F. Moroni

Nicotinylalanine Increases the Formation of Kynurenic Acid in the Brain and Antagonizes Convulsions

Abstract: Kynurenic acid (KYNA) was quantified in the extracellular spaces of the rat hippocampus using microdialysis and HPLC (fluorimetric detection) to study the possible role of this tryptophan metabolite in the modulation of the function of the N‐methyl‐D‐aspartate (NMDA) receptor. Addition of probenecid (1 mM), which is an inhibitor of the organic acid transport system, to the Ringers solution perfusing the dialysis probe increased the KYNA concentration in the dialysate from 10.4 ± 0.9 to 48 ± 6 nM. Addition of 2 mM aminooxyacetic acid, a nonspecific inhibitor of KYNA synthesis, reduced this concentration by 50%. These data suggest that KYNA is continuously synthesized in the rat hippocampus. Nicotinylalanine (NAL), 200–400 mg/kg i.p., an analogue of kynurenine that is able to direct the flow of tryptophan metabolites toward the synthesis of KYNA, significantly increased the KYNA concentration in the hippocampal dialysate and significantly potentiated the effect of tryptophan on the accumulation of KYNA in the brain and other organs. This increase resulted in pharmacological actions compatible with an antagonism of the NMDA receptors. In fact, NAL antagonized sound‐induced seizures and prevented death in DBA/2 mice. Pretreatment of the mice with D‐serine (100 μg intracerebroventricularly), a glycine agonist and a competitive antagonist of KYNA, completely prevented the anticonvulsive action of NAL. These data suggest that changes in the extracellular concentration of KYNA in the brain are associated with a modulation of NMDA receptor function.

Glutamate receptor antagonists protect against ischemia-induced retinal damage

The effects of intravitreal injections of excitatory amino acid receptor antagonists have been studied on the ischemic neuronal damage induced by photochemical occlusion of the retinal vessels. Rats were systemically injected with rose bengal fluorescein dye and one of their eyes was exposed to bright light. The activities of the enzymes, choline-acetyltransferase and glutamate decarboxylase, were measured as an index of neuronal loss in the lesioned tissue. Lesioned retinas had a 75 +/- 5% reduction in choline-acetyltransferase activity and a 72 +/- 8% reduction in glutamate-decarboxylase activity, suggesting that the lesion causes a massive loss of retinal neurons, which use acetylcholine or gamma-aminobutyric acid (GABA) as neurotransmitter. A single intravitreal injection of excitatory amino acid receptor antagonists, performed immediately after the lesion, significantly reduced this loss. Both alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate) (NMDA) types of ionotropic glutamate receptor antagonists were active in a dose-dependent manner. Almost complete protection was also obtained with relatively large doses of thiokynurenic acid (400 nmol), a non-selective antagonist of both AMPA and NMDA glutamate receptors, while 7-Cl-thiokynurenic acid, a potent and selective glycine receptor antagonist, was not active up to 200 nmol. These results strongly suggest that excitotoxic mechanisms are involved in ischemia-induced neuronal death in the retina and that appropriate treatments with antagonists of both AMPA and NMDA receptor types may significantly reduce this damage.

Ultrastructural and biochemical studies on the neuroprotective effects of excitatory amino acid antagonists in the ischemic rat retina.

The effects of glutamate receptor agonists were evaluated, by utilizing the electron microscope, in a photothrombotic occlusion model of rat retinal vessels in order to study the ischemic damage and its antagonism in each morphologically identified population of retinal neurons. Rats were systemically injected with rose bengal fluorescein dye and one of their eyes was then exposed to bright light. This treatment caused neuronal damage and reduced the activities of the neuronal marker enzymes, choline acetyltransferase and glutamate decarboxylase, by approximately 75%. A single intravitreal injection of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquinoxaline (NBQX, 10-50 nmol), an antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, or of thiokynurenate (100-400 nmol), which also antagonizes N-methyl-D-aspartate (NMDA) receptors, performed immediately after the lesion, significantly reduced this loss. The electron microscope examination showed major damage in each type of retinal neuron, the pigment epithelium, and the microvessels. NBQX or thiokynurenic acid reduced, in a comparable manner, the effects of ischemia on the pigment epithelium, the photoreceptors, and the bipolar and the horizontal cells. NBQX was particularly efficient in reducing the damage to the amacrine cells located in the inner nuclear layer. The displaced amacrine and ganglion cells were not protected by NBQX but were almost completely spared in animals treated with thiokynurenate. These results show that antagonism of AMPA receptors is sufficient to reduce ischemic damage in a large number of retinal neurons, but that neuroprotection in the ganglion cell layer may be obtained only with agents which also antagonize NMDA receptors.

The kynurenine metabolic pathway in the eye: studies on 3-hydroxykynurenine, a putative cataractogenic compound.

The rabbit lens has an elevated content of 3‐hydroxykynurenine (3OHKYN) in spite of a very low activity of the enzymes leading to its synthesis. The iris/ciliary body, on the contrary, has very high activity of 3OHKYN synthesizing enzymes but a content of 3OHKYN lower than that of the lens. These observations suggest that 3OHKYN is formed in the iris/ciliary body, released into the aqueous humor and then taken up into the lens where it may be used for the synthesis of UV filtering products. An excessive accumulation of 3OHKYN in the lens has been associated with cataract formation. We found that available selective inhibitors of kynurenine hydroxylase reduced 3OHKYN synthesis in both the lens and the iris/ciliary body.

Photochemically-induced lesion of the rat retina: a quantitative model for the evaluation of ischemia-induced retinal damage

The effects of ischemia-induced retinal damage were quantitatively evaluated in rats with the aim of obtaining a suitable model to study the pathogenesis of the loss of retinal neurons after ischemic episodes. Anaesthetized rats were injected with 80 mg/kg i.v. of the fluorescein rose bengal dye and one eye was exposed to cold light for different periods (from 5 to 30 min). The animals were sacrificed at different times (1 and 4 hr; 2 and 7 days) after the lesion and the photochemically-induced damage was evaluated. The damaged retinae appeared thicker, numerous neurons of the inner nuclear layers showed swelling of the perinuclear cytoplasm and the retinal vessels were enlarged. The activity of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD), two marker enzymes of the GABAergic and cholinergic neurons, significantly decreased, indicating a degeneration of GABAergic and cholinergic amacrine cells.