The Effect of Adenosine ASubscript2A/Subscript

Authors: Krystyna Gołembiowska Anna Dziubina

Publish Date: 2012/03/10

Volume: 22, Issue: 2, Pages: 150-157

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Abstract

It has been shown that a decreased vesicular monoamine transporter VMAT2 function and the disruption of dopamine DA storage is an early contributor to oxidative damage of dopamine neurons in Parkinson’s disease PD In our previous study we demonstrated that adenosine A2A receptor antagonists suppressed oxidative stress in 6hydroxydopaminetreated rats suggesting that this effect may account for neuroprotective properties of drugs In the present study rats were injected with reserpine 10 mg/kg sc and 18 h later the effect of the adenosine A2A receptor antagonists 83chlorostyrylcaffeine CSC and 427amino22furyl124triazolo23a135triazin5ylaminoethylphenol ZM 241385 on extracellular DA glutamate and hydroxyl radical formation was studied in the rat striatum using in vivo microdialysis By disrupting VMAT2 function reserpine depleted DA stores and increased glutamate and hydroxyl radical levels in the rat striatum CSC 1 mg/kg but not ZM 241385 3 mg/kg increased extracellular DA level and production of hydroxyl radical in reserpinised rats Both antagonists decreased the reserpineinduced increase in extracellular glutamate l34Dihydroxyphenylalanine LDOPA 25 mg/kg significantly enhanced extracellular DA had no effect on reserpineinduced hydroxyl radical production and decreased extracellular glutamate concentration CSC but not ZM 241385 given jointly with LDOPA increased the effect of LDOPA on extracellular DA and augmented the reserpineinduced hydroxyl radical production CSC and ZM 241385 did not influence extracellular glutamate level which was decreased by LDOPA It seems that by decreasing the MAOdependent DA metabolism rate CSC raised cytosolic DA and by DA autoxidation it induced hydroxyl radical overproduction Thus the methylxanthine A2A receptor antagonists bearing properties of MAOB inhibitor like CSC may cause a risk of oxidative stress resulting from dysfunctional DA storage mechanism in early PDProgressive degeneration of the dopaminecontaining neurons in the substantia nigra pars compacta results in deficiency of striatal dopamine DA and loss of neurochemical transport systems such as the dopamine transporter DAT and the vesicular monoamine transporter VMAT2 Miller et al 1997 1999 PET studies in a nonhuman primate model of Parkinson’s disease PD showed that a decreased VMAT2 function and the disruption of DA sequestration was an early and potent contributor to oxidative damage of dopamine neurons in PD pathogenesis Chen et al 2008DA is a highly reactive molecule that is capable of autoxidation to a quinone in the basic pH of the cytosol Furthermore cytosolic MAOdependent DA metabolism leads to the formation of aldehydes and peroxides Halliwell 2006 Regarding the role of oxidative stress in the pathogenesis of PD packing of cytosolic DA into synaptic vesicles by VMAT2 prevents its autoxidation and subsequent degeneration of dopamine neurons An animal model of PD mimicking the altered DA homeostasis by impaired DA storage mechanisms is based on the administration of the irreversible VMAT2 inhibitor reserpine to rats Carlsson et al 1957 Reserpine reduces vesicular storage and release of brain monoamines and leads to the accumulation of oxidative products of neurotransmitters Caudle et al 2007Recently antagonists of adenosine A2A receptors appeared as a new promising nondopaminergic therapy of PD Striatopallidal neurons are highly enriched in adenosine A2A receptors which occur there as heteromeric complexes with dopamine D2 receptors Ferré et al 1993 Behavioral studies in rodents and in nonhuman primates showed that A2A receptor antagonists reversed motor impairment induced by 6hydroxydopamine 6OHDA or MPTP Schwarzschild et al 2006 Morelli et al 2007 The mechanism of antiparkinsonian effects of A2A receptor antagonists is based on their ability to modulate GABA release and DAdependent cfos activation in the indirect striatopallidal pathway Pollack and Fink 1995 Ochi et al 2000 In addition presynaptic A2A receptors are able to control corticostriatal glutamatergic transmission by counteracting D2 receptor function Tozzi et al 2007 Several epidemiological and animal studies have suggested neuroprotective effects of caffeine and selective A2A adenosine receptor antagonists Ross et al 2000 Ascherio et al 2001 Xu et al 2005 Chen et al 2007 A protective effect of caffeine and more selective antagonists of A2A receptors similar to genetic inactivation of A2A receptors was observed in an animal MPTP neurotoxicity model Xu et al 2005 Chen et al 2007 or in ischemia and excitotoxic brain injury models Popoli et al 2004 Chen et al 2007 The mechanism allowing A2A receptor antagonists to protect dopaminergic neurons has not been fully explained yet but a variety of their effects on various types of neurons eg glutamatergic nerve terminals and glial or immune cells suggest its complex nature Chen et al 2007 In our earlier study we have shown that A2A receptor antagonists decreased the production of free radical and lowered extracellular glutamate level in 6OHDAtreated rats Gołembiowska et al 2009 Gołembiowska and Dziubina 2012 Moreover A2A receptor antagonists administered in combination with l34Dihydroxyphenylalanine LDOPA did not change inhibitory effect of LDOPA on free radical generation and glutamate enhancement in the striatum of 6OHDAtreated rats Gołembiowska and Dziubina 2012A class of A2A antagonists belonging to methylxanthine derivatives offers a neuroprotective benefit as MAOB inhibitors Castagnoli et al 2003 Inhibition of DA degradation by MAOB attenuates hydrogen peroxide formation but at the same time it increases the risk of DA autoxidation resulting from augmentation of the cytosolic DA pool Therefore in our present study we aimed to investigate whether two A2A receptor antagonists 83chlorostyrylcaffeine CSC and 427amino22furyl124triazolo23a135triazin5ylaminoethylphenol ZM 241385 belonging to different chemical classes methylxanthine and nonxanthine derivatives respectively can modulate extracellular level of DA and glutamate as cellular sources of hydroxyl radical in animals with reduced VMAT2 function after reserpine administration Since LDOPA may be toxic in the brain by promoting the formation of reactive species and neurotoxic quinones when cytosolic level of DA increases after disruption of DA storage mechanisms Halliwell 2006 we also studied the effect of A2A receptor antagonists given in combination with LDOPA in rats treated with reserpineMicrodialysis studies were conducted in male Wistar rats 250–300 g bred at the Institute of Pharmacology Polish Academy of Sciences Krakow Poland The rats were housed in temperature and humiditycontrolled rooms on a 12h light/dark cycle with free access to filtered tap water and standard pelleted laboratory chow throughout the study The experimental procedures and housing conditions used were in strict accordance with the Polish legal regulations concerning experiments on animals Dz U 0533289 All the experimental protocols were approved by the Local Bioethics Commission for Animal ExperimentsLDOPA CSC benserazide and phydroxybenzoic acid PBA were obtained from SigmaAldrich Poznań Poland reserpine was obtained from FlukaAnalytical Poland whereas 427amino22furyl124triazolo23a135triazin5ylaminoethylphenol ZM 241385 came from TOCRIS Warsaw Poland All the chemicals used for HPLC were purchased from Merck Warsaw Poland LDOPA and benserazide were dissolved in saline A solution of PBA was prepared in an artificial cerebrospinal fluid aCSF and was then adjusted to pH 74 with 01 M NaOH CSC was initially dissolved in dimethyl sulfoxide DMSO SigmaAldrich Poznań Poland and was then diluted in at least 20 vols of the vehicle consisting of a 2080 v/v mixture of Alkamulus EL620 RhonePoulenc Cranbury NJ and a phosphatebuffered saline ZM 241385 was dissolved in a small amount of DMSO and then was diluted in Cremophor EL SigmaAldrich Poznań Poland and 09 NaCl final concentration a 15 DMSO and a 15 Cremophor EL All injections were made by an intraperitoneal route ip Reserpine dissolved in a mixture of benzyl alcohol 2 ml citric acid 250 mg and Tween80 in H2O was given in a dose of 10 mg/kg 18 h before microdialysis experiment CSC 1 mg/kg and ZM 241385 3 mg/kg were given in single injections as indicated in the figures LDOPA 25 mg/kg was injected 20 min after the administration of A2A receptor antagonists together with benserazide 125 mg/kg Control animals received respective vehiclesFor the measurement of dopamine DA 34dihydroxyphenylacetic acid DOPAC and homovanilic acid HVA the brain tissue was homogenized in 01 M HClO4 centrifuged at 4°C for 5 min at 10000×g and the supernatant was filtered through 01 μm Millipore membranes An aliquot of 2–5 μl of each sample was then injected into a high performance liquid chromatograph HPLC with electrochemical detection

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