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Effect of ketamine on amino acid-evoked release of acetylcholine from rat cerebral cortex in vitro
Neuroscience Letters, 56 (1985) 371-375
371
Elsevier Scientific Publishers Ireland Ltd. NSL 03328
EFFECT OF KETAMINE ON AMINO ACID-EVOKED RELEASE OF A C E T Y L C H O L I N E F R O M RAT CEREBRAL C O R T E X IN VITRO
D. LODGE* and G.A.R. JOHNSTON
Department of Pharmacology, The University o['Sydney. Sydney, N S W 2006 (Australia) (Received November 12th, 1984; Revised version received and accepted February 26th, 1985)
Key words." ketamine-excitatory amino acids acetylcholine release
rat
N-Methyl-D-aspartate (NMDA), quisqualate, kainate and potassium increased, in a dose-dependent manner, the effiux of radioactivity from rat cerebral cortex minislices preincubated with 13H]choline. Ketamine (1-5/iM) and magnesium (0.1 1 mM) reduced only the release evoked by NMDA. The non-parallel shift of the NMDA dose-response curve suggests that ketamine is not acting as a competitive antagonist of NMDA.
On the basis of agonist and antagonist studies, it is generally accepted that there are three major receptor types for the excitatory amino acid transmitters, glutamate and aspartate, namely those activated by N-methyi-D-aspartate (NMDA), quisqualate and kainate [13]. Recent studies have shown that dissociative anaesthetics (e.g. ketamine, phencylclidine and the dioxolanes) and sigma opiates (e.g. cyclazocine and N-allylnormetazocine) reduce the excitatory action of N M D A on central mammalian neurones [1, 5]. This action is selective in that responses to quisqualate and to kainate are relatively unaffected by these drugs. Because their relative potency as N M D A antagonists parallels that in both phencyclidine/sigma binding [9, 14] and discrimination studies [3, 10], it has been proposed that N M D A antagonism may contribute to the psychotomimetic effects [2] common to the dissociative anaesthetics and the sigma opiates [5]. The nature of the interaction between ketamine and N M D A is, however, not known. The recent report that N M D A and other amino acids enhanced the release of acetylcholine (ACh) from rat brain slices [4] provided a model in which to examine in more detail the action of ketamine as an N M D A antagonist. Experiments were carried out using a modified release protocol, described in more detail elsewhere [12]. In brief, 200-~m slices of rat cortex were washed in standard magnesium-free buffer (in mM): NaCI 140, KC1 3.5, CaC12 2.5, NaHCO3 23.8, NaH2PO4 0.6 and glucose 11, equilibrated with 5~o CO2 in 02 to pH 7.4. The slices *Permanent address and address for correspondence: Department of Physiology, Royal Veterinary College, London NW1 0TU, U.K. 0304-3940/85/'$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
372
were then incubated with [3H]choline (50 nM) at 25'C for 30 min and later washed repeatedly in buffer for a further 30 min to allow the spontaneous release to reach a low and constant level. Aliquots of approximately 12 mg of tissue in 120/al of buffer were distributed to individual assay bottles containing 5 ml of buffer with 10/aM hemicholinium. To these had been added various concentrations of test substances, e.g. amino acids or KC1 to evoke release and ketamine as a potential inhibitor of this release. After a further 15-rain incubation, the slices were rapidly filtered, the fitrate collected and radioactivity in l-ml aliquots measured. The release was determined as the amount of radioactivity in the aliquot minus the appropriate zero time blank, and the effect of potential inhibitors of release was expressed as a percentage change from control values. In agreement with the results from the perfusion experiments of Lehmann and Scatton [4], N M D A (1-31.5/aM) produced a dose-dependent increase in the release of radioactivity with this modified release protocol, and the release was reduced both by magnesium and by organic N M D A antagonists, such as aminophosphonovalerate (APV). Dose-response curves (Fig. 1) show that the effect of N M D A reached a plateau at about 30/aM, at which concentration there was a 2-3-fold increase in release.
100-
0 Q
....
ii
W
i/I
KETAMIP 1 uM
/
w W * i i
W
, w
I
--w Z
CONTRC
x
I
0
~
O-
I i 3
, 10
~ 31
CONCENTRATION
i 100
'"l 316
(+)PCMI 0.1 uM
uM
OF N M D A
Fig. I. Dose-response curves showing the effect of N M D A on the increase in release of radioactivity from cortical slices preloaded with pH]choline. Each point on the graph represents the mean of at least 4 observations and t h e vertical bars represent S.E.M. The addition of ketamine (1 ~M) or ( + ) - P C M P (0.1#M) reduced the response to N M D A to a similar extent in a manner consistent with non-competitive antagonism. Ordinate: percent increase in release of radioactivity above spontaneous efflux. Abscissa: concentration of N M D A in 1 tLM.
373
NMDA 200 -
re
1 0 0 uM
KAINATE
KCI
2 8 0 uM
2 5 mM
"°I
-750
I(3 <
I
_o
Q ,( E LI. O U4 m ,( ILl ..I ILl E U4 G0 9[ lU a: U Z i
-1000
|U
"I I
1OO
-500
II I|
mg ||
i
01 |1
in ||
SO -
°"
'| !,
TI
i! el |U
ml
i|! II
0
Mg KET
-50-
SuM
~1
-250
B! |!
Mg KET
0.1
KET
SuM
mM
5uM
Fig. 2. Histograms showing the effect of ketamine (5 #M) and Mg 2+ (0.1 mM) on the increase in activity evoked by NMDA (100/tM), kainate (250 pM) and KCI (25 mM). The vertical bars indicate S.E.M. of at least 4 observations. Ordinate: percent increase in release of radioactivity above spontaneous efflux. Note the separate scale for KCl-induced release.
Similar increases in release were achieved by addition of kainate (125-500 pM) or KC1 (10-25 mM), but these effects were not reduced by the addition of Mg 2+ or APV at doses which antagonized NMDA. An example of this selective effect of Mg 2+ (0.1 mM) is shown in the histograms of Fig. 2. Ketamine (0,1-5 pM) also reduced the action of N M D A in a selective manner, neither the spontaneous release nor the enhanced release in the presence of kainate or KCI being affected. An example of this action of ketamine is illustrated in Fig. 2. In this experiment, 5 #M ketamine abolished the release evoked by NMDA. Concentrations of ketamine as low as 0.1/tM clearly inhibited the actions of N M D A and, although there was variation between individual experiments, dose-response curves for this antagonistic effect of ketamine suggest that the IC50 is between 0.1 and 0.5 /tM. Dose-response curves for NMDA-evoked release in the presence of ketamine (1 pM) showed a clear shift to the right with a decrease in slope and in the maximum effect of N M D A (Fig. 1). A similar non-parallel shift was also obtained with another
374 dissociative anaesthetic, (+)-phenyl-methyl-cyclohexylpiperidine ( P C M P ) (0.1 /~M; Fig. 1). Such changes suggest that the nature of the N M D A antagonism is n o n - c o m petitive. N-Allylnormetazocine (0.1-3 /~M), the prototypical sigma opiate [7], also reduced N M D A - e v o k e d A C h release, Estimates o f relative potency suggested that ( + ) - P C M P was about 10 times more potent than ketamine, whereas N-allylnormetazocine was about equipotent with ketamine. Our results support the view that N M D A - e v o k e d release o f A C h from rat brain slices has similar pharmacological characteristics to N M D A - e v o k e d excitation o f central neurones [4] and that dissociative anaesthetics/sigma opiates are selective N M D A antagonists in this release paradigm. They also suggest that ketamine does not act in a competitive manner at the N M D A recognition site, a finding in agreement with the failure o f excitatory amino acids to inhibit phencyclidine/sigma binding [9, 14] and of ketamine to inhibit binding at the N M D A receptor (J.C. Watkins and H.J. Olverman personal communication). Whether ketamine acts at the N M D A activated channel, e.g. in a manner similar to Mg 2+ [8], remains to be determined. Similarly, it is u n k n o w n to what extent N M D A antagonism contributes to be behavioural effects of dissociative anaesthetics and sigma opiates, which in m a n resemble those o f schizophrenia [2], but it is perhaps not without significance that the relative potencies o f ketamine, ( + ) - P C M P and N-allylnormetazocine in this study and o f phencyclidine, cyclazocine and N-allylnormetazocine in a similar study [1 1], are in close agreement with those obtained in behavioural studies [3, 10]. A potential synaptic substrate for such actions is the recently elucidated N M D A receptormediated synaptic excitation in the cerebral cortex which is reduced by Mg 2+, ketamine and A P V [6]. We are grateful for supplies of ketamine, ( + ) - P C M P and N-allylnormetazocine from W a r n e r - L a m b e r t , U.K. Ltd., Dr. R o n Browne and N I D A , respectively. This work was supported by the N H M R C o f Australia and by a Royal Society travel grant to D.L.
I Anis, N.A., Berry, S.C., Burton, N.R. and Lodge, D., The dissociative anaesthetics, ketamine and phencyclidine, selectively reduce excitation of central mammalian neurones by N-methylaspartate, Brit. J. Pharmacol., 79 (1983) 565 575. 2 Domino, E.F. and Luby, E.D., Abnormal mental states induced by phencyclidine as a model for schizophrenia. In E.F. Domino (Ed.), PCP (Phencyclidine): Historical and Current Perspectives, NPP Books, Ann Arbor, MI, 1981, pp. 401 413. 3 Herling, S. and Woods, J.H., Discriminative stimulus effects of narcotics: evidence for multiple receptor-mediated actions, Life Sci., 28 (1981) 1571 1587. 4 Lehmann, J. and Scanon, B,, Characterization of the excitatory amino acid receptor-mediated release of [3H]acetylcholinefrom rat striatal slices, Brain Res., 252 (1982) 77 89. 5 Lodge, D. and Berry, S.C., Psychotomimetic effects of sigma opiates may be mediated by block of central excitatory synapses utilising receptors for aspartate-like amino acids. In R. Bandler (Ed.), Modulation of Sensorimotor Activity during Alterations in Behavioural States, Alan R. Liss, New York, 1984, pp. 503-518.
375 6 Lodge, D., Thomson, A.M. and West, D.C., A Mg 2+ and ketamine-sensitive e.p.s.p, in slices of rat somatosensory cortex: a role for N-methylaspartate receptors, J. Physiol. (Lond.), 358 (1985) 22P. 7 Martin, W.R., Eades, C.G., Thompson, J.A., Huppler, R.E. and Gilbert, P.E., The effects of morphine- and nalorphine-like drugs in the nondependent and morphine dependent chronic spinal dog, J. Pharmacol. Exp. Ther., 197 (1976) 517-532. 8 Nowak, L., Bregestovski, P., Ascher, P,, Herbet, A. and Prochiantz, A., Magnesium gates glutamateactivated channels in mouse central neurones, Nature (Lond.), 307 (1984) 462-465. 9 Quirion, R., Hammer, R.P., Herkenham, M. and Pert, C.B., Phencyclidine (Angel Dust)/sigma 'opiate' receptor visualisation by tritium sensitive film, Proc. Natl. Acad. Sci, USA, 78 (1981) 5881-5885. 10 Shannon, H.E., Pharmacological evaluation of N-allylnormetazocine (SKF 10,047) on the basis of its discrimination stimulus properties in the rat, J. Pharmacol. Exp. Ther., 225 (1983) 144-152. 11 Snell, L.D. and Johnson, K.M., Phencyclidine antagonism of N-methyl-o-aspartate induced acetylcholine release from rat striatal slices, Fed. Proc., 43 (1984) 953. 12 Spence, I., Drew, C., Johnston, G.A.R. and Lodge, D., Acute effects of lead at central synapses in vitro, Brain Res., in press. 13 Watkins, J.C. and Evans, R.H., Excitatory amino acid transmitters, Ann. Rev. Pharmacol. Toxicol., 21 (1981) 165-204. 14 Zukin, R.S. and Zukin, S.R., Demonstration of [3H]cyclazocine binding to multiple opiate receptor sites, Molec. Pharmacol., 20 (1981) 246--254.
371
Elsevier Scientific Publishers Ireland Ltd. NSL 03328
EFFECT OF KETAMINE ON AMINO ACID-EVOKED RELEASE OF A C E T Y L C H O L I N E F R O M RAT CEREBRAL C O R T E X IN VITRO
D. LODGE* and G.A.R. JOHNSTON
Department of Pharmacology, The University o['Sydney. Sydney, N S W 2006 (Australia) (Received November 12th, 1984; Revised version received and accepted February 26th, 1985)
Key words." ketamine-excitatory amino acids acetylcholine release
rat
N-Methyl-D-aspartate (NMDA), quisqualate, kainate and potassium increased, in a dose-dependent manner, the effiux of radioactivity from rat cerebral cortex minislices preincubated with 13H]choline. Ketamine (1-5/iM) and magnesium (0.1 1 mM) reduced only the release evoked by NMDA. The non-parallel shift of the NMDA dose-response curve suggests that ketamine is not acting as a competitive antagonist of NMDA.
On the basis of agonist and antagonist studies, it is generally accepted that there are three major receptor types for the excitatory amino acid transmitters, glutamate and aspartate, namely those activated by N-methyi-D-aspartate (NMDA), quisqualate and kainate [13]. Recent studies have shown that dissociative anaesthetics (e.g. ketamine, phencylclidine and the dioxolanes) and sigma opiates (e.g. cyclazocine and N-allylnormetazocine) reduce the excitatory action of N M D A on central mammalian neurones [1, 5]. This action is selective in that responses to quisqualate and to kainate are relatively unaffected by these drugs. Because their relative potency as N M D A antagonists parallels that in both phencyclidine/sigma binding [9, 14] and discrimination studies [3, 10], it has been proposed that N M D A antagonism may contribute to the psychotomimetic effects [2] common to the dissociative anaesthetics and the sigma opiates [5]. The nature of the interaction between ketamine and N M D A is, however, not known. The recent report that N M D A and other amino acids enhanced the release of acetylcholine (ACh) from rat brain slices [4] provided a model in which to examine in more detail the action of ketamine as an N M D A antagonist. Experiments were carried out using a modified release protocol, described in more detail elsewhere [12]. In brief, 200-~m slices of rat cortex were washed in standard magnesium-free buffer (in mM): NaCI 140, KC1 3.5, CaC12 2.5, NaHCO3 23.8, NaH2PO4 0.6 and glucose 11, equilibrated with 5~o CO2 in 02 to pH 7.4. The slices *Permanent address and address for correspondence: Department of Physiology, Royal Veterinary College, London NW1 0TU, U.K. 0304-3940/85/'$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
372
were then incubated with [3H]choline (50 nM) at 25'C for 30 min and later washed repeatedly in buffer for a further 30 min to allow the spontaneous release to reach a low and constant level. Aliquots of approximately 12 mg of tissue in 120/al of buffer were distributed to individual assay bottles containing 5 ml of buffer with 10/aM hemicholinium. To these had been added various concentrations of test substances, e.g. amino acids or KC1 to evoke release and ketamine as a potential inhibitor of this release. After a further 15-rain incubation, the slices were rapidly filtered, the fitrate collected and radioactivity in l-ml aliquots measured. The release was determined as the amount of radioactivity in the aliquot minus the appropriate zero time blank, and the effect of potential inhibitors of release was expressed as a percentage change from control values. In agreement with the results from the perfusion experiments of Lehmann and Scatton [4], N M D A (1-31.5/aM) produced a dose-dependent increase in the release of radioactivity with this modified release protocol, and the release was reduced both by magnesium and by organic N M D A antagonists, such as aminophosphonovalerate (APV). Dose-response curves (Fig. 1) show that the effect of N M D A reached a plateau at about 30/aM, at which concentration there was a 2-3-fold increase in release.
100-
0 Q
....
ii
W
i/I
KETAMIP 1 uM
/
w W * i i
W
, w
I
--w Z
CONTRC
x
I
0
~
O-
I i 3
, 10
~ 31
CONCENTRATION
i 100
'"l 316
(+)PCMI 0.1 uM
uM
OF N M D A
Fig. I. Dose-response curves showing the effect of N M D A on the increase in release of radioactivity from cortical slices preloaded with pH]choline. Each point on the graph represents the mean of at least 4 observations and t h e vertical bars represent S.E.M. The addition of ketamine (1 ~M) or ( + ) - P C M P (0.1#M) reduced the response to N M D A to a similar extent in a manner consistent with non-competitive antagonism. Ordinate: percent increase in release of radioactivity above spontaneous efflux. Abscissa: concentration of N M D A in 1 tLM.
373
NMDA 200 -
re
1 0 0 uM
KAINATE
KCI
2 8 0 uM
2 5 mM
"°I
-750
I(3 <
I
_o
Q ,( E LI. O U4 m ,( ILl ..I ILl E U4 G0 9[ lU a: U Z i
-1000
|U
"I I
1OO
-500
II I|
mg ||
i
01 |1
in ||
SO -
°"
'| !,
TI
i! el |U
ml
i|! II
0
Mg KET
-50-
SuM
~1
-250
B! |!
Mg KET
0.1
KET
SuM
mM
5uM
Fig. 2. Histograms showing the effect of ketamine (5 #M) and Mg 2+ (0.1 mM) on the increase in activity evoked by NMDA (100/tM), kainate (250 pM) and KCI (25 mM). The vertical bars indicate S.E.M. of at least 4 observations. Ordinate: percent increase in release of radioactivity above spontaneous efflux. Note the separate scale for KCl-induced release.
Similar increases in release were achieved by addition of kainate (125-500 pM) or KC1 (10-25 mM), but these effects were not reduced by the addition of Mg 2+ or APV at doses which antagonized NMDA. An example of this selective effect of Mg 2+ (0.1 mM) is shown in the histograms of Fig. 2. Ketamine (0,1-5 pM) also reduced the action of N M D A in a selective manner, neither the spontaneous release nor the enhanced release in the presence of kainate or KCI being affected. An example of this action of ketamine is illustrated in Fig. 2. In this experiment, 5 #M ketamine abolished the release evoked by NMDA. Concentrations of ketamine as low as 0.1/tM clearly inhibited the actions of N M D A and, although there was variation between individual experiments, dose-response curves for this antagonistic effect of ketamine suggest that the IC50 is between 0.1 and 0.5 /tM. Dose-response curves for NMDA-evoked release in the presence of ketamine (1 pM) showed a clear shift to the right with a decrease in slope and in the maximum effect of N M D A (Fig. 1). A similar non-parallel shift was also obtained with another
374 dissociative anaesthetic, (+)-phenyl-methyl-cyclohexylpiperidine ( P C M P ) (0.1 /~M; Fig. 1). Such changes suggest that the nature of the N M D A antagonism is n o n - c o m petitive. N-Allylnormetazocine (0.1-3 /~M), the prototypical sigma opiate [7], also reduced N M D A - e v o k e d A C h release, Estimates o f relative potency suggested that ( + ) - P C M P was about 10 times more potent than ketamine, whereas N-allylnormetazocine was about equipotent with ketamine. Our results support the view that N M D A - e v o k e d release o f A C h from rat brain slices has similar pharmacological characteristics to N M D A - e v o k e d excitation o f central neurones [4] and that dissociative anaesthetics/sigma opiates are selective N M D A antagonists in this release paradigm. They also suggest that ketamine does not act in a competitive manner at the N M D A recognition site, a finding in agreement with the failure o f excitatory amino acids to inhibit phencyclidine/sigma binding [9, 14] and of ketamine to inhibit binding at the N M D A receptor (J.C. Watkins and H.J. Olverman personal communication). Whether ketamine acts at the N M D A activated channel, e.g. in a manner similar to Mg 2+ [8], remains to be determined. Similarly, it is u n k n o w n to what extent N M D A antagonism contributes to be behavioural effects of dissociative anaesthetics and sigma opiates, which in m a n resemble those o f schizophrenia [2], but it is perhaps not without significance that the relative potencies o f ketamine, ( + ) - P C M P and N-allylnormetazocine in this study and o f phencyclidine, cyclazocine and N-allylnormetazocine in a similar study [1 1], are in close agreement with those obtained in behavioural studies [3, 10]. A potential synaptic substrate for such actions is the recently elucidated N M D A receptormediated synaptic excitation in the cerebral cortex which is reduced by Mg 2+, ketamine and A P V [6]. We are grateful for supplies of ketamine, ( + ) - P C M P and N-allylnormetazocine from W a r n e r - L a m b e r t , U.K. Ltd., Dr. R o n Browne and N I D A , respectively. This work was supported by the N H M R C o f Australia and by a Royal Society travel grant to D.L.
I Anis, N.A., Berry, S.C., Burton, N.R. and Lodge, D., The dissociative anaesthetics, ketamine and phencyclidine, selectively reduce excitation of central mammalian neurones by N-methylaspartate, Brit. J. Pharmacol., 79 (1983) 565 575. 2 Domino, E.F. and Luby, E.D., Abnormal mental states induced by phencyclidine as a model for schizophrenia. In E.F. Domino (Ed.), PCP (Phencyclidine): Historical and Current Perspectives, NPP Books, Ann Arbor, MI, 1981, pp. 401 413. 3 Herling, S. and Woods, J.H., Discriminative stimulus effects of narcotics: evidence for multiple receptor-mediated actions, Life Sci., 28 (1981) 1571 1587. 4 Lehmann, J. and Scanon, B,, Characterization of the excitatory amino acid receptor-mediated release of [3H]acetylcholinefrom rat striatal slices, Brain Res., 252 (1982) 77 89. 5 Lodge, D. and Berry, S.C., Psychotomimetic effects of sigma opiates may be mediated by block of central excitatory synapses utilising receptors for aspartate-like amino acids. In R. Bandler (Ed.), Modulation of Sensorimotor Activity during Alterations in Behavioural States, Alan R. Liss, New York, 1984, pp. 503-518.
375 6 Lodge, D., Thomson, A.M. and West, D.C., A Mg 2+ and ketamine-sensitive e.p.s.p, in slices of rat somatosensory cortex: a role for N-methylaspartate receptors, J. Physiol. (Lond.), 358 (1985) 22P. 7 Martin, W.R., Eades, C.G., Thompson, J.A., Huppler, R.E. and Gilbert, P.E., The effects of morphine- and nalorphine-like drugs in the nondependent and morphine dependent chronic spinal dog, J. Pharmacol. Exp. Ther., 197 (1976) 517-532. 8 Nowak, L., Bregestovski, P., Ascher, P,, Herbet, A. and Prochiantz, A., Magnesium gates glutamateactivated channels in mouse central neurones, Nature (Lond.), 307 (1984) 462-465. 9 Quirion, R., Hammer, R.P., Herkenham, M. and Pert, C.B., Phencyclidine (Angel Dust)/sigma 'opiate' receptor visualisation by tritium sensitive film, Proc. Natl. Acad. Sci, USA, 78 (1981) 5881-5885. 10 Shannon, H.E., Pharmacological evaluation of N-allylnormetazocine (SKF 10,047) on the basis of its discrimination stimulus properties in the rat, J. Pharmacol. Exp. Ther., 225 (1983) 144-152. 11 Snell, L.D. and Johnson, K.M., Phencyclidine antagonism of N-methyl-o-aspartate induced acetylcholine release from rat striatal slices, Fed. Proc., 43 (1984) 953. 12 Spence, I., Drew, C., Johnston, G.A.R. and Lodge, D., Acute effects of lead at central synapses in vitro, Brain Res., in press. 13 Watkins, J.C. and Evans, R.H., Excitatory amino acid transmitters, Ann. Rev. Pharmacol. Toxicol., 21 (1981) 165-204. 14 Zukin, R.S. and Zukin, S.R., Demonstration of [3H]cyclazocine binding to multiple opiate receptor sites, Molec. Pharmacol., 20 (1981) 246--254.