Abstract 393
Poster Session III, Monday, 5/3 (poster 154)
Adenosine, a known neuromodulator, may play an important role in the prevention of excitotoxic injury in the newborn brain. Adenosine and postsynaptic A1 receptors may indirectly counteract NMDA-mediated depolarization by its effect on voltage-sensitive K+ currents. Our previous studies have shown that theophylline, an adenosine receptor antagonist, administration prior to hypoxia prevents the hypoxia-induced increase in free radical generation and lipid peroxidation. The present study tests the hypothesis that adenosine and its postsynaptic receptors are inhibitory to the NMDA receptor ion-channel function. Studies were conducted on six anesthetized normoxic newborn piglets, using adenosine and its antagonist, theophylline. P2 brain cell membranes fractions were prepared from the piglet cortices and 3H-MK-801 binding was used as an index of NMDA receptor activation. Binding studies were carried out at 32°C for 3 hours in a medium containing 10 mM HEPES buffer at pH 7.0, 75 µg membrane protein and 10 nM 3H-MK-801 in the presence and absence of the activators glutamate and glycine (1-10 µM). Non specific binding was performed in the presence of 10 µM unlabeled MK-801. Binding was also carried out in the presence of adenosine (1-100 µM), and the adenosine antagonists theophylline (1-100 µM). The glutamate and glycine dependent increase in 3H-MK-801 binding was 29 ± 26% at 1 µM and 118 ± 62% at 10 µM. Adenosine did not directly inhibit MK-801 binding. Theophylline increased MK-801 binding by 47.8 ± 30% independent of glutamate and glycine activation. In the presence of 100 µM theophylline and 100 µM adenosine, the 3MK-801 binding was 54 ± 22%, indicating that adenosine did not alter the theophylline-dependent increase in MK-801 binding. These in vitro results show that adenosine alone does not directly reduce NMDA receptor activation, however, the adenosine antagonist, theophylline, directly increases NMDA receptor activation independently from glutamate and glycine. The activation of NMDA receptor by theophylline was not blocked by adenosine. We conclude that postsynaptic adenosine receptors play a direct regulatory role on NMDA receptor-ion channel function. Since theophylline increased 3MK-801 binding without the presence of glutamate and glycine, we speculate that theophylline transduces its effect by interacting at a site other than the glutamate recognition and the glycine co-activator sites of the NMDA receptor and affecting the ion-channel opening.
