Potassium-evoked efflux of transmitter amino acids and purines from rat cerebral cortex

036I-9230193$6.00+ .@I copyright Q 1993Pergamon Press Ltd.

Brain ResearchB~~el~~,Vol. 31, pp. 54?-.%2, 1993 Printedin the USA. All rights reserved.

Potassium-Evoked Efflux of Transmitter Amino Acids and Purines From Rat Cerebral Cortex .I. W. PHILLIS,’

Received

L. M. PERKINS

20 July 1992; Accepted

AND

M. H. O’REGAN

14 December

1992

PHILLIS, J. W., L. M. PERKINS ANI) M. H. O’REGAN. Potassium-evoked eflux of transmitter amino acids andpurines j?om rat cerebral cortex. BRAIN RES BULL 31(5) 547-5.52, 1993.-Repeated applications of elevated K+ (50 or 75 mM) in cerebral cortical cup super&sates was used to evoke an e&x of y-aminobutyric acid (GABA), glutamate, aspartate, giycine, adenosine, and inosine frOM the in vivo rat cerebral cortex. K+ (SO mM) si~n~fi~ntly elevated GABA levels in cup superfusates but had Iittle effect on the e&x of glutamate, aspartate, glycine, adenosine, or inosine. K* (75 mM) sjgnj~~nt~y enhanced the e&x of GABA, aspatiate, adenosine, and inosine and caused nonsignificant increases in glutamate and gfycine ef%.ix. The adenosine A, receptor agonist N6-cyclo~ntyladenosine (CPA), applied in cup superfusatesat a concentration of IO-‘* M had no effect on either basal or K+-evoked release of any of the amino acids or purines measured. At 10e6 M CPA significantly enhanced aspartate reiease, and depressed GABA e&x. The selective AZadenosine receptor agonist 2-~Z-car~xyethyl) phenethylamj~o-S-N-ethylcarboxamidoadenosine (CGS 21680) (10-s M) was without effect on either basal, or K+-evoked, efflux of amino acids or purines. The enhancement of aspartate (an excitotoxic amino acid) efflux by higher concentrations of CPA is likely due to activation of adenosine AZ,, receptors. This observation may be of relevance when selecting adenosinergic agents to treat ischemic or traumatic brain injuries. Overall, the results suggest that effects of adenosine receptor agonists on K+-evoked et?lux of transmitter amino acids from the in vivo rat cerebral cortex may not be comparable to those observed with in vitro pre~ratio~s. Glutamate

Aspartate

GABA

Adenosine

Inosine

I~~NTI~~ATi~~ of the mechanisms by which the neurom~ulator, adenosine, regufates the release of physiolo~~lly active amino acids within the central nervous system (CNS) is clearly important for any rational development of purinergk therapies to minimize ischemic and traumatic brain injury. During brain ischemia and trauma a massive release of the excitotoxic amino acids, glutamate, and aspartate, occurs ( 1,9,13,16,26,33,34). These amino acids have been implicated in the induction of ischemic and traumatic injury to CNS tissues (22,X5). Adenasine levels are also elevated during cerebra1 ischemia and reperfusion. Extracellular levels of adenosine rise dramatically as adenosine, formed as ATP stores are utilized, escapes into the extracelfufar spaces (32,39). Adenosine analogs have been shown to be ~erebroprot~ve against ischemic and excitotoxic amino acid-induced injury (2,8,40). Amongst the several potential mechanisms that could account for its protective actions, the ability of adenosine to attenuate the release of giutamate and aspartate from nerve terminals has been considered to be of particular significance (6,7,10). The mechanism by which adenosine inhibits neurotransmitter release remains controversial f 11). In an attempt to define the effects of activation of the three currently recognized adenosine receptors (A,, Alat AZb) on amino acid neurotransmitter release, we recently assessed the abilities of selective adenosine

Cortex

Release

Potassium

agonists and antagonists to modulate glutamate, aspartate, and GABA release from the ischemic rat cerebral cortex. The results suggested that adenosine and its analogs suppressed ischemiaevoked release of glutamate and aspartate via high affinity A, receptors, whereas activation of low-affinity A2 receptors enhanced their release (27,37). GABA release was also depressed by activation of high-affinity A2 receptors (28). The present paper describes experiments in which the release of glutamate, aspartate, GABA, glycine, adenosine, and inosine from the rat cerebral cortex was elicited by the application of high concentrations of K+, replicating the increases in extracelMar K’, which occur during cerebral ischemia (3). The abilities of low f fO_” I&I;Al receptor activation) and high ( 10S6 M; A2 receptor activation) concentrations of ~6~~clo~nty~adenos~ne (CPA) and of the selective AZ agonist CGS 2 1680 to modulate amino acid release were also examined. METHOD

Male Sprague-Hawley rats were anesthetized with halothane. After insertion of a tracheal cannula, anesthesia was sustained with methoxyflurane in air. Body temperature was controlled at 37°C with a rectal probe and heating pad, One femoral artery was cannulated for the measurement of blood pressure and withdrawal of arterial blood samples for pH and blood gas measurement.

’ Requests for reprints shoutd be addressed to Dr. John W. Phi&.

547

54x

PHiLLfS. PERKINS AND O’REGAN

The dorsal surfaces of both cerebral hemispheres were exposed, and after reflection of the dura mater, oval cortical cups suspended in flexible mounting brackets were placed on both oortices (29,32). The dorsal surlace of the head around the cups was covered with a stabilizing gel of 4% agar in artificial CSF. A monopolar EEG electrode was placed in both cups. ECoGs and arterial blood pressure were recorded on a Grass Polygraph. Artificial CSF pipetted in the cortical cups was removed after a 30-min ~uilibmtion period and replaced with 300 gl of warmed (37°C) sterile artificial CSF that had been bubbled with a gas mixture of 5% carbon dioxide in nitrogen. The same gas mixture was bubbled into the corticai cups. Cup fluid was maintained at 37°C with a heat lamp. Cup fluid was collected at IO-min intervals and replaced with fresh artificial CSF. Depolarization of cortical tissues was elicited by the topical application of artificial CSF containing 50 mM or 75 mM K+, prepared by replacing the approp~at~ amount of NaCl in the artificial CSF with an equivalent amount of KCl. Two groups of rats (series 1)were used to determine the effects of topical applications of high K’ on amino acid and purine release into the cortical cups. Four rats (eight cortices) were tested with 50 mM I(’ and nine rats (I8 cortices) with 75 mM I(+. In both studies, after a basal 10 (Bi) min CSF (300 ~1) collection. the cups were filled with 300 ~1 of high K’ CSF for 10 min (Sri. followed by two more norma CSF collections. After a 2O-min break. this sequence was repeated twice, with a IO-min break between the sequences. For studies (series II and III) on the release of amino acids during CPA and CGS 21680 application, the initial sequence described above was followed by a 2(f-min period during which CPA (IO-‘” M or 1O-6M) or CGS 21680 (lo-* M} were applied (the cup solution was replaced 3X during this period). The second and third K’ depolarization sequences (S,, S,) were then repeated, with all solutions containing the appropriate concentration of CPA or CGS 2 1680. Five rats ( 10 cortices) were used to test for the effects of each concentration of CPA and four rats {eight corticesf for the effects ofCGS 2 1680. The con~entmt~ons at which CPA and CGS were used were selected on the basis of extensive previous ex~~men~t~on as being selective for the activation of A, or AZ receptors (27,28,37). The collected cortical perfusates were ejected into chilled microvials, centrifuged at 1,200 X g, and then stored at -20°C for later analysis. Quantitative determination of amino acids and purines in the perfusates was by HPLC using previously published procedures (29-32). Significant increases in amino acid or purine releases into the cortical cups during K+ application were determined by Student’s r-test, comparing the concentration during K+ exposure with that during the immediately preceding basal collection period. The effects of adenosine agonists on K’-evoked amino acid release are expressed as S,/Si or SJ$ ratios evoked during the three K’ application periods. In each instance, stimulated ef&.ix represents the increase in release above the immediately preceding basal release. The data were analyzed with the SPSS PC statistical program. A one-way analysis of variance (ANOVA) was performed and Student-Newman-Keuls @ < 0.05) or Sheffe’s (p < 0.01) tests were utilized to determine statistical significance, with comparisons between control and drug conditions or between the twodrug conditions. RESULTS

Physiological variables were monitored in ail groups of animals by recording of mean arterial blood pressure ~MA3P) and

electrocorticogram (ECoG) and through the determination of arterial pH and gas tensions in samples obtained during the initial and final basal collection periods of each experiment. There were no signi~~ant changes in MABP, arterial pH, PO2 or PCOZ as a result of the application of high K+ CSF, CPA, or CGS 2 1680. Application of both 50 and 7.5 mM K”-containing CSFs caused an initial marked depression of the ECoG, with some intermittent recovery (high-voltage, low-frequency potentials) becoming apparent during the latter phases of the IO-min application period. Electrical activity returned during the initial post-K+ collection period, with the graded onset of high-voltage. low-frequency potentials. Recovery to a pre-K’ flow voltage. higher frequency) ECoG occurred slowly over a subsequent IO20-min period.

Initial basal superfusate levels ofthe amino acids and purines for all groups were: aspartate, 278.1 c 3 1.9 nM; glutamate, 88 I .9 + I 15.3 nM; GABA, 76.1 f 5.77 nM; glycine. 3203.0 It 302.9 nM; adenosine, 34.6 2 3.0 nM; inosine, 30.8 rt 2.7 nM (mean i SEM, respectively). The effects of high-K+ CSF application on amino acid neurotransm~tter release are presented in Fig. I, A-D. K” (50 mM) failed to elevate super&sate levels of either glutamate or aspartate, but did sign~~~ntly elevate GABA levels above those in the preceding basal superfusate collections. K’ (75 mM) significantly elevated aspartate and GABA efflux. Glutamate release was also enhanced during the 75 mM K’ administration, but this increase was not significant. At both K’ concentrations glycine release rose continuously during and after the second and third K’ application sequences. This response was apparently elicited by the high K+ administration, because a comparable increase was not evident in control experiments in which glycine release was monitored in the absence of high K+ applications (3 I ).

K’ CSF, 75 mM, but not 50 mM, evoked repeatable increases in adenosine and inosine release into the cortical cups (Fig. 2). Inosine release continued to be significantly enhanced during the first post-K_’ collection period.

No-cyclo~ntyladenosine (lO-lo M) failed to alter either the basal or K+-evoked release of aspartate, glutamate, or GABA into the cortical superfusates (Table I). At 10s6 M CPA significantly enhanced K+-evoked release ofaspartate. Whereas basal levels of GABA release were unaffected, CPA ( 10d6 M) reduced K’-evoked GABA reIease, with this effect achieving significance during the 3rd K” appli~tion (Table 1). Swirs Ili. E@i?s

of C’GS 23680 fxx Amino Acid R&we

CGS 2 1680 ( 10sRM) failed to alter either basal or K’-evoked release of the amino acids (Table I ). DISCXJSSION

Although elevated K+ in the perfusion medium of slices or synaptosomes has long been recognized as an acceptable technique for eliciting transmitter release (4,25,38). its utilization for in vivo experiments is less well documented. Clark and Collins (5) were able to demonstrate a significant release of endogenous GABA, accompanied by a nons~gni~~ant decrease in glu-

K+-EVOKED EFFLUX

549

OF AMINO ACIDS AND PURINES

IWe_ t COLLECTION

OV 0

3

I

J

PERIOD

COLLECTION

f

3500 5

1

COLLECTION

PERIOD

PERIOD

0

1

!xl

COLLECTION

3

+

5

PERIOD

FIG. 1. (A-D) Potassium-evoked release of transmitter amino acids into rat cerebral cortical cup superfusates. Superfusates were collected at IO-min intervals and analyzed by HPLC. Data are presented as the means rt SEM of superfusate amino acids using pooled data from three successive sequences of exposure to artificial CSF containing either 50 or 75 mM K* applied during collection period 2. There was a 20-min break between the first two collection sequences and a IO-min gap between the second and third collection sequences. See the Method section for further details. (A} Aspartate; (B) glutamate; (C) GABA; and (D) glycine concentrations of cup superfusates. +p < 0.05; ++p < 0.01; +++p < 0.001 by Student’s t-test when K+-evoked &ease was compared with that in the preceding basal superfusate.

tamate and aspartate release from the rat visual cortex when a 50 mM K+ solution was placed in the cup, whereas 100 mM K+ additionally released glycine, aspartate, and glutamate. The K+-evoked release of these amino acids was Ca++-dependent, which would be consistent with their release from nerve terminals. In a separate series of experiments, 50 mM K’ enhanced the release of endogenous GABA, although decreasing that of glutamate, from the rat cerebral cortex (24). Paradoxically, the latter investigators recorded the opposite results if the K’ solution was applied epidurally, with elevated glutamate and decreased GABA releases. When measuring labeled purine release from the rat cortex, Jhamandas and Dumb~lle (18) were able to demonstrate a release of r3H~-adenosine derivatives during high K’ CSF application. An enhanced release of endogenous adenosine and inosine during high K+ (SO mM) perfusion through a microdialysis tube in the rat caudate nucleus has also been reported (39). The results of the present study are generally consistent with these previous observations. As reported by Clark and Collins (5), 50 mM K’-containing CSF evoked a release of GABA, but not of glutamate and aspartate, whereas 75 mM K+ also enhanced aspartate release, with a simultaneous tendency for glu-

tamate levels to increase. Both Clark and Collins (5) and Moroni et al. (24) have suggested that the fall in release of the two excitatory amino acid neurotransmitter candidates that accompanied the increase in GABA release evoked by 50 mM K” application was the result of GABAergic inhibition of glutamate and aspartate release. The reduction in release could be due to an action of GABA either on the presynaptic terminal or somatic receptors of glutamatergic neurons, inhibiting their release of neurotransmitter. However, in preliminary experiments in this laboratory, the administration of picrotoxin (GABA,+ receptor antagonist) or 2-hydroxy~clofen (GABAn receptor an~onist) failed to enhance either basal or K+-evoked release of excitatory amino acids into cortical superfusates. The failure of 50 mM K+ to elicit increases in glutamate and aspartate release comparable to those observed with GABA presents something of a conundrum. The basal levels of aspartate (278 nM), and especially glutamate (882 nM), in the cortical superfusates were substantially greater than those of GABA (76 nM). An even greater discrepancy (205-fold), between basal GABA and glutamate release was observed by Moroni et al. (24). it is, therefore, possible that any additional amounts of aspartate and glutamate, released as a result of K+-evoked de-

5%)

PHILLIS. PERKINS

20

L.

--

cl

@I

COLLECTION FIG.

1

-...__.~-I.._

1

3

+

20 5

PERIOD

1 (1

I

El

COLLECTION

AND O’REGAN

1

3

4

I

PERIOD

2. (A,B)

Potassium-evoked release of adenosine and inosine into rat cerebral cortical cups. See Fig. 1. legend for further details.

polarization. were simply obscured by this unstimulated basal release. Afthough marked changes in extracellular excitatory amino acids have been reported after ischemia and hypo~ycemia (3,16,33,34), the changes in the release of these amino acids during seizure states are not pronounced ( 15,2 1). The failure of elevated K+ to increase glutamate release from the cerebral cortex is, therefore, consistent with earlier observations of a lack of a demonstrable increase in release during other states of enhanced brain excitability. Glycine release increased steadily from the fourth to eleventh collection periods when cortices were exposed to either concentration of K+. This response pattern stands in sharp contrast to the stable levels of glycine in successive cup superfusate samples from cortices not exposed to elevated K* (34). The mechanism underlying the continuous increase in glycine release is unde~ned at this time. Application of 75 (but not 50) mM K’ evoked a si~ificant increase in both adenosine and inosine release into the cortical cup superfusates. The actual magnitude of these increases in purine levels (adenosine, 40-50 nM; inosine, 60-70 nM) were minimal in comparison with those which occur during cerebral ischemia (adenosine, 1 mM; inosine, 1 mM) (33,34). The small increases observed during K+ application, in comparison with those occurring during hypoxia and ischemia (32-34), indicate that cortical energy failure must be virtually absent, in spite of the increased demand for energy created by the K+-evoked depolarization of tissues. This may be comparable to the situation during status epilepticus, in which the rate of energy utilization increases to about 250% of control, but ATP levels are maintained at 97-99% of control (3). The lack of effect of CPA ( 10. ‘* M) on K’-evoked amino acid release in the present experiments clearly differs from its depressant action on ischemia-evoked efllux (37). However, the absence of a reduction in glutamate and aspartate release in the presence of low concentrations of CPA would be consistent with the frequently reported inability of adenosine A, agonists to reduce Ca2+ entry into K+-depolarized synaptosomes (12,23). In contrast Ca*’ entry into synaptosomes evoked by electrical stimulation is almost abolished by adenosine (14,36). Shinozuka et al. (36) have suggested that K+-induced depolarization may stimulate a mechanism of Ca2+entry that is adenosine-insensitive and different from the mechanism of Ca2+ entry occurring during electrical stimulation.

A different pattern of CPA effects on GABA release was evident in that while 10-‘” M CPA also failed to affect I&+-evoked release, lo-” M CPA signi~cantly reduced GABA levels in cortical superfusates. The failure of low concentrations of CPA to reduce GABA release may be related to the weak ability of adenosine agonists to prevent K+-elicited Ca” entry into nerve terminals described above. Clark and Collins (5) have reported a Ca2+ dependency of Kc-evoked GABA release from the cerebral cortex and CPA may have failed to attenuate the influx of Ca*+ into IS’.-depolarized nerve terminals. There is also evidence to suggest that adenosine A, receptor activation is less effective at reducing GABA release, in comparison with excitatory amino acid release, from depolarized nerve terminals (7,19,20,41). A lack of effect of lO_” M CPA in K+-evoked GABA release was not, therefore, unexpected. At a higher concentration ( 10e6 M), CPA did depress GABA release, a finding that is consistent with the obviation that the AZ receptor agonist, CGS 2 1680, at a con~ntration of 1O-’ M, depressed ischemia-evoked GABA release from the cerebral cortex (28). Hollins and Stone (17) have previousiy described an ability of high concentrations of adenosine ( 10e5- 10e3 M), which would have activated A2 receptors, to depress GABA release from rat cerebral cortical slices. Adenosine receptor agonists apparently modulate K+-evoked GABA release in a strikingly different manner than excitatory amino acid release. CGS 2 1680 (lo-* M) failed to alter K+-evoked efflux of glutamate, aspartate, GABA, or glycine. Again, the discrepancy between the resuits obtained with ischemia-evoked and F-evoked GABA efflux demonstrates the apparent existence of a different mechanism of Ca++-mobilization in the K+-evoked release of GABA. In conclusion, these experiments demonstrate certain potential dif&erencesbetween the characteristics of K+-evoked efflnx of different amino acids and of purines. K+ (50 mM) application elicited a robust release of GABA, but not of the other agents. At 75 mM, K+-application evoked releases of GABA, aspartate, adenasine, and inosine, but failed to significantly enhance ghttamate and glycine efflux. A possible explanation for this d@rence may reside in the high basal levels of glutamate and glycine efflux, which could potentially mask any additional release. Results obtained with K+stimulated transmitter amino acid efflux Born the in viva cerebral cortex have proven to be inconsistent with earlier findings on tihemiaevoked release, indicating that the stimulus for amino acid release during &hernia involves factors in addition to elevated K+.

K+-EVOKED

EFFLUX

TABLE SPONTANEOUS

Substance

Aspartate

Glutamate

AND 75 MM K+-EVOKED

Drug

EFFLUX

Present During S2and S,

CPA 10~” M CPA 1O-6M CGS 21680 IO-* M CPA

IO-”

M

CPA 10m6M CGS 21680 IO-* M GABA

Glycine

551

OF AMINO ACIDS AND PURINES

-

CPA lo-” M CPA lO-6 M CGS 21680 IO-* M CPA IO-” M CPA 1O-6M CGS 21680 IO-’ M

OF TRANSMITTER

1 AMINO ACIDS FROM THE IN VfVO RAT CEREBRAL Basal Efflux

CORTEX K+-Evoked Efflux

Basal Efflux

K+-Evoked Efflux

(B&I)

&/&l

1.42r?r0.28 1.oo rf: 0.09 1.17 f 0.16 2.72 rL:1.81 1.68 r 0.38 1.01 f 0.06 1.67 + 0.20 1.18 c 0.15

1.03 _+0.12 1.24 + 0.14 2.10 f 0.44* 0.97 * 0.18 1.02 _+0.09

1.27 + 1.31 + 1.52 + 1.46 + 1.82 *

1.01 f 0.06

1.40 * 0.11

1.22i 0.32

1.13 f 0.13 0.96 + 0.10

1.19 + 0.11

1.07 + 0.07

1.00 f 0.01 1.07 rf:0.07 0.77 + 0.06 1.70 ;t 0.18 0.88 rt 0.07 1.24rtO.11 1.23 rt: 0.19

1.39t 0.25 0.87 -t 0. I3 1.28 rf-0.16 1.04 + 0.06 1.25 + 0.05 2.62 It 1.47 1.07 t 0.06

2.40 i 0.38 1.94f 0.32 1.09* 0.08 1.oo _t 0.0 I 1.00 + 0.01 1.09 * 0.14 2.76 -t 0.52 1.24 rt 0.18 1.52 + 0.19 2.06 rtr0.40

1.22 + 0.19 0.95 to.11 1.13 rt 0.08 1.48 s 0.27 0.64 Z!Z O.lO* 1.01 + 0.26 1.06 + 0.79 1.32 + 0.14 3.20 + 1.81 1.22 * 0.22

(S,/S,)

@#I) 0.19 0.16 0.23 0.53 0.27

1.53 + 1.15 + 2.22 * 1.16 +

0.26 0.17 0.41 0.19

1.17 + 0.11

Effects of adenosine receptor agonists on the 75 mM K+-evoked efflux of aspartate, glutamate, GABA and glycine from the in vivo rat cerebral cortex. The left and right cortices were exposed three times (S,, Sr, S,) to artificial CSF containing elevated K+. Drugs were added to the artificial CSF 20 min prior to the collection of the basal (B,) superfusate preceding S2 and were present in the superfusate medium continuously thereafter. Changes in basal and K+-evoked efflux are expressed as the ratios (BdB,, B3/B,; S&l,; S,/S,) of amino acid release imm~iately prior to and during the three periods of 75 mM K+ application. The magnitude of I(+-evoked effluxes of amino acids was determined by subtra~ng the immediately preceding basal release from the I(+-evoked et&x. * Significantly different from control response; p < 0.05.

ACKNOWLEDGEMENT

Topical applications of K’ may provide an unsatisfactory model which to study the effects of adenosine on injury-evoked tram+

with

This study was supported by USPHS Grant NS269 12.

mitter amino acid release.

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PHILLIS,

30.

31. 32.

33.

34.

35. 36.

37.

38.

39.

40. 41.

PERKINS

AND O’REGAN

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