Ammonia toxicity is clinically important and biologically poorly understood. We reported previously that 3mM
ammonia chloride (
ammonia), a relevant concentration for
hepatic encephalopathy studies, increases production of endogenous
ouabain and activity of Na,K-
ATPase in astrocytes. In addition,
ammonia-induced upregulation of gene expression of α2
isoform of Na,K-
ATPase in astrocytes could be inhibited by
AG1478, an inhibitor of the
EGF receptor (EGFR), and by PP1, an inhibitor of Src, but not by
GM6001, an inhibitor of
metalloproteinase and shedding of
growth factor, suggesting the involvement of endogenous
ouabain-induced
EGF receptor transactivation. In the present cell culture study, we investigated
ammonia effects on phosphorylation of
EGF receptor and its intracellular signal pathway towards MAPK/ERK1/2 and PI3K/AKT; interaction between
EGF receptor, α1, and α2
isoforms of Na,K-
ATPase, Src, ERK1/2, AKT and
caveolin-1; and relevance of these signal pathways for
ammonia-induced cell swelling, leading to
brain edema, an often fatal complication of
ammonia toxicity. We found that (i)
ammonia increases
EGF receptor phosphorylation at EGFR(845) and EGFR(1068); (ii)
ammonia-induced ERK1/2 and AKT phosphorylation depends on the activity of
EGF receptor and Src, but not on
metalloproteinase; (iii) AKT phosphorylation occurs upstream of ERK1/2 phosphorylation; (iv)
ammonia stimulates association between the α1 Na,K-
ATPase isoform, Src,
EGF receptor, ERK1/2, AKT and
caveolin-1; (v)
ammonia-induced ROS production might occur later than EGFR transactivation; (vi) both
ammonia induced ERK phosphorylation and ROS production can be abolished by
canrenone, an inhibitor of
ouabain, and (vii)
ammonia-induced cell swelling depends on signaling via the Na,K-
ATPase/
ouabain/Src/
EGF receptor/PI3K-AKT/ERK1/2, but in response to 3mM
ammonia it does not appear until after 12h. Based on literature data it is suggested that the delayed appearance of the
ammonia-induced swelling at this concentration reflects required
ouabain-induced oxidative damage of the ion and water
cotransporter NKCC1. This information may provide new therapeutic targets for treatment of hyperammonic
brain disorders.