Elsevier

Brain Research

Volume 888, Issue 1, 5 January 2001, Pages 150-157
Brain Research

Research report
Inhibition of evoked glutamate release by the neuroprotective 5-HT1A receptor agonist BAY x 3702 in vitro and in vivo

https://doi.org/10.1016/S0006-8993(00)03074-2Get rights and content

Abstract

Brain ischemia provoked by stroke or traumatic brain injury induces a massive increase in neurotransmitter release, in particular of the excitotoxin glutamate. Glutamate triggers a cascade of events finally leading to widespread neuronal cell damage and death. The aminomethylchroman derivative BAY x 3702 is a novel neuroprotectant which shows pronounced beneficial effects in various animal models of ischemic brain injury. As shown previously BAY x 3702 binds to 5-HT1A receptors of different species in subnanomolar range and is characterized as a full receptor agonist. In this study we investigated the influence of BAY x 3702 on potassium-evoked glutamate release in vitro and ischemia-induced glutamate release in vivo. In rat hippocampal slices BAY x 3702 inhibited evoked glutamate release in a dose-dependent manner (IC50=1 μM). This effect was blocked by the selective 5-HT1A receptor antagonist WAY 100635, indicating that BAY x 3702 specifically acts via 5-HT1A receptors. In vivo, release of endogenous aspartate and glutamate was measured in the cortex of rats by microdialysis before and after onset of permanent middle cerebral artery occlusion. Single dose administration of BAY x 3702 (1 μg/kg or 10 μg/kg i.v.) immediately after occlusion reduced the increase and total release of extracellular glutamate by about 50% compared to non-treated animals, whereas the extracellular aspartate levels were not significantly affected. Inhibition of glutamate release may therefore contribute to the pronounced neuroprotective efficacy of BAY x 3702 in various animal models of ischemic brain damage.

Introduction

Ischemic brain damage caused by stroke or traumatic brain injury is characterized by an immediate depletion of cellular energy levels. Massive ion fluxes across the plasma membrane and breakdown of the energy-driven membrane potential induces liberation of neurotransmitters into the extracellular space, in particular the excitotoxin glutamate [2], [20], [36], [39], [42]. Excess of glutamate leads to a continuous activation of NMDA, AMPA and metabotropic glutamate receptors, resulting in massive calcium influx and mobilization of intracellular calcium stores [32]. An extensive and long-lasting rise in intracellular calcium levels represents a non-physiological stimulus, triggering various intracellular processes including activation of lipases and nitric oxide synthase, formation of oxygen free radicals and release of neurotransmitters as glutamate, dopamine, serotonin etc. [11], [19], [22]. As a result of this cascade neurons finally die and represent new sources of neurotoxic glutamate [40].

Based on the prominent role of glutamate, strategies for development of pharmacological principles interfering with the cascade of ischemia-mediated cell death have initially focused on receptors, which trigger the neurotoxic effects caused by glutamate, i.e., on NMDA receptors including their modulatory sites, and AMPA receptors [5]. Glutamate antagonists directly acting at the postsynaptic glutamate receptors show considerable side effects, including hypotension and psychotomimetic effects in humans, and can cause neurotoxic injury in animals [26], [32], [45]. So far, most glutamate receptor antagonists investigated in clinical trials revealed no therapeutic efficacy due to unfavorable risk–benefit ratio or lack of efficacy [13], [28]. However, new compounds acting via modulatory sites at the NMDA receptor like the glycine receptor antagonist GV 150526 do not produce neuronal vacuolization or cognitive disturbances. In addition, clinical studies revealed that this compound is well tolerated, even at plasma levels, which displayed neuroprotective efficacy in preclinical models of stroke.

Alternative strategies to attenuate glutamate-mediated toxicity have been set up. These approaches include inhibition of second messenger cascades involved in glutamatergic signaling and blockade of ion channels which may counteract excessive ischemia-induced neuronal depolarization. 5-HT1A receptor agonists have been tested in various models of ischemic damage and have revealed some neuroprotective efficacy in vivo and in vitro [14], [34], [35]. However, all compounds investigated so far showed only restricted 5-HT1A receptor selectivity, affinity and/or intrinsic activity. Recently, the aminomethylchroman derivative BAY x 3702 was described as a novel highly potent 5-HT1A receptor full agonist with pronounced neuroprotective properties in various animal models of ischemic brain injury [1], [14], [15], [16], [23], [24], [41].

In the present study we investigated the effects of BAY x 3702 on glutamate release both in vitro and in vivo in order to evaluate whether modulation of glutamatergic neurotransmission may be involved in the neuroprotective properties of this compound.

Section snippets

Chemicals and reagents

All chemicals were of highest commercially available purity and were purchased from E. Merck, Darmstadt, FRG. BAY x 3702 (R-(−)-2-{4-[(chroman-2-ylmethyl)-amino]-butyl}-1,1-dioxo-benzo[d]isothiazolone hydrochloride) and WAY 100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N(2-pyridinyl)cyclohexane carboxamide trihydrochloride) were obtained from the Chemistry Department of Bayer AG, Wuppertal, FRG. Isoflurane was obtained from Abbot GmbH, Wiesbaden, FRG.

Determination of glutamate release from hippocampus slices

Hippocampi were dissected from

Glutamate release in vitro

During incubation of hippocampus slices in physiological buffer glutamate is spontaneously released into the surrounding medium. Mean basal release was ∼0.5 μmol glutamate/mg protein/min. Transfer of the tissue pieces into high potassium buffer (75 mM K+) resulted in an 20–40% increase of glutamate accumulation in the medium. After wash out of excess of potassium, basal glutamate release recovered to about 95% of that observed prior to stimulation. A second exposure of hippocampal slices to

Discussion

The aminomethylchroman derivative BAY x 3702 is a novel neuroprotectant and has previously been characterized as a highly potent and selective 5-HT1A receptor full agonist which bound to human and rat 5-HT1A receptors with nearly identical affinities [16]. Strong neuroprotective efficacy of BAY x 3702 has been demonstrated in a number of different animal models of acute ischemic brain injury [1], [14], [23], [24].

Preclinical as well as clinical studies indicate that excitatory amino acids are

Acknowledgements

The excellent technical assistance of Mrs M. Borsch, H. Struppeck, and U. Wende is gratefully acknowledged.

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