Endothelial cells (EC) exhibit a rise in cytosolic Ca²⁺ (Ca_i) during hypoxia followed by an additional increase during reoxygenation, which is even more pronounced than under hypoxic conditions and is caused by a Ca²⁺ influx as well as a Ca²⁺ release from the ER via the IP3-receptor.

The aim of this study was to examine whether the reperfusion-induced initiation of the respiratory chain in the presence of oxygen and energy might cause ROS generation within the mitochondria. As a consequence a disturbance of the ATP production occurs and simultaneously ROS may also lead to PLCy activation, causing the reperfusion-induced Ca²⁺ release from the ER via the IP₃-receptor. Cardiac microvascular EC (CMEC) of hearts from humanely killed rats were exposed to 40 min acidic hypoxia (pH 6.4) followed by 40 min of reoxygenation (pH 7.4; 2.5 mM glucose). ROS generation or Ca²⁺ or ATP loss was detected via DCF or fura-2 or mag-fura, respectively. The data presented were taken from at least four different experiments (mean values s.e.m. in arbitrary units of fura-2 ratio and mag-fura or DCF fluorescence in % of end-anoxic value). All data were background-corrected.

During reoxygenation there is an increase in ROS, which is blocked by the scavenger trolox (DFC in %: 31254 reox. 40 min vs with trolox 14112*,* p<0.05). This effect is mimicked by the mitochondria-specific scavenger MitoQ (11018* vs ctr, *p<0.05), whereas apocynin, inhibitor of the NADPH-oxidase, had no effect (33244 vs ctr, n.s.). Within the first minutes of reoxygenation ATP is produced, but after 40min of reoxygenation a loss of ATP occurs, which is unchanged in the presence of apocynin (Mag-fura in a.u.: 1.150.02 reox. 40 min vs with apocynin 1.160,02 vs ctr, n.s.). But this ATP loss can be blocked by application of MitoQ (Mag-fura in a.u.: 1.150.02 reox. 40 min vs with MitoQ 1.040.01*,*p<0.05). TTFA, a complex II inhibitor, also reduces the ATP loss (1.070.02 vs ctr*, *p<0.05). Ca_i is blocked in the presence of MitoQ significantly (Fura-2 Ratio: 1.400.02 reox. 40 min vs with MitoQ 1.180.01*,* p<0.05). Apocynin had no effect on Ca_i (Fura-2 Ratio: 1.400.02 reox. 40 min vs with apocynin 1.440.01, n.s.) Western blot analysis showed that PLCy was phosphorylated during reoxygenation and this phosphorylation was reduced either by application of U73122 or after application of the scavenger trolox.

ROS are generated during reoxygenation within the mitochondrial respiratory chain and these ROS cause a disturbance of the starting ATP synthesis. The result is an ATP loss similar to ischemic conditions. PLCg is also activated via ROS, initiating the Ca²⁺ release from the ER. MitoQ is able to protect the EC against the reperfusion-induced Ca²⁺ overload pointing to new possibilities in the therapy of CMEC after myocardial infarct.