A stress-responsive, mitogen-activated protein kinase, p38, is activated by phosphorylation in response to adverse environmental insults. In the present study, the effects of hyperosmolarity on p38 activation and protein synthesis in the brain were examined. Hyperosmotic stress of rat brain slices, produced by addition of sorbitol to the incubation buffer, produced prolonged phosphorylation and activation of p38, most prominently in the hippocampus as compared to the cortex or cerebellum. In comparison, the prototypic mitogen-activated protein kinase, extracellular signal-regulated kinase, was transiently phosphorylated and another stress-activated protein kinase, c-Jun NH(2)-terminal kinase, was not phosphorylated above basal levels. Examination of downstream p38 signaling events revealed phosphorylation of the small heat shock protein 27 (HSP27) that was abolished by incubation with SB202190 [4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole], a p38 inhibitor. Concomitantly, hyperosmolarity diminished total levels of protein synthesis within hippocampal slices, as determined by incorporation of (35)S-labeled methionine/cysteine into protein during tissue incubation. However, synthesis of a 30-kDa protein, identified as 14-3-3epsilon with mass spectrometry, increased in response to hyperosmolarity. The synthesis of 14-3-3epsilon was dose-dependently induced by increasingly hyperosmotic conditions in a p38-independent manner. We conclude from these results that 14-3-3epsilon synthesis and p38-mediated HSP27 phosphorylation in the hippocampus are parallel responses to the hyperosmotic environment.