Regulation of Amino Acid Neurotransmitter Levels by 8-isopge2 in Bovine Retinae, ex vivo

Abstract

Purpose:: We have evidence that isoprostanes (IsoPs) can alter the release of exogenously applied radiolabeled excitatory amino acid neurotransmitters in isolated bovine retinae (Opere et al., Neurochem. Res. 30: 2005). It remains to be determined whether IsoPs can also regulate endogenous amino acid neurotransmitter concentrations in the bovine retina. The aim of the present study was to investigate the effects of intravitreally injected 8-isoprostaglandin (8-isoPGE₂) on endogenous levels of excitatory and inhibitory neurotransmitters in bovine retina, ex vivo.

Methods:: Freshly isolated bovine eyeballs were equilibrated in oxygenated Krebs buffer solution before being injected intravitreously with either IsoP or vehicle for control. After 30 minutes of incubation in Krebs buffer solution, retina was isolated and prepared for measurement of glutamate, glutamine, glycine and γ-aminobutyric acid (GABA) by HPLC-EC. The mobile phase was 75% 0.1M sodium phosphate with 25% methanol, pH 6.75 and was pumped at 0.6 ml/min. Samples were derivatised by o-phthaldialdehyde (OPA)/ 2-mercaptoethanol (ß ME) pre-column for detection. Amino acids were detected using homoserine as the internal standard and Shimadzu chromatographic software for peak area integration.

Results:: Intravitreally injected 8-isoPGE₂ (0.1nM-100µM) attenuated the levels of both glutamate and its metabolite, glutamine in a concentration-dependent manner in bovine retina, ex vivo. For instance, 8-isoPGE₂ (100 µM) decreased glutamate and glutamine levels by 48% (P<0.01) and 82% (p<0.001). Similarly, 8-isoPGE₂ (0.1nM-100µM) decreased the level of inhibitory amino acid neurotransmitter, glycine in bovine retina, with a maximal inhibitory effect of 40% (P<0.05) being observed at a concentration of 10nM. Interestingly, 8-isoPGE₂ (0.1nM-100 µM) had no significant effect on the level of the inhibitory neurotransmitter, GABA.

Conclusions:: We conclude that with the exception of GABA, 8-isoPGE₂ can regulate both excitatory and inhibitory amino acid neurotransmitter levels in bovine retina, ex vivo. The mechanisms underlying the observed changes in neurotransmitter level due to isoprostanes merits further investigation.

Keywords: retina • excitatory neurotransmitters • inhibitory neurotransmitters