increases in intracellular calcium levels with subsequent cytoskeletal disruption have been implicated in tissue injury caused by oxygen free radicals (OFRs). The purpose of these experiments was to study the kinetics of free intracellular calcium in endothelial cells on exposure to exogenous OFRs and investigate the mechanisms of this altered homeostasis. Monolayers of endothelial cells were labeled with a calcium-sensitive probe and exposed to exogenous OFRs generated with hypoxanthine and xanthine oxidase. Intracellular calcium changes were monitored dynamically by continuous measurement of fluorescence with a spectrofluorometer. A sustained rise in intracellular Ca++ levels reaching a peak at 5 minutes was observed. This effect was blocked by superoxide dismutase and catalase. Removal of calcium from the medium or chelating the extracellular calcium with ethyleneglycoldiamine tetraacetate significantly blunted the response to OFRs (p less than 0.05). Preincubation of the cells with verapamil did not alter the observed increase in Ca++i. Addition of another divalent cation (Mn++) to the medium partially blocked the rise in calcium levels (p less than 0.05). Membrane potential measurements assessed fluorometrically, with the fluorescent probe bisoxonol, demonstrate a transient hyperpolarization of the plasma membrane on exposure to OFRs, temporally associated with the rise in [Ca++]i. In summary, OFRs cause an increase of intracellular calcium in endothelial cells. This response is dependent on extracellular calcium and independent of voltage-sensitive calcium channels. The increase can be partially inhibited by other divalent cations. These results suggest that the transformation of the plasma membrane components (lipid peroxidation and cross-linking of proteins) caused by OFRs may produce cation ionophores.
Franceschi
D
D
Metrohealth Medical Center, Case Western Reserve University, Cleveland, Ohio.
Graham
D
D
Sarasua
M
M
Zollinger
R M
RM
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology; D002478:Cells, Cultured; D004594:Electrophysiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005609:Free Radicals; D007425:Intracellular Membranes / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology; D008564:Membrane Potentials; D010100:Oxygen / Q000494:pharmacology*; D011956:Receptors, Cell Surface / Q000502:physiology
D015220:Calcium Channels; D005609:Free Radicals; D011956:Receptors, Cell Surface; D008345:Manganese; D010100:Oxygen; D002118:Calcium