Astrocytic adenosine kinase regulates basal synaptic adenosine levels and seizure activity but not activity-dependent adenosine release in the hippocampus

doi:10.1016/j.neuropharm.2008.09.016

Neuropharmacology

Volume 56, Issue 2, February 2009, Pages 429-437

https://doi.org/10.1016/j.neuropharm.2008.09.016 Get rights and content

Abstract

Adenosine is an endogenous inhibitor of excitatory synaptic transmission with potent anticonvulsant properties in the mammalian brain. Given adenosine's important role in modulating synaptic transmission, several mechanisms exist to regulate its extracellular availability. One of these is the intracellular enzyme adenosine kinase (ADK), which phosphorylates adenosine to AMP. We have investigated the role that ADK plays in regulating the presence and effects of extracellular adenosine in area CA1 of rat hippocampal slices. Inhibition of ADK activity with 5′-iodotubercidin (IODO; 5 μM) raised extracellular adenosine, as measured with adenosine biosensors, and potently inhibited field excitatory post-synaptic potentials (fEPSPs) in an adenosine A₁R-dependent manner. In nominally Mg²⁺-free aCSF, which facilitated the induction of electrically-evoked epileptiform activity, adenosine biosensor recordings revealed that seizures were accompanied by the transient release of adenosine. Under these conditions, IODO also inhibited the fEPSP and greatly suppressed epileptiform activity evoked by brief, high-frequency stimulation. During spontaneous seizures evoked by the A₁R antagonist CPT, adenosine release was unaffected by IODO. This suggests that ADK activity does not limit activity-dependent adenosine release. On the basis of strong ADK immunoreactivity in GFAP-positive cells, astrocytes are likely to play a key role in regulating basal adenosine levels. It is this action of ADK on the basal adenosine tone that is permissive to seizure activity, and, by extension, other forms of activity-dependent neuronal activity such as synaptic plasticity.

Introduction

The purine nucleoside adenosine regulates many physiological and pathological processes via four G-protein coupled receptors (A₁, A_2A, A_2B, A₃) (Fredholm et al., 2005). These physiological processes range from regulation of transmitter release, in particular that of the major excitatory neurotransmitter, glutamate, to the induction of sleep following the gradual accumulation of adenosine during wakefulness (Basheer et al., 2004). In addition, the release of adenosine during a host of pathological events in the mammalian brain is believed to exert a strong neuroprotective influence via inhibition of glutamate release and neuronal and network activity, thereby reducing nutrient demand, as well as acting as a vasodilator during such insults as stroke, epileptic seizures and head injury (Cunha, 2005).

Given the powerful and ubiquitous nature of adenosine action within the CNS, basal levels of extracellular adenosine are carefully regulated and are estimated to be in the region of 30–300 nM (Fredholm et al., 2001). The two main pathways for the control of extracellular adenosine involve phosphorylation of cytosolic adenosine to AMP, which is mediated by adenosine kinase (ADK), or deamination by adenosine deaminase (ADA) resulting in the formation of inosine. In rat brain, K_m values are ∼2 μM for ADK and ∼17 μM for ADA (Phillips and Newsholme, 1979). This suggests that under normal physiological conditions adenosine is metabolised primarily via the action of ADK, with the contribution of ADA increasing as the extracellular adenosine concentration is elevated, for example, during metabolic stress (Lloyd and Fredholm, 1995, Latini and Pedata, 2001). These K_m values also suggest that adenosine kinase is the primary regulator of intracellular, and, most likely by virtue of the ubiquitous equilibrative nucleoside transporters (King et al., 2006), basal extracellular adenosine concentration (Boison, 2006).

Accordingly, inhibition of ADK, but not ADA, in hippocampal slices increased extracellular levels of adenosine and depressed excitatory synaptic transmission in an A₁R-dependent manner (Pak et al., 1994, Lloyd and Fredholm, 1995). More recently, hippocampal grafts of ADK-deficient myoblasts, fibroblasts or embryonic stem cells raised extracellular adenosine levels in vivo and were able to retard kindling-induced seizure activity in rats (Boison, 2006, Boison, 2007).

Thus, ADK is a key regulator of the concentration, and hence effects, of extracellular adenosine. In this study we have examined the role of ADK in an in vitro model of electrically-evoked epileptiform activity. We show that inhibition of ADK, which is located primarily in GFAP-positive astrocytes, elevates synaptic adenosine and greatly suppresses glutamatergic excitatory synaptic transmission in both control and nominally Mg²⁺-free aCSF in an A₁R-dependent manner. In addition, seizure activity evoked by brief, high-frequency stimulation was greatly attenuated. These data suggest that not only do astrocytes and ADK play a pivotal role in the regulation of synaptic adenosine under both physiological and pathological conditions, they also imply that basal ADK activity is permissive to seizure activity. Thus, factors influencing the activity or levels of ADK are therefore likely to have important consequences for activity-dependent neuronal function, such as synaptic plasticity, whilst targeting ADK may provide novel therapies for epilepsy and other neurological disorders.

Section snippets

Slice preparation

Hippocampal slices were obtained from 16 to 22 day-old Sprague–Dawley rats of either sex. The animals were killed by cervical dislocation in accordance with Schedule 1 of the UK Government Animals (Scientific Procedures) Act 1986 and with the approval of the local Ethical Review Committee. The animals were then decapitated and the whole brain rapidly dissected and placed in ice-cold artificial cerebrospinal fluid (aCSF) containing 11 mM Mg²⁺. Slices were cut using a Vibratome (Intracel,

ADK powerfully regulates extracellular adenosine levels

Treatment of 600 μm rat hippocampal slices with the ADK inhibitor 5′-iodotubercidin (IODO; 5 μM) resulted in a rapid and profound depression of the fEPSP slope to 6.1 ± 2.2% of control (n = 5; Fig. 1). To establish whether this depression of glutamatergic transmission was dependent on an increase in extracellular adenosine and activation of A₁Rs, we applied the A₁R antagonist CPT (1 μM). Under basal conditions CPT enhanced the fEPSP by 9.3 ± 2.5% (n = 5; data not shown) indicative of an

Adenosine as an endogenous anticonvulsant

Extracellular adenosine increases in the mammalian, including human, brain during seizure activity and both terminates ictal activity and mediates post-ictal refractoriness (During and Spencer, 1992). In the present study we provide the first continuous, on-line determinations of adenosine release during both evoked and spontaneous seizure activity. The former, typically of ∼10–20 s duration, elevates extracellular adenosine to approximately 1 μM above basal for several minutes, which is

Acknowledgements

We are grateful to the MRC, Epilepsy Research UK, Tenovus Scotland, Anonymous Trust, the Physiological Society and the Health Foundation for financial support. We thank Mr. Ján Lopatář for help with some of the experiments.

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Astrocytic adenosine kinase regulates basal synaptic adenosine levels and seizure activity but not activity-dependent adenosine release in the hippocampus

Abstract

Introduction

Section snippets

Slice preparation

ADK powerfully regulates extracellular adenosine levels

Adenosine as an endogenous anticonvulsant

Acknowledgements

J. Neurosci. Methods

Prog. Neurobiol.

Trends Pharmacol. Sci.

Prog. Neurobiol.

Brain Res.

Neurochem. Int.

Int. Rev. Neurobiol.

Eur. J. Pharmacol.

Exp. Neurol.

Epilepsy Res.

Trends Pharmacol. Sci.

Neurochem. Int.

Neuropharmacology

Neuroscience

Semin. Pediatr. Neurol.

Adenosine-mediated presynaptic modulation of glutamatergic transmission in the laterodorsal tegmentum

J. Neurosci.

Adenosine-based cell therapy approaches for pharmacoresistant epilepsies

Neurodegener. Dis.

Neonatal hepatic steatosis by disruption of the adenosine kinase gene

Proc. Natl. Acad. Sci. U.S.A.

Neuroprotection by adenosine in the brain: from A1 receptor activation to A2A receptor blockade

Purinergic Signal.

Direct measurement of adenosine release during hypoxia in the CA1 region of the rat hippocampal slice

J. Physiol.

Hypoxia-induced inhibition of adenosine kinase potentiates cardiac adenosine release

Circ. Res.

Extracellular adenosine concentrations in hippocampal brain slices and the tonic inhibitory modulation of evoked excitatory responses

J. Pharmacol. Exp. Ther.

Adenosine: a potential mediator of seizure arrest and postictal refractoriness

Ann. Neurol.

Neuroprotection by adenosine in the brain: from A₁ receptor activation to A_2A receptor blockade