Antioxidants and NOS inhibitors selectively targets manganese-induced cell volume via Na–K–Cl cotransporter-1 in astrocytes

• Mn, oxidants & NO donors significantly increased the oxidation/nitration of NKCC1.
• Total protein, phosphorylation, and activity of NKCC & cell volume were increased.
• Antioxidants and NOS inhibitor reversed manganese-induced alterations.
• Bumetanide as well as NKCC1-siRNA also reversed all these changes.
• Oxidation/nitration of NKCC1 may contribute to Mn-induced astrocytes swelling.

Manganese has shown to be involved in astrocyte swelling. Several factors such as transporters, exchangers and ion channels are attributed to astrocyte swelling as a result in the deregulation of cell volume. Products of oxidation and nitration have been implied to be involved in the pathophysiology of swelling; however, the direct link and mechanism of manganese induced astrocyte swelling has not been fully elucidated. In the current study, we used rat primary astrocyte cultures to investigate the activation of Na–K–Cl cotransporter-1 (NKCC1) a downstream mechanism for free radical induced astrocyte swelling as a result of manganese toxicity. Our results showed manganese, oxidants and NO donors as potent inducer of oxidation and nitration of NKCC1. Our results further confirmed that manganese (50 μM) increased the total protein, phosphorylation and activity of NKCC1 as well as cell volume (p<0.05 vs. control). NKCC1 inhibitor (bumetanide), NKCC1-siRNA, antioxidants; DMTU, MnTBAP, tempol, catalase and Vit-E, NOS inhibitor; l-NAME, peroxinitrite scavenger; uric acid all significantly reversed the effects of NKCC1 activation (p<0.05). From the current investigation we infer that manganese or oxidants and NO induced activation, oxidation/nitration of NKCC1 play an important role in the astrocyte swelling.