Optic nerve head astrocytes become abnormal in eyes that have elevated intraocular pressure, and cultured astrocytes display altered protein expression after being subjected for > or = 1 days to elevated hydrostatic pressure. Here we show that 2-h elevated hydrostatic pressure (15 or 30 mmHg) causes phosphorylation of ERK1/2, ribosomal S6 protein kinase (p90(RSK)), and Na/H exchanger (NHE)1 in cultured rat optic nerve head astrocytes as judged by Western blot analysis. The MEK/ERK inhibitor U0126 abolished phosphorylation of NHE1 and p90(RSK) as well as ERK1/2. To examine NHE1 activity, cytoplasmic pH (pH(i)) was measured with BCECF and, in some experiments, cells were acidified by 5-min exposure to 20 mM ammonium chloride. Although baseline pH(i) was unaltered, the rate of pH(i) recovery from acidification was fourfold higher in pressure-treated astrocytes. In the presence of either U0126 or dimethylamiloride (DMA), an NHE inhibitor, hydrostatic pressure did not change the rate of pH(i) recovery. The findings are consistent with NHE1 activation due to phosphorylation of ERK1/2, p90(RSK), and NHE1 that occurs in response to hydrostatic pressure. These responses may precede long-term changes of protein expression known to occur in pressure-stressed astrocytes.