Naunyn-Schmiedeberg’s Arch Pharmacol (2004) 369 : 300–304 DOI 10.1007/s00210-004-0872-2 300 O R I G I N A L A RT I C L E Katarzyna Wejksza · Wojciech Rzeski · Jolanta Parada-Turska · Barbara Zdzisinska · Robert Rejdak · Tomasz Kocki · Etsuo Okuno · Martyna Kandefer-Szerszen · Eberhart Zrenner · Waldemar A. Turski Kynurenic acid production in cultured bovine aortic endothelial cells. Homocysteine is a potent inhibitor Received: 13 November 2003 / Accepted: 15 January 2004 / Published online: 10 February 2004 © Springer-Verlag 2004 Abstract Kynurenic acid (KYNA) is a broad-spectrum antagonist at all subtypes of ionotropic glutamate receptors, but is preferentially active at the strychnine-insensitive glycine allosteric site of the N-methyl-D-aspartate (NMDA) receptor and is also a non-competitive antagonist at the alpha7 nicotinic receptor. KYNA occurs in the CNS, urine, serum and amniotic fluid. Whilst it possesses anticonvulsant and neuroprotective properties in the brain, its role in the periphery, however, is unknown. In this study we demonstrated the presence of kynurenine aminotransferase (KAT) I and II in the cytoplasm of bovine aortic endothelial cells (BAEC). BAEC incubated in the presence of the KYNA precursor L-kynurenine synthesized KYNA concentration- and time-dependently. KYNA production was inhibited by the aminotransferase inhibitor aminooxyacetic acid but was not affected by a depolarising concentration of K+ or by 4-aminopyridine. The glutamate agonists L-aspartate and L-glutamate depressed KYNA production significantly. The selective ionotropic glutamate receptor agonists α-amino-2,3-dihydro-5-methyl-3oxo-4-isoxazolepropionic acid (AMPA) and NMDA were ineffective in this respect. D,L-Homocysteine and L-homocysteine sulphinic acid lowered KYNA production in BAEC. Further investigations are needed to assess the role and importance of KYNA in vessels and peripheral tissues. Keywords Kynurenic acid · Kynurenine aminotransferase · Bovine aortic endothelial cells · Glutamate · Homocysteine Introduction T. Kocki · W. A. Turski (✉) Department of Pharmacology and Toxicology, Medical University, Jaczewskiego 8, 20-090 Lublin, Poland Fax: +48-81-7478646, e-mail: turskiwa@asklepios.am.lublin.pl K. Wejksza · W. Rzeski · B. Zdzisinska · M. Kandefer-Szerszen Department of Virology and Immunology, Institute of Microbiology and Biotechnology, Maria-Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland J. Parada-Turska Department of Rheumatology, Medical University, Jaczewskiego 8, 20-090 Lublin, Poland R. Rejdak · E. Zrenner Department of Pathophysiology of Vision and Neuro-Ophthalmologyl, Division of Experimental Ophthalmology, University Eye Hospital, Röntgenweg 11, 72076 Tübingen, Germany E. Okuno Department of Molecular Medicine, Wakayama Medical University, Wakayama, Japan W. A. Turski Department of Toxicology, Institute of Agricultural Medicine, Jaczewskiego 2, 20-950 Lublin, Poland Kynurenic acid (KYNA) is a broad-spectrum antagonist at all subtypes of ionotropic glutamate receptors (Perkins and Stone 1982) but acts preferentially at the strychnineinsensitive glycine allosteric site of the N-methyl-D-aspartate (NMDA) receptor (Birch et al. 1988). Recently, it has been demonstrated that KYNA is also a non-competitive antagonist at the alpha7 nicotinic receptor (Hilmas et al. 2001). Its presence has been documented in urine, serum, amniotic fluid (Milart et al. 1999), cerebrospinal fluid (Swartz et al. 1990), brain tissue (Turski et al. 1988; Swartz et al. 1990) and the retina (Rejdak et al. 2001). The tryptophan metabolite KYNA is converted from kynurenine by kynurenine aminotransferase (EC 2.6.1.7; KAT). In the rat liver, four enzymes responsible for this reaction have been described (Kido 1991). In the brain, two distinct enzymes, named KAT I and II, have been characterized in detail (Okuno et al. 1991). KAT I prefers pyruvate as a cosubstrate and has a pH optimum at 9.5. KAT II prefers oxoglutarate and has a pH optimum in the physiological range of 7.4 (Schmidt et al. 1993; Guidetti et al. 1997). Animal studies have shown that KYNA possesses anticonvulsant and neuroprotective properties (Foster et al. 301 1984; Moroni 1999) and that it may be involved in the pathophysiology of various brain disorders, e.g. Parkinson’s disease (Ogawa et al. 1992), Huntington’s disease (Beal et al. 1992) and epilepsy (Yamamoto et al. 1995). Only recently, it was reported that KYNA deficiency might be related causally to the pathology of excitotoxic retinal diseases (Rejdak et al. 2003). Its role in the periphery is, however, unknown. We investigated the presence of KAT I and II and synthesis of KYNA in endothelial cells derived from bovine aorta in culture. Materials and methods Bovine aorta endothelial cell culture. Bovine aorta endothelial cells (BAEC) were cultured as previously described (Zdzisinska et al. 2000). Vessels taken from freshly slaughtered steers and heifers were pooled, dissected from surrounding tissue, excised, rinsed with phosphate-buffered saline (PBS) and filled with 0.25% trypsin solution. After 30 min incubation at 37 °C, the endothelial cell suspension was collected and centrifuged at 250×g for 10 min. The cell pellet was resuspended in culture medium and transferred into 75-cm2 tissue culture flasks (Nunc, Roskilde, Denmark). The culture medium consisted of DMEM (Sigma, St Louis, Mo., USA), 10% fetal bovine serum (FBS) (Life Technologies, Karlsruhe, Germany), penicillin (100 U/ml) and streptomycin (100 µg/ml) (Sigma). Cells were grown at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Endothelial cell morphology was confirmed by typical cobble-stone morphology as well as by staining for von Willebrand factor (Dakopatts, Denmark). For experiments, BAEC from passages 5–8 were used. Immunocytochemistry. Cells plated on Lab-Tek Chamber Slides (Nunc) at a density of 1×105 cells/ml were allowed to grow for 24 h. After being washed the next day with PBS, cells were fixed with cold methanol/acetic acid (3:1) for 5 min and subsequently exposed to rabbit primary antibodies against KAT I and II at 4 °C overnight. Antibodies were detected and antigens visualized using a streptavidin horseradish peroxidase kit (STAR2004, Serotec, Oxford, Oxon., UK). Cells were counterstained with 0.1% Mayer’s haematoxylin solution. Negative controls were prepared from cells incubated with primary antibody diluent only. Primary antibodies were obtained from Prof. Etsuo Okuno, Japan (for specificity of antibodies see Okuno et al.1990, 1993). Specimens were examined using an Olympus BX51 system microscope (Olympus, Tokyo, Japan) and micrographs prepared using AnalySIS software (Soft Imaging System, Münster, Germany). KYNA production in endothelial cells. BAEC growing on culture flasks were trypsinized, suspended in fresh culture medium and Fig. 1A–C Localisation of kynurenine aminotransferase (KAT) I and II immunoreactivity in cultured bovine aortic endothelial cells (BAEC). A Control showing no staining; B, C Diaminobenzidine (DAB)-labelling of KAT I (B) and KAT II (C) showing the expression of both in the cytoplasm of all the cells in the monolayer transferred to 24-well plates (Nunc) at a density of 2×105 cells/ml. On the next day the culture medium was removed, and the cells were rinsed 4 times with Hank’s balanced salt solution (HBSS, Sigma), consisting of (in mM) CaCl2 1.27, KCl 5.4, K2HPO4 0.34, MgSO4 0.8, NaCl 138.0, NaHCO3 4.1, NaH2PO4 0.39 and glucose 5.5. After rising, the cells were exposed to L-kynurenine dissolved in HBSS and incubated at 37 °C in 95%/5% CO2. At the end of incubation, supernatants were collected, 50% tricarboxylic acid added and precipitated protein removed by centrifugation. Supernatants were applied to cation-exchange columns (Dowex 50 W+) and the KYNA content determined using an HPLC system with fluorescence detection (excitation: 334 nm; emission: 398 nm). Drugs. α-Amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropionic acid (AMPA), aminooxyacetic acid (AOAA), 4-aminopyridine, L-aspartic acid, L-glutamic acid, D,L-homocysteine, L-homocysteine sulphinic acid, kynurenic acid, L-kynurenine sulphate and NMDA were from Sigma (USA). All other drugs were of highest available purity. Data analysis. The significance of differences between means was determined using Student’s t-test. The drug concentration inducing 50% inhibition of KYNA synthesis (IC50) and the respective 95% confidence limits were calculated using computerised linear regression analysis of quantal log dose-probit functions according to the method of Litchfield and Wilcoxon (1949) and transformed subsequently into standard errors (SE) according to Porecca et al. (1990). Results KAT I and KAT II cellular expression Diaminobenzidine (DAB)-labelling of KAT I and II showed that both enzymes were clearly present in BAEC and were expressed strongly in the cytoplasm. All cells in the monolayer were stained. Control cultures showed no immunoreactivity and only a weak and diffuse background staining (Fig. 1). KYNA synthesis BAEC incubated in HBSS with L-kynurenine synthesized KYNA concentration- and time-dependently (Fig. 2A, B). The mean production of KYNA under standard conditions (L-kynurenine 5 µM; 2 h incubation) was 1.66±0.19 pmol KYNA/ 2×105 cells (n=11). KYNA production was depressed con- 302 Table 1 Effect of the depolarising agents 50 mM KCl and 4-aminopyridine on kynurenic acid (KYNA) production in bovine aortic endothelial cells (BAEC). Results are expressed as percentages of the respective control; means±SEM; n=6 experiments (P probability, NS not significantly different) Treatment [mM] KYNA production (%) P KCl 50 4-Aminopyridine 0.01 104.5±5.70 98.4±4.81 NS NS Table 2 Effect of excitatory amino acid agonists on kynurenic acid production in BAEC. Results are expressed as percentages of the respective control; means±SEM; n=6 experiments (NMDA N-methyl-D-aspartate, AMPA α-amino-3-hydroxy-5-metyloisoxazolo-4-propionate) Treatment [mM] KYNA production (%) P L-Aspartate 58.9±1.77 72.0±2.93 91.1±7.06 81.5±6.11 <0.001 <0.01 NS NS 0.1 0.1 AMPA 0.01 NMDA 0.01 L-Glutamate Fig. 2 Concentration (A) and time (B) dependence of kynurenic acid (KYNA) production in BAEC. A BAEC were incubated in the presence of L-kynurenine (1, 5 or 10 µM) for 2 h. B BAEC were incubated in the presence of L-kynurenine (5 µM) for 1, 2 or 4 h. Data are mean production of KYNA in pmol; means±SEM; n=6 independent experiments Fig. 3 Effect of aminooxyacetic acid on kynurenic acid production in BAEC. BAEC were incubated in the presence of L-kynurenine (5 µM) for 2 h. Results are expressed as percentages of control; means±SEM; n=6 independent experiments. The abscissa is a logarithmic scale, the ordinate is linear. The solid line is the linearlog concentration regression, calculated using GraphPad Software; y=–31.26x+67.18, r=0.99 centration-dependently in the presence of AOAA (IC50 3.20±2.89 µM, n=18, Fig. 3). KYNA synthesis was not affected by a depolarising concentration of K+ (50 mM) or by 4-aminopyridine (0.01 mM) (Table 1). The endogenous glutamate agonists L-aspartate and L-glutamate (both 0.1 mM) significantly lowered KYNA production (Table 2). The selective ionotropic glutamate receptor agonists AMPA Fig. 4 Effect of D,L-homocysteine (A) and L-homocysteine sulphinic acid (B) on kynurenic acid production in BAEC. BAEC were incubated in the presence of L-kynurenine (5 µM) for 2 h. Results are expressed as percentages of control; means±SEM; n=6 independent experiments; *P<0.05 vs. control and NMDA did not affect KYNA synthesis in BAEC (Table 2). D,L-Homocysteine concentration-dependently inhibited KYNA synthesis (IC50 0.054±0.045 mM, n=24, Fig. 4A), 303 while L-homocysteine sulphinic acid (0.1–0.5 mM) lowered KYNA production slightly (by 12–17%, Fig. 4B). Discussion Although KYNA is always present in the blood, its origin and role in the periphery is unknown. The present study revealed the presence of two enzymes responsible for KYNA synthesis, KAT I and II in BAEC and documented de novo synthesis of KYNA from kynurenine in these cells. Both enzymes were present in the cytoplasm of the cultured endothelial cells and all the cells in the monolayer were stained. In the rat brain KAT immunoreactivity has been found in both glial cells and neurons (Du et al. 1992; Roberts et al. 1992). In glia cells, labelling is associated mainly with the cytoplasmic matrix, whilst in neurons, most labelling is found in membrane-bound cytoplasmic organelles (Roberts et al. 1992; Kapoor et al. 1997). In the rat retina, KAT I is expressed preferentially in Müller cell endfeet, while KAT II is localised in cells within the ganglion cell layer and in both cell types the cytoplasm stains uniformly (Rejdak et al. 2001). BAEC incubated under standard conditions in the presence of L-kynurenine synthesized KYNA concentrationand time-dependently. The presence of KYNA in the incubation medium suggests that it was liberated freely from cells to the external milieu. Similarly, brain cortical and spinal cord slices liberate de novo synthesized KYNA readily under comparable conditions (Turski et al. 1989; Urbanska et al. 2000). AOAA, an inhibitor of aminotransferases concentration-dependently decreased KYNA synthesis, confirming its enzymatic origin. The effectiveness of the inhibitory action of AOAA in endothelial cells corresponds to that recorded in brain cortical slices (Turski et al. 1989). In brain cortical slices and C6 cells in culture, a high [K+] inhibits production of KYNA (Turski et al. 1989; Kocki et al. 2002). On the contrary, in BAEC the depolarizing agents 50 mM K+ and 4-aminopyridine did not affect KYNA synthesis. A similar effect has been reported in liver and kidney tissue (Gramsbergen et al. 1991; 1997). Our finding further supports the hypothesis that the decrease of KYNA formation may result from an inhibitory action of a factor released from depolarized cells in brain, but not in peripheral non-excitable tissues. Glutamate, a potent inhibitor of KYNA synthesis in brain cortical (Turski et al. 1989; Urbanska et al. 1997) and spinal cord slices (Urbanska et al. 2000), in astrocytes (Curatalo et al. 1996) and C6 cells in culture (Kocki et al. 2002), significantly decreased KYNA production also in BAEC. Similarly, aspartate reduces the formation of KYNA in brain slices (Urbanska 1997) and in BAEC (this study). In contrast, NMDA and AMPA, selective agonists at ionotropic glutamate receptors, influence KYNA synthesis neither in neuronal tissue (Urbanska et al. 1997, 2000) nor in BAEC (this study). Our findings add further support to the hypothesis that the effect of glutamate and aspartate is not due to their interaction with specific receptors at the cell membrane but may be exerted intracel- lularly (Kocki et al. 2002), presumably via the inhibition of KAT activity (Battaglia et al. 2000). D,L-Homocysteine concentration-dependently inhibited the synthesis of KYNA in BAEC. In comparison, L-homocysteine sulphinic acid was much less potent, inhibiting KYNA synthesis only modestly: about 17% at the highest concentration used (500 µM). Under the same conditions the IC50 for homocysteine was almost 10 times lower, reaching values as low as 54 µM. Interestingly, such concentrations of homocysteine may occur in plasma of humans. Homocysteine is being recognized increasingly as an important risk factor for vascular disease, including coronary atherosclerosis (Auer et al. 2001) and growing interest is being focused on the connection between hyperhomocysteineaemia and diabetes mellitus and chronic renal disease, including renal transplant recipients (Chiarelli et al. 2000). Epidemiological and experimental studies have linked increased homocysteine levels with several neurodegenerative conditions, e.g. stroke, Alzheimer’s disease and Parkinson’s disease (Mattson et al. 2003) and glaucoma (Bleich et al. 2002). It should be emphasized that homocysteine at 10 µM, a concentration that can be considered as a moderate risk predictor, lowered the production of KYNA significantly by 28%. The mechanism of the inhibitory action of homocysteine on KYNA synthesis is unknown. Homocysteine acts as an agonist at the glutamate binding site of the NMDA receptor (Lipton et al. 1997). Since, however, the NMDA receptor agonist NMDA (100 µM) did not affect production of KYNA in BAEC a direct interaction of homocysteine with NMDA receptors can be excluded. It is widely accepted that homocysteine causes acute and chronic endothelial dysfunction. In the present study we have shown that moderate and high concentrations of homocysteine inhibit KYNA synthesis in BAEC. This result is good reason for careful investigation of the role of KYNA in atherogenic processes initiated by homocysteine. In summary, BAEC express KATs and synthesise KYNA. Our results agree well with a recent report of Stazka et al. (2002) showing that rat aortic rings produce and liberate KYNA upon exposure to L-kynurenine. The presence of glutamate receptors and alpha7 nicotinic receptors in the periphery raises the question of whether KYNA, an antagonist at both these receptors, exerts effects on peripheral tissues. 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