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Induction of seizures in mice by intracerebroventricular administration of the calcium channel agonist BAY k 8644
Brain Research, 4(12(1987) 399-402
399
Elsevier BRE 22010
Induction of seizures in mice by intracerebroventricular administration of the calcium channel agonist BAY k 8644 Richard C. Shelton t, Jack A. G r e b b 2'3 and William J. Freed 4 l Department of Psychiatry, Vanderbilt University Medical School, Nashville, TN37232 (U.S.A.), 2Nathan Kline Institute, Orangeburg, NY 10962 (U.S.A.), ~Department of Psychiatry, New York University Medical Center, New York, N Y 10016 (U.S.A. ) and 4preclinical Neurosciences Section, Neuropsychiatrv Branch, National lnsitute of Mental Health, Saint Elizabeths Hospital, Washington, DC 20032 (U. S. A. )
(Accepted 16 September 1986) Key words: Calcium: Calcium channel; Calcium channel agonist; Calcium channel inhibitor: Seizure; Anticonvulsant
The calcium channel agonist BAY k 8644 was used to investigate the role of the calcium ion (Ca2+) in epileptogenesis, lntracerebroventricular administration of the compound induced murine seizures that were reversed by calcium channel inhibitors (CCIs) but not by anticonvulsants such as carbamazepine, pentobarbital, and diazepam. The seizures were exacerbated by phenytoin and valproic acid. Chronic administration of CCFs, previously shown to produce down-regulation of the binding of the CCI [3H]nitrendipine, resulted in augmentation of BAY k 8644-induced seizures.
A substantial body of literature suggests that calcium plays an important role in both epileptogenesis and the actions of anticonvulsants. Rapid influx of calcium ions (Ca e+) into neurons occurs prior to spike activity in the hippocampus Is, including that generated by penicillin ~'ls, barium 13, excitatory amino acids ~:, pentatetrazole ~, and electrical current 1~'~2 Seizure threshold can be raised by the application of 1,25-dihydroxyvitamin D 3 that modulates intracellular Ca e+ activity through the stimulation of synthesis of intraneuronal Ca 2+ binding proteins 22. Ca 2+ is apparently also a critical component in kindling 2~. Ca 2+ is required for long-term potentiation of synapses > and transient increases in extracellular Ca 2+ yield long-lasting potentiation and spike formation in kindled preparations -'5. Kindling itself specifically reduces Ca: Ucalmodulin-dependent protein phosphorvlation in rat brain e7. Finally, the reversal of seizures by calcium channel inhibitors (CCI's) in animal models <14-'s and humans I>, supports both the significance of Ca -~+ in epilepsy and the potential role of CCI's as anticonvulsant agents.
Anticonvulsant drugs are known to have effects on Ca2+-dependent mechanisms in the neuron. Phenytoin, for example, reduces Ca 2+ uptake into neurons 9'10'17'23 and inhibits Ca2+-dependent mechanisms such as guanosine-3',5'-monophosphate synthesis 24 and protein phosphorylation 3. Phenytoin has also been shown to inhibit binding of the dihydropyridine calcium channel inhibitor (CCI) [3H]nitrendipine to neuronal membranes, indicating that the inhibition of Ca 2+ flux might be via binding to calcium channel regulatory proteins <9. In order to investigate the relationship of calcium flux and seizure activity more directly, we have used B A Y k 8644, a calcium channel 'agonist' of the dihydropyridine class, thought to have its effects on voltage-dependent calcium channels in cell membranes 7's'tg-21, including those of neurons ~'1~. Recently, significant central effects of relatively low doses of the compound have been reported I. B A Y k 8644 inhibited the binding of [3H]nitrendipine to mouse brain membranes. When administered systemically to animals in dosages of 0.5-5.0 mg/kg, B A Y k 8644
Correspondence: R.C. Shelton, Vanderbilt University Medical School, Medical Center North, Room A-2215, Nashville, TN 37232,
U.S.A.
0006-8993/87,$II3.50© 1987 Elsevier Science Publishers B.V. (Biomedical Division)
400 induced ataxia, decreased motor activity, tail flexion, arched back. limb tonus and clonus, and hyperreactivity to sensory stimuli. The effects were blocked by the dihydropyridine calcium channel antagonist nifedipine (2 mg/kg), but not by verapamil (2 and 6 mg/kg). In the present study, we report that intracerebroventricular injections of small amounts of BAY k 8644 produced seizures, and that these seizures are susceptible to alteration by pharmacological manipulations. Female Swiss-Webster mice (Veterinary Resource Branch. National Institute of Health. Bethesda. MD) were used for all experiments. The sources of compounds were as follows: BAY k 8644: Miles Pharmaceuticals. West Haven. CT: carbamazepine: Ciba-Geigy, Summit. N J; diazepam: Roche Laboratories. Nutley N J; diltiazem: Marion Laboratories. Kansas City, MO; flunarizine: Janssen Pharmaceuncal. New Brunswick. NJ: nifedipine: Pfizer Laboratories. New York. NY: phenytoin: Sigma Chemical Co.. St. Louis. MO: valproic acid: Abbott Laboratories. North Chicago, IL: verapamil: Knoll Pharmaceutical Co.. Whippany, NJ. A dose-response curve for duration of seizures elicited in mice by intracerebroventricular BAY k 8644 was generated (Fig. 1). Mice (n = 8 in each group) were injected intracerebroventricularly 2 with a 5-/~1solution of BAY k 8644 in 10% Tween-80/saline in doses of 6.25. 12.5. 25 and 37.5/~g. The ani-
mals were placed in 16.3 x 23 cm polystyrene cylinders and observed for a total duration of seizures for 60 min postinjection: no seizure lasted longer than this time period. Seizure activity was noted to be of increasing duration and seventy with progressive dosage. Low doses produced tonic contractions with intermittent clonic jerks of the extrcmities and tail flexion. As the dose increased, head bobbing, complex grooming actions (licking of fur. scratching, etc. J. squeaking, jumping, and running occurred. A series of CCIs and anticonvulsant drugs were tested for their effects on BAY k 8644-induced seizures. Mice were pretreated with intraperimneat injections of nifedipine, verapamil, diltiazem, flunarizine. carbamazepme, pentobarbital, diazepam, valproic acid. or phenytoln mixed m a 10% Tween80/saline solution in doses listed m Fig. 2 All drugs were administered 30 min prior to intracerebroventricular injection of 37.5 ug BAY k 8644, except for pentobarbital which was given 5 rain before injection. As summarized in Fig. 2. the CCIs nifediplnc and verapamil completely blocked BAY k 8644-induced seizures and diltiazem m a r k e d b antagonized them Flunarizine. a piperidine-type calcium channel antagonist, had no effect, nor did pentobarbitat, carbamazepine, and diazepam. Valproic acid and phenytoin increased seizure intens~tx and duration: bar60--
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Fig. 1. D o s e - r e s p o n s e curve l m e a n 1- S . E . M . ) for seizures elicited in female S w i s s - W e b s t e r mice (n = 8 in each group), 26-46 g, injected intracerebroventricularly with B A Y k 8644.
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Fig. 2. Seizure duration ( m e a n _~ S.E.M.) m femalc S w i s s - W e b s t e r mice (n = 8 in each group), 14-30 g. pretreated with intraperitoneal injections of CCIs and anticonvulsants. Controls were injected withvehicle only. Significant differences from controls *P < 0.02. **P < 0.007, ***P < O.OO1 (two-tailed t-Test).
401
rel-rolling and some self-mutilation were observed. Three of eight mice pretreated with phenytoin had residual ataxia for at least 2 h after termination of overt seizure activity. A dose-response curve contrasting the effects of nifedipine and phenytoin on B A Y k 8644-induced seizures was generated and is summarized in Fig. 3. Mice were pretreated with 5, 25, and 50 mg/kg of nifedipine and phenytoin by intraperitoneal injection. After 30 min 37.5 #g of B A Y k 8644 was injected and observations were made as in the previous experiments. Chronic treatment of mice with verapamil or nifedipine (but not diltiazem) has been shown to decrease binding of [3H]nitrendipine to neuronal menbranes in a previous experiment 16. To determine whether these alterations in calcium channel antagonist binding were of functional significance, mice were administered nifedipine, verapamil, and diltiazem mixed with powdered food for 28 days. Absolute calculated dosages, corrected for intake, were nifedipine 0.39 rag/g/day, verapamil 0.30 mg/g/day, and diltiazem 0.39 mg/g/day. Animals were given free access to water. After the 26 days, the drugs were withdrawn for 4 days so that the mice could be tested in the absence of CCIs themselves. B A Y k 8644 was ad-
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Fig. 3. The effects of increasing doses of nifedipine and phenytoin on seizure duration in female Swiss-Webster mice (n = 8 in each group), 22-42 g. Mice were pretreated with 5, 25, and 50 mg/kg of nifedipine and phenytoin by intraperitoneal injection. After 30 min, BAY k 8644 37.5/~g was injected intracerebroventricularly. Comparison is to control animals pretreated with vehicle only. Significant differences: *P < 0.05, **P < 0.007 (two-tailed t-test).
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Fig. 4. Dose-response curves of seizure duration in female Swiss-Webster mice (n = 8 in each group), 16-38 g, after withdrawal of nifedipine, verapamil, and diltiazem administered orally for 26 days. Intracerebroventricular BAY k 8644 was administered 4 days after withdrawal of CCIs. Comparisons are to control animals given powdered food only. Significant differences: *P < 0.04, **P < 0.002 (two-tailed t-test).
ministered as in the previous experiments at 6.25, 12.5, and 25/~g, to the pretreated mice and a control group was given powdered food only. The results are given in Fig. 4. These findings give functional significance to the alteration of CCI binding, with the expected order of potency of nifedipine and verapamil being greater than diltiazem. Functionally distinct subtypes of (at least) dihydropyridine calcium channel receptors may exist with excitatory and inhibitory properties 5. Calcium channel agonists and antagonists may, in fact, actually function as partial agonists for the receptor subtypes. The down-regulation of calcium channel binding may, therefore, be predominantly of the inhibitory component, yielding a relative excess in excitatory receptors and, in this model, seizure induction. The findings of these experiments lend further credence to the concept that disturbances of central Ca 2+ regulation is involved in seizure mechanisms. The seizures induced by B A Y k 8644 were not blocked by standard anticonvulsants, but were reversed by the CCIs diltiazem, verapamil, and nifedipine, suggesting that the induction of seizures in this model is a specific calcium channel-mediated event. The exacerbation of seizure activity by valproic acid and phenytoin was an unexpected finding, particularly in view of the apparent binding of phenytoin to
402 n e u r o n a l c a l c i u m c h a n n e l s a n d i n h i b i t i o n o f C a 2÷
t h e i r e f f e c t o n n e u r o n s , t h e s e f i n d i n g s a r e f u r t h e r evt-
flux 9. T h e specific p h a r m a c o l o g i c a l a c t i o n o f t h e s e
d e n c e in s u p p o r t o f C C I t r e a t m e n t in s e i z u r e s a n d
agents requires further investigation. Regardless of by which the CCIs exert
s u g g e s t a p o t e n t i a l m o d e l for t h e m o d u l a t i o n o f activ a t i o n in epilepsy•
1 Boiger, G.T.. Weissman. B.A. and Skolnick. P.. The behavioral effects of the calcium channel agonist BAY k 8644 in the mouse: antagonism by the calcium channel antagorest nifedipine, Naunyn Schmeideberg's Arch. Pharrnacol.. 328 (1985) 373-377• 2 Clark. W.G., Vivonia• C.A. and Baxter. C.F.. Accurate freehand injections into the lateral brain ventricle of the conscious mouse• J. Appl. Physiol., 25 (1968) 319-321. 3 DeLorenzo. R.J.. Calmodulin systems m neuronal excitability: a molecular approach to epilepsy• Ann. Neurol.. 16 (1984] S104-Sl14 4 Desmendt. L.K.C.. Niemegeers• C.J.E. and Janssen P.A.J.. Anticonvulsant properties of cinnanzme and flunanzlne in rats and mice. Arzneim-Forsch (Drug Res. t. 25 (1975) 1408-1413. 5 Dube, G.P.. Baik. Y.H., Vaghy, P.L.. and Schwartz. A.. Nitrendipine potentiates BAY k 8644-induced contraction of isolated porcine coronary artery: evidence for functionally distinct dihydropyridine receptor subtypes. Biochem. Biophys. Res. Comm.. 128 (1985) 1295-1302. 6 Greenberg, D.A.. Cooper. E.C. and Carpenter. C.L.. Phenytoin interacts with calcium channels in brain membranes. Ann. Neurol.. 16 (1984) 616-617. 7 Greenberg, D.A., Cooper. E.C. and Carpenter. C.L.. Calcium entry activators: distinct sites of dihydropyridine and aminopyridine action. Neurosci. Len.. 50 (1984) 279-282. 8 Greenberg, D.A., Cooper. E.C. and Carpenter. C.L.. Calcium channel 'agonist' BAY k 6844 inhibits calcium antagonist binding to brain and PC12 cell membranes. Brain Research. 305 (1984) 365-368. 9 Harris. R.A.. Jones. S.B.. Bruno, P. and Bylund. D.B.. Effects of dihydropyridine derivitives and anticonvulsant drugs on [3H]nitrendipine binding and calcium and sodium fluxes in the brain. Biochem. Pharmacol.• 34 (1985) 2187-2191. 10 Hasbani. M., Pincus, J.H. and Lee. S.• Dephenylhydantoin and calcium movement in lobster nerves. Arch. Neurol.. 31 (1974) 250-254. 11 Heinemann. U. and Louvel. J.. Changes in [Ca2+]o and [K+]o during repetitive electrical stimulation and during pentetrazole-induced seizure activity in the sensorimotor cortex of cats. Pflfigers Arch.. 398 (1983) 310-317. 12 Heinemann. U. and Pumain. R.• Effects of tetrodotoxin on changes in extracellular free calcium induced by repetitive electrical stimulation and iontophoretic application of excitatory amino acids in the sensorimotor cortex of cats. Neurosci. Lett.. 21 (1981) 87-91. 13 Hotson. J.R. and Prince. D.A.. Penicillin- and barium-reduced epileptiform bursting in hippocampal neurons: actions on Ca ** and K* potential. Ann. Neurol.. 10 f1981) 11-17. 14 Meyer. F.B., Anderson. F.E.. Sundt, T.M. and Sharbrough, F.W.. Selective central nervous system calcium channel blockers a new class of anticonvulsant agents, Mayo Clin. Proc.. 61 (1986) 239-247. 15 Overweg J.. Binnie. C.D.. Meijer. J.W.A., Meinardi. H..
Nuijfen. S.T.M.• Schmaltz, S. and Wauquier. A.. Doubleblind, placebo-controlled trial of flunarizine as add-on therapy In epilepsy, Epilepsia, 25 (1984) 2t7-222. 16 Panza. G., Grebb. J.A., Senna, E.. Wright. A.G. and Hanbauer. I.. Evidence for down-regulation of [3H]nitrendipine recognition sites in mouse brain after long-term treatment with nifedipine or verapamil, Neuropharmacology. 24 (1985) 1113-1117. 17 Pincus. J. and Hsiao. K.. Calcium uptake mechanisms affected by some convulsant and anticonvulsant drugs, Brain Research. 217 11981) 119-129. Pumain. R.• Kurcewicz. I. and Louvel. J.. Fast extracellular calcium transients: involvement in epileptic processes. Science. 222 ~1983) 177-179. 19 Rampe, D., Janis, R.A. and Triggle, D.J.. BAY k 8644. a 1 4 d t 'h y dro py ndl " "he Ca 2+ channel activator: dissociation of binding and functional effects in brain synaptosomes. J. Neurochem•. 43 (1984) 1688-1692. 20 Schramm. M . Thomas, G., Towart. R. and Franchowiak_ G., Novel dihydropyridines with positive inotropic action through activation of Ca 2+ channels. Nature tLondon). 303 (1983) 535-537. 21 Schramm. M., Thomas, G.. Towart. R. and Franckowiak. G,. Activation of calcium channels by novel 1,4-dihydropyridines. Arzneim.-Forsch. Drug Res.. 33 (19831 t268-1272. 22 Siegel, A.• Malkowitz. L.. Moskovitz. M.J. and Cristakos. S.. Administration of 1,25-dihydroxyvitamin D 3 results in the elevation of hippocampal seizure threshold in rats. Brain Research, 298 (1984) 125-129. 23 Sohn, R.C. and FerrendeUi. J.A., Anticonvulsant drug mechamsms phenytoin, phenobarbitol, and ethosuxlmide and calcium flux in isolated presynaptic endings, Arch, Neurol.. 33 (1976) 626-629 24 Study, R.E.. Phenytoin inhibition of cyclic guanosine 3'.5'monophosphate (cGMP) accumulation m neurobtastoma cells by calcium channel blockade. J. Pharmacol. Exp. Ther. 215 (1980) 575-581. 25 Turner. R.W.. Baimbridge. K.G and Miller. J.J.. Calcium-induced long-term potentiation in the hippocampus, Neuroscience. 7 (1982) 1411-1416. 26 Wadman. W.J.. Heinemann. U., Konnerth, A. and New haus, S.. Hippocampal slices of kindled rats reveal calcium involved in epiteptogenesis. Exp. Brain Res.. 57 (19857 404-407. 27 Wasterlain. C.G. and Farber. D.B., Kindling alters the calciurn/ealmodulin-dependent phosphorylation of synaptic plasma membrane proteins in rat hippocampus. Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1253-1257. 28 Wauquier, A., Ashton, D.. Clincke. G. and Fransen J., 'Calcium entry blockers' as cerebral protecting agents: comparative activity in tests of hypoxia a n d hyperexcitability, Jap. J. Pharmacol., 38 (1985) 1-7 29 Wigstrom,H., Swarm, J.W. and Andersen, P.. Calcium dependency of synaptic long-lasting potentiation in the hippocampal slice, Acta. Physiol. Scand.. 105 (1979) 126-128.
the ultimate mechanism
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399
Elsevier BRE 22010
Induction of seizures in mice by intracerebroventricular administration of the calcium channel agonist BAY k 8644 Richard C. Shelton t, Jack A. G r e b b 2'3 and William J. Freed 4 l Department of Psychiatry, Vanderbilt University Medical School, Nashville, TN37232 (U.S.A.), 2Nathan Kline Institute, Orangeburg, NY 10962 (U.S.A.), ~Department of Psychiatry, New York University Medical Center, New York, N Y 10016 (U.S.A. ) and 4preclinical Neurosciences Section, Neuropsychiatrv Branch, National lnsitute of Mental Health, Saint Elizabeths Hospital, Washington, DC 20032 (U. S. A. )
(Accepted 16 September 1986) Key words: Calcium: Calcium channel; Calcium channel agonist; Calcium channel inhibitor: Seizure; Anticonvulsant
The calcium channel agonist BAY k 8644 was used to investigate the role of the calcium ion (Ca2+) in epileptogenesis, lntracerebroventricular administration of the compound induced murine seizures that were reversed by calcium channel inhibitors (CCIs) but not by anticonvulsants such as carbamazepine, pentobarbital, and diazepam. The seizures were exacerbated by phenytoin and valproic acid. Chronic administration of CCFs, previously shown to produce down-regulation of the binding of the CCI [3H]nitrendipine, resulted in augmentation of BAY k 8644-induced seizures.
A substantial body of literature suggests that calcium plays an important role in both epileptogenesis and the actions of anticonvulsants. Rapid influx of calcium ions (Ca e+) into neurons occurs prior to spike activity in the hippocampus Is, including that generated by penicillin ~'ls, barium 13, excitatory amino acids ~:, pentatetrazole ~, and electrical current 1~'~2 Seizure threshold can be raised by the application of 1,25-dihydroxyvitamin D 3 that modulates intracellular Ca e+ activity through the stimulation of synthesis of intraneuronal Ca 2+ binding proteins 22. Ca 2+ is apparently also a critical component in kindling 2~. Ca 2+ is required for long-term potentiation of synapses > and transient increases in extracellular Ca 2+ yield long-lasting potentiation and spike formation in kindled preparations -'5. Kindling itself specifically reduces Ca: Ucalmodulin-dependent protein phosphorvlation in rat brain e7. Finally, the reversal of seizures by calcium channel inhibitors (CCI's) in animal models <14-'s and humans I>, supports both the significance of Ca -~+ in epilepsy and the potential role of CCI's as anticonvulsant agents.
Anticonvulsant drugs are known to have effects on Ca2+-dependent mechanisms in the neuron. Phenytoin, for example, reduces Ca 2+ uptake into neurons 9'10'17'23 and inhibits Ca2+-dependent mechanisms such as guanosine-3',5'-monophosphate synthesis 24 and protein phosphorylation 3. Phenytoin has also been shown to inhibit binding of the dihydropyridine calcium channel inhibitor (CCI) [3H]nitrendipine to neuronal membranes, indicating that the inhibition of Ca 2+ flux might be via binding to calcium channel regulatory proteins <9. In order to investigate the relationship of calcium flux and seizure activity more directly, we have used B A Y k 8644, a calcium channel 'agonist' of the dihydropyridine class, thought to have its effects on voltage-dependent calcium channels in cell membranes 7's'tg-21, including those of neurons ~'1~. Recently, significant central effects of relatively low doses of the compound have been reported I. B A Y k 8644 inhibited the binding of [3H]nitrendipine to mouse brain membranes. When administered systemically to animals in dosages of 0.5-5.0 mg/kg, B A Y k 8644
Correspondence: R.C. Shelton, Vanderbilt University Medical School, Medical Center North, Room A-2215, Nashville, TN 37232,
U.S.A.
0006-8993/87,$II3.50© 1987 Elsevier Science Publishers B.V. (Biomedical Division)
400 induced ataxia, decreased motor activity, tail flexion, arched back. limb tonus and clonus, and hyperreactivity to sensory stimuli. The effects were blocked by the dihydropyridine calcium channel antagonist nifedipine (2 mg/kg), but not by verapamil (2 and 6 mg/kg). In the present study, we report that intracerebroventricular injections of small amounts of BAY k 8644 produced seizures, and that these seizures are susceptible to alteration by pharmacological manipulations. Female Swiss-Webster mice (Veterinary Resource Branch. National Institute of Health. Bethesda. MD) were used for all experiments. The sources of compounds were as follows: BAY k 8644: Miles Pharmaceuticals. West Haven. CT: carbamazepine: Ciba-Geigy, Summit. N J; diazepam: Roche Laboratories. Nutley N J; diltiazem: Marion Laboratories. Kansas City, MO; flunarizine: Janssen Pharmaceuncal. New Brunswick. NJ: nifedipine: Pfizer Laboratories. New York. NY: phenytoin: Sigma Chemical Co.. St. Louis. MO: valproic acid: Abbott Laboratories. North Chicago, IL: verapamil: Knoll Pharmaceutical Co.. Whippany, NJ. A dose-response curve for duration of seizures elicited in mice by intracerebroventricular BAY k 8644 was generated (Fig. 1). Mice (n = 8 in each group) were injected intracerebroventricularly 2 with a 5-/~1solution of BAY k 8644 in 10% Tween-80/saline in doses of 6.25. 12.5. 25 and 37.5/~g. The ani-
mals were placed in 16.3 x 23 cm polystyrene cylinders and observed for a total duration of seizures for 60 min postinjection: no seizure lasted longer than this time period. Seizure activity was noted to be of increasing duration and seventy with progressive dosage. Low doses produced tonic contractions with intermittent clonic jerks of the extrcmities and tail flexion. As the dose increased, head bobbing, complex grooming actions (licking of fur. scratching, etc. J. squeaking, jumping, and running occurred. A series of CCIs and anticonvulsant drugs were tested for their effects on BAY k 8644-induced seizures. Mice were pretreated with intraperimneat injections of nifedipine, verapamil, diltiazem, flunarizine. carbamazepme, pentobarbital, diazepam, valproic acid. or phenytoln mixed m a 10% Tween80/saline solution in doses listed m Fig. 2 All drugs were administered 30 min prior to intracerebroventricular injection of 37.5 ug BAY k 8644, except for pentobarbital which was given 5 rain before injection. As summarized in Fig. 2. the CCIs nifediplnc and verapamil completely blocked BAY k 8644-induced seizures and diltiazem m a r k e d b antagonized them Flunarizine. a piperidine-type calcium channel antagonist, had no effect, nor did pentobarbitat, carbamazepine, and diazepam. Valproic acid and phenytoin increased seizure intens~tx and duration: bar60--
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Fig. 1. D o s e - r e s p o n s e curve l m e a n 1- S . E . M . ) for seizures elicited in female S w i s s - W e b s t e r mice (n = 8 in each group), 26-46 g, injected intracerebroventricularly with B A Y k 8644.
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Fig. 2. Seizure duration ( m e a n _~ S.E.M.) m femalc S w i s s - W e b s t e r mice (n = 8 in each group), 14-30 g. pretreated with intraperitoneal injections of CCIs and anticonvulsants. Controls were injected withvehicle only. Significant differences from controls *P < 0.02. **P < 0.007, ***P < O.OO1 (two-tailed t-Test).
401
rel-rolling and some self-mutilation were observed. Three of eight mice pretreated with phenytoin had residual ataxia for at least 2 h after termination of overt seizure activity. A dose-response curve contrasting the effects of nifedipine and phenytoin on B A Y k 8644-induced seizures was generated and is summarized in Fig. 3. Mice were pretreated with 5, 25, and 50 mg/kg of nifedipine and phenytoin by intraperitoneal injection. After 30 min 37.5 #g of B A Y k 8644 was injected and observations were made as in the previous experiments. Chronic treatment of mice with verapamil or nifedipine (but not diltiazem) has been shown to decrease binding of [3H]nitrendipine to neuronal menbranes in a previous experiment 16. To determine whether these alterations in calcium channel antagonist binding were of functional significance, mice were administered nifedipine, verapamil, and diltiazem mixed with powdered food for 28 days. Absolute calculated dosages, corrected for intake, were nifedipine 0.39 rag/g/day, verapamil 0.30 mg/g/day, and diltiazem 0.39 mg/g/day. Animals were given free access to water. After the 26 days, the drugs were withdrawn for 4 days so that the mice could be tested in the absence of CCIs themselves. B A Y k 8644 was ad-
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Fig. 3. The effects of increasing doses of nifedipine and phenytoin on seizure duration in female Swiss-Webster mice (n = 8 in each group), 22-42 g. Mice were pretreated with 5, 25, and 50 mg/kg of nifedipine and phenytoin by intraperitoneal injection. After 30 min, BAY k 8644 37.5/~g was injected intracerebroventricularly. Comparison is to control animals pretreated with vehicle only. Significant differences: *P < 0.05, **P < 0.007 (two-tailed t-test).
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Fig. 4. Dose-response curves of seizure duration in female Swiss-Webster mice (n = 8 in each group), 16-38 g, after withdrawal of nifedipine, verapamil, and diltiazem administered orally for 26 days. Intracerebroventricular BAY k 8644 was administered 4 days after withdrawal of CCIs. Comparisons are to control animals given powdered food only. Significant differences: *P < 0.04, **P < 0.002 (two-tailed t-test).
ministered as in the previous experiments at 6.25, 12.5, and 25/~g, to the pretreated mice and a control group was given powdered food only. The results are given in Fig. 4. These findings give functional significance to the alteration of CCI binding, with the expected order of potency of nifedipine and verapamil being greater than diltiazem. Functionally distinct subtypes of (at least) dihydropyridine calcium channel receptors may exist with excitatory and inhibitory properties 5. Calcium channel agonists and antagonists may, in fact, actually function as partial agonists for the receptor subtypes. The down-regulation of calcium channel binding may, therefore, be predominantly of the inhibitory component, yielding a relative excess in excitatory receptors and, in this model, seizure induction. The findings of these experiments lend further credence to the concept that disturbances of central Ca 2+ regulation is involved in seizure mechanisms. The seizures induced by B A Y k 8644 were not blocked by standard anticonvulsants, but were reversed by the CCIs diltiazem, verapamil, and nifedipine, suggesting that the induction of seizures in this model is a specific calcium channel-mediated event. The exacerbation of seizure activity by valproic acid and phenytoin was an unexpected finding, particularly in view of the apparent binding of phenytoin to
402 n e u r o n a l c a l c i u m c h a n n e l s a n d i n h i b i t i o n o f C a 2÷
t h e i r e f f e c t o n n e u r o n s , t h e s e f i n d i n g s a r e f u r t h e r evt-
flux 9. T h e specific p h a r m a c o l o g i c a l a c t i o n o f t h e s e
d e n c e in s u p p o r t o f C C I t r e a t m e n t in s e i z u r e s a n d
agents requires further investigation. Regardless of by which the CCIs exert
s u g g e s t a p o t e n t i a l m o d e l for t h e m o d u l a t i o n o f activ a t i o n in epilepsy•
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