Neurochemical Research, VoL 17, No. 2, 1992, pp. 147-150

An Adenosine Uptake Blocker, Propentofylline, Reduces Glutamate Release in Gerbil Hippocampus Following Transient Forebrain Ischemia Kotaro Miyashita 1,2, Takashi Nakajima 1, Atsushi Ishikawa 1, and Tadashi Miyatake 1 (Accepted June 17, 1991)

In the present study, the effect of the adenosine uptake blocker, propentofylline (HWA 285) on the extracellular concentration of several amino acids including glutamate, glycine and taurine following 10 min of forebrain ischemia in gerbil hippocampus was investigated using in vivo microdialysis. Pretreatment with HWA 285 (20 mg/kg i.p.) significantly reduced the extracellular concentration of glutamate following ischemia but did not significantly alter levels of other amino acids such as glycine and taurine. These findings suggest that the neuroprotective effect of HWA 285 may be associated with inhibition of glutamate release in the gerbil hippocampus. KEY WORDS: Propentofylline;adenosine;gerbilhippocampus;glutamate;microdialysis;transientischemia.

INTRODUCTION

degeneration following brain ischemia (7). Intraventricular pretreatment with the adenosine receptor agonist, cyclohexyl adenosine (CHA) protects against neuronal death following ischemia (8), although systemic administration of CHA and other stable analogues of adenosine has strong cardiovascular side-effects such as hypotension and cardiodepression (9). In contrast, the novel xanthine derivative propentofylline (HWA 285) is adenosineuptake blocker which is known to raise levels of adenosine in the brain (10) without depressing systemic blood pressure and inducing other major circulatory side effects in the dose of less than 32 mg/kg treated intraperitoneally (11). It has recently been demonstrated that HWA 285 has a protective effect against delayed neuronal death (12) in the hippocampus following transient forebrain ischemia in Mongolian gerbils (11,13,14). In the present study the effect of HWA 285 on the extracellular concentration of several amino acids, incuding glutamate, glycine and taurine, in the gerbil hippocampus following transient forebrain ischemia was investigated using in vivo brain microdialysis (15).

Levels of excitatory amino acid (EAA) such as glutamate and aspartate are dramatically elevated during brain ischemia (1,2) and these change may underlie ischemic neurotoxicity (3). Previous studies reveal that reduced glutaminergic neurotransmission, e.g. by deafferentation of the EAA input to the hippocampus (4) or by the administration of a N-methyl-D-aspartate receptor blocker (5), protects the hippocampal neurons in several animal models of ischemia. A major neuromodulator, adenosine has various pharmacological effects including modulation of cyclic adenosine monophosphate (cAMP) levels (6) and inhibition of neurotransmitter release. Adenosine receptor antagonists have been reported to potentiate neuronal 1 Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan. 2 Address reprint requests to: Dr. K. Miyashita, Dept. of Neurology,

Brain ResearchInstitute,NiigataUniversity,Asahimachi-1,Niigata, 951, Japan. Tel (025)223-6161 ext. 5184, Fax (025)225-6458.

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0364-3190/92/0200-0147506.50/09 I992 PlenumPublishingCorporation

148

EXPERIMENTAL

Miyashita, Nakajima, Ishikawa, and Miyatake

PROCEDURE

Animal Preparation. Male Mongolian gerbils (75-90 g, n = 15) were anesthetized with halothane (2% for induction and 1.5% for maintenance) in 30% NzO and 70% Oz mixture and allowed to breathe spontaneously. During the experiments the body temperature was maintained between 36.8 and 37.5~ by means of heating lamp. Mierodialysis Methods. The microdialysis probe (CMA/10, membrane length of 2ram, cut-off M.W. of 20,000; Carnegie Medicin, Stockholm, Sweden) was used and the microdialysis system was perfused with Ringer's solution at a flow rate of 2 ~l/min by CMA100 microinjection pump (BAS, U.S.A.). Drug Administration and Reversible Forebrain Ischemia. The above mentioned probe was vertically implanted into the CAt sector and dentate gyms of the unilateral hippocampus (1.8 mm lateral, 2.3 mm dorsal to bregma, and 0.8 mm down from the cortical surface). Following a period of 2 h to achieve a steady state, samples were obtained every 5 min. Animals were injected intraperitoneally with either saline vehicle (the control group), 10 or 20 mg/kg propentofylline (HWA 285, 1,5-oxohexyl-3-methyl-7-propylxanthine,Hoechst AG Werk KalleAlbert, Wiesbaden, F.R.G.). After 15 min, both carotid arteries were clamped by Yasargil aneurysm microclips (Aesculap, F.R.G.) for a period of 10 min. One sample of dialysate was taken prior to injection of saline or HWA 285 and two samples were taken in the interval between drug administration and clamping of the carotid arteries. 2 samples of dialysate were taken during occlusion of the carotid arteries and 4 samples were taken following the removal of the arterial clamping. Samples were stored at -80~ until analyzed. At the end of the experiments, the animals were sacrificed and brains were removed and immersed in 4% formaldehyde solution. The location of the microdialysis probes in the hippocampus was verified by examining serial coronal brain sections. Three gerbils were excluded from the study because their probes were located outside of the hippocampus. This resulted in each group consisting of 4 animals. In 2 animals, the diffusion area around the probe membrane was investigated by means of post-ischemic microdialysis of Ringer's solution containing 1% Evans blue (Figure 1). Amino Acids Analysis. Following pre-column derivatization with

o-phthaldialdehyde/13-mercaptoethanol,amino acids in the dialysates were detected electrochemically.Using a CMA-200 autoinjector (BAS, U.S.A.) amino acids were separated on a Bid-Phase ODS-II reversephase column (3 Ism, 3.2 x 100 mm), coupled to a CMA400A analyser (BAS, U.S.A.). The nongradient mobile phase in this HPLC system consisted of 0.1M sodium acetate buffer (pH6.0) with 8% acetonitrile at a flow rate of 0.7 ml/min. Further details of the method were reported elsewhere (1). In this analysis system, the concentration of more than 0.05 IxM in each amino acid could be detected. Statistical Analysis. Data were compared by analysis of variance and differences between individual means were assessed by post-hoe Dunnett's test. Differences with probability values of less than 0.05 were deemed statistically significant.

RESULTS In three experimental groups, 9 amino acids including glutamate, asparagine, serine, histidine, glutamine, glycine, phosphoethanolamine, alanine, and taurine were clearly determined. Aspartate was not detected at the steady state level in our system. Table I shows the average concentrations in the dialysates at the steady state. The levels of these amino acids did not change before or after saline or H W A 285 administration. As shown in Table II and Figure 2, the concentrations of all the a m i n o acids detected, except glutamine and histidine, increased rapidly during ischemia, reaching m a x i m a l values at the end of the ischemic period and decreased p r o m p t l y after r e c i r c u l a t i o n . T a u r i n e reached the m a x i m a l level 5 m i n after recirculation and still showed about 4 times basal levels 25 rain after recirculation. G l u t a m i n e did not change during ischemia and decreased to about 0 . 4 times basal Ievels at 10 m i n after recirculation (data not shown). The glutamate concentration in H W A 285 20 mg/kg group was significantly lower compared to the control group at 10 rain of

Table I. Concentrations of Amino Acids in Microdialysates Prior to the Ischemia at the Steady State (txM) (n = 12) (mean_+SEM)

Fig. 1. The dark pigmented part of the left hippocampus of the gerbil brain section indicates the diffusion area around a 2 mm probe membrane after the dialysis system was perfused with the Ringer's solution containing 1% Evans blue at a flow rate of 2 ~l/min for 45 min following a series of ischemic experiment.

Amino acid

Basal levela

Glutamate Asparagine Serine Histidine Glutamine Glycine PEAb Alanine Taurine

0.81 _+0.15 0.37 -_+0.05 2.15 -2-_0.42 0.74 _+0.17 29.8 _ 3.68 0.97 _ 0.16 1.32--_0.22 : 1.83 ___0.46 1.36 - 0.24

"Concentration of the average contents in the dialysate samples 5 min before ischemia. ~'phosphoethanolamine

Propentofylline Reduces Glutamate Release Following Ischemia Table II. Maximum Times Basal Level" of Amino Acids in Each Groups(n= 4) (mean• SEM)

149

DISCUSSION

Amino acid"

Saline

Pretreatment w i t h HWA28510mg/kg

HWA285 20mg/kg

Glutamate Asparagine Serine Histidine Glutamine Glycine PEAc Alanine Taurine"

29.0 • 3.8 2.5 • 0.6 2.8 • 0.8 1.4__.0.3 1.8 • 0.5 5.5 ---1.7 2.5 • 0.7 4.5 • 1.1 17.9 • 4.0

16.3 ---3.4 2.1 - 0.3 2.0 • 0.2 1.1 • 1.2 • 0.1 4.7 • 0.7 1.8 -+0.2 3.1 - 0.4 13.2 • 1.8

14.5 • 3.6b 1.7_+0.2 2.3 • 0.2 1.2• 1.4 • 0.1 3.5 • 0.6 2.2 ---0.3 3.6 • 0.6 15.7 • 4.4

Intraperitoneal administration of H W A 285 (10 mg/ kg) has already been reported to increase extracellular levels of adenosine (10) and decrease glutamate levels in rat hippocampus (17). In the present study it is elucidated that H W A 285 (at a dose of 20 mg/kg) reduces the release of glutamate during the ischemic period in gerbil hippocampus as well as in the early post-ischemic period (this latter effect was also observed following a 10 mg/kg dose). Neither of these doses of H W A 285 influenced the extracellular levels of taurine, glycine or the other amino acids detected. DeLeo et al. (11) reported that 10 mg/kg of H W A 285 administered 15 min before ischemia is most efficacious in preventing delayed neuronal death and is still moderately effective even when administered lh after ischemia. Massive glutamate release during the ischemic period is reported to play a role in the initial step of the degradative process leading to delayed neuronal necrosis (12). The present data, as well as that of Andin6 et al. (17) indicate that reduction of glutamate release may be

"All the amino acids except taurine reached the peak level at the end of the ischemic period. Taurine only exhibited the peak level at 5 rain after the recirculation. bsignificantly different from saline group (p < 0.05). cphosphoethanolamine

ischemia. After 5 min of recirculation, the level of glutamate in both 20 and 10 mg/kg H W A 285 treated groups were lower than the control group. In contrast, the concentrations of amino acids other than glutamate were not influenced by H W A 285 pretreatment.

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Fig. 2. Time course of the concentrationsof glutamate (Gh), taurine (Wau), serine (Ser), and glycine (Gly) in the dialysate samples from gerbil hippocampusbefore, during, and after a 10 min bilateral carotid occlusion in control (o--o), 10 mg/kg HWA 285 (t . . . . . t), and 20 mg/kg HWA 285 (& ...... &) groups. Amino acid concentrations in each sample were calculated as a function of the baseline concentrations determined from the mean concentrations in the three samples collected before the transient forebrain ischemia. Data represent the mean- SEM from 4 amino acids in each group. Asterisks indicate statistically significantdifferences comparedto controlusing Dunnet's test (*p < 0.05; **p