Screening of anti-hypoxia/reoxygenation agents by an in vitro method. Part 2: Inhibition of tyrosine kinase activation prevented hypoxia/reoxygenation-induced injury in endothelial gap junctional intercellular communication.
In this study, we demonstrated that hypoxia/reoxygenation (H/R) induced an injury in gap junctional intercellular communication (GJIC) after 2 h of reoxygenation in cultured HUVEC. Free radical scavenger (DMSO) and antioxidant (SOD) did not prevent this GJIC injury at all. Protein kinase C inhibitor (calphostin C) partly blocked this injury. However, the protein tyrosine kinase (PTK) inhibitor genistein completely inhibited this GJIC injury. Compounds 1 [laxogenin-3-O-alpha-L-arabinosyl-(1-->6)- beta-D-glucopyranoside], 2 (macrostemososide A), 3 [laxogenin-3-O-beta-D-xylopyranosyl-(1-->4)-alpha- L-arabinopyranosyl-(1-->6)-beta-D-glucopyranoside], 4 (chinenoside II), 5 (beta-sitosterol), 6 (daucosterine), 7 (ginsenoside-Rd), 29 (isocumarine), 52 (icariin), 53 (icariside), and 54 (icaritin), which showed obvious influence on H/R-induced PTK activation as stated in Part 1 (except 1), were explored for their effects on GJIC. The results showed that compounds 2-7 and 52-57 partly protected H/R-induced GJIC injury. Compounds 5 and 6 (especially 5), which showed the strongest inhibitory effects on PTK activation, completely blocked H/R-provoked GJIC injury. Compound 1, which did not influence PTK activation, failed to prevent this GJIC injury. In contrast, compound 29, which significantly promoted PTK activation, enhanced this H/R-induced GJIC injury further. Western blotting of connexin 43, an important gap junctional protein for modulating GJIC in HUVEC, revealed that interference with the gap junctional protein might be the most direct mechanism for compounds 2, 5, 29, and 53 to affect H/R-injured GJIC.
Zhang
Y W
YW
Section of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, Japan.
Morita
I
I
Zhang
L
L
Shao
G
G
Yao
X S
XS
Murota
S
S
D002450:Cell Communication / Q000187:drug effects*; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004365:Drugs, Chinese Herbal; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D017629:Gap Junctions / Q000187:drug effects*; D006801:Humans; D066298:In Vitro Techniques; D010477:Perfusion; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015427:Reperfusion Injury / Q000201:enzymology*; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects / Q000201:enzymology
D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D011505:Protein-Tyrosine Kinases