ATP extracellular concentrations are increased in the rat striatum during in vivo ischemia

https://doi.org/10.1016/j.neuint.2005.05.014Get rights and content

Abstract

Interest is growing in the role of adenosine triphosphate (ATP) on P2 receptors during hypoxic/ischemic events in the brain. However, there is no direct evidence of an increase in extracellular ATP levels during cerebral ischemia in vivo. The aim of the present study was to evaluate ATP outflow from the rat striatum by the microdialysis technique associated with focal cerebral ischemia in vivo by intraluminal occlusion of the right middle cerebral artery (MCA). Between 1 and 4 h after ischemia, rats showed a clear turning behavior contralateral to the ischemic side. Twenty-four hour after MCA occlusion, ischemic rats had definite neurological deficit and striatal and cortical damage. The ATP concentration (mean ± S.E.M.) in the striatum of normoxic rats (n = 8) was 3.10 ± 0.34 nM. During 220 min after MCA occlusion, the extracellular ATP levels significantly increased two-fold, being 5.90 ± 0.61 nM (p < 0.01 versus normoxic level). ATP outflow showed a tendency to increase over time during the 220 min of ischemia.

Since extracellular ATP is rapidly metabolized to adenosine, we also assessed ATP outflow in the presence of the ecto-5′-nucleotidase inhibitor, α,β-methylene-adenosine diphosphate (AOPCP, 1 mM) directly perfused into the striatum. The ATP concentration in normoxic rats (n = 8) was increased three-fold in the presence of the ecto-5′-nucleotidase inhibitor (9.57 ± 0.26 nM). During 220 min of ischemia, extracellular ATP levels significantly increased 1.3-fold in AOPCP-treated rats (12.62 ± 0.65 nM, p < 0.01 versus normoxic level).

The present study confirms that ATP is continuously released in the brain and demonstrates for the first time that ATP outflow increases during ischemia in vivo. These results confirm that ATP may be an important mediator in brain ischemia.

Introduction

Adenosine triphosphate (ATP) plays a wide variety of important roles in cell to cell signaling acting on ionotropic P2X and G protein-coupled P2Y receptors in the mammalian brain (Ralevic and Burnstock, 1998). It acts as a neurotransmitter (Khakh, 2001) as a presynaptic neuromodulator (see Cunha and Ribeiro, 2000) it is also involved in neuron–glial interactions (Fields and Stevens, 2000) with a role in neuronal development, plasticity (Neary et al., 1996) and inflammation (Bodin and Burnstock, 2001).

Interest is growing in the role of ATP on P2 receptors during hypoxic/ischemic events. Recent data support the idea that ATP under such conditions exerts an excitotoxic role through activation of P2 receptors. Antagonists of P2 receptors have been proved protective against apoptotic and necrotic features induced by ATP (Amadio et al., 2002), by hypoglycemic, chemical-hypoxic insults (Cavaliere et al., 2001a, Cavaliere et al., 2001b) and by glutamate (Volontè and Merlo, 1996, Volontè et al., 1999) in primary cultures of cerebellar, striatal, cortical and hippocampal neurons. Furthermore, it was demonstrated that P2 antagonists protect in vivo from ATP-induced neuronal and glial cell death in the striatum (Ryu et al., 2002).

During oxygen and glucose deprivation, the vertebrate brain rapidly goes into energy failure which first results in loss of ionic gradients with consequent depolarization. Under these conditions, there is intracellular ATP depletion and adenosine production (Latini et al., 1999, Latini and Pedata, 2001, Rudolphi et al., 1992). In the model of focal ischemia in the rat, mitochondria appear to consume ATP (Takeda et al., 2004) and ATP content is decreased by about 30%, 15 min after ischemia induction (Madrigal et al., 2003). Despite its essential intracellular role, ATP can be released from cells. Evidence of an increased outflow of tritiated ATP from hippocampal slices during ischemic-like conditions was reported by Juranyi et al. (1999). In vivo it has been reported that extracellular ATP concentration increases in ischemic cardiac tissue (Kuzmin et al., 1998, Kuzmin et al., 2000) and in the striatum of the freshwater turtle during anoxia (Lutz and Kabler, 1997). However, although in vivo there is evidence of a continuous efflux of ATP from the cerebral cortex (Phillis et al., 1993, Phillis and O’Regan, 2002) there is no direct evidence of an increase in extracellular ATP concentration during hypoxic/ischemic episodes in the mammalian brain. Phillis et al. (1993) failed, in fact, to detect an increase in ATP from the rat cortex during hypoxia.

The aim of the present work was to assess whether an ischemic insult in vivo causes release of ATP. The ATP outflow from the rat striatum was investigated in the model of permanent focal cerebral ischemia induced by middle cerebral artery occlusion (MCAo) (Melani et al., 1999). Utilizing this method we have previously demonstrated that the extracellular concentrations of different transmitters, i.e. glutamate, aspartate, GABA, adenosine increase (Melani et al., 1999, Melani et al., 2003) and Shirotani et al. (1995) demonstrated that dopamine increases. In this study, ATP was quantified by a highly sensitive assay of luciferine/luciferase.

Section snippets

Animal housing and surgery

Male Wistar rats (Harlan, Italy) weighing 270–290 g were used. They were housed in groups of three with free access to food and water and kept on a 12-h light:12-h dark cycle. The guidelines of the European Community for animal experiments were followed. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.; Sigma–Aldrich, St. Louis, MO, USA) and placed in a stereotaxic frame (Kopf, USA). A

Results

Fig. 1 shows the time-course of ATP outflow in sham-operated, ischemic (vehicle- and AOPCP-treated) rats before and after permanent MCAo.

In the sham-operated rats, the extracellular levels of ATP remained stable through the entire sample collection period. In ischemic vehicle-treated rats, MCAo led to an increase in ATP level. The first outflow peak occurred 20 min after MCAo and it was a three-fold increase, evaluated as peak value over the pre-ischemic basal levels. The mean extracellular

Discussion

The present results confirm previous findings of Phillis et al. (1993) and Phillis and O’Regan (2002) that ATP is continuously released in the brain and demonstrate for the first time that ATP outflow increases under ischemia in vivo.

The extracellular ATP concentration is doubled in the first 220 min after ischemia and tends to increase over time. The concentration of ATP evaluated in our study in normoxic conditions is in the low nanomolar range and, when corrected for recovery of the

Acknowledgements

This investigation was supported by grants from MURST (ex 40% and FIRB founding) and from the “Ente Cassa di Risparmio” of Florence, Italy.

We thank Mrs. Laura Calosi for her collaboration in preparing histological specimens.

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