Free radicals are major determinants of oxidant injury during inflammation and post-ischemic reperfusion. Vascular endothelium is both a source and a target of reactive oxygen species. One such reactive oxygen species, hydrogen peroxide (H2O2) may cause endothelial cell damage by affecting intracellular signal transduction mechanisms and disrupting Ca2+ homeostasis.
The aim of the project is: 1) to characterize the effects of exposure of human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) to H2O2 in the range 0.1mM-1mM on endothelial cell Ca2+ homeostasis; 2) to define the role of cytosolic Ca2+ concentration, [Ca2+]i, in the pathophysiology of oxidative injury. In order to detect the effect of hydrogen peroxide on HAEC and HUVEC [Ca2+]i, indo-1 AM loaded endothelial cells grown on glass coverslips were bathed in a Hepes buffer (CaCl2 1.5mM, pH 7.4) containing H2O2 in the range 0.1mM-1mM. H2O2 slowly increased Cai and this effect was not reversible with wash- out. The increase in [Ca2+]i persisted in a Ca2+-free solution with 1mM EGTA suggesting that extracellular Ca2+ does not contribute to the increase in [Ca2+]i. In other experiments the effect of H2O2 on endothelial cell [Ca2+]i was examined following exposure to 1uM bradykinin, an agonist known to release Ca2+ from intracellular stores. Under these conditions, H2O2 still produced an increase in [Ca2+]i. Pre-exposure of HAEC to thapsigargin, an inhibitor of microsomal or endoplasmic reticulum (ER) Ca2+-ATPase which releases [Ca2+] from the endoplasmic reticulum, abolished the response to bradykinin (1uM) but did not suppress the subsequent response to H2O2. These results suggest that H2O2 releases [Ca2+] from a thapsigargin-insensitive intracellular store and increases [Ca2+]i via this mechanism.
