Effects of diazoxide pretreatment on expression of phosphatidylinositol 3-kinase mRNA and protein serine/threonine kinase mRNA in rat myocardial microvascular endothelial cells exposed to hypoxia-reoxygenation

Abstract

Objective To investigate the effects of diazoxide pretreatment on expression of phosphatidylinositoi 3-kinase（PI3K） mRNA and protein serine/threonine kinase（Akt） mRNA in rat myocardial microvascular endothelial cells exposed to hypoxia-reoxygenation （H/R）. Methods The SD rat myocardial microvascular endothelial cells were cultured. The cells were seeded in 96-well plates （100 μl/hole） or in 6 cm diameter dishes （2 ml/dish） with the density of 1 × 10^6/ml and randomly divided into 4 groups （ n = 25 each） ： normal control group （group C）, H/R group, diazoxide pretreatment group （group DZ） and diazoxide pretreatment ＋ 5-hydroxydecanoate （5-HD, a mitochondrial ATP-sensitive potassium channel blocker） group （group DZ ＋ 5-HD） . The cells were exposed to 2 h hypoxia followed by 2 h reoxygenation. Diazoxide 100 μmol/L and diazoxide 100 μmol/L ＋ 5-HD 100 μmol/L were added to the culture medium 2 h before hypoxia in groups DZ and DZ ＋ 5-HD respectively. The cell viability, apoptotic rate and expression of PI3K mRNA and Akt mRNA were detected at the end of reoxygenation. Results Compared with group C, the cell viability was significantly decreased, while the apoptotic rate increased and expression of PI3 K mRNA and Akt mRNA up-regulated in group H/R （P 〈 0.05 or 0.01 ）. Compared with group H/R, the cell viability was significantly increased, while the apoptotic rate decreased and expression of PI3K mRNA and Akt mRNA up-regulated in group DZ （P 〈 0.05 or 0.01 ）. 5-HD could inhibit diazoxide pretreatment-induced changes mentioned above （ P 〈 0.05 or 0.01 ）. Conclusion Diazoxide pretreatment can reduce H/R injury by promoting PI3K gene and Akt gene transcription through activation of mitochondrial ATP-sensitive potassium channels in rat myocardial microvascular endothelial cells. Key words: Diazoxide ; Heart ; Endothelium, vascular; Oxygen; Cell hypoxia ; 1- Phosphati-dylinositol 3-kinase ; Protein-serine-threonine kinases ; RNA, messenger