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

To investigate the possible cellular mechanisms of the ischemia-induced impairments of cerebral microcirculation, we investigated the effects of hypoxia/reoxygenation on the intracellular Ca²⁺ concentration ([Ca²⁺](i)) in bovine brain microvascular endothelial cells (BBEC). In the cells kept in normal air, ATP elicited Ca²⁺ oscillations in a concentration-dependent manner. When the cells were exposed to hypoxia for 6 h and subsequent reoxygenation 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 inhibited capacitative Ca²⁺ entry (CCE), which was evoked by thapsigargin (Δ[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 [Ca²⁺](i), were restored by superoxide dismutase and the inhibitors of mitochondrial electron transport, rotenone and thenoyltrifluoroacetone (TTFA). By using a superoxide anion (O₂/^-)-sensitive luciferin derivative MCLA, we confirmed that the production of O₂/^- was induced by hypoxia/reoxygenation and was prevented by rotenone and TTFA. These results indicate that hypoxia/ reoxygenation generates O₂/^- at mitochondria and impairs some Ca²⁺ mobilizing properties in BBEC.