Research reportDifferential roles of cyclooxygenase isoforms after kainic acid-induced prostaglandin E2 production and neurodegeneration in cortical and hippocampal cell cultures
Introduction
Cyclooxygenase (COX), the enzyme responsible for the conversion of arachidonic acid to prostaglandin H2, exists as two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed to produce prostaglandins required for normal physiological functions, while COX-2 is rapidly and transiently induced by various growth factors and cytokines [16]. In the brain, COX-2 is also induced in pathological conditions such as seizure, ischemia or other degenerative diseases [18], [28], [32]. However, the functional roles of COX isozymes have not yet been explored. Recently, in vivo experiment showed that indomethacin (a COX nonselective inhibitor), or NS398 (a COX-2 selective inhibitor), prevented or aggravated neuronal death following ischemia and kainic acid injection [2], [4], [22], [27]. Indomethacin increased cell viability in cerebellar granule cells stimulated by N-methyl-d-aspartate (NMDA) and kainic acid [7]. Despite these reports, the relative contributions of COX-1 and COX-2 to neuronal cell death and prostaglandin synthesis triggered by kainic acid are largely unknown.
Kainic acid is a cyclic analogue of the major excitatory neurotransmitter, glutamate. It is an agonist for the kainic acid receptor, which is a subfamily of glutamate receptors. Glutamate receptor-mediated excitotoxicity is believed to contribute to neuronal cell death in many neurodegenerative diseases including CNS trauma, cerebral hypoxia–ischemia and seizure [11], [24]. Systemic kainic acid administration in rats produced a seizure activity in limbic structure and selective neuronal cell death in hippocampal CA1 and CA3 neurons [2], and triggered the increase of COX-2 expression as well as prostaglandin formation in the brain [9]. In transgenic mice overexpressing neuronal COX-2, excitotoxicity was potentiated [20]. These reports consequently indicated that prostaglandin and COX might have an important role in neuronal cell death or survival.
To further understand the involvement of COX isoforms in kainic acid-induced neuronal cell death, we undertook studies on the production of PGE2 following kainic acid treatment in cortical or hippocampal neuronal culture. We also noted the differential effects of COX isoforms on kainic acid-induced neuronal death using COX inhibitors.
Section snippets
Cell culture
Primary cultured cortical or hippocampal neurons were prepared from fetal mice with 14–15-day gestation as previously described [10], [14]. Briefly, cortices or the hippocampi freed of meninges were mechanically dissociated with a flame-polished Pasteur pipette in MS (Eagle’s minimal essential medium supplemented with 20 μM glucose). Cells were placed in 24-multiwell plates (Primaria, Falcon, Bedford, MA) that had been coated previously overnight with poly-d-lysine (100 μg/ml) and laminin (100
Effect of COX inhibitors on kainic acid-induced PGE2 releases in cell cultures of cortical and hippocampal neurons
In cortical neurons, PGE2 was released in basal condition without stimuli, and kainic acid (100 μM) increased PGE2 release by 2.5-fold for 24 h. Indomethacin (10 μM) and aspirin (100 μM) inhibited PGE2 release by 25% and 54% in the basal and by 29% and 30% in kainic acid-stimulated cortical neurons. However, NS398 (10 μM) failed to inhibit PGE2 release in basal as well as in kainic acid-treated condition (Fig. 1A). We also examined other COX inhibitors such as celecoxib, a COX-2 selective
Discussion
In this study, we found kainic acid-induced PGE2 release closely correlated with neuronal death in primary cultured cortical or hippocampal neurons. Cortical neuron released PGE2 through COX-1 activity rather than COX-2 activity in basal as well as in kainic acid-stimulated conditions, while hippocampal neurons acted with more sensitive response to kainic acid through COX-2 de novo synthesis.
Apoptotic or necrotic mechanisms might participate in kainic acid-induced neuronal death. In particular,
Acknowledgements
This study was supported by KOSEF 1999-01-205-004-3 and KOSEF 2000-G-0102.
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