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Angiotensin II potentiates zinc-induced cortical neuronal death by acting on angiotensin II type 2 receptor.

Park MH, Kim HN, Lim JS, Ahn JS, Koh JY - Mol Brain (2013)

Bottom Line: Interestingly, addition of angiotensin II significantly enhanced the zinc-triggered neuronal death, while leaving astrocytic cell death relatively unchanged.Combined treatment with zinc and angiotensin II substantially increased the levels of superoxides in neurons compared to those induced by zinc alone.The present study shows that the angiotensin system, especially that involving AT2R, may have an oxidative injury-potentiating effect via augmentation of the activity of NADPH oxidase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neural Injury Research Lab, University of Ulsan College of Medicine, Seoul 138-736, South Korea. jkko@amc.seoul.kr.

ABSTRACT

Background: The angiotensin system has several non-vascular functions in the central nervous system. For instance, inhibition of the brain angiotensin system results in a reduction in neuronal death following acute brain injury such as ischemia and intracerebral hemorrhage, even under conditions of constant blood pressure. Since endogenous zinc has been implicated as a key mediator of ischemic neuronal death, we investigated the possibility that the angiotensin system affects the outcome of zinc-triggered neuronal death in cortical cell cultures.

Results: Exposure of cortical cultures containing neurons and astrocytes to 300 μM zinc for 15 min induced submaximal death in both types of cells. Interestingly, addition of angiotensin II significantly enhanced the zinc-triggered neuronal death, while leaving astrocytic cell death relatively unchanged. Both type 1 and 2 angiotensin II receptors (AT1R and AT2R, respectively) were expressed in neurons as well as astrocytes. Zinc neurotoxicity was substantially attenuated by PD123319, a specific inhibitor of AT2R, and augmented by CGP42112, a selective activator of AT2R, indicating a critical role for this receptor subtype in the augmentation of neuronal cell death.Because zinc toxicity occurs largely through oxidative stress, the levels of superoxides in zinc-treated neurons were assessed by DCF fluorescence microscopy. Combined treatment with zinc and angiotensin II substantially increased the levels of superoxides in neurons compared to those induced by zinc alone. This increase in oxidative stress by angiotensin II was completely blocked by the addition of PD123319. Finally, since zinc-induced oxidative stress may be caused by induction and/or activation of NADPH oxidase, the activation status of Rac and the level of the NADPH oxidase subunit p67phox were measured. Angiotensin II markedly increased Rac activity and the levels of p67phox in zinc-treated neurons and astrocytes in a PD123319-dependent manner.

Conclusion: The present study shows that the angiotensin system, especially that involving AT2R, may have an oxidative injury-potentiating effect via augmentation of the activity of NADPH oxidase. Hence, blockade of angiotensin signaling cascades in the brain may prove useful in protecting against the oxidative neuronal death that is likely to occur in acute brain injury.

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AT2R modulates the level of zinc-triggered oxidative stress in cortical cells. A) Cortical cells were loaded with H2-DCFDA (DCF) to detect ROS as an indicator of oxidative stress, and subsequently treated with zinc (Zn), zinc plus angiotensin II (+Ang), zinc plus PD123319 (+PD), or zinc plus losartan (+Losartan). Compared to sham-washed cultures, zinc treatment increased the level of DCF fluorescence in neurons, an effect that was further augmented by addition of angiotensin II. PD123319 markedly attenuated the zinc-induced increase in DCF fluorescence in neurons, whereas losartan had no effect. B) Bars denote relative DCF fluorescence in cortical cell cultures following 15 min exposure to zinc, zinc plus angiotensin II, zinc plus PD123319, or zinc plus losartan (*p < 0.05 versus zinc alone; two-tailed t-test). C) 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 NAC (1 mM). Addition of NAC blocked the potentiating effect of angiotensin II in zinc-induced neuronal cell death (*p < 0.05, **p < 0.01 for differences between indicated values; two-tailed t-test).
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Figure 4: AT2R modulates the level of zinc-triggered oxidative stress in cortical cells. A) Cortical cells were loaded with H2-DCFDA (DCF) to detect ROS as an indicator of oxidative stress, and subsequently treated with zinc (Zn), zinc plus angiotensin II (+Ang), zinc plus PD123319 (+PD), or zinc plus losartan (+Losartan). Compared to sham-washed cultures, zinc treatment increased the level of DCF fluorescence in neurons, an effect that was further augmented by addition of angiotensin II. PD123319 markedly attenuated the zinc-induced increase in DCF fluorescence in neurons, whereas losartan had no effect. B) Bars denote relative DCF fluorescence in cortical cell cultures following 15 min exposure to zinc, zinc plus angiotensin II, zinc plus PD123319, or zinc plus losartan (*p < 0.05 versus zinc alone; two-tailed t-test). C) 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 NAC (1 mM). Addition of NAC blocked the potentiating effect of angiotensin II in zinc-induced neuronal cell death (*p < 0.05, **p < 0.01 for differences between indicated values; two-tailed t-test).

Mentions: Although diverse intracellular events contribute to zinc-triggered cell death [34], oxidative stress is considered a major mechanism [35]. Hence, it is plausible that angiotensin II and AT2Rs specifically participate in oxidative stress mechanisms in the context of zinc neurotoxicity. To examine this possibility, we loaded cortical neurons with H2-DCFDA, a fluorescent indicator for superoxides, and examined ROS levels following different treatment regimens. Exposure to 300 μM zinc for 15 min substantially increased the levels of ROS in cultured cortical neurons, an effect that was markedly potentiated by the addition of 1 μM angiotensin II (Figure 4A). Co-incubation with the AT2R inhibitor PD123319 (1 μM) blocked the zinc-induced increases in ROS levels, whereas the AT1R inhibitor losartan was not effective. Quantitative measurements of H2-DCFDA fluorescence further confirmed that the inhibition of AT2R specifically reduced ROS levels in zinc-treated cells (Figure 4B). We also used N-acetyl-L-cysteine (NAC) to suppress oxidative stress in zinc-induced neuronal cell death, and found that suppression of oxidative stress fully negated the potentiating effect of angiotensin II (Figure 4C).


Angiotensin II potentiates zinc-induced cortical neuronal death by acting on angiotensin II type 2 receptor.

Park MH, Kim HN, Lim JS, Ahn JS, Koh JY - Mol Brain (2013)

AT2R modulates the level of zinc-triggered oxidative stress in cortical cells. A) Cortical cells were loaded with H2-DCFDA (DCF) to detect ROS as an indicator of oxidative stress, and subsequently treated with zinc (Zn), zinc plus angiotensin II (+Ang), zinc plus PD123319 (+PD), or zinc plus losartan (+Losartan). Compared to sham-washed cultures, zinc treatment increased the level of DCF fluorescence in neurons, an effect that was further augmented by addition of angiotensin II. PD123319 markedly attenuated the zinc-induced increase in DCF fluorescence in neurons, whereas losartan had no effect. B) Bars denote relative DCF fluorescence in cortical cell cultures following 15 min exposure to zinc, zinc plus angiotensin II, zinc plus PD123319, or zinc plus losartan (*p < 0.05 versus zinc alone; two-tailed t-test). C) 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 NAC (1 mM). Addition of NAC blocked the potentiating effect of angiotensin II in zinc-induced neuronal cell death (*p < 0.05, **p < 0.01 for differences between indicated values; two-tailed t-test).
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Figure 4: AT2R modulates the level of zinc-triggered oxidative stress in cortical cells. A) Cortical cells were loaded with H2-DCFDA (DCF) to detect ROS as an indicator of oxidative stress, and subsequently treated with zinc (Zn), zinc plus angiotensin II (+Ang), zinc plus PD123319 (+PD), or zinc plus losartan (+Losartan). Compared to sham-washed cultures, zinc treatment increased the level of DCF fluorescence in neurons, an effect that was further augmented by addition of angiotensin II. PD123319 markedly attenuated the zinc-induced increase in DCF fluorescence in neurons, whereas losartan had no effect. B) Bars denote relative DCF fluorescence in cortical cell cultures following 15 min exposure to zinc, zinc plus angiotensin II, zinc plus PD123319, or zinc plus losartan (*p < 0.05 versus zinc alone; two-tailed t-test). C) 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 NAC (1 mM). Addition of NAC blocked the potentiating effect of angiotensin II in zinc-induced neuronal cell death (*p < 0.05, **p < 0.01 for differences between indicated values; two-tailed t-test).
Mentions: Although diverse intracellular events contribute to zinc-triggered cell death [34], oxidative stress is considered a major mechanism [35]. Hence, it is plausible that angiotensin II and AT2Rs specifically participate in oxidative stress mechanisms in the context of zinc neurotoxicity. To examine this possibility, we loaded cortical neurons with H2-DCFDA, a fluorescent indicator for superoxides, and examined ROS levels following different treatment regimens. Exposure to 300 μM zinc for 15 min substantially increased the levels of ROS in cultured cortical neurons, an effect that was markedly potentiated by the addition of 1 μM angiotensin II (Figure 4A). Co-incubation with the AT2R inhibitor PD123319 (1 μM) blocked the zinc-induced increases in ROS levels, whereas the AT1R inhibitor losartan was not effective. Quantitative measurements of H2-DCFDA fluorescence further confirmed that the inhibition of AT2R specifically reduced ROS levels in zinc-treated cells (Figure 4B). We also used N-acetyl-L-cysteine (NAC) to suppress oxidative stress in zinc-induced neuronal cell death, and found that suppression of oxidative stress fully negated the potentiating effect of angiotensin II (Figure 4C).

Bottom Line: Interestingly, addition of angiotensin II significantly enhanced the zinc-triggered neuronal death, while leaving astrocytic cell death relatively unchanged.Combined treatment with zinc and angiotensin II substantially increased the levels of superoxides in neurons compared to those induced by zinc alone.The present study shows that the angiotensin system, especially that involving AT2R, may have an oxidative injury-potentiating effect via augmentation of the activity of NADPH oxidase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neural Injury Research Lab, University of Ulsan College of Medicine, Seoul 138-736, South Korea. jkko@amc.seoul.kr.

ABSTRACT

Background: The angiotensin system has several non-vascular functions in the central nervous system. For instance, inhibition of the brain angiotensin system results in a reduction in neuronal death following acute brain injury such as ischemia and intracerebral hemorrhage, even under conditions of constant blood pressure. Since endogenous zinc has been implicated as a key mediator of ischemic neuronal death, we investigated the possibility that the angiotensin system affects the outcome of zinc-triggered neuronal death in cortical cell cultures.

Results: Exposure of cortical cultures containing neurons and astrocytes to 300 μM zinc for 15 min induced submaximal death in both types of cells. Interestingly, addition of angiotensin II significantly enhanced the zinc-triggered neuronal death, while leaving astrocytic cell death relatively unchanged. Both type 1 and 2 angiotensin II receptors (AT1R and AT2R, respectively) were expressed in neurons as well as astrocytes. Zinc neurotoxicity was substantially attenuated by PD123319, a specific inhibitor of AT2R, and augmented by CGP42112, a selective activator of AT2R, indicating a critical role for this receptor subtype in the augmentation of neuronal cell death.Because zinc toxicity occurs largely through oxidative stress, the levels of superoxides in zinc-treated neurons were assessed by DCF fluorescence microscopy. Combined treatment with zinc and angiotensin II substantially increased the levels of superoxides in neurons compared to those induced by zinc alone. This increase in oxidative stress by angiotensin II was completely blocked by the addition of PD123319. Finally, since zinc-induced oxidative stress may be caused by induction and/or activation of NADPH oxidase, the activation status of Rac and the level of the NADPH oxidase subunit p67phox were measured. Angiotensin II markedly increased Rac activity and the levels of p67phox in zinc-treated neurons and astrocytes in a PD123319-dependent manner.

Conclusion: The present study shows that the angiotensin system, especially that involving AT2R, may have an oxidative injury-potentiating effect via augmentation of the activity of NADPH oxidase. Hence, blockade of angiotensin signaling cascades in the brain may prove useful in protecting against the oxidative neuronal death that is likely to occur in acute brain injury.

Show MeSH
Related in: MedlinePlus