Abstract:
:Vascular endothelial cells (EC), communicating with one another across gap junctions, are usually made dysfunctional by hypoxia and reoxygenation (H/R); however, very limited information exists regarding the effects of H/R on the endothelial gap junctions. We investigated whether H/R interferes with endothelial gap junctional intercellular communication (GJIC). After human umbilical vein EC had grown to confluence, they were exposed to hypoxia (pO2 < 0.1%) for 12-16 h and then returned to normal atmospheric conditions for reoxygenation. At 0-, 2-, 4-, 6-h reoxygenation, GJIC was detected by means of a fluorescence recovery after a photobleaching technique. The results demonstrated that a GJIC reduction (about 20% less than that under normoxia) was induced after 2 h of reoxygenation; after 4 h of reoxygenation, it began to recover (to about 10% less than that under normoxia); and after 6 h of reoxygenation, GJIC was restored to the normal level. Calphostin C (1 x 10(-7) mol/l), a specific protein kinase C inhibitor, partially inhibited the reduction in GJIC (resulting in a level about 10% less than that under normoxia), whereas the tyrosine kinase inhibitor genistein (10 micromol/L) completely blocked the reduction in GJIC. Vanadate (1.5 mmol/l), a tyrosine phosphatase inhibitor, amplified the inhibitory effect of H/R on GJIC (to about 40% less than that under normoxia). Immunofluorescence and immunoprecipitation showed that 2-h reoxygenation significantly stimulated tyrosine protein phosphorylation, and this phosphorylation event was obviously enhanced by vanadate. The results of Western blotting showed that the gap junctional protein connexin 43 (Cx43) was phosphorylated by H/R; moreover, immunoprecipitation demonstrated that 2-h reoxygenation induced a prominent increase of tyrosine phosphorylation of Cx43 compared with that under normoxia. These data indicate that H/R induces a transient endothelial GJIC dysfunction through the activation of tyrosine kinase and phosphorylation of tyrosine residues of Cx43.
journal_name
J Cell Physioljournal_title
Journal of cellular physiologyauthors
Zhang YW,Morita I,Nishida M,Murota SIdoi
10.1002/(SICI)1097-4652(199909)180:3<305::AID-JCP1keywords:
subject
Has Abstractpub_date
1999-09-01 00:00:00pages
305-13issue
3eissn
0021-9541issn
1097-4652pii
10.1002/(SICI)1097-4652(199909)180:3<305::AID-JCP1journal_volume
180pub_type
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