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The Relationship Between Anticonvulsant Activity and Receptor Affinity of N-methyl-D-aspartate Antagonists in Epileptic Fowl
Neuropharmacology Vo1.28, No.7, ~~~753-756, 1989 Printed in Great Britain
PRELIMINARY
0028-3908/89 $3.00+0.00 Pergamon Press plc
NOTES
The Relationship Between Anticonvulsant Activity and Feceptor Affinity of N-methyl-D-aspartats Antagonists in Epileptic Fowl S.C.J.
Pedder, R. Wilcox, J.M. Tuchek, D.D. Johnson* and R.D. Crawford
**
Department of Pharmacology College of Medicine and Department of Animal & Poultry Science** University of Saskatchewan Saskatoon, Saskatchewan S7N OK0 Canada (Accepted 14 Aphie 1989) The N-methyl-D-aspartate (NNDA) receptor antagonists [(3-(i)2-carboxypiperazin-4yl)-propyl-l-phosphonic acid (CPP), 5 Z-amino-7-phosphonoheptanoic acid (2AP7). + 2-amino-5-phosphcnovaleric acid (2AP51, D-o-aminoadipic acid (a AA), and fo ,e-diaminopimelic acid (DAP)] were tested for anticonvulsant activity in epileptic chickens. There was a high correlation between anticonvulsant potencies (ED,U) and the affinity for the NMDA receptor measured by displacement of L-r HIglutamate from synaptosomal membranes. The high seizure susceptibility is not due to abnormalities in the KNDA receptor as comparison of KD, B and K. values in synaptosomal preparations from epileptic and non-epileptic chickens in%'dated nh differences in NMDA receptor binding receptor characteristics. Key Nerds:
NMDA receptor antagonists, Anticonvulsant Activity, Epileptic Chickens
Evidence that both glutamate and aspartate are major excitatory neurotransmitters in the mammalian central nervous system is continuing to accumulate (Robinson and Coyle, 1987). At least three distinct receptors appear to exist for the excitatory amino acids and these have been named after the relatively selective agonists: N-methyl-D-aspartate (NMDA), quisqualate, and kainate. A fourth receptor, which selectively binds 2-amino-4-phosphonobutyric acid (2AP4), has been prOFOSed (Fagg, Foster, Elena and Cotman, 19RZ). Although it is now clear that NMDA antagonists are potent anticonvulsants in a number of animal models of epilepsy (Neldrum, 1985) to our knowledge there have been no attempts to correlate ligand affinity for the NMDA receptor pith anticonvulsant potency. This may be due to low specific binding observed when using L-[ Hlglutamate for NMDA binding studies. A NMDA selective binding asSay has been described (Monahan and Michel, 1987) which results in a high level of specific L-[ HIglutamate binding to the NNDA sits. Using this procedure we examined the ability of a series of NMDA antagonists to displace L-[ HIglutamate from synaptosomal membranes prepared from epileptic chicken brains and correlated these results with their anticonvulsant potency determined in viva. Since it is possible that the cause of the high seizure susceptibility in epileptic fowl?nzes abnormalities in NMDA receptors, the binding characteristics at this site were compared in epileptic and non-epileptic chickens using Scatchard analyses. The high seizure susceptibility in epileptic chickens is due to an autosomal recessive mutation (Crawford, 1970). Epileptifonn seizures can be produced in homozygotes (epileptics) by intermittent photic stimulation (1PS.Ibut not in heterosygote (carrier) hatchmates (Crichlow and Crawford, 1974). METHODS Qileptic chickens and administratio-n of drugs The chickens were classified as homcsygous (epileptic) or heterosygous (carrier) on the basis of their response to IPS (Crichlow and Crawford, 1974). For the determination of anticonvulsant potency each NMDA antagcnist was administered i.v., in a vclume not exceeding 0.2 ml, thirty minutes before exposure to IPS. At least eight birds were used at each dose and a minimum of 4 doses were used with each drug. In each trial 8 vehicle-treated control epileptic chickens were tasted for seizure activity by exposure to IPS. The mean effective dose (BD50) for each drug was deteminjd by probit analysis. L-r HIglutamate (51.9 Ci/mmol) was purchased from New England Nuclear (Boston, MA, USA). Kainete, D-(XFA, DL-DAP, DL-2AP4 (2-amino-4-phosphonobutyric acid) and non-radioactive L-glutamate were from Sigma (St. Louis, USA) while racemate 2~P5, 2AP7, CPP, AMPA (a-amino-3-hydroxy-S-methylI-isoxazolepropionic acid) and NMDA were purchased from Tocris Neuramin (Essex, UK). The drugs were dissolved in either 0.9% saline (2AP5, 2AP7, CPP) or dimethyl sulphoxide (CLAA, DAP). Receptor binding studies ____ Synaptic plasma membranes were prepared from epileptic and carrier avian brains using the procedure described by Monahan and Niche1 (1987). The membranes were stored at -60°C or at least s 24 hours before being used for receptor binding studies. The assay conditions for L-L HIglutamate binding and displacement studies were also as described by Eronahan and Michel (1987). Under these conditions, L-L HIglutamate binds primarily to the NNDA receptor subtype. Briefly, after incubating the membranes at 37OC for 30 min in 20 vol 50 mm Tris/acetate pH 7.4 buffer and centrifuging (repeated 3x), the final pellet was resuspended in double distilled H 0 to produce a protein concentration of 8.0 mg/ml. Aliquots of this preparation (0.4 mg towry pro Fein) were incubated in 50 mM Tris/acetate , pH 7.4 at O°C for 10 min with 2.0 nM L-1 HIglutamate and Varying concentrations of either cold glutamate, NMDA, AMPA, kainate, 7AP4 or one of the NMDA antagonists 753
Preliminary Notes
754
in a final volme of 1.0 ml. The membranes were pelleted by centrifugation at 20,000 xg, superficially washed and solubilized by heating at 6O-"C for 30 min in 150 ~1 tissue solubilirer (NCS, Amersham). A 100 !.I1aliquot was taken for liquid scintillation counting analysis using 10 ml F~r~?ula 963 (NEN) as scintillant. Nonspecific binding was determined in the presence of either 0.1 r&l glutamate or 0.5 mM NMDA. The K. values were determined from the Cheng-Prusoff eqaaticn. At least five determinations, performed'in duplicate were carried cut for each drug. RESULTS To demonstrate that L-[3H]glutamate was binding mainly to the NMDA receptor subtype, selective ligands for four subclasses of glutamate receptors JNMDA, AMPA, kainate and 2AP4) were used in the displacement studies. The characteristics of L-[ HIglutamate binding were found to be similar to those reported previously (Monahan and Michel, 1967) as shown in Fig.1.
Fig. 1. The ability of glutamate (closed circles), NMDA (open circles), AMPA (open triangles), kainate (closed triangles) and 2AP4 (closed boxes) to displace3the specific binding of 2nM L-[ HIglutamate frcm synaptosomal membranes from the cerebral hemispheres of epileptic chickens. Data presented are averages from the total of 5 separate experiments, each performed in duplicate. DRUG CONC.
I,Ml
Both glutamate (K.=1.3 DM) and NMDA (K.=18.4 DM) potenfly displaced L-13Hlglutamate. AMPA, kainate and 2AP4 khowed little ability'to displace L-[ Hlglutamate even at high (0.1 mM) concentrations. Specific binding determined in the presence of 0.1 mM non-radioactive glutamate or 0.5 mM KMDA was approximately 90% of total binding. All of the NMDA antagonists were found to have anticonvulsant activity in epileptic fowl. 2AP7 and CPP were found to be most potent with EDSO and 95% confidence limits values of 4.97 (1.70-13.0) and 5.62 (1.22-25.9) I~mol/kg respectively, while 2AP5 had an ED value of 48.4 (16.1-145.7) Dmol/kg. UPA and DAP were found to be significantly less poten'? with EDGE values of 354.7 (118.0-963.6) and 397.5 (132.0-1024.7) Hmol/kg respectively. All NMDA antagonists displaced L-[ HIglutamate from the NKDA binding site in synaptosomal membranes. CPP and 2AP7 displayed the highest affinity with K. values of 11.6 J.IMand 15.6 DM respectively. ZAP5 was found to have a slightly lower affinitt (Ki=29.5 NM) while 8AA (129.3 PM) and DAP (149.0 2M) were the least potent. As indicated in Fig. 2, a high correlation (r=0.982) was found between in viva potency and -7 the affinity to the NMDA binding site determined -in vitro suggesting that this Interaction mediates the anticonvulsant effect.
Fig. 2. Correlation between the anticonvulsant potency and binding affinity for a series of NMDA antagcnists in epileptic chickens.
Preliminary Notes
755
To determine if an abno_-alit3 in NFDA binding sites is involved in the seizure susceptibility of epileptic chickens, L-1 HIglutamate binding characteristics for the h?lDA receptor were compared in synaptosomal membranes prepared from epileptic and carrier (non-epileptic) chickens. Results from displacement studies with NIrgA and CPP indicated no differences in K. v lues from 4 epileptic chickens (data not shown). Scatchard analyses (Fig.3) of NMDA-speCifi&L[ HIglutamate binding indicated no differences in B (7.95t1.95 pmol/mg protein) and KD (2.16f0.48 PM) values max between epileptics and carriers.
6 6
70
4-
2
3-
s
h’1g.3. Scatchard analysis of L-f3H1glutamate binding to synaptosomal preparations from epileptic (closed circles) and carrier (open circles) cerebral hemispheres. The data ore from a single representative experiment (from a total of three separate experiments each performed in duplicate).
21~ I BOUNO
[p mole/mgl
DISCUSSION The relative anticonvulsant activity of a series of excitatory amino acid receptor antagonists was first described in audiogenic seizure susceptible mice (Crouchar, Collins and Meldrum, 1962). NMDA receptor antagonists were subsequently reported to have anticonvulsant activity in photosensitive baboons (Meldrum, Croucher, Badman and Collins, 19831, kindled seizures in rats (Peterson, Collins and Bradford, 19831, BS well as against a variety of convulsant chemicals ;csuczvar and Meldrum, 1982). The most potent and selective antagcnists were the D-isomers of longer chain analogues of the dicarboxylic acids with the :!I-carboxylgroup substituted by a phosphono group. 2AP7 showed the greatest anticonvulsant activity (Meldrum, 1985). Subsequently several authors reported that CPP, a 4-substituted piperasine-2-carboxylic acid, was a potent NMDA receptor antagonist in electrophysiological and receptor binding studies and that it had more potent anticonvulsant activity than 2AP7 (Davies, Evans, Herrling, Jones, Olverman, Pook and Watkins, 1986). 3 the present study. ZAP7 and CPP were found to be approximately equipotent in respect to both affinity for the NMDA site and anticonvulsant activity. The failure to observe a major difference between 2AP7 and CPP in this n,odel may be the result of differences in peripheral metabolism and ability to pass the blood brain barrier. However, the relative potency of 2AP5 to 2AP7 in epileptic chickens is similar to that reported in audiogenic seizure susceptible mice (Croucher, Collins and b!eldrum, 1982) and in the kindled amygdala model of epilepsy (Peterson, Collins and Bradford, 1983). oAA and DAP were used in early studies examining excitatory neurotransmission. Although they are selective antagonists at the NMDA site, the present results confirm previous reports that they are weak antagonists with low anticonvulsant activity. The present results suggest that affinity to the LYDA site is a determinant of anticonvulsant potency. The strong correlation (r=0.982, Fig. 2) between the inhibition constants (affinity) of a series of NMDA antagonists and the anticonvulsant activity provides strong support for the hypothesis that this site is a pharmacologically relevant receptor. An atnormality in excitatory amino acid receptors and/or their associated ion channels could conceivably be a cause of the high seizure susceptibility. The lack of differences in binding characteristics between epileptics and carriers suggests that the NMDA receptor is not the site of en abnormality causing the increased seizure susceptibility of the epileptic birds. Acknowledgements: MFR604.
This project was funded by the Medical Research Council of Canada, Grant
REFERENCES Crawford R.D. (1970) Cpileptiform seizures in dcmestic fowl. J. Hered. 61: 185-188. Crichlow E.C. and Crawford R.D. (1974) Epileptic seizures in domestic fowl II. Intermittent light stimulation and the electroencephalogram. Can. J. Physiol. Pharmacol. 52: 424-429. Croucher M.J., Collins J.F. and Meldrum B.S. (1982) Anticonwlsant action of excitatory amino acids antagonists. Science 216: 899-901. Csucswar S.J. and Meldrum B.S. (1982) Protection against chemically induced seizures by 2-amino-7-phosphonoheptanoic acid. Fur. J. Pharmacol. 83: 335-338. Davies J., Evans R.H., Herrling P.L., Jones A.M., Olverman H.J., Pook P. and Watkins J.C. (1986) CCP, a new potSnt and selective NMDA antagonist. Depression of central neuron responses, affinity for [ HID-AP5 binding sites on brain membranes and anticonvulsant activity. Brain Res. 382: 169-173.
Preliminary Notes
Fagg G.E., Foster A.C., Mena E.G. and Cotman C.K. (1982) Chloride and calcium ions reveal a pharmacologically distinct population of L-glutamate binding sites in synaptic membranes: correspondence between biochemical and electrophysiological data. J. Eeurosci. 2: 958-965. I,!eldrumB.S., Croucher E.J., Badman G. and Collins J.F. (1983) Antiepileptic action of excitatory amino-acid antagonists in the photosensitive baboon, Papio papio Neurosci. Lett. 39: 101-104. Meldrum B. (1985) Possible therapeutic applications of antagonists of excitatory amino acid neurotransmitters. Clin. Sci. 68: 113-172. Eonahan J.B. and Eichel J. (1987) Identification and characterization of an E!-methyl-D-aspartatespecific L-['HIglutamate recognition site in synaptic plasma membranes. J. Neurochem. 48: 1699-1768. Peterson D.R., Collins J.F. and Bradford H.F. (1983) The kindled amygdala model of epilepsy: anticonvulsant action of amino acid antagonists. Brain Res. 275: 169-172. Robinson B.B. and Coyle J.T. (1987) Glutamate and related acidic excitatory neurotransnitters: from basic science to clinic application. FASEB J. 1: 446-455.
PRELIMINARY
0028-3908/89 $3.00+0.00 Pergamon Press plc
NOTES
The Relationship Between Anticonvulsant Activity and Feceptor Affinity of N-methyl-D-aspartats Antagonists in Epileptic Fowl S.C.J.
Pedder, R. Wilcox, J.M. Tuchek, D.D. Johnson* and R.D. Crawford
**
Department of Pharmacology College of Medicine and Department of Animal & Poultry Science** University of Saskatchewan Saskatoon, Saskatchewan S7N OK0 Canada (Accepted 14 Aphie 1989) The N-methyl-D-aspartate (NNDA) receptor antagonists [(3-(i)2-carboxypiperazin-4yl)-propyl-l-phosphonic acid (CPP), 5 Z-amino-7-phosphonoheptanoic acid (2AP7). + 2-amino-5-phosphcnovaleric acid (2AP51, D-o-aminoadipic acid (a AA), and fo ,e-diaminopimelic acid (DAP)] were tested for anticonvulsant activity in epileptic chickens. There was a high correlation between anticonvulsant potencies (ED,U) and the affinity for the NMDA receptor measured by displacement of L-r HIglutamate from synaptosomal membranes. The high seizure susceptibility is not due to abnormalities in the KNDA receptor as comparison of KD, B and K. values in synaptosomal preparations from epileptic and non-epileptic chickens in%'dated nh differences in NMDA receptor binding receptor characteristics. Key Nerds:
NMDA receptor antagonists, Anticonvulsant Activity, Epileptic Chickens
Evidence that both glutamate and aspartate are major excitatory neurotransmitters in the mammalian central nervous system is continuing to accumulate (Robinson and Coyle, 1987). At least three distinct receptors appear to exist for the excitatory amino acids and these have been named after the relatively selective agonists: N-methyl-D-aspartate (NMDA), quisqualate, and kainate. A fourth receptor, which selectively binds 2-amino-4-phosphonobutyric acid (2AP4), has been prOFOSed (Fagg, Foster, Elena and Cotman, 19RZ). Although it is now clear that NMDA antagonists are potent anticonvulsants in a number of animal models of epilepsy (Neldrum, 1985) to our knowledge there have been no attempts to correlate ligand affinity for the NMDA receptor pith anticonvulsant potency. This may be due to low specific binding observed when using L-[ Hlglutamate for NMDA binding studies. A NMDA selective binding asSay has been described (Monahan and Michel, 1987) which results in a high level of specific L-[ HIglutamate binding to the NNDA sits. Using this procedure we examined the ability of a series of NMDA antagonists to displace L-[ HIglutamate from synaptosomal membranes prepared from epileptic chicken brains and correlated these results with their anticonvulsant potency determined in viva. Since it is possible that the cause of the high seizure susceptibility in epileptic fowl?nzes abnormalities in NMDA receptors, the binding characteristics at this site were compared in epileptic and non-epileptic chickens using Scatchard analyses. The high seizure susceptibility in epileptic chickens is due to an autosomal recessive mutation (Crawford, 1970). Epileptifonn seizures can be produced in homozygotes (epileptics) by intermittent photic stimulation (1PS.Ibut not in heterosygote (carrier) hatchmates (Crichlow and Crawford, 1974). METHODS Qileptic chickens and administratio-n of drugs The chickens were classified as homcsygous (epileptic) or heterosygous (carrier) on the basis of their response to IPS (Crichlow and Crawford, 1974). For the determination of anticonvulsant potency each NMDA antagcnist was administered i.v., in a vclume not exceeding 0.2 ml, thirty minutes before exposure to IPS. At least eight birds were used at each dose and a minimum of 4 doses were used with each drug. In each trial 8 vehicle-treated control epileptic chickens were tasted for seizure activity by exposure to IPS. The mean effective dose (BD50) for each drug was deteminjd by probit analysis. L-r HIglutamate (51.9 Ci/mmol) was purchased from New England Nuclear (Boston, MA, USA). Kainete, D-(XFA, DL-DAP, DL-2AP4 (2-amino-4-phosphonobutyric acid) and non-radioactive L-glutamate were from Sigma (St. Louis, USA) while racemate 2~P5, 2AP7, CPP, AMPA (a-amino-3-hydroxy-S-methylI-isoxazolepropionic acid) and NMDA were purchased from Tocris Neuramin (Essex, UK). The drugs were dissolved in either 0.9% saline (2AP5, 2AP7, CPP) or dimethyl sulphoxide (CLAA, DAP). Receptor binding studies ____ Synaptic plasma membranes were prepared from epileptic and carrier avian brains using the procedure described by Monahan and Niche1 (1987). The membranes were stored at -60°C or at least s 24 hours before being used for receptor binding studies. The assay conditions for L-L HIglutamate binding and displacement studies were also as described by Eronahan and Michel (1987). Under these conditions, L-L HIglutamate binds primarily to the NNDA receptor subtype. Briefly, after incubating the membranes at 37OC for 30 min in 20 vol 50 mm Tris/acetate pH 7.4 buffer and centrifuging (repeated 3x), the final pellet was resuspended in double distilled H 0 to produce a protein concentration of 8.0 mg/ml. Aliquots of this preparation (0.4 mg towry pro Fein) were incubated in 50 mM Tris/acetate , pH 7.4 at O°C for 10 min with 2.0 nM L-1 HIglutamate and Varying concentrations of either cold glutamate, NMDA, AMPA, kainate, 7AP4 or one of the NMDA antagonists 753
Preliminary Notes
754
in a final volme of 1.0 ml. The membranes were pelleted by centrifugation at 20,000 xg, superficially washed and solubilized by heating at 6O-"C for 30 min in 150 ~1 tissue solubilirer (NCS, Amersham). A 100 !.I1aliquot was taken for liquid scintillation counting analysis using 10 ml F~r~?ula 963 (NEN) as scintillant. Nonspecific binding was determined in the presence of either 0.1 r&l glutamate or 0.5 mM NMDA. The K. values were determined from the Cheng-Prusoff eqaaticn. At least five determinations, performed'in duplicate were carried cut for each drug. RESULTS To demonstrate that L-[3H]glutamate was binding mainly to the NMDA receptor subtype, selective ligands for four subclasses of glutamate receptors JNMDA, AMPA, kainate and 2AP4) were used in the displacement studies. The characteristics of L-[ HIglutamate binding were found to be similar to those reported previously (Monahan and Michel, 1967) as shown in Fig.1.
Fig. 1. The ability of glutamate (closed circles), NMDA (open circles), AMPA (open triangles), kainate (closed triangles) and 2AP4 (closed boxes) to displace3the specific binding of 2nM L-[ HIglutamate frcm synaptosomal membranes from the cerebral hemispheres of epileptic chickens. Data presented are averages from the total of 5 separate experiments, each performed in duplicate. DRUG CONC.
I,Ml
Both glutamate (K.=1.3 DM) and NMDA (K.=18.4 DM) potenfly displaced L-13Hlglutamate. AMPA, kainate and 2AP4 khowed little ability'to displace L-[ Hlglutamate even at high (0.1 mM) concentrations. Specific binding determined in the presence of 0.1 mM non-radioactive glutamate or 0.5 mM KMDA was approximately 90% of total binding. All of the NMDA antagonists were found to have anticonvulsant activity in epileptic fowl. 2AP7 and CPP were found to be most potent with EDSO and 95% confidence limits values of 4.97 (1.70-13.0) and 5.62 (1.22-25.9) I~mol/kg respectively, while 2AP5 had an ED value of 48.4 (16.1-145.7) Dmol/kg. UPA and DAP were found to be significantly less poten'? with EDGE values of 354.7 (118.0-963.6) and 397.5 (132.0-1024.7) Hmol/kg respectively. All NMDA antagonists displaced L-[ HIglutamate from the NKDA binding site in synaptosomal membranes. CPP and 2AP7 displayed the highest affinity with K. values of 11.6 J.IMand 15.6 DM respectively. ZAP5 was found to have a slightly lower affinitt (Ki=29.5 NM) while 8AA (129.3 PM) and DAP (149.0 2M) were the least potent. As indicated in Fig. 2, a high correlation (r=0.982) was found between in viva potency and -7 the affinity to the NMDA binding site determined -in vitro suggesting that this Interaction mediates the anticonvulsant effect.
Fig. 2. Correlation between the anticonvulsant potency and binding affinity for a series of NMDA antagcnists in epileptic chickens.
Preliminary Notes
755
To determine if an abno_-alit3 in NFDA binding sites is involved in the seizure susceptibility of epileptic chickens, L-1 HIglutamate binding characteristics for the h?lDA receptor were compared in synaptosomal membranes prepared from epileptic and carrier (non-epileptic) chickens. Results from displacement studies with NIrgA and CPP indicated no differences in K. v lues from 4 epileptic chickens (data not shown). Scatchard analyses (Fig.3) of NMDA-speCifi&L[ HIglutamate binding indicated no differences in B (7.95t1.95 pmol/mg protein) and KD (2.16f0.48 PM) values max between epileptics and carriers.
6 6
70
4-
2
3-
s
h’1g.3. Scatchard analysis of L-f3H1glutamate binding to synaptosomal preparations from epileptic (closed circles) and carrier (open circles) cerebral hemispheres. The data ore from a single representative experiment (from a total of three separate experiments each performed in duplicate).
21~ I BOUNO
[p mole/mgl
DISCUSSION The relative anticonvulsant activity of a series of excitatory amino acid receptor antagonists was first described in audiogenic seizure susceptible mice (Crouchar, Collins and Meldrum, 1962). NMDA receptor antagonists were subsequently reported to have anticonvulsant activity in photosensitive baboons (Meldrum, Croucher, Badman and Collins, 19831, kindled seizures in rats (Peterson, Collins and Bradford, 19831, BS well as against a variety of convulsant chemicals ;csuczvar and Meldrum, 1982). The most potent and selective antagcnists were the D-isomers of longer chain analogues of the dicarboxylic acids with the :!I-carboxylgroup substituted by a phosphono group. 2AP7 showed the greatest anticonvulsant activity (Meldrum, 1985). Subsequently several authors reported that CPP, a 4-substituted piperasine-2-carboxylic acid, was a potent NMDA receptor antagonist in electrophysiological and receptor binding studies and that it had more potent anticonvulsant activity than 2AP7 (Davies, Evans, Herrling, Jones, Olverman, Pook and Watkins, 1986). 3 the present study. ZAP7 and CPP were found to be approximately equipotent in respect to both affinity for the NMDA site and anticonvulsant activity. The failure to observe a major difference between 2AP7 and CPP in this n,odel may be the result of differences in peripheral metabolism and ability to pass the blood brain barrier. However, the relative potency of 2AP5 to 2AP7 in epileptic chickens is similar to that reported in audiogenic seizure susceptible mice (Croucher, Collins and b!eldrum, 1982) and in the kindled amygdala model of epilepsy (Peterson, Collins and Bradford, 1983). oAA and DAP were used in early studies examining excitatory neurotransmission. Although they are selective antagonists at the NMDA site, the present results confirm previous reports that they are weak antagonists with low anticonvulsant activity. The present results suggest that affinity to the LYDA site is a determinant of anticonvulsant potency. The strong correlation (r=0.982, Fig. 2) between the inhibition constants (affinity) of a series of NMDA antagonists and the anticonvulsant activity provides strong support for the hypothesis that this site is a pharmacologically relevant receptor. An atnormality in excitatory amino acid receptors and/or their associated ion channels could conceivably be a cause of the high seizure susceptibility. The lack of differences in binding characteristics between epileptics and carriers suggests that the NMDA receptor is not the site of en abnormality causing the increased seizure susceptibility of the epileptic birds. Acknowledgements: MFR604.
This project was funded by the Medical Research Council of Canada, Grant
REFERENCES Crawford R.D. (1970) Cpileptiform seizures in dcmestic fowl. J. Hered. 61: 185-188. Crichlow E.C. and Crawford R.D. (1974) Epileptic seizures in domestic fowl II. Intermittent light stimulation and the electroencephalogram. Can. J. Physiol. Pharmacol. 52: 424-429. Croucher M.J., Collins J.F. and Meldrum B.S. (1982) Anticonwlsant action of excitatory amino acids antagonists. Science 216: 899-901. Csucswar S.J. and Meldrum B.S. (1982) Protection against chemically induced seizures by 2-amino-7-phosphonoheptanoic acid. Fur. J. Pharmacol. 83: 335-338. Davies J., Evans R.H., Herrling P.L., Jones A.M., Olverman H.J., Pook P. and Watkins J.C. (1986) CCP, a new potSnt and selective NMDA antagonist. Depression of central neuron responses, affinity for [ HID-AP5 binding sites on brain membranes and anticonvulsant activity. Brain Res. 382: 169-173.
Preliminary Notes
Fagg G.E., Foster A.C., Mena E.G. and Cotman C.K. (1982) Chloride and calcium ions reveal a pharmacologically distinct population of L-glutamate binding sites in synaptic membranes: correspondence between biochemical and electrophysiological data. J. Eeurosci. 2: 958-965. I,!eldrumB.S., Croucher E.J., Badman G. and Collins J.F. (1983) Antiepileptic action of excitatory amino-acid antagonists in the photosensitive baboon, Papio papio Neurosci. Lett. 39: 101-104. Meldrum B. (1985) Possible therapeutic applications of antagonists of excitatory amino acid neurotransmitters. Clin. Sci. 68: 113-172. Eonahan J.B. and Eichel J. (1987) Identification and characterization of an E!-methyl-D-aspartatespecific L-['HIglutamate recognition site in synaptic plasma membranes. J. Neurochem. 48: 1699-1768. Peterson D.R., Collins J.F. and Bradford H.F. (1983) The kindled amygdala model of epilepsy: anticonvulsant action of amino acid antagonists. Brain Res. 275: 169-172. Robinson B.B. and Coyle J.T. (1987) Glutamate and related acidic excitatory neurotransnitters: from basic science to clinic application. FASEB J. 1: 446-455.