Inhibition of Ischemia-Induced [3H]D-Aspartate Release in Bovine Retinae by Cannabinoids

Abstract

Abstract: : Purpose: There is evidence that cannabinoid receptor agonists can protect rat hippocampal neurons against ischemia-induced, glutamate-mediated excitotoxicity (Nagayama et al., J. Neurosci. 19:2987, 1999). Although cannabinoid receptors subtypes have been localized in the mammalian retina (Lu et al., Vis. Neurosc. 17: 91, 2000), their role in neurocircuitry and neurotransmission is unknown. In the present study, we investigated the effect of selective cannabinoid CB₁-receptor agonists on ischemia-induced [₃H]D-aspartate release from bovine retinae in vitro. Methods: Isolated neural retinae were incubated in oxygenated Krebs solution containing 200 nM of [₃H]D-aspartate for 60 mins and then prepared for studies of neurotransmitter release using the superfusion method. Release of [₃H]D-aspartate was evoked by K₊(50 mM) stimuli applied at 90 mins (S₁) and ischemia (induced by exposure of tissues to glucose-deficient Krebs buffer solution gassed with 95% N₂:5% CO₂) at 108 mins (S₂) after the onset of superfusion. Under these experimental conditions, [₃H]D-aspartate has been reported to be a valid marker of endogenous glutamate in retinal neurons (Santos et al., Neurochem. Res. 21: 361, 1996). Results: Under conditions of ischemia, the pO₂ in glucose-deprived buffer solution was 0.54 ± 0.04 (n = 20). Exposure of bovine retinal tissues to ischemic buffer solution increased the spontaneous efflux of [₃H]D-aspartate by 22%. In the concentration range, 0.1 µM to 1 µM, methananamide, a selective cannabinoid CB₁-receptor agonist caused a concentration-dependent inhibition of ischemia-induced [₃H]D-aspartate overflow without affecting basal tritium efflux. The IC₅₀ (effective concentration of drug that caused 50% inhibition of the ischemic response) values obtained for methanandamide was 0.68 µM ± 0.1 (n = 4). Arachidonyl-2’-chloroethylamide (1 µM), another selective cannabinoid CB₁-receptor agonist, also attenuated [₃H]D-aspartate release by 30%. Conclusion: We conclude that selective cannabinoid CB₁-receptor agonists can inhibit ischemia-induced [₃H]D-aspartate release from bovine retinae. These compounds may have a neuroprotective role against glutamate excitotoxicity in the neural retina.

Keywords: 489 neuroprotection • 514 pharmacology • 556 retina: neurochemistry