High Glucose–Induced Oxidative Stress Increases Transient Receptor Potential Channel Expression in Human Monocytes
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- FIG. 1.
Changes of mRNA expression of (A) TRPC3, (B) TRPC6, (C and D) TRPC1, TRPC5, TRPM6, TRPM7, and TNF-α induced by high glucose and ONOO. Exposure of monocytes to high glucose (HG; 30 mmol/l d-glucose) or ONOO (100 μmol/l) for 4 h significantly increased the TRPC3 and TRPC6 mRNA expression. The effect of high glucose is attenuated by superoxide dismutase mimetic TMP (100 μmol/l). Administration of l-glucose showed no significant effect. Control (open bar) indicates control glucose (5.6 mmol/l d-glucose). The expression of each factor is normalized to GAPDH expression. Data are mean ± SEM from at least four to six independent experiments. *P < 0.05; **P < 0.01 compared with control conditions. E: Bar graph showing TRPC3 mRNA and TRPC6 mRNA expression (normalized ratio) in monocytes from 28 age-matched control subjects (open bars) and from 18 patients with type 2 diabetes (filled bars). *P < 0.05 compared with control.
- FIG. 2.
Quantitative in-cell Western assay of TRPC3 and TRPC6 channel protein expression in monocytes treated with HG (30 mmol/l d-glucose) in the absence and presence of TMP (100 μmol/l), ONOO (100 μmol/l), l-glucose (30 mmol/l), or control (5.6 mmol/l d-glucose). A: Representative conventional Western assay (TRPC3 protein and GAPDH protein for loading control with top values indicating densitometric analysis of blots). The TRPC3/GAPDH ratio (fold over control) is also indicated. B and D: Representative in-cell Western assays and (C and E) summary data are shown. Data are means ± SEM from at least eight independent experiments. **P < 0.01 compared with control conditions.
- FIG. 3.
Recordings of fura-2 fluorescence in human monocytes. A: OAG-induced calcium influx was measured under control conditions (5.6 mmol/l d-glucose, ○) and in the presence of high glucose (30 mmol/l d-glucose) without (■) and with (▲) TMP (100 μmol/l). B: Summary data showing intracellular calcium levels after treatment of monocytes with high d-glucose for 4 h (control) stimulated with ONOO in the absence and presence of the membrane-permeable TRPC blocker, 2-APB. **P < 0.01.
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