Hypoxia/Hypoglycemia-Induced Amino Acid ReleaseI

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Biochemical and Biophysical Research Communications 276, 134–136 (2000)

doi:10.1006/bbrc.2000.3443, available online at http://www.idealibrary.com on

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s Decreased in Vitro by Preconditioning

isa Johns,*,† Alan J. Sinclair,† and John A. Davies*,1

Department of Pharmacology, Therapeutics and Toxicology, University of Wales College of Medicine, Heath Park, CardiffF14 4XN, United Kingdom; and †Academic Department of Geriatric Medicine, University of Birmingham,ayward Building, Selly Oak Hospital, Birmingham, B29 6JD, United Kingdom

eceived August 14, 2000

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The aim of this study was to investigate the effects ofreconditioning on amino acid neurotransmitter re-

ease, induced by hypoxia/hypoglycaemia, from ratrain cortical slices. Tissue, perfused with artificialerebrospinal fluid (aCSF) at 37°C with zero glucosend gassed with 95% nitrogen and 5% carbon dioxide,howed a fivefold increase in glutamate release withittle effect on g-aminobutyric acid (GABA) release.reconditioning, with three 5-min periods of hypoxia/ypoglycaemia preceding continuous hypoxia/hypo-lycaemia, significantly decreased glutamate releasehilst significantly elevating GABA release. These re-

ults suggest that GABA may reduce the release oflutamate and consequently decrease the neurotoxicffects of glutamate. © 2000 Academic Press

Key Words: hypoxia/hypoglycaemia; brain slices;mino acid release; preconditioning.

Murry et al. (1) first described the phenomenon ofardiac preconditioning in that transient ischaemia af-orded protection against subsequent ischaemic andeperfusion injury in dogs. This protection also occursn the human myocardium (2).

In the brain, periods of short ischaemic insults havelso been shown to have neuroprotective effects to sub-equent ischaemia both in vitro (3) and in vivo (4).euronal toxicity following ischaemia is accepted to bes a result of excessive synaptic glutamate due to fail-re of adequate energy-dependent removal from thextracellular space (5). This increased level of gluta-ate leads to stimulation of a-amino-3-hydroxy-

-methyl-4-isoxazole propionic acid (AMPA) and-methyl-D-aspartate (NMDA) receptors and subse-

uent excessive depolarization of the postsynaptic neu-one and entry of calcium. This increased intracellularalcium concentration is considered to be the factor

1 To whom correspondence should be addressed. Fax: 144 29-0748316. E-mail: daviesja8@cf.ac.uk.

134006-291X/00 $35.00opyright © 2000 by Academic Pressll rights of reproduction in any form reserved.

ltimately leading to cell death (6). A recent paper (7)as reported that preconditioning of cultured corticaleurones afforded protection against subsequent isch-emia and this protection was dependent upon NMDAeceptor stimulation, nitric oxide production and cal-ium entry through voltage-dependent channels which,n turn, induced p21RAS formation.

The aim of this present study was to investigate theffects of preconditioning on hypoxia/hypoglycaemia-nduced release of glutamate and g-aminobutyric acidGABA) from rat cortical slices.

ATERIALS AND METHODS

Coronal cortical slices (250 mm) were prepared from male Wistarats weighing 300–350 g using a McIlwain tissue chopper. The slicesere immediately transferred into ice-cold artificial cerebrospinaluid gassed with 95% oxygen and 5% carbon dioxide (normal aCSF).he slices were then placed in a sealed tissue chamber and perfusedith normal aCSF at 37°C at a flow rate of 1 ml/min. The slices werellowed 1 h to equilibrate. Following equilibration the protocol forhe experiment consisted of 10 min perfusion with normal aCSFollowed by 30 min perfusion with aCSF containing no glucose andassed with 95% nitrogen and 5% carbon dioxide (hypoxic/ypoglycaemic aCSF). For the preconditioning experiments the tis-ues were subjected to three 5-min periods of hypoxia/hypoglycaemianterspersed with 10 min of perfusion with normal aCSF. Followinghe final 10-min perfusion with normal aCSF the tissues were per-used for 30 min with hypoxic/hypoglycaemic aCSF.

One-minute samples of perfusate were collected and assayed byigh-performance liquid chromatography following pre-column der-

vatization with o-phthaldialdehyde with subsequent fluorometricetection for glutamate and GABA. Full details of the assay methodsre given elsewhere (8).Composition of normal aCSF in mM: NaCl 124, KCl 5, NaH2PO4

.25, CaCl2 2, MgSO4 2, NaHCO3 26, D-glucose 10 and the pH was

.4. A corresponding increase in NaCl was made to compensate forhe lack of glucose in the hypoxic/hypoglycaemic aCSF in order toaintain osmolarity.

Statistical analysis. Differences in glutamate and GABA releasever the 40-min experimental period were analysed with 2-wayNOVA followed by Tukey’s post hoc test.

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ESULTS

Under control conditions, with perfusion of normalCSF for 40 min, both glutamate and GABA showed noignificant change from basal release (Figs. 1 and 2).erfusion with hypoxic/hypoglycaemic aCSF inducedn increase in the release of glutamate within 10 minhich progressively increased over the following 20in (Fig. 1). At the end of this perfusion the increase in

lutamate release was approximately 5-fold above con-rol release (P , 0.001). GABA release was notignificantly affected by perfusion with hypoxic/ypoglycaemic aCSF (Fig. 2). Following precondition-

ng with 3 3 5 min periods of perfusion with hypoxic/ypoglycaemic aCSF, glutamate release, to theubsequent 30-min period of hypoxia/hypoglycaemia,as significantly reduced when compared to the re-

ease in the absence of preconditioning (P , 0.001,ig. 1). However, this release was still significantlylevated above control release (P , 0.001, Fig. 1).onversely, GABA release, to the subsequent 30-mineriod of hypoxia/hypoglycaemia, following precondi-ioning, was significantly increased to approximately00% of control release and the release observed in thebsence of preconditioning (P , 0.001 in both cases,ig. 2). The periods of hypoxia/hypoglycaemia duringreconditioning had no measurable effect on the re-ease of either amino acid.

ISCUSSION

The present study demonstrates that periods ofreconditioning have a significant effect on the re-

FIG. 1. The effect of preconditioning on hypoxia/hypoglycaemia-nduced glutamate release from rat cortical slices. Consecutive 1-minamples of perfusate were collected. Preconditioning was carried outy perfusing hypoxic/hypoglycaemic aCSF 3 3 5 min interspersedith 10 min perfusion with normal aCSF prior to the start of thexperiment. Tissues were perfused with hypoxic/hypoglycaemicCSF for 30 min (bar in fig., *** P , 0.001 from both hypoxia/ypoglycaemia and control, N 5 6).

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rolonged periods of hypoxia/hypoglycaemia in corti-al slices.The mechanisms underlying the neuroprotective

ffect of preconditioning on subsequent periods ofxtended hypoxia/hypoglycaemia are not well under-tood. In the heart, experimental evidence suggestshat adenosine-activated KATP channels are impli-ated in the adaptive mechanisms involved in pre-onditioning (9) and that these channels, by hyper-olarizing the myocyte, limit Ca21 influx duringeperfusion following hypoxia. A similar mechanismould account for the neuroprotective effect of pre-onditioning in nervous tissue. A study (10) involv-ng anoxia-induced depolarizations in rat hippocam-al slices demonstrated that three periods of 1 min ofnoxia separated by 10 min was sufficient to improvehe recovery of evoked potentials following a pro-onged anoxic insult. This same study also showedhat the potassium channel activator, pinacidil, alsoarkedly improved the recovery of evoked potentialshen perfused 30 min prior to the anoxic insult.otassium channel (KATP) activators have beenhown to decrease neurotransmitter release, in par-icular glutamate, from brain slices probably byaintaining the channel in its open configuration

nd thus inducing hyperpolarization (11). Such anction elicited by preconditioning would be compati-le with the results described in this present paper.reconditioning had a dramatic effect on the releasef GABA in this study. Hypoxia/hypoglycaemia hadittle effect on GABA release when the tissue was notreconditioned, however, following preconditioningABA release was increased 2-fold whilst at the

ame time glutamate release was significantly de-reased. The decrease in hypoxia/hypoglycaemia-nduced glutamate release following preconditioning

FIG. 2. The effect of preconditioning on hypoxia/hypoglycaemia-nduced GABA release from rat cortical slices. Legend as for Fig. 1.*** P , 0.001 from both hypoxia/hypoglycaemia and control, N 5 6).

could be explained by an action of GABA on KATP

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hannels. It is well documented that GABA, actinghrough GABAB heteroreceptors, modulates KATP

hannel function to decrease neurotransmitter re-ease (12).

Brief periods of hypoxia/hypoglycaemia are probablyufficient to have a marked effect on extracellular con-entrations of glutamate through decreased removalrom the extracellular space. Synaptic concentrations oflutamate are of the order of 0.5–1 mM, transient in-reases are sufficient to excessively stimulate AMPA re-eptors and thus remove the voltage-dependent Mg21

lock on NMDA receptors and consequently initiate theascade of events leading to increased calcium entry anditric oxide synthesis. Another mechanistic possibility, asescribed in a recent paper (7), is that preconditioning isMDA-, nitric oxide-, and calcium-dependent. Applica-

ion of the NMDA channel blocker dizocilpine (MK801) orhe binding site antagonist 2-amino-5-phosphono-entanoic acid (AP5), at the same time as the precondi-ioning event, reduced the protective effect of precondi-ioning. Similarly, application of the voltage-dependentalcium channel antagonists, nifedipine and nimodipine,lso blocked the beneficial effects of preconditioning. Inur study, the preconditioning stimuli possibly releaselutamate (and/or GABA) but at a level below our limitsf detection. If this is so, the released glutamate wouldtimulate NMDA receptors and so have a marked effectn calcium entry and consequently on nitric oxide syn-hesis. Gonzalez-Zulueta et al. (7) concluded that thisequence of events led to an increase in RAS activityownstream and that this was the critical factor under-ying the effects of preconditioning.

In conclusion, we report here that preconditioning,ith short periods of hypoxia/hypoglycaemia, of rat cor-

ical slices reduces the subsequent release of glutamatelicited by prolonged hypoxia/hypoglycaemia. Whilst thexact mechanism of action of the neuroprotective effect ofreconditioning stimuli is still debatable, the role of glu-

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hat are currently being proposed.

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