Prostaglandin E2 receptor 1 activity regulates cell survival following hypoxia in cultured rat cortical neurons

Abstract

The clinical side-effects of increased cyclooxygenase (COX) activity induced by pathologic conditions have raised concerns recently. However, a better understanding of the mechanisms underlying the subsequent neurotoxicity requires knowledge of pathways downstream of COX, especially prostaglandin E2 (PGE2) and its receptors. Therefore, this study was performed to investigate the effects of PGE2 receptor 1 (EP1) activity on neuronal cell death resulting from hypoxia/reoxygenation (Hyp). As cyclinD1 activity has been shown to regulate neuronal apoptosis as well, the role of cyclinD1 was investigated, as well. Cortical neural cells isolated from fetal Wistar rats were cultured for 12 d and exposed to Hyp conditions to establish an in vitro Hyp model. To determine the effects of EP1 activity on Hyp-induced neurotoxicity, cells were treated with 17-phenyl trinor-PGE2 (17-pt), a synthetic EP1 agonist, or sc-51089, an EP1 antagonist, then exposed to hypoxic conditions for 3 h and reoxygenated for 21 h. Following Hyp, cell viability was quantified by MTT assays, and apoptosis was assessed by flow cytometry. Protein expression levels of caspase-3 and cyclinD1 were examined by Western blot analysis. Treatment of cultured cortical neurons with 17-pt significantly decreased the survival rate of Hyp-treated neurons ( p < 0.05), while treatment with sc-51089 increased the survival rate. Treatment with 17-pt also led to increased expression levels of caspase-3, further supporting a role for EP1 in the observed neurotoxicity. However, cyclinD1 expression levels were unchanged following treatment with either 17-pt or sc-51089. Therefore, EP1 may play an important role in Hyp-induced neuronal apoptosis, but this neurotoxic activity is unlikely to involve cyclinD1.