Figure 5

AT2R modulates zinc-triggered NADPH oxidase activation in cortical cells. A) Western blotting for the cytosolic subunit of the NADPH oxidase p67^phox in cytosolic and membrane fractions of cortical cell cultures, sham-washed (CTL) or after 15 min exposure to 300 μM zinc, zinc plus 1 μM angiotensin II, angiotensin II alone, zinc plus 1 μM PD123319, or PD123319 alone. B) Bars denote the relative density of p67^phox bands normalized to corresponding actin bands (mean ± SEM, n = 11) in the above Western blot experiments. Whereas angiotensin II or PD123319 alone had no effect on the level or distribution of p67^phox, zinc exposure substantially increased the levels of p67^phox in both cytosolic and membrane fractions, an effect that was significantly potentiated by addition of angiotensin II. Notably, p67^phox increased to a greater degree in the membrane fraction, indicating that zinc exposure not only induced, but also activated, NADPH oxidase. Addition of PD123319 inhibited increases in p67^phox in the membrane fraction induced by zinc plus angiotensin II (*p < 0.05, **p < 0.01 for differences between indicated values; two-tailed t-test). C) Bars denote the level of Rac activation as assessed by G-LISA assay. Consistent with the pattern of NADPH oxidase subunit translocation, zinc treatment increased Rac activity in cortical cells, an effect that was increased by angiotensin II and decreased by PD123319. Again, neither angiotensin II nor PD123319 alone had any effect on the level of Rac activation (*p < 0.05, **p < 0.01 for differences between indicated values; two-tailed t-test). D) LDH release (mean ± SEM, n = 4) in cortical cell cultures after 15 min exposure to 300 μM zinc, zinc plus angiotensin II (1 μM), and zinc plus angiotensin II and apocynin (500 μM) (**p < 0.01 for differences between indicated values; two-tailed t-test).