Effect of Probenecid in the Levels of Kynurenic Acid and ... · EFFECT OF PROBENECID IN THE LEVELS...
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EFFECT OF PROBENECID IN THE LEVELS OF KYNURENIC ACID AND CATECHOLAMINES IN DIFFERENT BRAIN REGIONS Reyes-Ocampo J.G. 1 , García E. 2 , Pineda B. 2 , Rangel-López E. 3 Pérez-de la Cruz V. 3 , Santamaría A. 3 1. Departamento de Ciencias de la Salud, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana-Iztapalapa, Avenida San Rafael Atlixco 186, Colonia Vicentina Iztapalapa, Mexico D.F., 09340, México. 2. Laboratorio de Neuroinmunología, Instituto Nacional de Neurología y Neurocirugía México D.F., México. 3. Laboratorio de Aminoácidos Excitadores, Instituto Nacional de Neurología y Neurocirugía México D.F., México. 0 1 2 3 4 0 10000 20000 30000 * Time (h) KYNA (fmoles/mg tissue) 0 1 2 3 4 0 5 10 15 20 25 Time (h) DA content ( g/ mg wet tissue) 0 1 2 3 4 0 100000 200000 300000 400000 500000 * Time (h) KYNA (fmoles/mg tissue) 0 1 2 3 4 0.0 0.5 1.0 1.5 2.0 Time (h) DA content ( g/ mg wet tissue) 0 1 2 3 4 0 20000 40000 60000 80000 100000 * * * * Time (h) KYNA (fmoles/mg tissue) 0 1 2 3 4 0.0 0.2 0.4 0.6 0.8 1.0 Time (h) DA content ( g/ mg wet tissue) Cortex Hippocampus Ctrl 20X BREGMA 1.8 mm Ctrl 20X BREGMA 2.2 mm 2 h 20X BREGMA 1.8 mm 2 h 20X BREGMA 2.2 mm Striatum Results Ctrl 20X BREGMA 1.7 mm 2 h 20X BREGMA 1.7 mm Introduction Kynurenic acid (KYNA) is an antagonist for the α7-nicotinic acetylcholine receptor (nAChR) and the glycine co-agonist site of the N-methyl-D-aspartate receptor at nanomolar concentrations 1 . KYNA is synthesized in the brain from kynurenine (KYN) through irreversible transamination by kynurenine aminotransferases (KATs). In turn, KYN can be synthesized within the brain from L-tryptophan, or transported across the brain-blood barrier (BBB) by the large neutral amino acid uptake system. It has been estimated that 40% of KYN is synthesized in the brain, while the remaining comes from the periphery. Its activity on excitatory amino acid receptors and nAChR might account for its neuromodulatory and neuroprotective effects in the CNS. Thus, fluctuations in brain KYNA levels under physiological and pathological conditions could modulate the activity of cholinergic and glutamatergic transmission. Furthermore, KYNA, at nanomolar concentrations, can decrease the extracellular level of dopamine in the striatum of rat. Neuroprotective effects of KYNA had been addressed through pharmacologic manipulations leading to increased brain concentrations of KYNA, which have shown anti-epileptic effects. Probenecid (PROB), a drug widely used for the treatment of the gout, inhibitor of the organic acids transporter (OAT), and found in the BBB, has been previously shown to increase brain concentrations of KYNA. PROB has shown to prevent OAT- mediated transport of KYNA towards the cerebrospinal fluid, the blood torrent and finally, out of the circulatory system as urine in experiments in which its precursor KYN was co-administrated 2,3 . However, there is no direct evidence showing if the increase in KYNA concentrations by PROB action, without its precursor, is dose-dependent, and if this accumulation also differs through the time and brain region. Materials and methods Animals Male Wistar bred-in-house rats (270 - 300 g) were used throughout the study. Animals (6 per group) were randomly assigned to six experimental groups as follows: After the time provided, striatal, hippocampus, and the cortex tissues were collected by decapitation and immediately dissected out on ice and preserved for limited times at −70 °C. During dissection, all efforts were made to minimize animal suffering. Measurement of KYNA levels KYNA content was measured in freshly tissues homogenates obtained from animals of experimental groups, and determined according to established high performance liquid chromatography (HPLC) procedures 4 , with a mobile phase containing 200 mM of zinc acetate and 5% acetonitrile (pH 6.2) was used for KYNA content and detected fluorimetrically (excitation/emission wavelength 344/398 nm). Measurement of DA levels DA levels were measured in freshly obtained tissues from control and experimental groups, according to previous reports 5 . The brain DA tissue content was analyzed by HPLC with electrochemical detection, as previously described 6 . All samples were analyzed in duplicate; the concentrations of DA and its metabolites were first calculated as μg per gram of wet tissue. KYNA Immunohistochemical Assay Polyclonal antibody against KYNA (Sigma-Aldrich, Inc. St. Louis, MO, USA) was used to examine the cellular location in rat brain sections. The expression of KYNA was examined using an immunohistochemical approach. This assay was performed after 1, 2, 3 hours after PROB (50mg/kg, i.p.) Formalin-fixed, paraffin-embedded archived tissues were sectioned (thickness of 4 μm) 7 . The sections were then dewaxed, rehydrated and goat serum albumin-blocked. Following blocking, the sections were incubated with the antibody for 2 h at room temperature (1:80 dilution). The slides were incubated with a secondary FITC-labeled antibody (1:1000 dilution). Finally, the slides were VECTASHIELD-mounted. Images were obtained in an inverted microscope (Leica Microsystems, Heidelberg, Germany) using an ArKr laser at 488 and 520 nm to define the intracelular location of KYN. GROUP ADMINISTRATION CONTROL NaOH 0.1 N, pH 9.0 adjusted 30 min PROB (50 mg/kg, i.p.) compound was dissolved in NaOH 0.1 N pH 9.0 adjusted 1 hour 2 hours 3 hours 4 hours References 1. Hilmas C., Pereira E. F., Alkondon M., Rassoulpour A., Schwarcz R. and Albuquerque E. X. (2001) The brain metabolite kynurenic acid inhibits alpha7 nicotinic receptor activity and increases non-alpha7 nicotinic receptor expression: physiopathological implications. J. Neurosci. 21, 7463–7473. 2. Hesselink MB, Smolders H, Eilbacher B, De Boer AG, Breimer DD, Danysz W. 1999. The Role of Probenecid-Sensitive Organic Acid Transport in the Pharmacokinetics of N-Methyl-D-Aspartate Receptor Antagonists Acting at the GlycineB-Site: Microdialysis and Maximum Electroshock Seizures Studies. J Pharmacol Exp Ther, 290: 543-550. 3. Silva-Adaya, D.; Pérez-De La Cruz, V.; Villeda-Hernández, J.; Carrillo-Mora, P.; González-Herrera I.; Esperanza García, E.;Colín- Barenque, L.; Pedraza-Chaverrí, J.; Santamaría, A. Protective effect of L-kynurenine and probenecid on 6-hydroxydopamine-induced striatal toxicity in rats: Implications of modulating kynurenate as a protective strategy. 4. Swartz KJ, During MJ, Freese A, Beal MF (1990) Cerebral synthesis and release of kynurenic acid: an endogenous antagonist of excitatory amino acid receptors. J Neurosci 10:2965–2973. 5. Blandini F, Levandis G, Bazzini E, Nappi G, Armentero MT. Time-course of nigrostriatal damage, basal ganglia metabolic changes and behavioural alterations following intrastriatal injection of 6-hydroxydopamine in the rat: new clues from an old model. Eur J Neurosci 2007;25:397–405. 6. García E, Limón D, Pérez-De La Cruz V, Giordano M, Díaz-Muñoz M, Maldonado PD, et al. Lipid peroxidation, mitochondrial dysfunction and neurochemical and behavioural deficits in different neurotoxic models: protective role of S-allylcysteine. Free Radic Res 2008;42:892–902. 7. Rodríguez E, Méndez-Armenta M, Villeda-Hernández J, Galván-Arzate S, BarrosoMoguel R, Rodríguez F, et al. Dapsone prevents morphological lesions and lipid peroxidation induced by quinolinic acid in rat corpus striatum. Toxicology 1999;139: 111–8. Conclusions Our data show a time-dependent increase of KYNA levels in cortex and striatum after administration of PROB, effect not observed in hippocampus, this increase is inversely related to DA levels in time. Clearly PROB in CNS has effect on the OAT resulting in an accumulation of KYNA, and may be a valid therapeutic tool for regulating the glutamatergic and cholinergic transmission.
Transcript of Effect of Probenecid in the Levels of Kynurenic Acid and ... · EFFECT OF PROBENECID IN THE LEVELS...
EFFECT OF PROBENECID IN THE LEVELS OF KYNURENIC ACID AND CATECHOLAMINES IN DIFFERENT BRAIN REGIONS
Reyes-Ocampo J.G.1, García E.2, Pineda B.2, Rangel-López E.3 Pérez-de la Cruz V.3, Santamaría A.3
1. Departamento de Ciencias de la Salud, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana-Iztapalapa, Avenida San Rafael Atlixco 186, Colonia Vicentina Iztapalapa, Mexico D.F., 09340, México.2. Laboratorio de Neuroinmunología, Instituto Nacional de Neurología y Neurocirugía México D.F., México. 3. Laboratorio de Aminoácidos Excitadores, Instituto Nacional de Neurología y Neurocirugía México D.F., México.
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Cortex
Hippocampus
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Ctrl 20X BREGMA 2.2 mm
2 h 20X BREGMA 1.8 mm
2 h 20X BREGMA 2.2 mm
Striatum
Results
Ctrl 20X BREGMA 1.7 mm 2 h 20X BREGMA 1.7 mm
Introduction
Kynurenic acid (KYNA) is an antagonist for the α7-nicotinic acetylcholine receptor (nAChR) and theglycine co-agonist site of the N-methyl-D-aspartate receptor at nanomolar concentrations1. KYNA issynthesized in the brain from kynurenine (KYN) through irreversible transamination by kynurenineaminotransferases (KATs). In turn, KYN can be synthesized within the brain from L-tryptophan, ortransported across the brain-blood barrier (BBB) by the large neutral amino acid uptake system. Ithas been estimated that 40% of KYN is synthesized in the brain, while the remaining comes fromthe periphery. Its activity on excitatory amino acid receptors and nAChR might account for itsneuromodulatory and neuroprotective effects in the CNS. Thus, fluctuations in brain KYNA levelsunder physiological and pathological conditions could modulate the activity of cholinergic andglutamatergic transmission. Furthermore, KYNA, at nanomolar concentrations, can decrease theextracellular level of dopamine in the striatum of rat. Neuroprotective effects of KYNA had beenaddressed through pharmacologic manipulations leading to increased brain concentrations ofKYNA, which have shown anti-epileptic effects. Probenecid (PROB), a drug widely used for thetreatment of the gout, inhibitor of the organic acids transporter (OAT), and found in the BBB, hasbeen previously shown to increase brain concentrations of KYNA. PROB has shown to prevent OAT-mediated transport of KYNA towards the cerebrospinal fluid, the blood torrent and finally, out ofthe circulatory system as urine in experiments in which its precursor KYN was co-administrated2,3.However, there is no direct evidence showing if the increase in KYNA concentrations by PROBaction, without its precursor, is dose-dependent, and if this accumulation also differs through thetime and brain region.
Materials and methods
Animals
Male Wistar bred-in-house rats (270 - 300 g) were used throughout the study. Animals (6 pergroup) were randomly assigned to six experimental groups as follows:
After the time provided, striatal, hippocampus, and the cortex tissues were collected bydecapitation and immediately dissected out on ice and preserved for limited times at −70 °C.During dissection, all efforts were made to minimize animal suffering.
Measurement of KYNA levels
KYNA content was measured in freshly tissues homogenates obtained from animals of experimentalgroups, and determined according to established high performance liquid chromatography (HPLC)procedures4, with a mobile phase containing 200 mM of zinc acetate and 5% acetonitrile (pH 6.2)was used for KYNA content and detected fluorimetrically (excitation/emission wavelength 344/398nm).
Measurement of DA levels
DA levels were measured in freshly obtained tissues from control and experimental groups,according to previous reports5. The brain DA tissue content was analyzed by HPLC withelectrochemical detection, as previously described6. All samples were analyzed in duplicate; theconcentrations of DA and its metabolites were first calculated as μg per gram of wet tissue.
KYNA Immunohistochemical Assay
Polyclonal antibody against KYNA (Sigma-Aldrich, Inc. St. Louis, MO, USA) was used to examine thecellular location in rat brain sections. The expression of KYNA was examined using animmunohistochemical approach. This assay was performed after 1, 2, 3 hours after PROB (50mg/kg,i.p.) Formalin-fixed, paraffin-embedded archived tissues were sectioned (thickness of 4 μm)7. Thesections were then dewaxed, rehydrated and goat serum albumin-blocked. Following blocking, thesections were incubated with the antibody for 2 h at room temperature (1:80 dilution). The slideswere incubated with a secondary FITC-labeled antibody (1:1000 dilution). Finally, the slides wereVECTASHIELD-mounted. Images were obtained in an inverted microscope (Leica Microsystems,Heidelberg, Germany) using an ArKr laser at 488 and 520 nm to define the intracelular location ofKYN.
GROUP ADMINISTRATION
CONTROL NaOH 0.1 N, pH 9.0 adjusted
30 min
PROB (50 mg/kg, i.p.) compound was dissolved in NaOH 0.1 N pH
9.0 adjusted
1 hour
2 hours
3 hours
4 hours
References
1. Hilmas C., Pereira E. F., Alkondon M., Rassoulpour A., Schwarcz R. and Albuquerque E. X. (2001) The brain metabolite kynurenicacid inhibits alpha7 nicotinic receptor activity and increases non-alpha7 nicotinic receptor expression: physiopathologicalimplications. J. Neurosci. 21, 7463–7473.2. Hesselink MB, Smolders H, Eilbacher B, De Boer AG, Breimer DD, Danysz W. 1999. The Role of Probenecid-Sensitive Organic AcidTransport in the Pharmacokinetics of N-Methyl-D-Aspartate Receptor Antagonists Acting at the GlycineB-Site: Microdialysis andMaximum Electroshock Seizures Studies. J Pharmacol Exp Ther, 290: 543-550.3. Silva-Adaya, D.; Pérez-De La Cruz, V.; Villeda-Hernández, J.; Carrillo-Mora, P.; González-Herrera I.; Esperanza García, E.;Colín-Barenque, L.; Pedraza-Chaverrí, J.; Santamaría, A. Protective effect of L-kynurenine and probenecid on 6-hydroxydopamine-inducedstriatal toxicity in rats: Implications of modulating kynurenate as a protective strategy.4. Swartz KJ, During MJ, Freese A, Beal MF (1990) Cerebral synthesis and release of kynurenic acid: an endogenous antagonist ofexcitatory amino acid receptors. J Neurosci 10:2965–2973.5. Blandini F, Levandis G, Bazzini E, Nappi G, Armentero MT. Time-course of nigrostriatal damage, basal ganglia metabolic changesand behavioural alterations following intrastriatal injection of 6-hydroxydopamine in the rat: new clues from an old model. Eur JNeurosci 2007;25:397–405.6. García E, Limón D, Pérez-De La Cruz V, Giordano M, Díaz-Muñoz M, Maldonado PD, et al. Lipid peroxidation, mitochondrialdysfunction and neurochemical and behavioural deficits in different neurotoxic models: protective role of S-allylcysteine. Free RadicRes 2008;42:892–902.7. Rodríguez E, Méndez-Armenta M, Villeda-Hernández J, Galván-Arzate S, BarrosoMoguel R, Rodríguez F, et al. Dapsone preventsmorphological lesions and lipid peroxidation induced by quinolinic acid in rat corpus striatum. Toxicology 1999;139: 111–8.
ConclusionsOur data show a time-dependent increase of KYNA levels in cortex and striatum afteradministration of PROB, effect not observed in hippocampus, this increase is inversely related toDA levels in time.Clearly PROB in CNS has effect on the OAT resulting in an accumulation of KYNA, and may be avalid therapeutic tool for regulating the glutamatergic and cholinergic transmission.
