There is increasing evidence that cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostanoid synthesis, is involved in the mechanism of ischemic brain injury. However, the reaction products responsible COX-2-mediated neurotoxicity have not been defined. Prostaglandin E2 (PGE2) is the major COX-2 derived prostanoid produced following brain injury and is involved in the mechanisms of glutamate excitotoxicity (Ann. Neurol. 55, 668). PGE2 acts on four receptor subtypes (EP1-4). While activation of EP2 receptors is neuroprotective (J. Neurosci. 24, 257), EP1 receptors mediate injury in other organs (Hypertension 42, 1183). Therefore, in this study we tested the hypothesis that EP1 receptors mediate the neurotoxicity exerted by COX-2.
Methods
Brain lesions were produced by injecting NMDA into the parietal cortex of wild-type mice or mice lacking EP1 receptors (EP1−/−). Lesion volumes were determined 24 hours later in thionine-stained sections. The COX-2 inhibitor NS398 (NS) (20 mg/kg) or the EP1 antagonist SC51089 (SC) (10 μg/kg) was administered systemically. Organotypic hippocampal slices from newborn mice were used after 2 weeks in culture. Slices were exposed to oxygen-glucose deprivation (OGD) for 1 h and assayed for cell death 24 hours later. Intracellular free Ca++ was determined in cortical mixed cultures by ratiometric fluorography using Fura-2 as an indicator.
Results
The COX-2 inhibitor NS and the EP1 receptor antagonist SC produced comparable reductions in NMDA-induced lesions (figure) A. Co-administration of NS and SC did not produce added protection (figure 1A). However, the free radical scavenger SOD (100mU) reduced lesion volume further (figure A), indicating that the injury was not maximally reduced. EP1−/− mice showed a reduction in lesion volume similar to SC-treated wild-type mice. OGD-induced cell death in hippocampal slices was reduced by 32.9±2.4% by NS and by 29.2±2.1% by SC (p<0.05 from vehicle; n=15/group). Co-treatment with NS and SC did not confer added protection (p>0.05 from NS or SC alone; n=15/group). To gain insight into the mechanism by which EP1 receptors promote neuronal cell death, we monitored intracellular free Ca++ in cultures exposed to NMDA (100 μM) in the presence or absence NS or SC. NS or SC significantly attenuated NMDA-induced rise in intracellular free Ca++ (figure B).
Conclusions
EP1 receptors contribute to the neurotoxicity exerted by activation of NMDA receptors or OGD. The observation that the protection conferred by COX-2 or EP1 receptor inhibition are not additive is consistent with the hypothesis that they exert their deleterious effects through the same cell death pathway and support the idea that EP1 receptors mediate the deleterious effects of COX-2. EP1 receptors may promote cell death by facilitating the Ca++ overload produced by excitotoxicity.