Brain Research, 551 (1991) 331-333 (~) 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 A D ON1S 000689939124702D
331
BRES 24702
Calcium channel blockers and excitatory amino acids Ralph Karler, Larry D. Calder and Stuart A. Turkanis Department of Pharmacology, University of Utah School of Medicine, Salt Lake City, UT 84132 (U.S.A.)
(Accepted 12 March 1991) Key words: Calcium channel blocker; Excitatory amino acid; N-Methyi-D-asparticacid; Kainic acid; Anticonvulsant; Neurodegenerative
disease; Epilepsy
The calcium channel blockers (CCB), diltiazem, verapamil and nifedipine, antagonize in mice both N-methyl-DL-aspartate-(NMDLA) and kainate-induced convulsions, which were not affected by carbamazepine and ethosuximide. The CCB, on the other hand, were ineffective against convulsions induced by bicuculline, pentylenetetrazol and electroshock. The results suggest that the CCB may be efficacious in the treatment of those neurodegenerative diseases putatively caused by the excitatory amino acids.
The excitatory amino acids (EAA) have assumed considerable significance in recent years for at least two reasons: first, they are considered to be the neurotransmitters for a major component of CNS function, the excitatory system; and, secondly, they have been implicated in a variety of pathological states 9. For these reasons, a number of E A A antagonists - - ranging from competitive to non-competitive - - have been investigated; however, most of these agents are experimental drugs that are not currently available for clinical use. Our present report on the antagonism of E A A effects by the calcium channel blockers (CCB) thus provides an opportunity for clinical evaluation of these commonly used drugs in disorders that putatively involve the E A A . The studies were conducted on male CF-1 mice (20-25 g). All drugs were prepared in isotonic saline solution; bicuculline required acidification, while nifedipine and carbamazepine were suspended with the aid of 1% Tween and ultrasound 13. Anticonvulsants were administered intraperitoneally 30 min prior to a convulsant test. Chemical convulsants were administered by tail-vein infusion 6. Rates of infusion for N-methyl-DL-aspartate, (NMDLA) (20 mg/ml), kainate (15 mg/ml) and pentyienetetrazol (3 mg/ml) were 0.54 ml/min; for bicuculline (0.1 mg/ml), 0.2 ml/min. The maximal electroshock threshold (MEST) was determined as previously described 5. The data in Table I illustrate that the CCB nonselectively antagonize convulsions induced by either N M D L A or kainate. These results contrast with the
selective antagonism exhibited by CPP and DNQX; CPP antagonizes only NMDLA- while D N Q X blocks only kainate-induced convulsions. Carbamazepine and ethosuximide, two antiepileptic prototypes, lack activity against either of the E A A agonists. Fig. 1 illustrates the dose-response relationships between the 3 CCB and their anticonvulsant activity against N M D L A - and kainate-induced convulsions. The results indicate that the responses are proportional to the dose within the dosage ranges investigated, and they provide a basis for the selection of doses in subsequent experiments. The data in Table II illustrate that diltiazem, in a dose that produces a marked anticonvulsant effect against N M D L A and kainate, lacks anticonvulsant activity in 3 standard convulsive tests. As expected, carbamazepine was selective in the MEST test and ethosuximide was selective against pentylenetetrazo112. None of the drugs in their respective anticonvulsant doses affected the convulsive threshold to bicuculline, the G A B A antagonist. Previous studies have demonstrated the existence of high-affinity binding sites in the CNS for the dihydropyridine-type of CCB4;however, the function of these sites is not known, even though the CCB have been reported to produce a number of behavioral effects3. That these drugs can produce CNS effects is not surprising in view of the important role of calcium in neuronal functions 7. The data presented in the work described above demonstrate that the CCB can antagonize the convulsant effects elicited by the E A A , N M D L A and kainate. This
Correspondence: R. Karler, Department of Pharmacology, Rm. 2C219, University of Utah School of Medicine, Salt Lake City, UT 84132,
U.S.A.
332 60O
.... _T ~,r,lil
I'ABLE II
%
7oo i
~,~
FI
]
Comparative anticonvulsant activity of diltiazem, carbamazepine and ethosuximide
500
Anticonvulsants were administered i.p. 30 min prior to convulsive threshold tests; doses in parentheses represent mg/kg. Bicucullinc and pentylenetetrazol minimal convulsive thresholds determined by i.v. administration; MEST determined with corneal electrodes. Values represent means and standard deviations from groups of 10 animals.
400 o']
500
E]
,,"
300 400
Z
Anticonvulsants
200
300 i 0
,
i 10
,
i 20
,
i 30
,
i 40
i 50
J
mg/kg
,
i 10
,
i 2O
,
i 3O
,
r 4O
,
i 5O
mg/kg
Fig. 1. Dose-response curves for the anticonvulsant activity of diltiazem, verapamil and nifedipine against NMDLA- and kainateinduced convulsions. Each point represents a mean threshold for 10 animals; the vertical bars represent S.D. © = nifedipine; [] = diitiazem; ~ = verapamil.
Control Diltiazem (30) Carbamazepine (30) Ethosuximide (200)
Convulsive threshold Bicuculline (mg/kg)
Pentylenetetrazol (mg/kg)
MEST (mA)
0.7 + 0.1 0.7 _+0.1 0.7 +_0.1 0.6 + 0.1
32 _+6 34 +_5 35 + 4 61 + 9*
7.2 i 0.4 6.7 _+0.5 11.4 + 3.5* 7.6 _+0.5
* Significantlydifferent from control by one-way analysis of variance and least significant difference test (P < 0.01). generalized antagonism of both N M D A and n o n - N M D A receptor activation is in contrast to the differential effects of the selective antagonists, CPP and D N Q X , which block N M D L A and kainate, respectively. The anticonvulsant activity of the CCB appears to be relatively selective for the E A A because diltiazem, in the doses studied, lacked anticonvulsant activity in 3 standard convulsant tests, bicuculline, pentylenetetrazol and the M E S T tests. Furthermore, the prototype antiepileptic drugs, carbamazepine and ethosuximide, in anticonvul-
TABLE I Anticonvulsant activity of the calcium channel blockers against EAA-induced convulsions
Anticonvulsants were administered i.p. 30 min prior to convulsive threshold tests; doses in parentheses represent mg/kg. Minimal convulsive thresholds determined for NMDLA and kainate by i.v. administration. Each value represents the mean and standard deviation obtained from 10 animals. Anticonvulsants
Convulsive threshold (mg/kg) NMDLA
Kainate
Control Diltiazem (30) Control Verapamil (30) Control Nifedipine (30)
417 _+52 705 + 61" 332 + 22 557 + 63* 349 + 48 475 + 33*
256 + 30 429 + 19" 234 + 33 409 + 52* 241 + 16 393 + 38*
Control CPP (4) DNQX (100)
369 + 51 495 + 95* 385 +_27
236 + 45 278 _+34 326 _+57*
Control Carbamazepine (30) Ethosuximide (200)
333 _+34 326 __+38 342 + 25
285 + 35 320 + 55 288 + 50
* Significantlydifferent from control by one-way analysis of variance and least significantdifference test (P < 0.01).
sant doses, were ineffective against E A A - i n d u c e d convulsions, which illustrates the relative selectivity of these agents. The ability of the CCB to antagonize E A A induced convulsions suggests that calcium influx participates in the mechanism of the convulsions and that the calcium influx involves L-type channels. The potential role of the E A A and voltage-sensitive calcium channels in epilepsy has been considered by many investigatorsS'l°'u; and one of the CCB, flunarizine, has been clinically tested in epileptics by other investigators t. In all instances, the trials have been limited to add-on therapy; nevertheless, the drug was generally reported to produce a significant reduction in seizure frequency, which implies that at least some clinical seizures may involve the E A A system, and, if so, then the CCB as a class may be clinically useful antiepileptic agents. The neurotoxicity of the E A A has been implicated in the pathogenesis of a wide variety of diseases of the CNS2; these disorders result from acute brain injuries due to status epilepticus, stroke, hypoglycemia and physical trauma. E A A toxicity may also be involved in chronic degenerative diseases, including neurolathyrism, G u a m amyotrophic lateral sclerosis/parkinsonism/dementia complex, classical amyotrophic lateral sclerosis, sulfite oxidase deficiency, olivoponto cerebellar degeneration, H u n t i n g t o n ' s disease, Alzheimer's disease, parkinsonism, Wernicke/Korsakoff syndrome and J a k o b - C r e u t z feldt syndrome. The potential role of the E A A in these disorders suggests that their antagonists may ultimately contribute significantily to our therapeutic armamentarium; however, most of the potentially useful antagonists currently u n d e r laboratory study are years away from
333 clinical trial. In contrast, the CCB are currently available clinically, and because they have a long track record of
safety, they can be tested immediately for clinical efficacy
1 Binnie, C.D., Potential antiepileptic drugs. Flunarizine and other calcium entry blockers. In R. Levy, R. Mattson, B. Meldrum, J.K. Penry and RE. Dreifuss (Eds.), Antiepileptic Drugs, 3rd edn., Raven, New York, 1989, pp. 971-982. 2 Choi, D., Glutamate neurotoxicity and diseases of the nervous system, Neuron, 1 (1988) 623-634. 3 Hoffmeister, F., Benz, U., Heize, H., Krause, P. and Neuser, U., The behavioral effects of nimodipine in animals, Arzneimittelforschung, 32 (1982) 347-360. 4 Janis, R.A., Bellemann, P., Sarmiento, J.G. and Triggle, D.J., The dihydropyridine receptors. In A. Fleckenstein and C. van Breemen (Eds.), Cardiovascular Effects of Dihydropyridine Cad+ Channel Antagonists and Agonists, Springer, Berlin, 1985, pp. 140-156. 5 Karler, R., Calder, L.D. and Turkanis, S.A., Prolonged CNS hyperexcitability in mice after a single exposure to delta9-tetrahydrocannabinol, Neuropharmacology, 25 (1986) 441446. 6 Karler, R., Chaudhry, I.A., Calder, L.D. and Turkanis, S.A., Amphetamine behavioral sensitization and the excitatory amino aids, Brain Research, 537 (1990) 76-82.
7 Kennedy, M.B., Regulation of neuronal function by calcium, Trends Neurosci., 12 (1989) 417-420. 8 Meldrum, B., Excitatory amino acids and epilepsy. In T.P. Hicks, D. Lodge and H. McLennan (Eds.), Excitatory Amino Acid Transmission, Liss, New York, 1987, pp. 189-196. 9 Olney, J.W., Excitotoxic amino acids and neuropsychiatric disorders, Annu. Rev. Pharmacol. Toxicol., 30 (1990) 47-71. 10 Prince, D.A. and Connors, B.W., Mechanisms of interictal epileptogenesis, Adv. Neurol., 44 (1986) 275-299. 11 Sato, K., Morimoto, K. and Okamoto, M., Anticonvutsant action of a non-competitive antagonist of NMDA receptors (MK801) in the kindling model of epilepsy, Brain Research, 463 (1988) 12-20. 12 Swinyard, E.A., Woodhead, J.H., White, H.S. and Franklin, M.R., General principles, experimental selection, quantification and evaluation of anticonvulsants. In R. Levy, R. Mattson, B. Meldrum, J.K. Penry and F.E. Dreifuss (Eds.), Antiepileptic Drugs, 3rd edn., Raven, New York, 1989, pp. 85-102. 13 Turkanis, S.A., Cely, W., Olsen, D.M. and Karler, R., Anticonvulsant properties of cannabidiol, Res. Commun. Chem. Pathol. Pharmacol., 8 (1974) 231-246.
in the CNS disorders that putatively involve the E A A .
331
BRES 24702
Calcium channel blockers and excitatory amino acids Ralph Karler, Larry D. Calder and Stuart A. Turkanis Department of Pharmacology, University of Utah School of Medicine, Salt Lake City, UT 84132 (U.S.A.)
(Accepted 12 March 1991) Key words: Calcium channel blocker; Excitatory amino acid; N-Methyi-D-asparticacid; Kainic acid; Anticonvulsant; Neurodegenerative
disease; Epilepsy
The calcium channel blockers (CCB), diltiazem, verapamil and nifedipine, antagonize in mice both N-methyl-DL-aspartate-(NMDLA) and kainate-induced convulsions, which were not affected by carbamazepine and ethosuximide. The CCB, on the other hand, were ineffective against convulsions induced by bicuculline, pentylenetetrazol and electroshock. The results suggest that the CCB may be efficacious in the treatment of those neurodegenerative diseases putatively caused by the excitatory amino acids.
The excitatory amino acids (EAA) have assumed considerable significance in recent years for at least two reasons: first, they are considered to be the neurotransmitters for a major component of CNS function, the excitatory system; and, secondly, they have been implicated in a variety of pathological states 9. For these reasons, a number of E A A antagonists - - ranging from competitive to non-competitive - - have been investigated; however, most of these agents are experimental drugs that are not currently available for clinical use. Our present report on the antagonism of E A A effects by the calcium channel blockers (CCB) thus provides an opportunity for clinical evaluation of these commonly used drugs in disorders that putatively involve the E A A . The studies were conducted on male CF-1 mice (20-25 g). All drugs were prepared in isotonic saline solution; bicuculline required acidification, while nifedipine and carbamazepine were suspended with the aid of 1% Tween and ultrasound 13. Anticonvulsants were administered intraperitoneally 30 min prior to a convulsant test. Chemical convulsants were administered by tail-vein infusion 6. Rates of infusion for N-methyl-DL-aspartate, (NMDLA) (20 mg/ml), kainate (15 mg/ml) and pentyienetetrazol (3 mg/ml) were 0.54 ml/min; for bicuculline (0.1 mg/ml), 0.2 ml/min. The maximal electroshock threshold (MEST) was determined as previously described 5. The data in Table I illustrate that the CCB nonselectively antagonize convulsions induced by either N M D L A or kainate. These results contrast with the
selective antagonism exhibited by CPP and DNQX; CPP antagonizes only NMDLA- while D N Q X blocks only kainate-induced convulsions. Carbamazepine and ethosuximide, two antiepileptic prototypes, lack activity against either of the E A A agonists. Fig. 1 illustrates the dose-response relationships between the 3 CCB and their anticonvulsant activity against N M D L A - and kainate-induced convulsions. The results indicate that the responses are proportional to the dose within the dosage ranges investigated, and they provide a basis for the selection of doses in subsequent experiments. The data in Table II illustrate that diltiazem, in a dose that produces a marked anticonvulsant effect against N M D L A and kainate, lacks anticonvulsant activity in 3 standard convulsive tests. As expected, carbamazepine was selective in the MEST test and ethosuximide was selective against pentylenetetrazo112. None of the drugs in their respective anticonvulsant doses affected the convulsive threshold to bicuculline, the G A B A antagonist. Previous studies have demonstrated the existence of high-affinity binding sites in the CNS for the dihydropyridine-type of CCB4;however, the function of these sites is not known, even though the CCB have been reported to produce a number of behavioral effects3. That these drugs can produce CNS effects is not surprising in view of the important role of calcium in neuronal functions 7. The data presented in the work described above demonstrate that the CCB can antagonize the convulsant effects elicited by the E A A , N M D L A and kainate. This
Correspondence: R. Karler, Department of Pharmacology, Rm. 2C219, University of Utah School of Medicine, Salt Lake City, UT 84132,
U.S.A.
332 60O
.... _T ~,r,lil
I'ABLE II
%
7oo i
~,~
FI
]
Comparative anticonvulsant activity of diltiazem, carbamazepine and ethosuximide
500
Anticonvulsants were administered i.p. 30 min prior to convulsive threshold tests; doses in parentheses represent mg/kg. Bicucullinc and pentylenetetrazol minimal convulsive thresholds determined by i.v. administration; MEST determined with corneal electrodes. Values represent means and standard deviations from groups of 10 animals.
400 o']
500
E]
,,"
300 400
Z
Anticonvulsants
200
300 i 0
,
i 10
,
i 20
,
i 30
,
i 40
i 50
J
mg/kg
,
i 10
,
i 2O
,
i 3O
,
r 4O
,
i 5O
mg/kg
Fig. 1. Dose-response curves for the anticonvulsant activity of diltiazem, verapamil and nifedipine against NMDLA- and kainateinduced convulsions. Each point represents a mean threshold for 10 animals; the vertical bars represent S.D. © = nifedipine; [] = diitiazem; ~ = verapamil.
Control Diltiazem (30) Carbamazepine (30) Ethosuximide (200)
Convulsive threshold Bicuculline (mg/kg)
Pentylenetetrazol (mg/kg)
MEST (mA)
0.7 + 0.1 0.7 _+0.1 0.7 +_0.1 0.6 + 0.1
32 _+6 34 +_5 35 + 4 61 + 9*
7.2 i 0.4 6.7 _+0.5 11.4 + 3.5* 7.6 _+0.5
* Significantlydifferent from control by one-way analysis of variance and least significant difference test (P < 0.01). generalized antagonism of both N M D A and n o n - N M D A receptor activation is in contrast to the differential effects of the selective antagonists, CPP and D N Q X , which block N M D L A and kainate, respectively. The anticonvulsant activity of the CCB appears to be relatively selective for the E A A because diltiazem, in the doses studied, lacked anticonvulsant activity in 3 standard convulsant tests, bicuculline, pentylenetetrazol and the M E S T tests. Furthermore, the prototype antiepileptic drugs, carbamazepine and ethosuximide, in anticonvul-
TABLE I Anticonvulsant activity of the calcium channel blockers against EAA-induced convulsions
Anticonvulsants were administered i.p. 30 min prior to convulsive threshold tests; doses in parentheses represent mg/kg. Minimal convulsive thresholds determined for NMDLA and kainate by i.v. administration. Each value represents the mean and standard deviation obtained from 10 animals. Anticonvulsants
Convulsive threshold (mg/kg) NMDLA
Kainate
Control Diltiazem (30) Control Verapamil (30) Control Nifedipine (30)
417 _+52 705 + 61" 332 + 22 557 + 63* 349 + 48 475 + 33*
256 + 30 429 + 19" 234 + 33 409 + 52* 241 + 16 393 + 38*
Control CPP (4) DNQX (100)
369 + 51 495 + 95* 385 +_27
236 + 45 278 _+34 326 _+57*
Control Carbamazepine (30) Ethosuximide (200)
333 _+34 326 __+38 342 + 25
285 + 35 320 + 55 288 + 50
* Significantlydifferent from control by one-way analysis of variance and least significantdifference test (P < 0.01).
sant doses, were ineffective against E A A - i n d u c e d convulsions, which illustrates the relative selectivity of these agents. The ability of the CCB to antagonize E A A induced convulsions suggests that calcium influx participates in the mechanism of the convulsions and that the calcium influx involves L-type channels. The potential role of the E A A and voltage-sensitive calcium channels in epilepsy has been considered by many investigatorsS'l°'u; and one of the CCB, flunarizine, has been clinically tested in epileptics by other investigators t. In all instances, the trials have been limited to add-on therapy; nevertheless, the drug was generally reported to produce a significant reduction in seizure frequency, which implies that at least some clinical seizures may involve the E A A system, and, if so, then the CCB as a class may be clinically useful antiepileptic agents. The neurotoxicity of the E A A has been implicated in the pathogenesis of a wide variety of diseases of the CNS2; these disorders result from acute brain injuries due to status epilepticus, stroke, hypoglycemia and physical trauma. E A A toxicity may also be involved in chronic degenerative diseases, including neurolathyrism, G u a m amyotrophic lateral sclerosis/parkinsonism/dementia complex, classical amyotrophic lateral sclerosis, sulfite oxidase deficiency, olivoponto cerebellar degeneration, H u n t i n g t o n ' s disease, Alzheimer's disease, parkinsonism, Wernicke/Korsakoff syndrome and J a k o b - C r e u t z feldt syndrome. The potential role of the E A A in these disorders suggests that their antagonists may ultimately contribute significantily to our therapeutic armamentarium; however, most of the potentially useful antagonists currently u n d e r laboratory study are years away from
333 clinical trial. In contrast, the CCB are currently available clinically, and because they have a long track record of
safety, they can be tested immediately for clinical efficacy
1 Binnie, C.D., Potential antiepileptic drugs. Flunarizine and other calcium entry blockers. In R. Levy, R. Mattson, B. Meldrum, J.K. Penry and RE. Dreifuss (Eds.), Antiepileptic Drugs, 3rd edn., Raven, New York, 1989, pp. 971-982. 2 Choi, D., Glutamate neurotoxicity and diseases of the nervous system, Neuron, 1 (1988) 623-634. 3 Hoffmeister, F., Benz, U., Heize, H., Krause, P. and Neuser, U., The behavioral effects of nimodipine in animals, Arzneimittelforschung, 32 (1982) 347-360. 4 Janis, R.A., Bellemann, P., Sarmiento, J.G. and Triggle, D.J., The dihydropyridine receptors. In A. Fleckenstein and C. van Breemen (Eds.), Cardiovascular Effects of Dihydropyridine Cad+ Channel Antagonists and Agonists, Springer, Berlin, 1985, pp. 140-156. 5 Karler, R., Calder, L.D. and Turkanis, S.A., Prolonged CNS hyperexcitability in mice after a single exposure to delta9-tetrahydrocannabinol, Neuropharmacology, 25 (1986) 441446. 6 Karler, R., Chaudhry, I.A., Calder, L.D. and Turkanis, S.A., Amphetamine behavioral sensitization and the excitatory amino aids, Brain Research, 537 (1990) 76-82.
7 Kennedy, M.B., Regulation of neuronal function by calcium, Trends Neurosci., 12 (1989) 417-420. 8 Meldrum, B., Excitatory amino acids and epilepsy. In T.P. Hicks, D. Lodge and H. McLennan (Eds.), Excitatory Amino Acid Transmission, Liss, New York, 1987, pp. 189-196. 9 Olney, J.W., Excitotoxic amino acids and neuropsychiatric disorders, Annu. Rev. Pharmacol. Toxicol., 30 (1990) 47-71. 10 Prince, D.A. and Connors, B.W., Mechanisms of interictal epileptogenesis, Adv. Neurol., 44 (1986) 275-299. 11 Sato, K., Morimoto, K. and Okamoto, M., Anticonvutsant action of a non-competitive antagonist of NMDA receptors (MK801) in the kindling model of epilepsy, Brain Research, 463 (1988) 12-20. 12 Swinyard, E.A., Woodhead, J.H., White, H.S. and Franklin, M.R., General principles, experimental selection, quantification and evaluation of anticonvulsants. In R. Levy, R. Mattson, B. Meldrum, J.K. Penry and F.E. Dreifuss (Eds.), Antiepileptic Drugs, 3rd edn., Raven, New York, 1989, pp. 85-102. 13 Turkanis, S.A., Cely, W., Olsen, D.M. and Karler, R., Anticonvulsant properties of cannabidiol, Res. Commun. Chem. Pathol. Pharmacol., 8 (1974) 231-246.
in the CNS disorders that putatively involve the E A A .
