European Journal of Pharmacoiogy,224 I19921 Sf7- 124 Q 1992 Elsevier Scic-rtcr Publishws R.V. All rights resented ~14-2999,/92/$GS.GG

117

EJP 52784

Protective effect of vinconate on isch~~ia-induces in the rat hippocampus

neuronal ~a~a~~

Toshiaki Iino, Masashi Katsura and Kinya Kuriyama Departmentof P~la~~~olo~, KyotoPrefemrai Unir*ersity of Medicine, Kawara~ac~li-~~ro~~‘i, Kan@yo-Ku, Kyoto602, Japan Received 9 April 1992, revised MS received 31 July 1992, accepted 8 September 1992

The prntective effect of vinconate, a vinca alkaloid derivative, on ischemia-induced neuronal damage was investigated using a model of rat forebrain ischemia caused by occlusion of four vessels. Hippocampal cell loss was observed histologicahy and neur~hemically 5 days after 10 min of ischcmia. Treatment with vinconate (50 and 200 mg/kg ip.) before cerebral ischemia significantly suppressed neuronal cell loss in the hippocampal CA1 region and the decrease in the content of neuroaczive amino acids in the hippocampus. The release of neuroactive amino acids in the hippocampus was significantly increased by cerebral &hernia. Pretreatment with vinconate (SO and 200 mg/kg i.p.1 significantly attenuated the increased release of glutamic acid and aspartic acid, but not the release of y-aminobutyric acid IGABA), taurine and glycine. This suppressive effect of vinconatc was antagonized by scopolamine (low5 ML The addition of vinconate (10-“-10-4 MI had no effect on the binding of [‘HJMK-gO1. These results indicate that pretreatment with vinconatc attenuafcs the ischemia-induced release of excitatory amino acids into the extraccllular space of the hippocampus via the stimulation of presynaptic muscarinic acetylcholinc receptors. The present results also suggest that this suppressive effect of vinconatc on the rclcasc of excitatory amino acids (glutamic acid and aspartic acid) may play a crucial role in the protective action of this agent against ischcmia-induced neuronal damage in the hippocampus. Ischemia; ~xcitato~

amino acids; ~~~~~ronaldamage; Vincon~tc; Muscarinic receptors (prcsynaptic)

( f I-Methyl-3-cthyl-2,3,3a,4-tetrahydro-lH-indolo3,2,1-de][ 1,5]naphthyridine-6carboxylate hydrochloride (vinconate~ is a vinca alkaloid derivative and was dcveloped to enhance cerebral metabolism and/or to protect against ischemia-induced neuronal damage in the brain. Previous investigations indicted that vinconate significantly prolonged the duration of cansciousness and significantly delayed the appearance of electroencephalographic disturbances observed under hypoxic conc’itions ~Thiebauld st al., 1983). Oral administration of vinconate is also known to improve psychomotor activity, cognitive function, attention and concentration in elderly subjects (Saletu et al., 1984). Furthermore, it has been reported that vinca alkaloids, such as vin-

Corrcspon~encc to: K, Kuriyama, Department of ~harnl~lcology, Kyota Prefectural University of Medicine. Kllwurnmschi-llimkoji. Kamiyyo-Ku, Kyooo602, Japan. Tel. 81-75-251-5333,fax Xl-75241. 11824.

camine, exert a protective effect against hypoxia-induced death in mice (King, 1987) and have cerebral vasodilating actions (Karpati and Szorny, 1976; Caravaggi et al., 1977). Recent investigations have reported that vinconate prevents ischemia-induced neuronal damagr in the hippocampus of rats or Mongolian gerbils (Araki et al., 198% Araki and Kogure, 1989). The detailed mechanisms underlying the protective effect of vinconate on ischemic neuronal damage have, however, not yet been clearly defined. It has been suggested that glutamic acid, an excitatory amino acid, may play an important role in the pathogenesis of ischemic neur~nal damage (Rothman and Olney, 1986;Onodera et al., 19861 Benveniste et al., 1989). Previous reports indicate that an abnormal release of glutamic acid during ischcmia induces an excessive influx of Cat+ by activation of N-methyl-D-aspartate (NMDAI receptor-operated Ca*+ channels and subsequently causes the non-specific activation of phospholipases involved in the hydrolytic breakdown of membrane structural lipids (Nedergaard, 1988; Westerbcrg ct al., 1987). Mario and Mauri& (1989) have reported that the relcnsc of glutamic acid

i PS in the rat

~~pp~~~ampus is rn~dul~It~d by muscarinic

~~~s~~~l~ti~

receptors in an in~ibit~~~ rn~nn~r. In addi-

tion, it has also hccn reported affinity

that vinccnate has an

for muscarinic receptors

(Koda et al., 19x9). In this study, we examined the effect of vinconate on the release of neuroactive amino acids into the extraceliular space of the hippocampus during and after tramient forebrain &hernia to cl&y the mechanism undcrlyin~ the protective action of vinconate against ischcmin-induced neuronal damage. In addition. we also examined the effect of scopolamine on the protective action of vineonate to clarify whcthcr or not the effect of vinconate is related to an action on mus~rinic presynaptic receptors.

Male Wistar rats chased from Japan The animats had ad (MF, Oriental Yeast water.

weighing ZOO-3(~) g wet-c purSLC, Inc. (Hamamatsu, Japan). libitum access to laboratory chow Co., Ltd., Chiba, Japan) and tap

Vinconate (Tokyo Tanahc Co, Ltd., Tokyo, Japan) was dissolved and diluted in distilled water. Vinconate (5. SO and 200 mg/lO ml per kg) and the same volume of vehicle were administered i.p. if) min before the induction of cerebral ischcmia. In the cxpcrimcnt with [‘H]( + )-S-methyl-IO,1 I-dihydro-5H-dibcnzo[a,d]cycloheptenS,IO-iminc f['H]MK-801 ) binding, the reaction mixture w”as in~u~~~ed at 30°C for fro min in the presence or absence of vinconate (11)-“-10-4 MI or (+I-MK-801 110-‘t-IO-“M). 2.3. Cerebraf isclremia nmdel

Rats were divided into two groups, sham-operated and ischemia groups. Rats in the ischemia group were subjected to forebrain ischemia according to the method of Pulsinelli and Brierly (1979), with minor modifications. Briefly, rats were anesthetized wilh 0.5% halothane in a mixture of 30% 0, and 70% N,O, and the vertebral arteries were electrocauterized. The next day, the rats were re-anesthetized under spontaneous respiration, and the bilateral common carotid arteries were occluded with aneurysm clips. In each experiment, we used rats showing isoelectronic waves on their electroencephalograms. Ten minutes after this 4-vessel occlusion, the clips were removed and the carotid arteries were rcpcrfuscd. The rectal temperature of rats was maintained at 37 f 0.5% by

using a tcmperat!trc controller @AS, CMA/150). In the sham-operated rats, the vertebral arteries were electrocauterizcd and the common carotid artcries were exposed as in the ischemia group, but ischemia was not applied (Matsumoto et al., 1991). 2.4. Histological examinations Rats were anesthetized with ether 5 days after the induction of isehemia or the sham operation. They were briefly perfused with saline via the left ventricle of the heart, followed by perfusion fixation with 300 ml of 10% foreign-salute. The skull was opened I h after fixation and the brain was rcmovcd and fixed for several days in 20% formalin. After dehydration and paraffin embedding, 4+m coronal sections were cut and stained with hematoxylin-eosin.

Rats were killed by focused microwave irradiation on the head (5 kW, 1.1 s) on the 5th day after cerebral ischemia or sham operation (Katsura et ai., 19921, and the hippo~ampus was dissected out according to the method of Glowinski and Iversen (1966). For the measurcmcnt of ncuroactive amino acids, the tissue samples were extracted with 50 mM perchloric acid and diluted wirh 0.1 N HCI.

The synaptic membrane fraction was prepared by a modification of the method of Yoncda and Ogita tl9~9a~. A crude synaptic membrane fraction was obtained from rat whole brain, excluding the cerebellum, pons and medulla oblongata, which have a low density of NMDA receptors. The membrane fraction was washed three times at 4°C with SC mM T&-acetate buffer (pH 7.4) by suspension and subsequent eentrifugation at 50000 X g for 20 min. The buffer used in this study was sterilized immediately before use by filtration through a nitr~cellulose membrane filter (0.45~pm pores) to avoid microbial artifacts (Yoneda and Ogita, 1989b1b). The pellet was suspended in 0.32 M suerose and stored at -80°C until use. On the day of the experiment, the frozen suspension was thawed at room temperature and was centrifuged at ~00~~ x g for 20 min at 4°C. The resultant pellet was washed three times with the same buffer as described above, The membrane preparation was then suspended in the same buffer and used in the binding assay. The protein content of the membrane preparation was dctcrrnincd by the method of Lowry ct al. (1951), with bovine serum albumin as standard.

IIY

2.7. Measuremw of 13HIMK-SO1binding

maintained at 37 f 0.5”C with a temperature controller. The skull was exposed and a hole was drilled for the implantation of a microdialysis probe in the hippocampus (Matsumoto et al., 1991). The stereotaxic coordinates were A, -3.0, L: 2.0 and V: 4.0 (mm) according to the stereotaxic atlas of Pellegrino et al. (1979). Vertical-type microdialysis probes !CMA-IO), with an outer diameter of 0.5 mm, length of 2 mm and molecular weight cut-off of 20000 Da, were obtained from Carnegie Medicin (Stockholm, Sweden). The microdialysis probe was perfused with Ringer solution (mM: NaCl 147, KCI 4, CaCI, 4.5) at a flow rate of 2.0 Fl/min. The collection of lO-min samples of perfusate was started 120 min after the onset of perfusion.

The binding assay was performed in a total volume of 0.5 ml of T&acetate buffer (50 mM, pH 7.4) ~ntaining 100-300 pg of membrane protein, [“H]MK801 (5 nM) and vinconate. The reaction mixture was incubated at 30°C for 60 min; incubations were terminated by the addition of the same buffer (2°C) and filtration through a Whatman GF/B glass filter. The filter was washed four times with 3 ml of the same buffer, and the radioactivity retained on the filter was measured in a liquid scintillation spectrometer using a triton-toluene scintillator. Non-specific binding was defined by adding 0.1 mM unlabeled ( + )-MK-801 and accounted for 10 to 15% of the total binding.

2.9. ~ete~ination of neuro~ct~i~e amino acids

2.8. Brain microdialysis

The content of neuroactive amino acids in the cerebral tissue homogenates and in the perfusates obtained by brain microdialysis were analyzed by high-performance liquid chromatography (HPLC) with fluorescent detec?ion following ~st-coIumn labeling with ophthalaldehyde by the method of Ida and Kuriyama (1983).

One or two days after electrocauterization of the vertebral arteries, the rats were anesthetized with 0.5% halothane in a mixture of 30% 0, and 70% N,O. The anesthetized rats were placed in a stereotaxic frame (Narishige, Tokyo, Japan), and rectal temperature was

s-7. *

*

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:

‘.

b

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-a’

1 -*

f

I

1

II

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Fig. 1. Effect of vinconatc on histolo&.A changes in the rut hippocnmpus induced by ischcmia. (A nnd B) Sham-oie;uted. The hippocilmpalCA1 neurons WC inluct. (C und D) On the 5th day after ccrcbrtil ischcmiu for IO min. Almost ail hippocampll CA1 neurons iwe deycner:~led, (fz :md Hcmatoxylin-cosin slain. FI Treatment wittr vinconute (50 mg/kg i.p.) 10 min bcforc idlcmiu. Almost aI1 hippncampel CA1 nwons MC intucl. Bur = IW ym. DG: dcntutc gyrus.

T& reagents used were halothane (Koechst Japan Ltd.. Tokyo, Japan); [‘HIMK-ROI. [[3-‘HI( + I-MK-801, g-12.5 ~Bq/mmol~ l~ew England Nuclear, Boston, I_.I.S.A.I;f + f-MK-#Ol hydrogen maleate (Merck Sharpe and D&me, Inc., Harlow, U.K.); amino acids standard solution H-type (Wake Chemicals, Osaka, Japan); ya~~nobu~ric acid IGABA) (Sigma Chemical CO., St. Louis, U.S.A.). Scopolamine hydrabromide, o-phthalaldehyde and 2-mercaptoethanol were purchased from Nacalai Tesque inc. (Kyoto, Japan). Taurine was obtained from Kanto ~herni~a~ Co., Inc. (Tokyo, Japan). Other reagents used were of the highest purity available.

Results are expressed as means rt S.E. Statistical significance was assessed with an analysis of variance followed by Dunnett’s test and Scheffe‘s test. Comparisons between two independent treatments were made with Cochran-Cox’s t-test. In fig. 4, the concehtration of amino acids in the dialy~te (% of basal) was calculated based on the average value of two or three samples obtained prior to the onset of ischemia (basal release?. In figs. 5 and 6, the total increase in the release of each neuroactive amino acid is expressed as a percentage of the average of the release obtained before the onset of ischemia and that obtained after release had returned to baseline from the ischemia-induced increase.

Tau

6IY

ilu

ASP

GMA

Fig. 2. Effect of vinconate on changes in rhe content of neuroactive amino acids &urine fTau), aspartic acid (Asp), glutamic acid (Glu), glycine IGiy) and GABA) in the hippocampus after ischemia. Rats were killed by microwave irradiation on the head on the 5th day after 10 min of ischemia. The amino acid content of the cerebral tissue homogenate was analyzed by HPLC with fluorescent detection after past-column labeling with o-phfhalaldehyde, The-content of neuroactive amino acids wus measured in the contrci group iUl. ischemic group (#I 1and ischemic group pretreated with vinconate (50 my/kg i.p.) ( S ). Each column represents the mean + S.E. (N = 5-8). * P < 0.05 and * * P < 0.01, compared with each controt value (CochranCnx’s t-test).

acid 44%; aspartic acid 24%; taurine 40%; glycine 51%; fig. 2). In the ather brain areas, the content of these amino acids was not significantly altered by 10 min of ischemia (data not shown). The administration of vinconate (50 mg/kg i.p.1 10 min before cerebral ischemia significantly prevented these decreases in the hippocampus (gfutamic acid 13%; aspartic acid 6%; taurine 12%; glycine 31%; fig. 2). In normal rat hippocampus, the same treatment with vinconate had no effect on the content of neuroactive amino acids (data not shown).

3. ResuRs To examine the effect of vinconate on the NME)A receptor, a glutamate receptor subtype known to be associated with the occurrence of ischemia~indu~ed On the 5th day after 10 min of cerebral ischemia, neuronal damage was localized in the hippocampal region oc!y in comparison with the damage in shamoperated rats (fig. lA, ‘8, C and D). Treatment with vinconate (50 mg/kg i.p.) before cerebral &hernia prevented this neuronal damage (fig. 1E and F). The same effect was obtained after treatment with 200 mg/kg of vinconatc, but no effect was observed after treatment with 5 mg/kg of vinconate (data not shown).

I

100

I

B*

*f* *

L

11 10

’

’

9

Canc~ntration

On the 5th day after 10 min of cerebral ischcmia, all

ncuroactive amino acids measured, except CABA, were significantly decrcascd in the hippocampus (ytutamic

f-tog

Yl

Fig. 3. Effect of vinconatc on [“H]MK-801 binding, The preparalion of crude synaptic membranes was incubated with 5 nM (“HJMK4W PI 3@‘C for 60 min in Tris-~tcetilte buffer containing i + )-MK-#~I (01 or vinconalc (@Q.Each value represems the mean f&E, (N = 3).

121

neuronal damage, the binding Qf ( f )-MK-801 to crude synaptic membranes was investigated. [ “H]MK-801 binding was disptaced by (+ )-MK-801 (IC& = iOp8 Mf; the addition of vinconate (lo-” lo-“’ lo-“, 10-7, lo-“, lo-” and 10v4 MI had no eifezii this binding (fig. 3). 3-4. Effect of uinccnate on the ischemia-induced release of neu~oacti~~e amino acids ne basal release of neuroactive amino acids in the hippocampus, measured by microdialysis, was as follows (pmol/min): glutamic acid 2.35 k 1.25; aspartic acid 0.53 I): 0.05; GABA 0.23 + 0.03; taurine 7.19 f 0.61; glycine 2.58 2 0.01. The administration of vinconate had no effect on the basal release of these amino acids (data not shown). The release of these amino acids was significantly increased by 10 min of cerebra9 ischemia and reached peak levels 10 min after ischemia. At the peak level, the increased release of ea, 1 ne~roa~tive amino acid (expressed as a percentage of basal release) was as follows: glutamic acid 777 f 197; aspartic acid 510 + 141; GABA 1172 rt 327; taurine 739 f 111; glycine 253 f, 46. Treatment with vinconate (50 and 200 mg/kg i.p.1 10 min before cerebral ischemia significantly attenuated the increased release of glutamic acid (figs. 4 and 5A). Similarly, the increased release of aspartic acid was significantly suppressed by treatment with vinconate (50 and 200 mg/kg i.p., fig. 5B). The release of GABA, taurine and glycine was not altered by

Fig. 4. Release of giutamic acid before, during and after &hernia for 10 min (filled bar) in the hippocampus. The release of glutamic acid is expressed as a percentage of basal release (the average value of two or three samples obtained prior to the onset of ischemia~. Each symbol represents the control group fol or the vinconate-treated groups (5 (*I, 50 I Cl) and 200 (0 ) mg/kg). ilinconate or vehicle was administered i.p. IO min before ischem~ marrow}, Basal release in the control groupwas l.Y7+0.41 and in the vinconatc-treated groups (5, 50 and 200 mg/ky) was 1.54f0.45, 1,8211:0.34 and I.YOf0.52 (pmol/minI, rcspcctively. Euch vaiuc reprcscnts the mean. (N = 5IOf. * P < 0.05, compured with the control value ~with~)trtvinc~~llate~ (Dunnclt’s tcstl.

treatment with vinconate, although GABA release showed a tendency to decrease after treatment wirh a higher dose of vinconate (fig. SC, D and E). Vincor,& at 5 mg/kg had no significant effect on the increased release of any neuroactive amino acid (figs. 4 and fig. 5). The suppressive effect of vinconate on the ischemia-induced increase in excitatory amino acid release was antagonized by lo-’ M scopolamine (fig. 6).

4. Discussion It is we99recognized that hippocampal CA1 pyramidal cells are especially vulnerable to transient cerebral ischemia (Kirino et al., 1984, Munekata and Hossman, 1987; Pulsinelli et al., 1982). Although detailed mechanisms underlying this neuronal death in the hippocamPUS have not been identified, the involvement of glutamic acid in the pathogenesis has been proposed (Rothman and Olney, 1986; 1587; Onodera et al., 1986). Rothman (1983; 1984) reported that anoxic injury of cultured neurons required synaptic activity and could be prevented by antagonists of ex~itato~ amino acids. It has been demonstrated that there is a massive release of glutamic acid into the extracellular space during cerebra9 ischemia (Benveniste et al., 1984; Globus et al., 1988). Furthermore, the preventive effect of deafferentiation of excitatory circuits CWieloch et al,, 9985; Jorgensen et al., 1987) and/or the administration of glutamic acid antag0nist.s (Meldrum, 1989) on ischemic neuronal death has been reported. However, it has also been reported that the abnormal release of glutamic acid during cerebra9 ischemia induces an excessive influx of Ca ‘+ through NMDA receptor-operated CaZf channels and/or voltage-dependent Car* channels, resulting in the non-specific activation of phospholipases and thus in the increase of the hydrolytic breakdown of membrane pho~pholipids (Nedergaard, 1988; Westerberg et al,, 1987). In addition, it has been reported that NMDA receptor antagonists provide partial protection against neuronal death in the CA1 region (Boast et al., 1988; Gill and Woodruff, 1990; Swan and Meldrum, 1990). although contradictor results have also been reported (Bu~han and Pulsinelli, 1990; Lanier et al., 1990). It has recently been reported that pretreatmenl with vinconate protects against ischemia-induced neurona death in the hippocampal CA1 region. (Araki et al., 1989; Araki and Kogwc, 1989). However, the detailed mechanisms that underlie the protective effect Of vin__...._-_ damage arc stilt UiXlcar. conate on is&emic fica*u*~*i We therefore attempted to clarify the effect of vinconate on the release of neuroactive amino acids in the hippocampus by usitlg microdialysis. The ischcmia-induccd release of excitatory amino acids was WParentfY attcnuatcd by pretrcatm~nt with vinconatc (50 and 2m

A, Glutaaic Acid

g. Aspartic Acid

Contrct

200

50

5

50

ZOO

Yinconata irglkgl

Yinconatelagikgl

L

C

w i

dU0

f E

-E 5

5

Control

200

5B

0

Control

15QDf

1

i

50

200

Yinconata lag/kg1

~inWat8 lag/kg1

z H

5

t. rsurlne .

roila

Li

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9

dc 500 B ._ %x4 2 0

5

Control

200

50

Fig.5. E&XV of vinconatc on the ;scbem~-induced release of ncuro~~c~iv~amino acids. The effect of v~ncona~e($50 and 200 mg/ky i.p.) on the ischemia-induced release of (A) glutamic acid. \B) aspartic acid, (Cl GABA, fD) glycine and (El taurine is expressedas a percentage (% increase) of the average of the release obtained beFore the onset of ischemia and that obtained after the release had returned to baseline from the ischemia~~nduced increase. E&h column represents the mean f SE. (N = 5- 10). * P < 0.05, compared with each control value (without vincunstc) (ScheffiYs test).

g. Aspaftir hid

1. 6lutaaic Acid

Scapoleains

F

Vin~an6ta Scopalsaino

Fio. h. Effect of scopolamine on vincona~e-induced suppression of ~schemja-induced release of 6%) gfutumic a& and (Bl aspartic acid. Vinconate ~m~/k~ i.p.t was admiois~ered IO min before ischemia. S~o~iamine (@MB was added to the perfusion fluid from 20 min before ischemia. The release of each amino acid is expressed as a pcrcenta~e of total increase f% increase) as shawn in fig. 5. Each column rcprcsenta the mean it SE. fN = 5- 10). ’ P c: 0.05. compared with each control value (without vincnnate and scapolnminc) tScheff6‘s test).

123

mgfkg i.p.). This treatment

had no effect on the ischemia-induced release of GABA, taurinc or giyeine, although the release of GABA showed a tendency to decrease after treatment with a higher dose of vinconate. These results suggest that vinconate may act selectively on presynaptic structures of glutamatergic neurons and that this suppressive effect of vinconate is probably not due to non-specific effects such as membranc stabilization. It has been reported that ncrvc terminals possess presynapti~ receptors that are sensitive to the same neurotransmitter released from nerve terminals (autoreceptor) or to a different neurotransmitter released from nerve terminals (heteroreceptor) (Langer, 1980). Glutamatergi~ nerve terminals in various areas of the rat brain are believed to possess presynaptic heteroreceptors that modulate the release of glutamic acid in an inhibitory manner. It has also been reported that these presynaptic heteroreceptors are muscarini~ Mz type in the hippocampus (Mario and Maurigio, 19891, dopamine D, and cholinergic types in the striatum ~Maura et al., 1988; Godulhin et al., 19841, S-hydroxyt~ptamine SHT, type in the cercbellum (Raiteri et al., 198636) and GABA, and adenosine A, types in the nucleus accumbens Wchimura and North, 1991). Vinconate is reported to inhibit the binding of E”H]quinuclidinyl benzilate([3H]QNBl, a sclectivc muscarinic a~etyl~holinc receptor ligand, more potently than that of carbachol, and therefore this drug may enhance dopamine release in the rat striatum via the stimulation of striatal muscarinic acetylcholine rcceptors (Koda et al., 1989). In the present study, we also found that the inhibitor effect of vinconatc on the excessive release of excitatory amino acids in rat hippocampus induced by ischemia was antagonized hy scopolamine, a muscarinic acetylcholine receptor antagonist. These results suggest that vinconate may have the capacity to attenuate the excessive ischemia-induced release of excitatory amino acids via stinlulation of hippocampal muscarinic acetylcholine receptors. The enteral absorption factor of vinconate was 92% after oral administration to rat. The concentration of vinconate in the rat brain reached its maximal level 5 min after oral administration, which was I.8 times higher than that in plasma Wnbara and Satou, 1992). Araki ct al. (1989) reported that vinconatc penetrated the blood-brain barrier and distributed to various brain areas including the hippocampus. Vinconatc may therefore act directly on ~ippocampal neurons to exert its protective effects against ischemic neuronal damage. However, vinconate had no effect on the binding of [3H]MK-801, which is a known blocker of NMDA receptors. Therefore, blockade of NMDA rcccptor-operated Ca” channels may not be involved in the protective effect of vinconate against ischemia-induced ne~~ron~l damage,

In conclusion, we demonstrated that pretreatment with vinconatc, a vinca alkaloid derivative which was developed to enhance cerebral metabolism, attenuates the ischemia-induced release of excitatory amino acids in the hippo~ampus via stimulation of hippocampal muscarinic acetylcholine receptors. It seems likely that this action underlies the abilhy of vinconatc to act as an enhancer of cerebral metabolism and/or a protective agent against cerebral ischemia-induced neuronal damage.

References Araki. T. and K. Kogure. 1989. Prevention of delayed neuronal death in gerbil hippocampus by a novel vinca alkaloid derivative tvinconate). Mol. Chem. Neuropathol. 1I, 33. Araki. T.. K. Nishioka, S. Yuki and K. Kogure. 198~). Vfnconate prevenls ischemie neuronal damage in the rat hippocampus, Acta Neurol. Stand. 81. 173. Benveniste, H., J. Drejer. A. Shousboe and N.H. Diemer. lY84. Elevation of the extracellular concentrations of glutamate and aspartate in rat hipp~ampus during transient cerebral ischemia monitored hy intracerebral microdialysis. 3. Neurochem. 4.7, l.iuY. Benveniste, H., M.B. Jorgensen, M. Sandberg, T. Christensen. H. Elagberg and NH. Diemer, 19X9, isehemic damage in hippocampal CA1 is dependent on glutamate release and intact innetvation from CA% J. Cereb. Blood Flow Metah. 9. 629. Boast. C.A., SC. Gerhardt, G. Pastor. J. Lehmann. P.E. Etienne and J.M. Liebman. IYgX, The N-methyl-D-aspartate antagonists CGS 19735 and CPP reduce ischemic damage in gerbils, Brain Res. 442. 345. B&an, A. and W.A. Pulsinelli, IYYO, Hypothermia but not the N-methyl-D-asp~irtate anta~~~nist,MK-801, attenll~ltes neuronai damage in gerbils subjected to transient global ischemia. J. Neurosci. IO. 31 I. Ci~r~i~E~i. A.M., A, Sardi. E. Baldoli, G.F. Di Franccsco and C. Luca, lY77, Hemodynamic profile of 11 new cerebral vasodihitor. vincamine and of its derivatives. apovincaminc acid ethylester (RGH-440% 11.Arch. Int. Pharmacol. 22% 139, Gill. R. and G.N. Woodruff, 1090, The nemoprotective actions of kynurenic acid and MK-X01 in gerbils are synergistic and not related to t~yp‘~tl~errni~~, Eur. J. Ph~~rmacol.176, 143. Globus, M.Y.T., R. Busto, W.D. Dietrich. R. Martinez, 1. Valdes and M.D. Ginsberg. 198X, Effect on ischemia on the in vivo release of striatal dopamine, glutamate. and ~aminobutyric acid studied by intracerebral microdialysis, J. Neurochem. 51, 1455. Giowinski, J. and L.L. lversen, 1906. Regional studies of cathccholamines in the rat brain - I, J. Neurochem. 13, 6555. Godulhin. O.V., A.Yu. Budantsev, O.V. Selifanova and V.N. Agapova. 1984, Effect of ch(;linomimetics on the release and uptake of L-[.‘H]glutamic acid in rat ncostriatum, Cell. Mol. Neurohiol. 4. 1 17. f&t, S. and K. Kuriyama, IOi(3, Simultaneous deterniinali(~;l of CSA and CA in rat bruin Iry high pcrfwmancc liquid cliromatwraplly. Anal Biochem. 130. Y5. Jorgensen, MB., F.F. Johansen and N.f 1. Diemer. lY87, Remow:t of the entorhinal cortex protects hippocampal CA1 neurons from ischemrc damage, Acta N~llr~)p~lth~)l.7.1, iX9. Karpati, E. and L. Szomy, lY76, General nnd ccrchral hemtdynamic activity of ethyl apovincaminate, Arzneim. Fnrsch. 26. 1Yttg. Katsura, M,, T. firto and K, Kl~riy;lmil~ iYY2. Changes in COntCntOf neurouctive aminn acids and acetylcholine in the rat hipwampus f(jll()wing trensicnt forchrnin ischemia. Neur~xhcm. lnt. 21. 243.

~~nbara, hf. and Y. Satou. 1942. Pharmacokineric studies of vinconate hydrochloridetOM-XS3XIV) Plasma concentration and transport into brain in ratsand dogs (abstract in English). fyakuhin Kenhyu 23. 266. King. GA.. 1987. Protective effects of vinpocetine and structurally related drugs on the lethal consequences of hypoxia in mice. Arch. Int. Pharmacodyn. 286. 299. Kirino, T., A. Tamura and K. Sano, 1984, Delayed neuronaf death in the rat h~p~ampus following transient forebrain ischemia. Acta Neuropathof. 64. 139. Koda, H.. T. Hashimoto and K. Kuriyama, 1989, Muscarinic receptor-mediated regulation of ON-853 enhanced dopamine release in stristum of rat, Eur. J. Pharmacol. 162, SC!. Langer, S.Z.. 19X0. Presynaptic regulation of the release of catechofamines. Pharmacol. Rev. 32, 337. Lamer, W.L.., W.J. Perkins, B.R. Karfsson, J.H. Milde, B.W. Scheithauer, G.T. Sherman and J.D. ~ichen~elder, 1990, The effects of dizocilipine mafcate tMK-801). an antagonist of the N-methyfD-aspartate receptor, on neurologic recovery and histopathofogy following complete cerebral ischemia in primates. J. Cereb. Blood Flow Metab. 10, 252. Lowry, OH.. N.J. Rosehrough. A.L. Farr and R.J. Randall, 1951, Protein n~easurement with the Fofin phenol reagent, J. Biof. Chem. 193, 265. Mario. M. and R. Maurigio, 1989. fnteraction acetyfcholine-gfutamate in rat hip~campos: involvement of two subtypes of M-2 muscarinic receptors, J. Pharmacol. Exp. Ther. 24Y, 1255. Matsumoto. K., S. Ueda, T. Hashimoto and K. K~riyama, 1991, Ischemic ncuronal injury in the rat hippocampus folfowing transient forebrain ischemia: evaluation using in vivo microdialysis, Brain Res. 543, 236. Mama, Cf., A. Giardi and M. Roiteri, 19XS, Release-regulating D-2 dopamine receptors are located on striatal glutamatergic nerve terminals, J. Pharmacol. Exp. Ther. 247,680. Meldrum. B.. 1989. Excit;:toxicity in ischemia, in: An Overview, in Cerebrovascular Diseases (Research (Princeton) Conferences on Cerebrovascular Disease, 16th). eds. M.D. Ginsberg and W.D. Dietrich (Raven Press, New York) p. 47. Munekata, K. and K.A. Hossmann, 1987, Effect of S-minute ischemia on regional pH and energy state of the gerbil brain: Refation to selective vulnerability of the hippocampus, Stroke f& 412. Nedergaard, M., 19X%,Mechanisms of brain damage in total cerebral ischemia. Acta Neurol. Stand. 77. 81. Gnodera, H.. G. Sato and K. Kogure, 1986, Lesions to schaffer coffateta!s prevent ischemic death of CAI pyramidal cells, Neurosci. Len. 68, 169. Peffegrino, L.J., A.S. Pelfegrino and A.J. Cushmttn, fS9tJ, A Stereotaxic Atlas of the Rat Brain (Plenum Press, New York),

Pufsinelli, W.A. and J.B. Brierley, 1979, A new model of bilateral hemisphere ischemia in the unanesthetized rat, Stroke IO, 267. Pulsinelli, W.A., J.B. Brierley and F. Plum, 1982, Temporal profile of neurons1 damage in a model of transient forebrain ischemia, Ann. Neurof. 11,491. Raiteri, M., Cf. Naura, G. Bonanno and A. Pittafuga. 1986, Differential pharmacology and fmtction of two 5HT, receptors modufating transmitter release in rat cerebellum, J. Pharmacol. Exp. Ther. 237,644. Rothman, S.M.. 1983. Synaptic activity mediates death of Rypoxic neurons, Science 220, 536. Rothman, SM., 1984, Synaptic release of excitatory amino acid nemotransmitter mediates anoxic neuronal death, J. Neurosci. 4, 18114. Rothman, SM. and J.W. Olney, 1986, Glutamate and pathophysiofogy of hypoxic-ischemic brain damage, Ann. Neurof. 19, 105. Rothmac, S.M. and J.W. Olney, 1987, Excitotoxicity and the NMDA receptor, Trends Neorosci. 10, 299. Safetu, B., J. Grfinberger, L. Linzmayer and R. Wittek, 1984, Classification and determination of pharmacofodynamics of a new antihypoxic drug, vinconate, by pharmaco-EEG and psychometry, Arch. Gerontol. Ceriat. 3, 127. Swan, J.H. and B.S. Mefdrum, 1990, Protection by NMDA antagonists against selective cell loss following transient ischemia, J. Cereb. Blood Flow Metab. IO, 343. Thiebaufd, C., J. Van Muller, L. Lintermans and P. Sprrtmont, 1983, Testing a hypobaric chamber drugs claimed to improve brain function, Lancet 2, 225. Uchimura, N. and R.A. North, 1991. Baclofen and adenosine inhibit svnaptic potentials mediated by y-aminobutyric acid and gfutamate release in rat nucleus accumbens, J. P~rmaeof. Exp. Ther. 258,663. Westerberg, E., J.K. Deshpande and T. Wiefoch, 1987, Regional differences in arachidanic acid release in rat hippocampus CA1 and CA3 regions during cercbraf iachemia, J. Cereb. Blond Flow Metab. 7, 189. Wieloch, T., 0. Lindrall, P. Blomqvist and F.H. Gage, 1985, Evidence for amelioration of ischemic neuronal damage in the hippocampaf formation by legions of the perforant path, Neurol. Res. 7, 24. Yoneda, Y. and K. Ggita, fY59a, fabefing of NMDA receptor channels by [~H]MK-~1 in brain synaptic membranes treated with Triton X-100, Brain Res. 499, 305. Yoneda, Y. and K. Oyita, 1989b, Microbial methodological artifacts in [~H~lutamate receptor binding assays, Anal. B&hem. 177, 250.