Endothelial cells exhibit a rise in cytosolic Ca2+ during hypoxia followed by an additional increase during reoxygenation, which is even more pronounced than under hypoxic conditions. Former studies have shown that this reperfusion-induced increase is caused by a Ca2+ influx as well as a Ca2+ release from the ER via the IP3-receptor. The aim of the study was, to analyse the role of the phospholipase C (PLC) within this process. Coronary EC of rat hearts were exposed to 40 min acidic hypoxia (pH 6.4) followed by 40 min of reoxygenation (pH 7.4; 2.5 mM glucose). Under control conditions the fura-2 ratio increased during reoxygenation from an end-anoxic value of 1.27±0.03 to 1.40±0.03 after 40 min (p<0.05 vs. end-anoxia). The PLC inhibitor U73122 (1 mM) applied during reoxygenation suppressed this increase (fura-2 ratio: 1.29±0.03; p<0.05 vs. control), whereas the inactive analogue U73433 (1 mM) had no effect (1.43±0.02; ns vs. control). Application of the scavenger trolox (500 mM) reduced the increase during reperfusion significantly (1.33±0.03; p<0.05 vs. control). Western blot analysis showed that PLC was phosphorylated during reoxygenation and this phosphorylation was reduced either by application of U73122 or after application of trolox.

In conclusion: PLC is activated during reoxygenation and this activation can be reduced by application of scavengers, which also reduces the Ca2+ increase during reoxygenation.