Mechanisms of glutamate and aspartate release in the ischemic rat cerebral cortex

BRAIN RESEARCH ELSEVIER

Brain Research 730 (1996) 151)-164

Research report

Mechanisms of glutamate and aspartate release in the ischemic rat cerebral cortex J.W. Phillis *, M.H. O'Regan Department of Physiology. Wayne State Uniz,ersity School of Medicine, 540 E. Ca~!)qeld, Detroit, MI 48201, USA

Accepted 2 April 1996

Abstract Elevated levels of glutamate and aspartate have been implicated in the pathogenesis of neural injury and death induced by ischemia. The mechanism(s) whereby they escape into the extracellular environment have been a subject of controversy. This study evaluated the contribution of phospholipases and protein kinases to ischemia-evoked glutamate and aspartate release from the ischemic/reperfused rat cerebral cortex. Changes in the extracellular levels of these amino acids during four-vessel occlusion elicited global cerebral ischemia were examined using a conical cup technique, lschemia-evoked amino acid release was compared in control vs. drug treated animals, in which selective inhibitors of phospholipases and protein kinases were applied topically onto the cerebral cortex. The phospholipase inhibitors tested included 4-bromophenacyl bromide, a non-selective inhibitor: 7,7-dimethyleicosadienoic (DEDA), an inhibitor of secretory type phospbolipase A 2 (PLA:); AACOCF3, an inhibitor of the Ca- -dependent cytoplasmic form of PLA 2, HELSS, which inhibits a Ca2+-independent cytoplasmic PLA> and U73122, a selective inhibitor of phospholipase C (PLC). All five phospholipase inhibitors significantly attenuated glutamate and aspartate release into the extracellular milieu, indicating the possibility that several forms of the enzyme are likely to be involved. The protein kinase C (PKC) inhibitor, chelerythrine chloride, also reduced excitatory amino acid effiux, whereas the PKC activator phorbol 12-myristate 13-acetate (PMA) enhanced their release. The non-selective kinase inhibitor. staurosporine, and H-89, which selectively inhibits protein kinase A, did not reduce ischemia-evoked amino acid efflux. These results suggest that ischemia-evoked release of the excitatory transmitters amino acids is a result, in part, of the activation of phospholipases A~ and C, with PKC involvement in the transduction process. Destabilization and deterioration of the plasma membrane, as a consequence of phospholipid hydrolysis, may allow these transmitter amino acids to diffuse down their concentration gradients into the extracellular fluid. Kevwords." Cerebral ischemia: Stroke: Excitotoxic amino acid: Glutamate: Phospholipase: Protein kinase

1. Introduction Glutamate, a major excitatory amino acid neurotransmitter in the brain, plays an important role in the pathogenesis of neuronal injury and death induced by cerebral ischemia [52]. Extracellular brain concentrations of glutamate, and of the related excitatory amino acid aspartate, increase rapidly during ischemic episodes, contributing to neuronal depolarization and the elevation of intracellular calcium levels [4,9,57]. There is, however, a controversy regarding the mechanisms responsible for the increase in the extracellular levels of these excitotoxic amino acids. Depolarization induced, calcium dependent, exocytotic release of glutamate and aspartate [55] will undoubtedly

* Corresponding author. Fax: + I (313) 577-5494.

contribute to their initial accumulation. However, considering the rapid inactivation and desensitization of both voltage- and glutamate-receptor-gated calcium channels [13,31,34] this type of calcium entry may be insufficient to induce the progressive, continuous, increases in glutamate release which occur during ischemia. Other studies have indicated that a calcium-independent release of glutamate occurs during anoxia or ischemia [22,54]. In this instance, it has been proposed that glutamate could be released from the cytoplasm into the extracellular space as a result of a depolarization-induced reversal of the sodium-dependent high-affinity acidic amino acid plasma membrane transporter [2,12,36,62]. Evidence obtained in studies on the effects of glutamate transport inhibitors on the ischemiaevoked release of glutamate and aspartate would be consistent with the suggestion that a reversal of the transporter makes a substantial contribution to the efflux of these

0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII $0006-8993(96)00434-9

J. W. Phillis, M.H. O'Regan / Brain Re~'earch 730 (1996) 150-164

amino acids [44,50] although this result could not be confirmed in a subsequent study [18]. Yet another suggestion has been the possibility that endogenous amino acids are released, in amounts proportional to their concentration gradients across the cell membrane, as a consequence of phospholipase-induced plasma membrane disruption [39]. The levels of brain free fatty acids increase rapidly after Ihe onset of ischemia due to the deacylation of membrane phospholipids by phospholipases A . and C and the disturhance in reacylation of membrane phospholipids resulting from adenosine triphosphate depletion [1,14,64]. Composilional alteration of the plasma membrane would alter its structural integrity allowing intracellular constituents, including amino acids, to diffuse down their concentration gradients. Evidence in support of this concept of an early disruption of the plasma membrane was furnished by experiments on the release of lactate dehydrogenase (LDH) into the cxtracellular space of ischemic rat cerebral cortex. Increases in the LDH concentration in cortical superfusates become evident during a 30 min period of ischemia and the levels continued to rise during a subsequent 2 h reperfusion period [44]. Preliminary evidence for an involvement of phospholipases in the ischemia-evoked release of glutamate and aspartate was liwthcoming in experiments demonstrating lhat the phospholipase inhibitors mepacrine and 4bromophenacyl bromide significantly decreased excitotoxic amino acid release from the ischemic rat cerebral cortex [39] whereas topical application of phospholipases (7 and A~ to the non-ischemic cerebral cortex resulted in a significant inc,ease in the extracellular levels of glutamate and aspartate [39]. The present experiments were designed to further study the role of phospholipases in ischemia-evoked release of glutamate and aspartate in the rat cerebral cortex. Attention was also focused on the intracellular mechanisms underlying the enhancement of phospholipase activity. A preliminary report of these studies was presented at the 1995 Annual Meeting of the Society for Neuroscience [41].

2. Materials and methods 108 Male Sprague-Dawley rats (Harlan Sprague-DawIcy, Indianapolis, 300-400 g) were used in these experiments. Halothane was employed to induce anesthesia and. after insertion of a tracheal cannula, it was maintained with methoxyflurane. Body temperature was controlled at 37°C with an abdominal heating pad and rectal probe. One femoral artery was cannulated for measurement of arterial blood pressure and withdrawal of blood samples for pH and blood gas measurements. Cerebral ischemia was induced by coagulating the vertebral arteries and pulling on tapes placed around the carotid arteries (for further details see [47]). The dorsal surfaces of both cerebral hemispheres were

151

exposed and, after reflection of the dura mater, oval cortical cups suspended in flexible mounting brackets were placed on both cortices. The dorsal surface of the head around the cups was covered with a stabilizing gel of 4cA agar in artificial CSF (aCSF). The artificial CSF consisted of N a ' 155.8 m E q / L , K ~ 2.95 mEq/L, Ca -'÷ 2.5 mEq/L, Mg e~ 1.85 m E q / L . CI 141.13 m E q / L , HCO 22 m E q / L , dextrose 66.5 mg/dl, urea 40.2 mg/dl (302 mOsm/L). A monopolar EEG electrode was placed in each cup. EEGs and MABP (mean arterial blood pressure) were recorded on a Grass polygraph. Artificial CSF pipetted into the cortical cups was removed after a 30 rain period of equilibration and replaced with 200 txl of warmed (37°C) sterile artificial CSF, which had been bubbled with a gas mixture of 5cA carbon dioxide in nitrogen. The same gas mixture was bubbled into the cortical cups. Cup fluid was maintained at 37°C with a heat lamp. Cup lluids were collected at 10 rain intervals and replaced with fresh artificial CSF. Cerebral ischemia was elicited bs, occluding the carotid arteries for 20 rain. Successful occlusion and induction of cerebral ischemia was evident from a rapid flattening of the EEG traces. After 20 rain, the carotid snares w'ere withdrawn and reperfusion verified by' visual inspection of the pial vasculature within the cups. Results from 18 groups of rats are presented in this report. One group (n = 19) comprised the control (nontreated) ischemic animals. The remaming 17 groups were used to stud}, the effects of specific drug interventions on ischemia-evoked glutamate and aspartate release. Two of these were the controls tor drugs that were solubilized in dimethyl sulfoxide (0.05 or 0.55~ in aCSF). For all of these animals, after two basal (10 min) aCSF collections the animals received the appropriate drug treatment, w'ith agents applied topically in aCSF. A 20 rain period was allowed for equilibration during which the cortical superfusates were replaced three times with fresh drug-containing aCSF. Exposure to drug-containing aCSF was then continued for the duration of the experiments, during which two more basal samples were collected, followed by' a 20 rain period of ischemia (two superfusate samples collected) and then four 10 rain reperfusion samples. The control ischemic animals were exposed Io an identical schedule of superfusate sample collections, including the 20 min equilibration period between the two sets of basal samples. The sequence of collections ensured that the cerebral cortices were exposed to the appropriate test compounds for 40 min prior to the initiation of ischemia. The collected superfusate samples were ejected into chilled microvials, centrifuged at 1200 x ,~, and stored at -20°C. HPLC analyses of the perfusate amino acid contents were conducted within a few hours using previously published procedures [47]. Statistical differences between amino acid releases from control and drug treated animals were analyzed by' ANOVA and Student-Newman-Keuls ( P < 0.05) or Scheffe's (P < 0.01) tesls w'ith contrasts

152

J. W. Phillis, M.H. O'Regan / Brain Research 730 ( 19961 150 164

between the appropriate control group and each treatment group (SPSS PC statistical package). A probability of < 0.05 was accepted as denoting a significant difference. Sources of the chemicals used were: Sigma Chemical Co., (MO), 4-bromophenacyl bromide (BPB), staurosporine, dimethyl sulfoxide (DMSO), phorbol 12-myristate 13-acetate (PMA), nordihydroguaiaretic acid (NDGA); Calbiochem, (CA), chelerythrine chloride, N-t2(( p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulphonam±de hydrochloride (H-89); Biomol Research Laboratories Inc., (PA), AACOCF 3, E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (HELSS), U73122; L.C. Laboratories, (MA), 7,7-dimethyleicosadienoic acid (DEDA). The concentrations of drugs selected for use in these experiments were based on their K~ values and previously published results.

3. Results Physiological variables were monitored in all groups of animals by recording of MABP and EEG and through determination of arterial pH and blood gas tensions in samples obtained at the beginning and end of each experiment (Table 1). There were no significant differences in PO 2 values during the course of the experiments. Small changes in pCO 2 or pH values were observed in some groups. Mean arterial blood pressure generally increased during the ischemic period, then fell below basal values at the onset of reperfusion, and had recovered to basal levels by the end of the experiments, the exceptions being small reductions in arterial blood pressure in the groups treated with AACOCF> HELSS and BPB (5.0 txM). Occlusion of the carotid arteries consistently resulted in an isoelectric

Table I Physiological variables of experimental groups Treatment

n

Before

After

BP

pH

pCO 2

pO 2

BP

pH

pCO 2

pO 2

Saline control

19

100.0+5.5

7.40_+0.01

35.6+- 1.5

80.8_+4.1

102.3+3.3

7.39_+0.01

35.4+ 1,3

86.4_+3.1

PLA 2 inhibitors HELSS 1 txM AACOCF3 50 >M

6 6

113.0_+3.0 99.1 _ + 3 . 2

7.43_+0.06 7.40+0.01

31.8+0.6 34.4_+ 1.0

101.6±3.4 115.3 ! 3 . 1

94.5-+4.4 89.3_+2.0 :'

7.33_+0.05 7.26-+0.05 ~

41.9+5.9 44.0_+5.8

99.2+5.1 103.1 _+5.6

PKC inhibitor Chelerythrine 5 t~M

5

113.3 ± 6.6

7.39_+0.02

35.9_+2.0

104.8_+8.6

111.3+7.5

7.30_+0.03

39.8-+2.2

99.7+9.2

PKA inhibitor H-89 0.1 p~M

5

108.0+6.4

7.40+-0.01

38.2-+ 1.8

95.4+-5.5

100.3+4.2

7.36_+0.04

38.1 -+2.6

100.6+9.0

0.5% DMSO

9

94.7_+ 12.4

7.38_+0.01

39.6_+ 1.5

86.5_+4.2

101.5+4.3

7.34_+0.02

3 5 . 8 _ +1.8

91.6_+5.1

Lipoxygenase inhibitor NDGA 0.1 IxM NDGA 30 IxM NDGA 100 IxM

4 4 4

99.4-+3.9 100.7-+6.1 101.0_+1.9

7.41 +0.03 7.42_+0.02 7.42_+0.03

33.3 _+2.2 33.9_+ 1.7 39.2+4.3

98.1 _+6.4 98.2_+3.3 83.1 +9.5

99.7_+5.3 88.7+4.1 99.(1+4.5

7.41 _+0.04 7.39_+0.03 7.43+_0.01

34.5_+5.(/ 34.5_+3.4 99.0+4.5

102.3 _+9.b 99.2_+6.1 85.6-+- 12.5

PLC inhibitor U73122 10 btM

6

105.7+9.8

7.45_+0.01

30.0_+0.3

116,6_+3.4

80.0_+8.(/

7.41 +(I.04

3(/.1 -+2.6

100.8-+7.2

0.05% DMSO

8

98.7-+5.9

7.39__+0,01 34.9__+1.3

76.9_+4.6

106.4+3.2

7.39+0.01

35.32_+ 1.0 83.2±3.2

PLA 2 inhibitors BPB 0.5 IxM BPB 5.0 txM DEDA20 IxM

4 8 4

97.5-+ 10.2 109,5-+4.1 112,8+8.6

7.42+_0.01 7.40+0.01 7.37+0.02

38.7-+2.7 43.3+1.9 38.7_+ 1.8

113.8 + 5 . 6 92.0+2.5 95.3-+6.2

87.5+3.2 90.8+5.4 * 91.5-+ 1 2 . 8

7.39_+0.(t4 7.37_+0.02 7.42_+0.05

33.3-+ 1.7 126.5+4.0 37.9,+3.1 108.4+ ll.l 32.9+ 1.4 ' 100.37_+9.3

Protein kinase inhibitor Staurosporine0.01 IxM 4 Staurosporine 0.1 ~M 4 Staurosporine 2.0 btM 4

100.8_+5.8 110.8+5.5 95.4+4.5

7.46_+0.00 7.39-+0.02 7.41 +0.04

36.5_+ 1.1 35.0_+ 1.6 34.8+ 1.3

110.8_+9.5 95.4_+7.19 108.3_+5.9

78.5+8.5 7.9_+7.5 91.0+9.4

7.33-+0.04 7.33+0.01 * 7.36_+0.03

43.2_+6.4 31.2-+2.4 36.8+5.2

99.4-+ 13.6 101.0i 1(I.3 108.1 +8.t~

PKC activator PMA (10 p.M)

85.5+3.5

7.39_+0,02

36.6_+0.2

114.7_+0.6

77.0 _+ 3 . ( 1

7.38_+0.0(I

33.7_+3.4

101.8 + 2.5

4

Data are mean _+ S.E.M. values for each set of animals. The before values, obtained prior to drug administration, were compared to values ohtained a! the end of the experiment (after) using a Student's t-test. * P < 0.05: * * P < 0.01. n = number of animals per group.

,I. W. Phillis, M.H. O'Regan /Brai; Research 730 (1996) /50 /64

EEG during the entire period of ischemia, with some recovery of electrical activity occurring by the end of the experiment. Basal superfusate levels of aspartate and glutamate in the control animals were 329 + 40 nM and 1912 + 283 nM respectively. In the control animals, a 20 min period of cerebral ischemia resulted in a rapid elevation in cortical superfusate levels of both amino acids. Maximal percentage increases above basal values were: aspartate 1166% and glutamate 1088%. Superfusate concentrations of the amino acids then declined during the two reperfusion collection periods. To simplify comparisons between control ischemic responses and those following drug treatments, release in the initial ischemic collection period has been calculated as the release above basal levels and is presented as a percent of the release in the appropriate control group. Total amounts of each amino acid released during the two ischcmic and two reperfusion periods were

15

summed and compared as a percentage of that in the appropriate control group (Table 2).

3.1. Effects of drug treatments 3.1.1. Phost)holipase inhibitors The non-selective phospholipase inhibitor, BPB, was tested at concentrations of 0.5 and 5.0 IzM. No effect on non-ischemic release of glutamate and aspartate was observed at either concentration. At a 0.5 txM concentration BPB significantly depressed the ischemia-evoked release of glutamate during both the ischemic and reperfusion periods (Fig. 1) and aspartate release was significantly depressed during the reperfusion period (Fig. I). Both the initial release and total release of glutamate were depressed in comparison with control releases (Table 2). The total ischemia-ew&ed release of aspartate was inhibited following administration of BPB ((/.5 taM). BPB (5.0

Fable 2 Iscllemia-evoked transmitter amino acid release from cerebral cortex rreatment

Asparlate

Ghltamate

Total release (~7( of control)

Initial release

Initial release ((7; of control)

Total release (c.~ o f control)

( (i of control l

60.0+41.1

P I . A , inhibilors

Non-selective BPB(0.5 FM) BPB (5.0 #,M)

38.4+8.6

~"

188.7 + 34.(/ "

94.8 + 24.6

43.7 + 9.3 . . . . 199.4 + 20.3 "

32.8 ~ 15.9 t~3 5 -4 2(I.7

DELIA 120 # M ) cy tosolic

51.7 + 7.1

39.5 _+ 8.6

5 5 4 + 8.8

3 6 8 + 6.4

A A C O C F 3 (50 p M I cytosolic

32.2 + 5.18 ....

35.8 + 10.7

24.3 + 4.0 "

tIELSS ( I i x X l )

73.6 + 12.6

1,5.4 + 6.4 " " "

63.5 4:10.1

55.1 _+ 7.0 '

31.0 + 10.7

38.{I + 4.5 '

23.2 4 6.3

87.0 _+ I 1.6 70.1 _+ 33.0 59.2_+ 16.1

262.5 _+ 90.3 79.0 + 43.6 36.1 _+ ._.~; "~'~ _

146.4 + 20.0 162.2 + 73.4 71.8 + 20.4

20(I.4 + 52.5 175.0 ~ 08.4 4_.S ~ ' ~ 21 .I

2 9 . ( / + 3.4 . . . .

6.4 + 3.9 . . . .

4 1 . 0 + 6.0 "

4.1 ~ t ~

PKA inhibitor H-89 10.1 b~M)

112.2 + 34.4

130.9 + 55.4

112.6 + 39.5

0 7 ~ + 17.7

PKC activator PMA (tO p M )

201.8 - 51.0

91.4 + 24.2

192.9 + 32.0

~7~+

95.1 --+31.0 128.2 _+ 20.1 265.8 + 32.7 ......

89.1 _4_ 13.5 191.2 _4_43.9 48.1 + 24.3

811.2_+ 15.4 190.9 _+ 37.1 366.6 + 41.6

40.0 e 17.4 127.0 ± 23.4 __8.6 :L-

Secretor3, form ~

'

C a 2 ~ -dependcm

4 9 4 :~ 10.1

C a 2 ~ -independent

PI.C inhibitol U73122 Ill) pX,1) Non-selecli\e protein kinase inhibitor Staurosporine (0.01 ,u. M ) Staumsporine (0. I IxM ) Staurospori ne ( 2.0 bL M ) P K C inhibitor

Chclervthrine (5 ,u. M )

17.7

IJpoxygcnabc inhibitor N D G A (0. I g M ) N D G A (30 F M I N D G A (100 ~ M )

Release of aspartate and glutamate during and following ischemia as a percent of the appropriate control release. The initial release was calculated as the release above basal levels during the first ischemic period, as a percent of the release in the appropriate control group• Total release represents the sum ol amounts of each amino acid released during the two ischemic and the initial two reperfusion collection periods. P < (I.05; P < (1.01: P < 0.(1(II 2Ol/ipared to appropriate control group.

J. IV. Phillis, M.H. O'Re~an / Brain Research 730 (1996) 150 164

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COLLECTION PERIOD Fig. 1. Ischemia-evoked release of glutamate and aspartale into rat cerebral cortical superfusates, Line plots show the time course of changes m cortical superfusate concentration (nM) of aspartate and glutamate before, during, and alter a 20 rain period of four-vessel cerebral ischemia (collections 5 and 6, open box). 4-Bromophenacyl bromide (BPB; I , 0.5 ixM; or T, 5.0 IxM), a non-selective phospholipase inhibitor, was added to the artificial CSF after the initial two basal samples had been collected and 20 min later collection of sample 3 was initiated. BPB administration continued for the remainder of the experiment. The data are presented in comparison with those from the appropriate control ischemic animals (©). Data are presented as means _+ S.E.M. Statistically significant differences between superfusate amino acid concentrations in control and BPB treated animals were determined by ANOVA with Student-Newman-Keuls or Scheffe's tests. * P < 0.05: * * P < 0.01: * * * P < 0.001.

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COLLECTION PERIOD Fig. 2. Effects of an inhibitor of secretory-type phospholipase A2, DEDA (20 IxM), on basal and ischemia-evoked release of aspartate and glutamate from the rat cerebral cortex. DEDA was administered topically in aCSF after the collection of sample 2 and 20 rain prior to the start of collection of sample 3. See legend for Fig. 1 for further details.

J. W. Phillis. M.H. O'Regan / Brai, Research 730(19961 150 164

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tig. 3. El'l~cts of inhibitors of Ca 2 - d e p e n d e n t and -independent cytosolic Forms of PLA • AACOCF~ ( x . 50 ~ M ) and HEI,SS t v . I ~ M ) applied in aCSF on basal and ischemia-evoked release of aspartate and glutamate from the rat cerebral cortex, Drugs were added to 1he artificial CSF after collection ¢,t" sample 2 and 20 rain prior to the start of collection of sample 3. See legend For Fig. I ~ r further details.

laM), although it did not affect release during ischemia, significantly enhanced the release of both amino acids during the reperfusion period.

DEDA preferentially inhibits the calcium independent, secretory, type of phospholipase A, [20,26]. At a concentration of 20 txM. it significantly attenuated ischemia/re-

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Fig. 4. Effect of the phospholipase C inhibitor U73122 (10 IxM) applied topically in aCSF on basal and ischemia-evoked release of asparlate and glutamate t'rom the rat cerebral cortex. See legend lk~r Fig. I for further details.

J.W. Phillis. M,H. O'Regan / Brain Research 730 ( 1996J 150 164

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COLLECTION PERIOD Fig. 5. Effects of the non-selective protein kinase inhibitor staurosporine ( a , l0 nM: I . (/. 1 t~M; LX. 2.0 I-tM) on basal and ischemia-evoked release of aspartate and glutamate from the rat cerebral cortex. See legend for Fig. I for further details.

perfusion evoked release of both glutamate and aspartate (Fig. 2). This resulted in significant reductions in both the initial release of glutamate and total release of both amino acids (Table 2).

AACOCF 3 is a selective inhibitor of the cytosolic, Ca 2+-dependent, form of phospholipase A, [60]. AACOCF3 (50 ~M), whilst not affecting basal release, markedly reduced ischemia/reperfusion evoked release of

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Fig. 6. Effects of chelerytbrine chloride (5.0 p.M) a selective inhibitor of protein kinase C on basal and ischemia-evoked release of aspartate and glutamate From the rat cerebral cortex. See legend for Fig. I For further details.

,I.W. Phillis, M.H. O'Regan / B;ain Research 730 (19961 150 164

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glutamate and aspartate (Fig. 3) depressing both initial and lotal release of both amino acids (Table 2). HELSS, an inhibitor of the Ca-~+-independent cytosolic form of PEA, [17], at a concentration of 1 IxM inhibited

both aspartate and glutamate release during the two ischemic collections, but did not significantly reduce release during reperfusion (Fig. 3). HELSS did not alter basal, non-ischemic, release of either amino acid. Initial release

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of both amino acids and total release of glutamate was significantly attenuated. U73122 is a selective inhibitor of phospholipase C [59,68]. At a concentration of l0 IxM, this compound significantly attenuated the release of both aspartate and glutamate during ischemia and reperfusion (Fig. 4), but did not affect basal release. Both initial, and total, releases of both amino acids were significantly depressed (Table 2).

3.1.2. Protein kinase inhibitors Staurosporine, a non-selective inhibitor of PKA and PKC, was tested at three concentrations (0.01, 0.1 and 2 IxM). No significant effects were observed on basal, nonischemic, release at any of these concentrations, and ischemia/reperfusion evoked releases were not affected (Fig. 5 and Table 2). Chelerythrine chloride, a selective inhibitor of PKC, at

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a concentration of 5 ixM, inhibited ischemia/reperfusion evoked release of both glutamate and aspartate (Fig. 6); significantly attenuating both initial, and total, release of the two amino acids (Table 2). H-89 (0.1 ~M) a selective inhibitor of PKA, failed to depress the ischemia/reperfusion evoked release of glutamate and aspartate, indeed it appeared to enhance release during the reperfusion period (Fig. 7). Basal, pre-ischemic release was unaffected. Protein kinase C activation by PMA (10 I~M) led to striking increases in the release of glutamate and aspartate during the reperfusion collections, although pre-ischemic and ischemia-evoked releases were not affected (Fig. 8). Total release of glutamate was significantly elevated, whilst the increase m total aspartate release, although double that in the controls, failed to achieve significance (Table 2).

ixM it significantly enhanced both ischemia and postischemia evoked release of glutamate and elevated total release of glutamate (Fig. 9; Table 2). At 100 p+M it significantly elevated the ischemia/reperfusion evoked releases of the two amino acids, significantly increasing the total release of both and the initial release of glutamate. For summary of results, see Table 3. which includes the proposed involvement of these enzymes in ischemic dam+ age.

4. Discussion

Cerebral ischemia precipitates large increases in the levels of glutamate and aspartate in the extracellular fluid. These amino acids have neurotoxic properties and there is compelling evidence to support the conclusion that they are involved in the sequence of ischemia/reperfusion events which ultimately lead to neuronal death. Thus. ischemic injury to hippocampal CA1 pyramidal cells appears to be dependent upon an intact glutamatergic innervation from the CA_~ region [5] and blockade of glutamate receptors alleviates ischemic injury in some experimental

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160

,/. W. Phillis. M.H. O'Re~an / Br~fin Research 7 3 0 ( 1996~ 150 164

models [7,16,25,52,58,61]. In contrast to the many studies which have documented ischemia-evoked glutamate release, comparatively little attention has been paid to the potential intracellular changes that underlie this effiux of glutamate into extracellular space (Fig. 10). The elevation in extracellular glutamate concentrations during ischemia could be a result of several effects, occurring independently or in conjunction. Glutamate could be released from the neurotransmitter pool in presynaptic terminals [11] by a calcium dependent exocytotic process. Calcium-dependent exocytotic release declines rapidly during ischemia [35] being highly dependent on the A T P / A D P ratio [55], and is therefore unlikely to account for the large amounts of glutamate which efflux, in a Ca-'+-independent manner, into the extracellular space during ischemias of more than 2 - 3 min duration. It is tempting to speculate that the early brief peak in extracellular glutamate during cerebral ischemia, detected during on line monitoring of glutamate by microdialysis with enzyme-amperometric analysis, represents the Ca2+-dependent excocytotic release, whereas the subsequent progressive and sustained glutamate release was of metabolic (cytoplasmic) origin [69]. Another explanation for the increase in extracellular glutamate seen during ischemia could be that it is due to impairment of the high affinity reuptake system for this amino acid. The efflux of glutamate from a metabolic pool under in vitro ischemic conditions has been shown in synaptosomes [24,54] and in cultured astrocytes [38]. Reuptake of glutamate and aspartate into neurons and glia by the high affinity acidic amino acid transporter is dependent on the maintenance of an ATP-driven Na + gradient, and will fail during ischemia as ATP supplies are depleted. In addition to preventing reuptake, ATP-depletion during ischemia can contribute to the efflux of glutamate and aspartate, by inhibiting N a / K ATPases, with a reduction in the gradients of these ions and actual reversal of the glutamate transporter [2,12,27,36,62]. Experimental evidence in support of this concept has been obtained in in vitro and in vivo studies with glutamate transport inhibitors. £~H-Daspartate release from ischemic rat hippocampal slices was reduced by 55% following exposure to two competitively transported inhibitors of the Na-dependent glutamate transporter, D,L-threo-[3-hydroxyaspartate (THA) and L-transpyrrolidine-2,4-dicarboxylate (tPDC), but not by the nontransported inhibitor dihydrokainate (DHK) [50]. THA failed to reduce ischemia-evoked release of glutamate and aspartate from the in situ rat cerebral cortex, whereas DHK and tPDC were effective in reducing efflux [44]. In this instance the cerebral cortex was superfused with DHK (2.5 raM) for 40 min prior to ischemia to allow the inhibitor sufficient time to equilibrate across the cell plasma membrane. The failure of THA (1-2 mM) to depress glutamate and aspartate release was attributed to the potent depolarizing action of this compound at glutamate receptors in comparison with DHK [10], which could have negated its action on the transporter. Dunlop et al. [12] have previ-

ously demonstrated that DHK can, in fact, inhibit transporter-mediated efflux of [3H]-D-aspartate from cultured cerebellar granule cells. Heron et al. [18] failed to observe an inhibitory action of THA on ischemia-evoked glutamate release from the rat hippocampus in vivo, even though it partially blocked veratridine-evoked glutamate release. They concluded that reversal of Nan-dependent uptake is not involved in the increase in extracellular glutamate during ischemia, and proposed instead that excitotoxic oedema induced by ischemia could lead to glutamate leakage out of the cells. D,L-Threo-13-hydroxyaspartate is actively transported into cells and subsequently competes with glutamate and aspartate for release by the transporter under conditions of energy deprivation. As a result, prior exposure to THA reduced the amounts of glutamate and aspartate released front ATP-depleted rat hippocampal slices [29]. The failure of THA to reduce in vivo glutamate release from ischemic brain tissues [18,44], whereas it was effective in in vitro preparations, could be a reflection of a failure to achieve adequate intracellular concentrations of THA in intact tissues, although the observation that THA did reduce veratridine-evoked release of glutamate [18] sheds doubt on this explanation. The question of the cellular source of the glutamate remains controversial and the answer undoubtedly inw)lves the duration of the ischemic episode. Mitani et al. [33] postulate that the ischemia-evoked accumulation of glutamate in the CA I field of the hippocampus during a 5 rain period of ischemia originates primarily from neuronal elements, whereas astrocytes may contribute to the accumulation during longer periods of ischemia, hnmunoreactivity for glutamate was decreased in the CA1 pyramidal cell bodies following a 20 min period of ischemia whilst the levels in astrocytes was increased, suggesting that ischemia results in a redistribution of glutamate from neurons to gila [63]. The elevated levels of glutamate in glial cells indicates that the capacity of glia to metabolize glutamate to glutamine is exceeded during ischemia, as the glutamine synthetase in glia is dependent on ATP. This failure of the glial cells to metabolize glutamate could play a significant role in the chain of events leading to 'excitotoxic" cell death. The concept of a "leakage' of transmitter amino acids across the plasma membrane alluded to above [18], has been invoked by other investigators. Bradford et al. [6] proposed that glutamate and aspartate are released from striatal tissues in a continuous general efflux or leak across the surface of non-ischemic neurons and glial cells. Under ischemic conditions, with inhibition of high and low affinity energy-dependent reuptake processes, glutamate and aspartate would accumulate in the extracellular space, reaching concentrations at which they are likely to be neurotoxic. Leakage of cytoplasmic glutmnate occurs across cerebrocortical synaptosomal membranes [49] and would again normally be compensated for in normoxic

,I. W. Phillis. M.H. O'Re£,at ,/Br~ il Research 730 (/996) 150 164

tissues by the active uptake pathway. Madl and Burgesser [29] observed that, in contrast to the release of glutamate and aspartate, release of the non-excitatory amino acids asparagine and glutamine did not occur with ATP-deplefion in their hippocampal slices and concluded that this evidence suggests that there is not a general increase in membrane permeability that would allow all amino acids to escape into the extracellular space. In point of fact, when the ratios of whole brain amino acid concentrations I reflecting primarily intracellular levels [42]) are compared with microdialysate levels (extracellular space [65]) it is apparent that the highest ratios are for the three transmitter amino acids (glutamate, aspartate and y-aminobutyric acid). Taurine, a putative inhibitory neurotransmitter, also exists with a marked concentration across the plasma membrane, as does phosphoethanolamine. The ratios (i.e. concentration gradients) for other amino acids, including asparagine, glutamine, ,,'aline and phenylalanine are very low in comparison, potentially accounting for the failure of their levels in the extracellular space to increase dramatically during ischemia [8,29]. Conversely 3,-aminobutyric acid. taurine, and phosphoethanolamine levels in the extracellular fluid are substantially enhanced during ischemia [8,47.48,56]. A curious aspect of the results obtained in vivo with glutamate transport inhibitors, was the failure of THA, DHK and tPDC to affect basal, non-ischemic, glutamate and aspartate release [18,44]. This finding suggests that the rate of efflux from normoxic cells may be sufficiently low to be handled by non-saturable, low-affinity, glutamate transporters, insensitive to these inhibitors. Ischemia resuits in a rapid increase in the efflux of glutamate and aspartate which, coupled with the failure of the Na+-de pendent high affinity uptake system, leads to a dramatic accumulation of these amino acids in the extracellular space. As stated above, it is unlikely that Ca2+-dependent cxocytotic release coupled with a reversal of the Na-dependent transporter can fully account for this phenomenon. What other mechanisms could be involved'? Evidence that cerebral ischemia resulted in a rapid increase in the levels of brain free fatty acids [1,15] leads to the suggestion [64] that agents inhibiting phospholipase (~ or phospholipase A~ activity could protect against ischemic neuronal injuries by preventing membrane destruction by these enzymes. It became evident from this insight that a compositional alteration of the plasma membrane, due to activation by phospholipases by intracellular Ca 2+. could modify its structural integrity allowing intracellular amino acids, such as glutamate and aspartate, to diffuse down their concentration gradients into the extracellular space. The results presented in this report support this suggestion. As described by Abe et al. [1] and Umemura et al. [64]. il appears that the release of free fatty acids during the initial 1-2 rain of ischemia can be attributed mostly to the action of phospholipase C, which further influences the

161

activation of phospholipase A, through the subsequent inositol triphosphate-mediated C-a-"~ release. Translocation of phospholipase A, from cytosol to membranes in rat brain is induced by Ca 2. ions [67]. The activation of phospholipase A_~ occurs predominantly after 2 rain of ischemia. In addition, since de now~ synthesis and replacement of phospholipids by the reacylation pathway is an energy-dependent, active process requiring ATP. recovery of the membrane phospholipids will be inhibited during the ischemic episode, but could occur during reperfusion as ATP levels are restored. Reconstitution of the cenular membranes during reperfusion would account liar the rapid decline in extracellular levels of glutamate and aspartate following a limited (20 min) period of ischemia, whereas recovery was protracted when the ischemia was extended to 40 min [46]. Post-ischemia rates of recovery of cerebral ATP are known to be prolonged lollowing an extended ischemic period [37]. The possibility that phospholipase activation could account ik~r a substantial portion of the elflux of glutamate and aspartate received initial support from the observation that topical application of phospholipases A, or C onto the cerebral cortex could elicit a release of glutamate and aspartate [39]. Further, inhibitors of phospholipases. mepacrine and indomethacin, depressed excitatory amino acid efflux from the ischemic rat cerebral cortex [39 45]. A study of the effects of the products ol: phospholipase A activity, arachidonic acid and lysophosphatidylcholine, on excitatory neurotransmitter amino acid release from the ischemic cerebral cortex generated interesting new information [40]. Lysophosphatidylcholine, a detergent con> pound with membrane-lytic activity, signii:icantly enhanced basal, non-ischemic, glutamate release from the cerebral cortex. Aspartate release was also elevated, but the increase did not achieve significance. Arachidonic acid. at concentrations as lo,a as 0.5 ~M significantly depressed ischemia-evoked excitatory amino acid release. Arachidonic acid is known to exhibit a wide variety of actions, both directly and through its lipoxygcnase and cyclooxygenase products [23]. At micromolar concentrations, arachidonic acid can alter the activity of ion channels [32] and at 100 p,M it can inhibit glutamate reuptake [66]. Inhibition of the glutamate transporter could explain the suppression of ischemia-evoked release, however this effect is only observed at high arachidonic acid concentrations. A more likely explanation for the potent ability of this free fatty acid to block the ischemia-evoked release of glutamate and aspartate may inw~lve its inhibition of phospholipase A,. 50c/~ inhibition of platelet PLA_, is achieved by 0.2 buM arachidonic acid [3], a concentration comparable to that required to suppress glutamate and aspartate release. Arachidonic acid also inhibits the 4-aminopyridine-evoked release of glutamate from cerebrocortical synaptosomes [19], but in this instance its action was thought to be a consequence of chan,,es~_ in K channel permeability.

162

J.W. Phillis, M.H. O'Regan / Brain Research 730 (1996) 150 164

Insight into the nature of the phospholipases involved in glutamate and aspartate release has been gained from experiments with selective inhibitors. 7,7-Dimethyleicosadienoic acid (DEDA) is selective for the secretory, rather than the cytoplasmic, form of phospholipase A 2, with IC~0 values in the range of 6 - 2 0 ~M [26]. When applied topically onto the rat cerebral cortex, this inhibitor significantly reduced the ischemia-evoked release of glutamate and aspartate. HELSS (haloenol lactone suicide substrate) [17] possesses 1000 fold selectivity for the calcium-independent versus calcium-dependent cytosolic PLA 2. At a concentration of 1 p~M this agent also reduced glutamate and aspartate release, as did AACOCF 3, a highly selective inhibitor of the calcium-dependem cytosolic form of PLA., [59]. AACOCF 3 (50 o~M) was one of the most effective inhibitors of ischemia-evoked glutamate and aspartate release from the ischemic rat cerebral cortex tested. These results obtained with selective inhibitors for secretory and cytoplasmic forms of the enzyme would appear to implicate both secretory as well as the Ca 2+-dependent and independent forms of cytosolic PLA 2 in the ischemiaevoked release of glutamate and aspartate. U-73122, a selective inhibitor of phospholipase C [59,68], at a concentration of 10 p~M, also effectively depressed glutamate and aspartate release, confirming earlier suggestions [1,64] that PLC is involved in excitatory amino acid release during ischemia. The potential significance of phospholipase activation in the causation of ischemia/reperfusion neuronal injury and death has now been demonstrated in studies on stroked Mongolian gerbils [43]. Administration of mepacrine (10 m g / k g ) , a non-selective PLA 2 inhibitor, to unanesthetized gerbils prior to a 5 min period of forebrain ischemia induced by bilateral carotid artery occlusion significantly reduced the degree of hippocampal CA1 injury 5 days later as assessed by reduced increases in locomotor hyperactivity and CA1 pyramidal cell destruction [43]. A complex interrelationship may exist between phospholipase A 2 and phospholipase C. Activation of protein kinase C, produced by stimulation of phospholipase C, results in the modulation of phospholipase A 2 activity, either by direct phosphorylation of this enzyme or by phosphorylation of its regulatory proteins [15]. Evidence for an involvement of protein kinase C in ischemia-evoked glutamate and aspartate release was obtained by demonstrating an inhibitory effect of the potent and selective PKC inhibitor chelerythrine chloride (5 ~M). In contrast, the selective protein kinase A inhibitor, H-89 (0.1 txM) did not affect ischemia-evoked amino acid release. Staurosporine, a highly potent (low nM) inhibitor of a variety of kinases, including PKA and PKC, had no effect on transmitter amino acid release at concentrations of 0.01 and 0.1 ~M. At a high concentration (2 I-tM), it non-significantly depressed glutamate and aspartate release. The failure of staurosporine to depress excitotoxic amino acid release at a concentration of 0.01 ~M, which should have

been adequate to inhibit PKC, raises a potential questionnamely does inhibition of another kinase(s) stimulate amino acid neurotransmitter release. There is a suggestion in Fig. 7 that inhibition of PKA by H-89 enhanced ischemiaevoked glutamate and aspartate release during reperfusion. The possibility therefore arises that PKA activation may confer cerebroprotection by depressing glutamate release. Maiese et al. [30] have previously suggested that whereas PKC contributes to ischemic neuronal death, activation rather than inhibition of PKA increases hippocampal neuronal survival during nitric oxide exposure. This interpretation would be consistent with the above findings. The observation that a PKC activator, phorbofl2-myristate-13-acetate (PMA), significantly enhanced ischemiaevoked glutamate and aspartate release, lends further support to the concept that PKC may play a role in the activation of PLA, and excitatory amino acid transmitter release. Our pharmacological data in support of a role of PKC in ischemia-evoked glutamate release, confirms an earlier in vitro study showing that accumulation of glutamate is regulated by PKC in rat hippocampal slices exposed to ischemic states [28] since glutamate accumulation was attenuated by H7 (an inhibitor of PKA and PKC) and facilitated by phorbof 12,13-dibutyrate. The last agent to be considered is nordihydroguaiaretic acid (NDGA), a selective inhibitor of lipoxygenases over cyclooxygenase [21]. NDGA also inhibits microsomal cytochrome P-450 and phospholipase A~. NDGA (1-30 b~M) attenuated N-methyl-D-aspartate neurotoxicity of cultured rat hippocampal neurons, but at concentrations above 30 ~M, it was directly toxic [53]. NDGA (0.1 ~M) failed to alter ischemia-evoked glutamate and aspartate from the ischemic rat cerebral cortex and at higher concentrations (30 I~M, 100 ~M) it significantly enhanced amino acid release. Whilst it is apparent that the increase in glutamate and aspartate release during exposure to higher concentrations of NDGA could be related to the toxic effects of observed by Rothman et al. [53], it is uncertain as to the underlying mechanism(s) of action involved. Inhibition of its breakdown would precipitate an increase in the levels of arachidonic acid, which can have neurotoxic effects [23]. Not surprisingly, addition of NDGA worsens the neurotoxicity of exogenous arachidonic acid for cultured neurons, probably by limiting its breakdown [51]. It is possible that the NDGA-evoked neuroprotection observed with weak NMDA insults to these cultured preparations was due to PLA 2 inhibition, whereas with stronger insults lipoxygenase and cyclooxygenase inhibition leads to the accumulation of toxic levels of arachidonic acid. In conclusion, the present results demonstrate that ischemia/reperfusion precipitates a massive efflux of the excitotoxic neurotransmitters, glutamate and aspartate, into the extracellular space of the rat cerebral cortex. Evidence is presented documenting the role of phospholipases A, and C in the triggering of this effiux, with protein kinase C

J. 144 Phillis. M.H. 0 'Regan / Brain Research Z~O (1996) 150-164

also involved in the cascade of events. Destabilization and deterioration of the plasma membrane, as a consequence of phospholipid hydrolysis would allow for the diffusion of transmitter amino acids down their concentration gradients into the extracellular fluid. Thus, the release of glutamate during cerebral ischemia may both initially contribute to, and be a consequence of, the disruption of plasma membrane integri U.

Acknowledgements This work was supported by USPHS Grant 26912-06 and a grant from the American Heart Association. The expert technical assistance of M. Smith-Barbour and L.M. Perkins with these studies is appreciated.

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I (G

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