Regulation of astrocyte gap junctions by hypoxia-reoxygenation

Confluent cultures of rat cortical astrocytes were subjected to 12-h hypoxia (<1% O(2)) followed by reoxygenation. Just after hypoxia, the cellular distribution, phosphorylation state and levels of connexin43 (Cx43), as well as the extent of dye coupling were as in control conditions. Nonetheless, 15-30 min after reoxygenation, dye coupling was transiently reduced by approximately 70%. The reduction in dye coupling occurred without changes in the state of phosphorylation or levels of Cx43. Nevertheless, it was correlated with a decrease in Cx43 reactivity found at membrane appositions and the appearance of intracellular Cx43-positive vesicle-like structures of variable size, suggesting internalization of gap junction channels. Reoxygenation-induced cellular uncoupling and redistribution of Cx43 were prevented by melatonin (500 microM), a potent-free radical scavenger, or indomethacin (50 microM), an inhibitor of the cyclooxygenase-dependent arachidonic acid metabolism. In astrocytes cultured under normoxia, the state of phosphorylation of Cx43 was not affected by antimycin A, a blocker of the mitochondrial oxidative metabolism, but phosphorylation was drastically reduced by iodoacetate, a blocker of anaerobic glycolysis. Thus, these results strongly suggest that reoxygenation-induced uncoupling is mediated by arachidonic acid byproducts that induce, at least, disorganization of Cx43 gap junction channels.