Carmela Speciale

Uptake of Kynurenine into Rat Brain Slices

Abstract: The transport of [3H]kynurenine ([3H]KYN) into slices from rat tissue was examined in vitro. Brain accumulated KYN seven to eight times more effectively than any of several peripheral organs. Of all the organs tested, only the brain exhibited a sodium‐dependent component of the uptake process. After an incubation period of 1 h, sodium‐dependent transport amounted to 60% of total uptake. Both processes were abolished by prior sonication of the tissue and significantly inhibited by inclusion of metabolic blockers in the incubation medium. Time resolution showed that the sodium‐independent uptake occurred rapidly and reached saturation within 30 min. In contrast, sodium‐dependent transport was linear for at least 2 h of incubation. Brain regional analysis revealed a sevenfold difference between the areas of highest (cortex) and lowest (cerebellum) uptake. With the exception of cerebellar tissue, the ratio between sodium‐dependent and sodium‐independent processes was consistent among brain regions. Kinetic analyses were performed on striatal slices and revealed a Km of 927 μM and a Vmax of 18 nmol/h/mg of protein for the sodium‐dependent process, and a Km of 3.8 mM and a Vmax of 38 nmol/10 min/mg of protein for the sodium‐independent transport. The transporters were equally amenable to inhibition by KYN and tryptophan, indicating that KYN entry into the cell may be mediated by neutral amino acid uptake sites. No strict stereoselectivity existed, but L enantiomers were clearly more active than the D forms. Studies in ibotenate‐lesioned striata showed a 70%decrease in the sodium‐dependent process and a concomitant 60%increase in the sodium‐independent transport, suggesting differential cellular associations of the two uptake sites. Cellular uptake of KYN may constitute a critical step in the disposition of KYN metabolites such as quinolinic acid and kynurenic acid in the brain.

(R,S)-3,4-dichlorobenzoylalanine (FCE 28833A) causes a large and persistent increase in brain kynurenic acid levels in rats

Kynurenic acid is an endogenous excitatory amino-acid receptor antagonist with neuroprotective and anticonvulsant properties. We demonstrate here that systemic administration of the new and potent kynurenine 3-hydroxylase inhibitor (R,S)-3,4-dichlorobenzoylalanine (FCE 28833A) causes a dose-dependent elevation in endogenous kynurenine and kynurenic acid levels in rat brain tissue. In hippocampal microdialysates, peak increases of 10- and 80-fold above basal kynurenic acid concentrations, respectively, were obtained after a single oral or intraperitoneal administration of 400 mg/kg FCE 28833A. After intraperitoneal treatment with FCE 28833A, extracellular brain kynurenic acid levels remained significantly elevated for at least 22 h, rendering this compound a far more effective enhancer of kynurenic acid levels than the previously described kynurenine 3-hydroxylase blocker m-nitrobenzoylalanine. FCE 28833A and similar molecules may have therapeutic value in diseases which are linked to a hyperfunction of excitatory amino-acid receptors.

Molecular cloning of rat kynurenine aminotransferase: Identity with glutamine transaminase K

The enzyme kynurenine aminotransferase (KAT) catalyses the conversion of l‐kynurenine to kynurenic acid. A combination of polymerase chain reaction techniques and hybridization screening was used to isolate a cDNA clone encompassing the entire coding region of KAT from rat kidney. Identification of the cDNA as coding for KAT was based both on the comparison of amino acid sequences obtained from purified rat KAT and on the expression of KAT activity in COS‐1 cells transfected with the cDNA. RNA blot analysis indicated that KAT mRNA is widely expressed in rat tissues. Cultured cells transfected with the cDNA for KAT also showed glutamine transaminase K activity. Based mainly on sequence data, these results demonstrate that rat kidney KAT is identical with glutamine transaminase K.

Determination of extracellular kynurenic acid in the striatum of unanesthetized rats: Effect of aminooxyacetic acid

Kynurenic acid (KYNA) production from its bioprecursor L-kynurenine (KYN) was assessed in vivo by intrastriatal microdialysis in freely moving rats. In the absence of KYN, the extracellular concentration of KYNA was below the limit of assay sensitivity (i.e. less than 8 pmol/30 microliters). In the presence of KYN (50-2000 microM), KYNA concentration in the dialysate increased continuously to reach steady-state levels after 2h of perfusion. Introduction of the unspecific transaminase inhibitor aminooxyacetic acid (AOAA) through the dialysis probe caused a progressive decrease of extracellular KYNA, which reached dose-dependent minimal levels within 2 h. One mM AOAA caused an almost complete depletion of KYNA in the dialysate. These data demonstrate that extracellular KYNA can be assessed by microdialysis and that AOAA can be used as a tool to examine the neurobiology of KYNA in awake, freely moving animals.

Production of extracellular quinolinic acid in the striatum studied by microdialysis in unanesthetized rats

Striatal microdialysis was performed in awake rats in an attempt to produce extracellular quinolinic acid (QUIN) from its putative bioprecursors L-tryptophan, L-kynurenine and 3-hydroxyanthranilic acid (3HANA). Test compounds were included in the microperfusion solution. QUIN concentrations in the dialysate remained below the assay sensitivity (i.e. less than 20 nM) under baseline conditions or after extensive perfusion with 1 mM L-tryptophan or L-kynurenine. 3HANA (10-300 microM) caused dose-dependent increases in extracellular QUIN, which attained steady-state concentrations after 4 h. The initial rate of QUIN production was significantly increased in the ibotenate-lesioned striatum, suggesting a pivotal role of astroglia in the deposition of brain QUIN.

On the production and disposition of quinolinic acid in rat brain and liver slices

Abstract: The de novo production and subsequent disposition of the endogenous excitotoxin quinolinic acid (QUIN) was investigated in vitro in tissue slices from rat brain and liver. Incubation of tissue with QUINs immediate bioprecursor 3‐hydroxyanthranilic acid (3‐HANA) in oxygenated Krebs‐Ringer buffer yielded measurable amounts of QUIN both in the tissue and in the incubation medium. Saturation was reached between 16 and 64 μM 3‐HANA (166 pmol of QUIN formed per milligram of protein after a 60‐min incubation with 64 μM 3‐HANA). In the brain, more QUIN was recovered from the tissue than from the incubation medium at all time points examined (5 min to 4 h). In contrast, the tissue‐to‐medium ratio for QUIN in parallel experiments with hepatic slices was ≪ 1. The disposition of newly synthesized QUIN was further elaborated in tissue slices that had been preincubated for 60 min with 64 μM 3‐HANA. Subsequent incubation of brain tissue in fresh buffer revealed a steady but relatively slow efflux of QUIN from the cellular compartment, with >30% remaining in the tissue after a 90‐min incubation. Analogous experiments with liver slices showed that >93% of newly synthesized QUIN had entered the extracellular compartment within 30 min. Striatal and nigral slices obtained 7 days after an intrastriatal ibotenic acid injection showed severalfold increases in QUIN production compared with control tissues, in all likelihood due to astrogliosis and associated large increases in 3‐hydroxyanthranilic acid oxygenase activity. In addition, the apparent tissue‐to‐medium ratio was markedly reduced in striatal slices from lesioned animals. Taken together, these data indicate that both brain and liver cells have a rather limited capacity to retain QUIN, and that 3‐hydroxyanthranilic acid oxygenase activity is a critical determinant controlling extracellular QUIN concentrations in both organs. Changes in the activity of QUINs biosynthetic enzyme in the brain can therefore be expected to influence the possible function of QUIN as an endogenous agonist at the N‐methyl‐D‐aspartate receptor in health and disease.

Alternative Splicing at the C-terminal but not at the N-terminal Domain of the NMDA Receptor NR1 is Altered in the Kindled Hippocampus

Several lines of evidence suggest that N‐methyl‐D‐aspartate (NMDA) receptors significantly contribute to the development of kindling. In addition, a lasting enhancement of the NMDA receptor function has been suggested to play a significant role in the chronic hyperexcitability occurring in the hippocampus after kindling epileptogenesis. We have investigated whether hippocampal kindling induces changes in the NMDA receptor at the molecular level by assessing the expression of mRNAs of the different spliced variants at the N‐terminal (exon 5) and C‐terminal (exon 21) position of the NMDA receptor 1 (NR1) gene by means of the reverse transcription‐polymerase chain reaction. Alternative splicing at exon 5 confers different sensitivity of the NMDA receptor to polyamines while exon 21 encodes a 37‐amino acid insert containing the major phosphorylation sites for protein kinase C. One week after the acquisition of stage 5 of kindling in rats (generalized tonic‐clonic seizures), the relative abundance of the two alternatively spliced forms at the C‐terminal domain, respectively containing (+) or lacking (−) exon 21, was reversed compared to controls (implanted with electrodes but not stimulated) in the dorsal hippocampus ipsilateral and contralateral to the electrical stimulation. The exon 21+/exon 21− mRNA ratio for controls was 1.3 ± 0.04 (mean ± SE); for ipsilaterally kindled rats it was 0.64 ± 0.05 (P < 0.05), and for contralaterally kindled rats it was 0.48 ± 0.07 (P < 0.01). Similar bilateral effects were observed in the ventral hippocampus (temporal pole). No changes were found 4 weeks after stage 5 seizures and 1 week after the induction of a single afterdischarge. No significant alterations were induced by kindling in the relative abundance of the spliced variants containing or lacking exon 5. Our findings show selective changes in alternative splicing of the NR1 gene after repeated application of an epileptogenic stimulus. This may generate receptors with different functional properties, which may contribute to the increased sensitivity for the induction of generalized seizures during kindling.

Increased quinolinic acid metabolism following neuronal degeneration in the rat hippocampus

The excitotoxic brain metabolite quinolinic acid (QUIN) has been hypothetically linked to the pathogenesis of seizure disorders and other neurodegenerative events affecting the hippocampal formation. Its biosynthetic enzyme, 3-hydroxyanthranilic acid oxygenase (3-HAO) and its catabolic enzyme, quinolinic acid phosphoribosyltransferase (QPRT), can be used as markers for the cellular localization of the brains QUIN system. Measured between 2 days and 2 months following intrahippocampal ibotenic acid injections, the activities of both enzymes increased at the lesion site due to the synthesis of new enzyme protein. The time course of the increase in 3-HAO activity coincided with that of the known astrocytic proliferation following excitotoxic insults. It is less obvious if the elevation in QPRT activity, too, is related to an increase in the number of reactive glial cells. No changes in the activity of hippocampal 3-HAO or QPRT were noted 7 or 60 days after cholinergic deafferentation by fornix-fimbria transection nor were any changes observed in the contralateral hippocampus at any time-point following the ibotenate lesion. These data raise the possibility that a feed-forward mechanism, resulting in ever increasing amounts of QUIN in the brain, may be operant in situations of progressive hippocampal nerve cell loss.

Derivatives of kynurenine as inhibitors of rat brain kynurenine aminotransferase

Abstract The structural requirements of the catalytic site of kynurenine aminotransferase (KAT), the enzyme responsible for the conversion of l -kynurenine (KYN) to kynurenic acid (KYNA), were examined using analogs and derivatives of KYN. KYNA production from KYN was monitored in rat brain homogenates and brain tissue slices. Modification of KYNs acylalanine side chain or its ring amino group resulted in compounds which did not substantially affect KYNA synthesis. Ring chlorination in positions 3, 4, 5 and 6 yielded KYN analogs which interfered with KYNA production. l -5-Cl-KYN was the most active of the chlorinated kynurenines, and one of the most potent of several other 5-substituted kynurenines. l -5-Cl-KYN was an excellent substrate of KAT, yielding 6-Cl-KYNA. Finally, in kinetic studies, l -5-Cl-KYN ( K i = 5.4 μM) was found to have an approximately five times higher affinity to the enzyme than the natural substrate KYN ( K m = 28 μM).

Kynurenic Acid-Enhancing And Anti-Ischemic Effects of the Potent Kynurenine 3-Hydroxylase Inhibitor Fce 28833 in Rodents

When injected into the brain of experimental animals, quinolinic acid (QUIN) and kynurenic acid (KYNA) act as a neurotoxin and neuroprotectant, respectively, and these effects are mediated by excitatory amino acid (EAA) receptors (Stone, 1993). Since their identification in the mammalian brain, QUIN and KYNA have therefore been proposed to play roles as endogenous modulators of EAA receptors. Both compounds are metabolites of the kynurenine pathway in the periphery and in the brain, and a large number of studies have been performed to examine their source, metabolism and disposition in the central nervous system in physiological and pathological conditions (see Stone, 1993, for review). In particular, an increased formation of QUIN was observed in several models of nerve cell damage, such as excitotoxin lesions (Speciale et al., 1987), spinal cord trauma (Popovich et al., 1994) and global ischemia (Heyes and Nowak, 1990; Saito et al., 1993), and in humans infected with the HIV virus (Heyes et al., 1991). This enhancement of QUIN production was suggested to contribute to the loss of neurological function. On the other hand, KYNA was found to be likely involved in cerebral self-defence (Schwarcz et al., 1992). This is supported both by occurrence of cell death following the pharmacologically induced decrease in brain KYNA (McMaster et al., 1991) and by the increase in cerebral KYNA production following acute excitotoxic (Schwarcz et al., this volume) or convulsive (Wu and Schwarcz, 1994; Baran et al., 1995) insults.