Ammonia-induced activation of p53 in cultured astrocytes: Role in cell swelling and glutamate uptake

Cytotoxic brain edema, due principally to astrocyte swelling, is a major neurological complication of the acute form of hepatic encephalopathy (HE) (acute liver failure, ALF), a condition likely caused by elevated levels of brain ammonia. Potential mediators of ammonia-induced astrocyte swelling include oxidative/nitrosative stress (ONS), the mitochondrial permeability transition (mPT), mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-κB), since blockade of these factors reduces the extent of astrocyte swelling. As p53, a tumor suppressor protein and transcription factor, is a downstream target of ONS and MAPKs, we examined its potential role in the mechanism of ammonia-induced astrocyte swelling. Astrocytes exposed to NH₄Cl (5 mM) showed increased phosphorylation (activation) of p53^(Ser392) at 1 h and such phosphorylation was significantly reduced by inhibitors of MAPKs (ERK1/2, JNK and p38-MAPK), antioxidants (vitamin E, catalase, PBN, desferoxamine, MnTBAP), as well as by L-NAME, an inhibitor of nitric oxide synthase, indicating a key role of oxidative/nitrosative stress and MAPKs in the ammonia-induced activation of p53. Since p53 is known to induce the mPT and to activate NF-κB (factors leading to ONS and implicated in ammonia-induced astrocyte swelling), we examined whether inhibition of p53 activation blocked mPT induction, NF-κB activation, as well as cell swelling. Pifithrin-α (PFT), an inhibitor of p53, blocked these processes. Impairment of astrocytic glutamate uptake is another important feature of HE and hyperammonemia. We therefore examined the potential role of p53 in the ammonia-induced inhibition of glutamate uptake and found that PFT also reversed the ammonia-induced inhibition of glutamate uptake. Our results indicate that a potentially important downstream target of ammonia neurotoxicity is p53, whose activation contributes to astrocyte swelling and glutamate uptake inhibition, processes likely a consequence of ONS derived from the mPT and activation of NF-κB.