Hypoxia-reoxygenation inhibits gap junctional communication in cultured human umbilical vein endothelial cells

We studied the change in gap junctional intercellular communication (GJIC) on human umbilical vein endothelial cells (HUVEC) under hypoxia-reoxygenati on (H-R) conditions by the fluorescence redistribution after photobleaching (FRAP) method. Confluent HUVEC monolayers were exposed to hypoxia (pO2<0.1 %) for 12 hours, and then were returned to normal atmospheric conditions fo r reoxygenation. Contrast microscopic observation showed no significant cha nges in the morphology of the HUVEC at any times after H-R. Reoxygenation f ollowing hypoxia caused time-dependent decrease in GJIC, that is, GJIC redu ction was induced after 2 hours and reached maximum at 4<similar to>6 hours which recovered to normal levels after 18 hours. Oxidant sensitive fluores cence dye assay revealed that the generation of intracellular free radicals increased during the first 2 hours after reoxygenation. Hydroxyl radical s cavengers (MCI-186, DMSO) and an iron chelator (deferoxamine) abolished the reduction of GJIC due to H-R. However, SOD, catalase and probucol were ess entially inactive on this reduction. These data suggest that ischemia-reper fusion injury may be caused by a functional defect of GJIC induced by react ive oxygen radicals.