Quinolinic acid formation in immune-activated mice: studies with (m-nitrobenzoyl)-alanine (mNBA) and 3,4-dimethoxy-[-N-4-(-3-nitrophenyl) thiazol-2yl]-benzenesulfonamide (Ro 61-8048), two potent and selective inhibitors of kynurenine hydroxylase
Introduction
Quinolinic acid (QUIN), a tryptophan (TRP) metabolite present in the brain (Moroni et al., 1984, Wolfensberger et al., 1984), is able to interact with glutamate receptors of the NMDA type and to cause convulsions or neurodegeneration (Stone and Perkins, 1981, Foster et al., 1983, Schwarcz et al., 1983). Its synthesis occurs along the so-called kynurenine metabolic pathway, which also leads to the formation of other neuroactive compounds such as 3-hydroxykynurenine (3OH-KYN), another metabolite able to cause neuronal damage (necrosis and apoptosis) in cell cultures (Eastman and Guilarte, 1989, Okuda et al., 1998) and kynurenic acid (KYNA), an antagonist of excitatory amino acid receptors (Perkins and Stone, 1982, Foster et al., 1984, Moroni et al., 1988).
The rate limiting step in the formation of the above mentioned neuroactive compounds is the oxidative cleavage of the pyrrole ring of TRP (Yoshida and Hayashi, 1987). This cleavage is catalyzed by two enzymes, tryptophan dioxygenase [E.C. 1.13.11.11], which is present only in liver, and indoleamine dioxygenase (IDO) [E.C. 1.13.11.17], which has a wider distribution and is also present in brain. The other enzymes required for the synthesis of QUIN are kynurenine hydroxylase [E.C. 1.14.13.9], kynureninase [E.C 7.3.1.3] and 3-hydroxyanthranilic acid dioxygenase [E.C. 1.13.11.6] (see Fig. 1). A large increase in the content of kynurenine (KYN) metabolites, including QUIN, has been repeatedly shown in blood and in central nervous system (CNS) of mammals affected by inflammatory neurological disorders (Heyes et al., 1991, Heyes et al., 1992a, Heyes et al., 1993, Moffet et al., 1993, Saito et al., 1993b) since these enzymes are strongly induced in immune-activated macrophages and microglia (Werner et al., 1988, Saito et al., 1992, Saito et al., 1993a). It has also been proposed that QUIN accumulation in the CNS may have neuropathological significance because, in the course of several encephalopathies, a direct correlation between its levels in cerebrospinal fluid (CSF) and the severity of the neurological deficits has been found (Heyes et al., 1991, Heyes et al., 1992a, Flanagan et al., 1995, Blight et al., 1997). We have previously studied the neuropharmacological and biochemical effects of a number of kynurenine hydroxylase and/or kynureninase inhibitors (Moroni et al., 1991, Russi et al., 1992, Carpenedo et al., 1994, Chiarugi et al., 1995, Chiarugi et al., 1996) since compounds able to inhibit the kynurenine pathway enzymes could be of therapeutic importance (Kerr et al., 1997). In particular we characterized m-nitrobenzoylalanine (mNBA; Fig. 1), a selective kynurenine hydroxylase inhibitor (IC50 1 μM) able to increase the convulsive threshold in mice, cause sedation and mild analgesia, and increase KYN and KYNA content in the rodent brain (Carpenedo et al., 1994). Recently, a new selective, specific and potent kynurenine hydroxylase inhibitor was described, 3,4-dimethoxy-[N-4-(-3-nitrophenyl)thiazol-2-yl]-benzenesulfonamide (Ro 61-8048; IC50 0.04 μM;) (Roever et al., 1997). We took advantage of the availability of this molecule to study, in vitro and in vivo, the importance of KYN hydroxylation in QUIN synthesis.
Section snippets
Materials
mNBA and Ro 61-8048 were kindly provided by Drs S. Roever and A. Cesura (Hoffman-La Roche, Basel, CH). [137C]-QUIN was provided by Dr J.F. Reinhard Jr. (Research Triangle Park, North Carolina, USA).The TRP-depleted medium was from GIBCO Life Technologies Inc. (Gaithersburg, MD, USA). l-KYN, KYNA, QUIN and pokeweed mitogen (PWM) were from Sigma (St. Louis, MO, USA). Hexafluor-2-propanol and trifluoroacetyl-imidazole were from Aldrich (Milan, Italy). All remaining compounds were from Merck
Effects of mNBA and Ro 61-8048 on the content of KYN metabolites in brain and blood of PWM-treated mice
Kynurenine hydroxylase inhibitor administration has been previously shown to increase KYN and KYNA content in rat and gerbil brains (Carpenedo et al., 1994, Speciale et al., 1996, Roever et al., 1997). Table 1 shows that the administration of mNBA (400 mg/kg, i.p.) or Ro 61-8048 (60 mg/kg, i.p.) increased brain KYN content by two or three fold and brain KYNA content by nine or 15-fold, respectively in mice as well. To our surprise, brain QUIN content did not change after administration of the
Discussion
The most interesting finding of the present study is the demonstration that repeated treatments with large doses of mNBA or Ro 61-8048, two potent and selective kynurenine hydroxylase inhibitors, diminish the PWM-induced increase of QUIN content in blood and brain of mice (see Fig. 3). We also showed that, mNBA or Ro 61-8048 do not modify basal QUIN content, but significantly increase KYN and KYNA content in the blood and brain of saline pretreated animals (see Table 1). This observation could
Acknowledgements
This work was supported by: University of Florence; MURST: CNR, European Union Grants No B104-CT96-0049 and B104-CT960752 and Research Project on Multiple Sclerosis of the Istituto Superiore di Sanità, Rome, Italy.
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