adenosine-5--phosphorothioate and diphenyleneiodonium

To investigate the role of adenosine 5'-triphosphate (ATP)-induced generation of reactive oxygen species (ROS) and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the production of transforming growth factor-beta1 (TGF-beta1) in cultured rat glomerular mesangial cells under high-glucose conditions.. Subconfluent glomerular mesangial cells were serum-starved for 24 h and pretreated with suramin, diphenylenechloride iodonium (DPI) or PD98059 followed by stimulation with a high concentration of glucose (30 mmol/L D-glucose) or ATP (300 micromol/L). Extracellular and total ATP and ROS production were detected using commercially available kits. Phosphorylation of ERK1/2 was evaluated by Western blot. TGF-beta1 mRNA expression was examined by real-time PCR.. Suramin had a dose-dependent inhibitory effect on the generation of ROS induced by high glucose. Extracellular ATP production by mesangial cells increased markedly after a 2-h incubation with high glucose. ROS production was upregulated in mesangial cells after 5 min incubation with 300 micromol/L ATP and was sustained for 120 min. ERK1/2 was significantly activated after 5 min incubation of mesangial cells with ATP, this activation was partially inhibited by DPI. The effects of high glucose on TGF-beta1 mRNA were markedly inhibited by suramin, DPI or PD98059.. Our results suggest that a high concentration of glucose increases the extracellular levels of ATP in mesangial cells within a short time-frame. ATP, in turn, activates ERK1/2, an effect which is at least partially dependent on ROS, which results in the upregulation of TGF-beta1.

Topics: Adenosine Monophosphate; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Flavonoids; Glucose; Mesangial Cells; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitric Oxide Synthase; Onium Compounds; Phosphorylation; Rats; Reactive Oxygen Species; Signal Transduction; Suramin; Thionucleotides; Transforming Growth Factor beta1; Up-Regulation