Hypoxia-induced [³H]D-aspartate release from isolated bovine retina: Modulation by calcium-channel blockers and glutamatergic agonists and antagonists

Purpose. The aim of the present study was two-fold: (a) to examine the effect of hypoxia on [³H]D-aspartate release from isolated bovine and human retinae, and (b) to investigate the regulation of hypoxia-induced neurotransmitter release by glutamate receptor agonists and antagonists. Methods. Isolated neural retinae were incubated in oxygenated Krebs buffer solution containing [³H]D-aspartate and then prepared for studies of neurotransmitter release using the superfusion method. Release of [³H]D-aspartate was evoked by K⁺ (50 mM) applied at 90 minutes (S₁) and hypoxia (induced by exposure of tissues to solutions pre-gassed with 95% N₂: 5% CO₂ for 60 minutes) at 108 minutes (S₂) after onset of superfusion. Results. Under hypoxic conditions, pO₂ in normal Krebs buffer solution was reduced from 14.53 ± 0.26ppm (n = 6) to 0.54 ± 0.04ppm (n = 9) after one hour of gassing with 95% N₂: 5% CO₂. Exposure to hypoxia elicited an overflow of [³H]D-aspartate yielding S²/S¹ ratios of 0.62 ± 0.06 (n = 12) and 0.54 ± 0.03 (n = 8) in bovine and human tissues respectively. In isolated bovine retinae, L- and N-calcium-channel antagonists diltiazem, nitrendipine, verapamil and ω-conotoxin significantly (p <0.01 or higher) attenuated hypoxia-induced [³H]D-aspartate release. L-glutamate (30 μM) significantly (p <0.001) potentiated hypoxia-induced [³H]D-aspartate release whereas kainate (30 μM) inhibited this response. NMDA (in concentrations up to 1 mM) had no effect on hypoxia-induced [³H]D-aspartate release. Antagonists of glutamate receptors and the polyamine site on the NMDA receptor inhibited hypoxia-induced release of [³H]D-aspartate in bovine retina with the following rank order of activity: ifenprodil ≅ MCPG > L-AP3 > MK-801. At an equimolar concentration (10 μM), L-AP3 but not ifenprodil, MCPG, MK 801 or arcaine, caused a significant (p <0.001) inhibition of hypoxia-induced [³H]D-aspartate release from human retinae. Conclusions. Hypoxia can induce the release of [³H]D-aspartate from isolated bovine retinae by a calcium-dependent process. Hypoxia-induced [³H]D-aspartate release from isolated bovine retinae can be regulated by glutamate receptor agonists/antagonists and blockers of polyamine site on the NMDA receptor.