Hypoxia-induced alterations in Ca2+ mobilization in brain microvascular endothelial cells

To investigate the possible cellular mechanisms of the ischemia-induced imp airments of cerebral microcirculation, we investigated the effects of hypox ia/reoxygenation on the intracellular Ca2+ concentration ([Ca2+](i)) in bov ine brain microvascular endothelial cells (BBEC). In the cells kept in norm al air, ATP elicited Ca2+ oscillations in a concentration-dependent manner. When the cells were exposed to hypoxia for 6 h and subsequent reoxygenatio n for 45 min, the basal level of [Ca2+](i) was increased from 32.4 to 63.3 nM, and ATP did not induce Ca2+ oscillations. Hypoxia/reoxygenation also in hibited capacitative Ca2+ entry (CCE), which was evoked by thapsigargin (De lta [Ca2+](i-CCE): control, 62.3 +/- 3.1 nM; hypoxia/ reoxygenation, 17.0 /- 1.8 nM). The impairments of Ca2+ oscillations and CCE, but not basal [Ca 2+](i), were restored by superoxide dismutase and the inhibitors of mitocho ndrial electron transport, rotenone and thenoyltrifluoroacetone (TTFA). By using a superoxide anion (O-2(-))-sensitive luciferin derivative MCLA, we c onfirmed that the production of O-2(-) was induced by hypoxia/ reoxygenatio n and was prevented by rotenone and TTFA. These results indicate that hypox ia/ reoxygenation generates O-2(-) at mitochondria and impairs some Ca2+ mo bilizing properties in BBEC.