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Effect of cocaine, lidocaine kindling and carbamazepine on batrachotoxin-induced phosphoinositide hydrolysis in rat brain slices
Brain Research, 614 (1993) 185-190
185
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 18916
Effect of cocaine, lidocaine kindling and carbamazepine on batrachotoxin-induced phosphoinositide hydrolysis in rat brain slices R u s s e l l L. M a r g o l i s , D e - M a w C h u a n g , D o u g l a s Dick, S u s a n R . B . W e i s s a n d R o b e r t M. P o s t Biological Psychiatry Branch, National Institute of Mental Health, Bethesda, MD 20892 (USA) (Accepted 12 January 1993)
Key words." Phosphoinositide; Kindling; Cocaine: Lidocaine; Anesthetic; Na; Batrachotoxin; Ibotenate
Repeated administration of a subconvulsant dose of a local anesthetic will eventually induce seizures, a phenomenon similar to electrical kindling. We have investigated the effect of repeated lidocaine and cocaine administration on the phosphoinositide (PI) hydrolysis induced by batrachotoxin (BTX), a specific Na channel activator. Rats were injected with cocaine or saline daily for 6 days and PI hydrolysis was assayed in sliced frontal cortex. Cocaine treatment had no effect on BTX-induced PI hydrolysis while in vitro cocaine blocked the BTX effect. In a second experiment, rats received daily injections of lidocaine or saline. After a rat developed at least two seizures, it was sacrificed together with a rat receiving lidocaine injections which had never seized and a rat receiving saline injections. Basal, BTX and ibotenic acid (IBO; a glutamate receptor agonist)-stimulated PI hydrolysis did not differ among the three groups in slices of either hippocampus (HC) or piriform cortex (PC) though IBO-stimulated PI hydrolysis was much greater in the HC than in the PC. Neither in vitro nor in vivo carbamazepine altered the effect of cocaine on BTX-induced PI hydrolysis. These results demonstrate that local anesthetic kindling does not alter PI hydrolysis coupled to Na channel or IBO activation.
INTRODUCTION
Repeated administration of local anesthetics, including cocaine and lidocaine, gradually leads to the development of seizures, a phenomenon known as pharmacological kindling t9'2°'22'24. The process is parallel to electrical kindling, in which repeated subthreshold stimulation of focal brain regions through an implanted electrode also leads to the development of seizures 5. Some of the effects of repeated cocaine administration, such as increased motor activity and stereotopy (termed sensitization or reverse tolerance), apparently stem from the blockade of catecholamine re-uptake by cocaine 1°'21'25. However, the available evidence suggests that this property of cocaine does not account for kindling to a seizure endpoint. The dose required for cocaine kindling is higher than that required to induce sensitization28. Further, other local anesthetics, such as lidocaine, which are not psychomotor stimulants and do not block amine re-uptake, induce kindled seizures. Finally, the development of cocaine-kindled seizures is
attenuated by chronic treatment with the anticonvulsant and mood-stabilizer carbamazepine (CBZ) but CBZ has little effect on behavioral sensitization 28. The mechanisms accounting for local anesthetic kindling are unknown but could involve a change in the function of voltage-gated Na channels. Local anesthetics inhibit nerve conduction when ceils are firing rapidly or undergoing prolonged depolarization 9,H. This effect is achieved through the stabilization of Na channels in an inactive state, thereby blocking Na conductance 27. CBZ has a similar use-dependent effect on Na conductance ~5'29 and CBZ and lidocaine share a common binding site on this channel 3~. A substantial literature documents the impact of various receptor ligands on receptor number and effector response. Na channels are also susceptible to this type of regulation; e.g., chronic treatment of rats with mexiletine, a Na channel blocker functionally similar to lidocaine, upregulates Na channel binding sites in cardiac myocytes26. Na channels may also be influenced by events related to seizures. Electrical kindling re-
Correspondence: D.-M. Chuang, Biological Psychiatry Branch, National Institute of Mental Health, Building 10, Room 3N212, 9000 Rockville Pike, Bethesda, MD 20892, USA. Fax: (1X301)4020052.
186 duces the number of binding sites for the Na channel antagonist tetrodotoxin in frog brain ~6, suggesting Na channel down-regulation. Tottering mice, in which a single gene mutation is associated with hyperexcitability and spontaneous seizures, have fewer brain Na channels but more Na influx per channel than normal mice 3°. To test the possibility that local anesthetic kindling alters Na channel function, we chose to examine the effect of local anesthetic kindling on batrachotoxin (BTX)-induced phosphoinositide (PI) hydrolysis. BTX is a potent neurotoxin with high affinity and specificity for the site 2 receptor on voltage-gated Na channels 3. BTX prevents or vastly slows normal Na channel inactivation, thus locking channels into an open state and facilitating a massive influx of Na. BTX also stimulates PI breakdown through activation of phospholipase C (PLC)6-s; the mechanism for this activation may be either Na dependent (induction of the pathway through Na influx) TM a n d / o r independent (alteration of Na channel conformation leading to the activation of PLC) 4. Since an earlier effort at identifying the effects of local anesthetic kindling on Na channels by a binding assay failed to yield consistent results, we elected to re-examine this problem by measuring BTX-induced PI hydrolysis in rat brain slices after chronic local anesthetic or CBZ administration.
concentration of 2.25 g C B Z / k g of food was increased after 4 days to 5 g/kg. Previous studies have found that this incremental increase in CBZ is well tolerated and leads to minimal weight loss 2s. The blood levels of CBZ and its active 10,11-expoxide metabolite achieved with this regimen are similar to the therapeutic levels used to treat human patients 23 though the ratio of the two compounds is reversed in the rat 13.
PI hydrolysis assay
PI hydrolysis was measured according to the method of Berridge et al. 2, with slight modifications. Briefly, brain regions were rapidly dissected on ice, cross-chopped at 400 /xm and dispersed into preoxygenated K r e b s - H e p e s buffer (118 mM NaCI, 4.8 mM KCI, 1.2 mM KH2PO4, 1.3 mM CaC12.2H20, 1.2 mM MgSO4, 11 mM glucose and 33.3 mM Hepes; titrated to pH 7.65), re-oxygenated and then equilibrated at 37°C for 40 min (lidocaine experiments) or 30 min (cocaine experiments). Buffer was then aspirated and replaced with pre-oxygenated buffer containing [3H]myoinositol (2.5/xCi/ml). The slices were re-oxygenated and incubated for 60 min, washed three times and re-incubated with 5 mM lithium chloride in 40-/zl aliquots in an oxygenated chamber. Agonists were added after 25 min and the reaction was stopped with a methanol:chloroform solution 45 min later. In appropriate experiments, cocaine was added 15 min prior to agonists and CBZ was added 15 min prior to cocaine. [3H]inositol-monophosphate (IP l) was isolated by adding water, separating aqueous and organic phases by centrifugation at 3000 rpm for 5 min and applying the aqueous layer to a Bio-Rad 1 × 8 formate phase column. After two rinses with water and three rinses with 5 mM disodium tetraborate/60 mM sodium formate, IP l was eluted with 0.2 M ammonium formate and quantified by standard liquid scintilation techniques. The incorporation of [3H]inositol into total inositol containing lipids was measured by evaporating the organic phase and counting the residual radioactivity. Results are expressed as the ratio of IP 1 to total inositol incorporated into inositol lipids.
RESULTS MATERIALS
AND METHODS
Materials
BTX was a generous gift of J.W. Daly, National Institute of Health (Bethesda, MD). Ibotenic acid (IBO) was obtained from ICN Biochemicals (Irvine, CA), lithium chloride and cocaine hydrochloride from Sigma (St Louis, MO), CBZ for in vitro use from Research Biochemical (Natick, MA) and [3H]myoinositol (16.5 mCi/mmol) from New England Nuclear (Boston, MA). Dietary CBZ was formulated by Bioserve (Frenchtown, NJ). Lidocaine and cocaine treatment
All experiments used adult male Sprague-Dawley rats, weighing 250-300 g at the beginning of the experiment. The rats were grouphoused with a 12-h alternating light-dark cycle and free access to food and water. Cocaine hydrochloride (40 mg/kg) or saline were injected i.p. once daily. Previous work in our laboratory has demonstrated that seizure-induced mortality is high with cocaine-kindled seizures ( ~ 70-100% after one or two injection-induced bouts of seizures). We, therefore, chose to sacrifice all animals 24 h after their sixth injection. Several rats sustained a single seizure during the course of injections; data from these rats were compared with rats which did not have seizures. Lidocaine hydrochloride (initially 70 mg/kg, increasing by 10 m g / k g every 10 injections) or saline was injected i.p. once daily 5 days/week. After a rat receiving lidocaine had at least two consecutive seizures (6-48 daily injections), it was sacrificed along with a saline-injected control rat and a rat that had received an identical number of lidocaine injections but had never developed seizures. CBZ treatment
Rats were fed a diet of rat chow containing CBZ or an inert binding agent for 7 days and then were sacrificed. The initial
Our initial experiment (Fig. 1) was an attempt to confirm, in slices of rat cerebral cortex, a previous report that in vitro cocaine could block BTX-induced PI hydrolysis. The concentration of BTX selected for this experiment, 0.05 tzM, provided maximal stimulation of PI hydrolysis under our assay conditions (unpubl. data; see also Fig. 2), similar to previously published results of BTX-induced PI hydrolysis in cortical slices lz. The use of higher concentrations of BTX to achieve maximal stimulation of PI hydrolysis in synaptoneurosome preparations 8 or cell culture systems 4 (1 /~ M) probably reflects the intrinsic differences between these preparations and the more interconnected, compartmentalized and heterogenous tissue slice preparation 12. The IC5o for cocaine in our experiment was ~ 100 /zM, almost identical to that previously identified in mouse cerebral cortex 12 while basal PI hydrolysis was unaffected by cocaine in the concentration range examined. We next examined the effect of repeated cocaine injections on BTX-induced PI hydrolysis; no change in BTX-induced PI hydrolysis was observed at any concentration of BTX (Fig. 2). Results from the rats which experienced a seizure did not significantly differ from the other rats (data not shown).
187
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Fig l. In vitro cocaine inhibits BTX-induced PI hydrolysis in rat cortical slices. Cortical slices from untreated rats were pooled and the effect of cocaine on basal (open circles) and 0.05 /xM BTXstimulated (closed circles) IP 1 formation was assessed. Results are means_+ S.E.M. of four independent experiments performed in triplicate.
In lidocaine-treated animals and matched controls, we assayed PI hydrolysis in the piriform cortex (PC) and hippocampus (HC) rather than frontal cortex (which included a portion of the PC) since these regions have been more specifically linked to the development of kindling. Lidocaine-kindled seizures or repeated lidocaine injections did not affect BTX-induced PI hydrolysis in either region (Fig. 3; A N O V A for HC: F = 0.302, P = 0.74; A N O V A for PC: F = 0.548, P = 0.58). No consistent effect of lidocaine kindling on BTX-induced PI hydrolysis was oberved at lower doses of BTX (data not shown). Since electrical kindling of the amygdala has been reported to increase IBO-induced PI hydrolysis t, we also examined the influence of lidocaine kindling on this measure in the H C and PC. Again, no effect was observed. However, similar to previous reports, a regional difference in IBO-induced PI hydrolysis was apparent; response in the H C was much greater than in the PC (Fig 4; A N O V A for HC: F = 0.245, P = 0.78; A N O V A for PC: F = 0.178, P =
0.84). Finally, we evaluated both the the direct impact of C B Z and the interaction between C B Z and cocaine on BTX-induced PI hydrolysis. Even a high concentration of CBZ (100 /zM) applied in vitro to slices of rat frontal cortex had no effect on BTX-induced PI hydro-
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Fig 2. Repeated cocaine injections do not alter BTX-induced PI hydrolysis in rat cortical slices. Rats received 40 m g / k g cocaine (open circles; n = 8) or saline (closed circles; n = 4) i.p. daily for 6 days and cortical slices from each rat were assayed for BTX-induced IP l formation 24 h after the final injection. Results are m e a n s + S.E.M.; two independent assays were performed, each with unpooled slices from four treated and two control rats.
lysis and did not alter the ability of cocaine to decrease BTX-induced PI hydrolysis (Fig. 5). Cocaine and BTX, evaluated by separate A N O V A , each had a significant effect ( F = 12.05, P = 0.0032 and F = 9.80, P = 0.0064,
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Fig 3. Lidocaine kindling does not affect BTX-induced PI hydrolysis in rat PC or HC. PC and H C were sliced and assayed for 0.05 tzM BTX-induced PI hydrolysis 24 h after the last injection in lidocainekindled, lidocaine-injected and saline-injected rats matched for number of injections. Results are means_+ S.E.M. of nine separate assays each performed in triplicate. Values are calculated as percent of basal PI hydrolysis in each rat.
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Fig 4. Lidocaine kindling does not affect IBO-induced PI hydrolysis in rat PC or HC. PC and HC were sliced and assayed for IBO (1 mM)-induced PI hydrolysis 24 h after the last injection in lidocainekindled, lidocaine-injected and saline-injected rats matched for number of injections. Results are m e a n s + S.E.M. of six separate assays each performed in triplicate. Values are calculated as percent of basal PI hydrolysis in each rat.
respectively) while CBZ did not ( F = 1.24, P = 0.28). No significant differences emerged when all comparison were combined in a three-factor A N O V A ( F = 0.037, P = 0.85). Similar results were obtained in slices of frontal cortex of rats which were treated with a CBZ diet for 7 days (Fig. 6). A N O V A results again indicate a significant effect of BTX ( F = 26.67, P = 0.0001), a trend toward a significant effect of cocaine ( F = 2.157, P = 0.1549) but no significant effect of CBZ ( F = 0.05,
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Fig 5, In vitro CBZ does not affect basal PI hydrolysis, BTX-induced PI hydrolysis or the attenuation by cocaine of BTX-induced PI hydrolysis. Frontal cortex from untreated rats was sliced and assayed for PI hydrolysis in the presence and absence of 100/zM CBZ, 300 ,ttM cocaine and 0.05 p.M BTX. Results are means + S.E.M. of three independent assays each performed with three to six replicates.
0/0
0/.05
COCAINE/BTX
300/0 CONCENTRATION
300/.05 (~M)
Fig 6. Chronic CBZ treatment in vivo does not affect basal PI hydrolysis, BTX-induced PI hydrolysis or the attenuation by cocaine of BTX-induced PI hydrolysis. Frontal cortex from rats treated for 7 days with CBZ (solid bars; n = 4) or a control diet (white bars; n = 4) was sliced and assayed for PI hydrolysis in the presence and absence of 300 p.M cocaine and 0.05 /~M BTX. Results are means+S.E.M. of four independent assays each performed with three to six replicates.
P = 0.8252; 0.9801).
three-factor
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DISCUSSION The data demonstrate that, although in vitro administration of cocaine attenuates BTX-induced PI hydrolysis in rat frontal cortex, neither repeated cocaine injections nor lidocaine kindling alter BTX-induced PI hydrolysis in rat cortex or HC. Lidocaine kindling did not influence IBO-induced PI hydrolysis. The anticonvulsant drug CBZ had no effect when administered in vivo or in vitro on BTX-induced PI hydrolysis in frontal cortex and did not reverse the cocaine-induced attenuation of this phenomenon. These results suggest that neither repeated local anesthetic administration nor CBZ affected the coupling of the BTX-binding site to PI metabolism. We detected attenuation of BTX-induced PI hydrolysis by in vitro cocaine with an IC5o almost identical to that previously reported in slices of mouse cortex ]2. Others, using a dilute solution of guinea pig synaptoneurosomes and much higher concentrations of BTX, have reported an IC50 value > 10-fold higher t8, presumably reflecting the same difference in sensitivity between synapotoneurosome and tissue slice preparations observed in response to BTX. We observed a regional variation in the extent of IBO-induced PI hydrolysis similar to that previously reported 17, confirming the ability of our method to detect alterations in PI hydrolysis. The difference in BTX-induced PI hydrolysis be-
189
tween frontal cortex ( ~ 200% over control; Figs. 1, 2, 5, 6) and HC and PC ( ~ 400% over control; Fig. 3) may reflect an intrinsic regional difference in the coupling of PI hydrolysis to Na channels similar to that observed for IBO. Our results also imply that local anesthetics and CBZ do not alter the number or affinity of BTX-binding sites and therefore that Na channels are not up- or down-regulated by these agents though this possiblity will need confirmation by more direct methods. Overall, the data from this study suggest that Na channel-coupled PI hydrolysis is not altered by local anesthetic kindling. It remains possible that functional properties of Na channel conductance other than induction of PI hydrolysis may have some role in local anesthetic kindling and/or the anticonvulsant properties of CBZ, or that these phenomena alter Na channels in brain regions or at time points other than those assessed in the present study. Unlike electrical kindling 1, lidocaine kindling did not potentiate the PI response to IBO. This difference between the two types of kindling implies either that such a response is not central to the kindling process (i.e., that it is a unique by-product of electrical kindling) or that local anesthetic and electrical kindling are distinct processes produced by divergent mechansims. Acknowledgements. This work was supported in part by a PRAT fellowship from the National Institute of General Medical Sciences to R.L. Margolis. 1 Akiyama, K., Yamada, N. and Otsuki, S., Lasting increase in excitatory amino acid receptor-mediated polyphosphoinositide hydrolysis in the amygdala/pyriform cortex of amygdala-kindled rats, Brain Res., 485 (1989) 95-101. 2 Berridge, M.J., Downes, C.P. and Hanley, M.R., Lithium amplifies agonist-dependent phosphatidylinositol response in brain and salivary glands, Biochem. J., 206 (1982) 587-595. 3 Brown, G.B., Tieszen, S.C., Daly, J.W., Warnick, J.E. and Albuquerque, E.X., Batrachotoxinin-A 20-a-benzoate: a new radioactive ligand for voltage sensitive sodium channels, Cell Mol. Neuro: bioL, 1 (1981) 19-40. 4 Chuang, D.-M., Regulation by batrachotoxin, veratridine, and monensin of basal and carbachol-induced phosphoinositide hydrolysis in neurohybrid NCB-20 cells, Neurochem. Res., 15 (1990) 695-704. 5 Goddard, L.S., Mclntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 6 Gurwitz, D. and Sokolovsky, M., Dual pathways in muscarinic receptor stimulation of of phosphoinositide hydrolysis, Biochem/stry, 26 (1987) 633-638. 7 Gusovsky, F., Hollingsworth, E.B. and Daly, J.W., Regulation of phosphatidylinositol turnover in brain synatoneurosomes: stimulatory effects of agents that enhance influx of sodium ions, Proc. Natl. Acad. Sci. USA, 83 (1986) 3003-3007. 8 Gusovsky, F., McNeal, E.T. and Daly, J.W., Stimulation of phosphoinositide breakdown in brain synaptoneurosomes by agents that activate sodium influx: antagonism by tetrodotoxin, saxitoxin, and cadmium, Mol. Pharmacol., 32 (1987) 479-487. 9 Hondeghem L.M. and Katzung, B.G., Time- and voltage-depen-
dent interactions of antiarrhythmic drugs with cardiac sodium channels, Biochim. Biophys. Acta, 472 (1977) 373-398. 10 Kelly, P.H. and Iversen, S.D., Selective 6-OHDA-induced destruction of mesolimbic dopamine neurons: abolition of psychostimulant-induced locomotor activity in rats, Eur. J. Pharmacol., 40) (1975) 45-56. 11 Khodorov, B., Shishkovak, L., Peganov, E. and Revenko, S., Inhibition of sodium currents in frog Ranvier node treated with local anesthetics: role of slow sodium inactivation, Biochim. Biophys. Acta, 433 (1976) 409-435. 12 Kim, S.S. and Reith, M.E.A., Inhibition by cocaine of inositol phospholipid hydrolysis induced by the sodium channel activator batrachotoxin in mouse cerebral cortex, Biochem. Pharmacol., 37 (1988) 773-775. 13 Marangos, P.J., Weiss, S.R.B., Montgomery, P., Patel, J., Narang, P.K., Cappabianca, A.M. and Post, R.M., Chronic carbamazepine treatment increases brain adenosine receptors, Epilepsia, 26 (1985) 493-498. 14 McDonough, P.M., Goldstein, D. and Brown, J.H., Elevation of cytoplasmic calcium concentration stimulates hydrolysis of phosphatidylinositol bisphosphate in chick heart cells: effect of sodium channel activators, Mol. Pharmacol., 33 (1988) 310-315. 15 McLean, M.J. and Macdonald, R.L., Carbamazepine and 10,11epoxycarbamazepine produce use- and voltage-dependent limitation of rapidly firing action potentials of mouse central neurons in cell culture, J. Pharmacol. Exp. Ther., 238 (1986) 727-738. 16 Moneta, M.E., de la Fuente, M., Liberona, J.L. and Jaimovich, E., Sodium pathway markers in normal and kindled frog brains, Neurosci. Lett., 65 (1986) 331-335. i7 Nicoletti, F., Meek, J.L., ladarola, M.J., Chuang, D.-M., Roth, B.L. and Costa, E., Coupling of inositol phospholipid metabolism with excitatory amino acid recognition sites in rat hippocampus, J. Neurochem., 46 (1986) 40-46. 18 Nishizawa, Y., Gusovsky, F. and Daly, J.W., Local anesthetics: comparison of effects on batrachotoxin-elicited sodium flux and phosphoinositide breakdown in guinea pig cerebral cortical synaptoneurosomes, Mol. Pharmacol., 34 (1988) 707-713. 19 Post, R.M., Progressive changes in behavior and seizures following chronic cocaine administration: relationship of kindling and psychosis. In E.H. Ellinwood and M.M. Kilbey (Eds.), Advances in Behavioral Biology: Cocaine and Other Stimulants, Plenum Press, New York, 1977, pp. 352-353. 20 Post, R.M., Lidocaine kindled limbic seizures: behavioral implications. In J.A. Wada (Ed.), Kindling 2, Raven Press, New York, 1981, pp. 149-160. 21 Post, R.M. and Contel, N.R., Cocaine-induced behavioral sensitization: a model.for recurrent manic illness. In C. Perris et al. (Eds.), Biological Psychiatry, 1981, Elsevier, Amsterdam, 1981, pp. 746-749. 22 Post, R.M., Kopanda, R.T. and Lee, A., Progressive behavioral changes during chronic lidocaine administration: relationship to kindling, Life Sci., 17 (1975) 943-950. 23 Post, R.M., Uhde, T.W., Ballenger, J.C., Chatterji, D.C., Greene, R.K. and Bunney, W.E., Jr., Carbamazepine and its -10,11epoxide metabolite in plasma and CSF: relationship to antidepressant response, Arch. Gen. Psych., 40 (1983) 673-676. 24 Post, R.M., Weiss, S.R.B. and Clark, M., Amygdala versus local anesthetic kindling: differential anatomy, pharmacology and clinical implications. In J.A. Wada (Ed.), lO'ndling 4, Raven Press, New York, in press. 25 Scheel-Kruger, J., Braestrup, C., Nielson, M., Golembrowska, K. and Mogilnicka, F., Cocaine: discussion on the role of dopamine in the biochemical mechanism of action. In E.H. Ellinwood and M.M. Kilbey (Eds.), Advances in Behavioral Biology, Vol. 21, Cocaine and Other Stimulants, Plenum Press, New York, 1977, pp. 373-408. 26 Taouis M., Sheldon, R.S. and Duff, H.J., Upregulation of the rat cardiac sodium channel by in vivo treatment with a class I antiarrhythmic drug, J. Clin Invest., 88 (1991) 375-378. 27 Wang, G.K., Cocaine-induced closures of single batrachotoxinactivated Na + channels in planar lipid bilayers, J. Gen. Physiol., 92 (1988) 747-765.
190 28 Weiss, S.R.B., Post, R.M., Costello, M., Nutt, D.J. and Tandeciarz, S., Carbamazepine retards the development of cocaine-kindled seizures but not sensitization to cocaine-induced hyperactivity, Neuropsychopharmacology, 3 (1990) 273-281. 29 Willow, M., Kuenzel, E.A. and Catterall, W.A., Inhibition of voltage-sensitive sodium channels in neuroblastoma cells and synaptosomes by the anticonvulsant drugs diphenyl-hydantoin and carbamazepine, Mol. Pharmacol., 25 (1984) 228-234.
30 Willow, M., Taylor, S.M., Catterall, W.A, and Finnell, R.H., Down regulation of sodium channels in nerve terminals of spontaneously epileptic mice, Cell Mol. Neurobiol., 6 (1986) 213-220. 31 Zimanyi, I., Weiss, S.R.B., Lajtha, A., Post, R.M. and Reith, M.E.A.., Evidence for a common site of action of lidocaine and carbamazepine in voltage-dependent sodium channels, Eur. J. Pharrnacol., 167 (1989) 419-422.
185
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 18916
Effect of cocaine, lidocaine kindling and carbamazepine on batrachotoxin-induced phosphoinositide hydrolysis in rat brain slices R u s s e l l L. M a r g o l i s , D e - M a w C h u a n g , D o u g l a s Dick, S u s a n R . B . W e i s s a n d R o b e r t M. P o s t Biological Psychiatry Branch, National Institute of Mental Health, Bethesda, MD 20892 (USA) (Accepted 12 January 1993)
Key words." Phosphoinositide; Kindling; Cocaine: Lidocaine; Anesthetic; Na; Batrachotoxin; Ibotenate
Repeated administration of a subconvulsant dose of a local anesthetic will eventually induce seizures, a phenomenon similar to electrical kindling. We have investigated the effect of repeated lidocaine and cocaine administration on the phosphoinositide (PI) hydrolysis induced by batrachotoxin (BTX), a specific Na channel activator. Rats were injected with cocaine or saline daily for 6 days and PI hydrolysis was assayed in sliced frontal cortex. Cocaine treatment had no effect on BTX-induced PI hydrolysis while in vitro cocaine blocked the BTX effect. In a second experiment, rats received daily injections of lidocaine or saline. After a rat developed at least two seizures, it was sacrificed together with a rat receiving lidocaine injections which had never seized and a rat receiving saline injections. Basal, BTX and ibotenic acid (IBO; a glutamate receptor agonist)-stimulated PI hydrolysis did not differ among the three groups in slices of either hippocampus (HC) or piriform cortex (PC) though IBO-stimulated PI hydrolysis was much greater in the HC than in the PC. Neither in vitro nor in vivo carbamazepine altered the effect of cocaine on BTX-induced PI hydrolysis. These results demonstrate that local anesthetic kindling does not alter PI hydrolysis coupled to Na channel or IBO activation.
INTRODUCTION
Repeated administration of local anesthetics, including cocaine and lidocaine, gradually leads to the development of seizures, a phenomenon known as pharmacological kindling t9'2°'22'24. The process is parallel to electrical kindling, in which repeated subthreshold stimulation of focal brain regions through an implanted electrode also leads to the development of seizures 5. Some of the effects of repeated cocaine administration, such as increased motor activity and stereotopy (termed sensitization or reverse tolerance), apparently stem from the blockade of catecholamine re-uptake by cocaine 1°'21'25. However, the available evidence suggests that this property of cocaine does not account for kindling to a seizure endpoint. The dose required for cocaine kindling is higher than that required to induce sensitization28. Further, other local anesthetics, such as lidocaine, which are not psychomotor stimulants and do not block amine re-uptake, induce kindled seizures. Finally, the development of cocaine-kindled seizures is
attenuated by chronic treatment with the anticonvulsant and mood-stabilizer carbamazepine (CBZ) but CBZ has little effect on behavioral sensitization 28. The mechanisms accounting for local anesthetic kindling are unknown but could involve a change in the function of voltage-gated Na channels. Local anesthetics inhibit nerve conduction when ceils are firing rapidly or undergoing prolonged depolarization 9,H. This effect is achieved through the stabilization of Na channels in an inactive state, thereby blocking Na conductance 27. CBZ has a similar use-dependent effect on Na conductance ~5'29 and CBZ and lidocaine share a common binding site on this channel 3~. A substantial literature documents the impact of various receptor ligands on receptor number and effector response. Na channels are also susceptible to this type of regulation; e.g., chronic treatment of rats with mexiletine, a Na channel blocker functionally similar to lidocaine, upregulates Na channel binding sites in cardiac myocytes26. Na channels may also be influenced by events related to seizures. Electrical kindling re-
Correspondence: D.-M. Chuang, Biological Psychiatry Branch, National Institute of Mental Health, Building 10, Room 3N212, 9000 Rockville Pike, Bethesda, MD 20892, USA. Fax: (1X301)4020052.
186 duces the number of binding sites for the Na channel antagonist tetrodotoxin in frog brain ~6, suggesting Na channel down-regulation. Tottering mice, in which a single gene mutation is associated with hyperexcitability and spontaneous seizures, have fewer brain Na channels but more Na influx per channel than normal mice 3°. To test the possibility that local anesthetic kindling alters Na channel function, we chose to examine the effect of local anesthetic kindling on batrachotoxin (BTX)-induced phosphoinositide (PI) hydrolysis. BTX is a potent neurotoxin with high affinity and specificity for the site 2 receptor on voltage-gated Na channels 3. BTX prevents or vastly slows normal Na channel inactivation, thus locking channels into an open state and facilitating a massive influx of Na. BTX also stimulates PI breakdown through activation of phospholipase C (PLC)6-s; the mechanism for this activation may be either Na dependent (induction of the pathway through Na influx) TM a n d / o r independent (alteration of Na channel conformation leading to the activation of PLC) 4. Since an earlier effort at identifying the effects of local anesthetic kindling on Na channels by a binding assay failed to yield consistent results, we elected to re-examine this problem by measuring BTX-induced PI hydrolysis in rat brain slices after chronic local anesthetic or CBZ administration.
concentration of 2.25 g C B Z / k g of food was increased after 4 days to 5 g/kg. Previous studies have found that this incremental increase in CBZ is well tolerated and leads to minimal weight loss 2s. The blood levels of CBZ and its active 10,11-expoxide metabolite achieved with this regimen are similar to the therapeutic levels used to treat human patients 23 though the ratio of the two compounds is reversed in the rat 13.
PI hydrolysis assay
PI hydrolysis was measured according to the method of Berridge et al. 2, with slight modifications. Briefly, brain regions were rapidly dissected on ice, cross-chopped at 400 /xm and dispersed into preoxygenated K r e b s - H e p e s buffer (118 mM NaCI, 4.8 mM KCI, 1.2 mM KH2PO4, 1.3 mM CaC12.2H20, 1.2 mM MgSO4, 11 mM glucose and 33.3 mM Hepes; titrated to pH 7.65), re-oxygenated and then equilibrated at 37°C for 40 min (lidocaine experiments) or 30 min (cocaine experiments). Buffer was then aspirated and replaced with pre-oxygenated buffer containing [3H]myoinositol (2.5/xCi/ml). The slices were re-oxygenated and incubated for 60 min, washed three times and re-incubated with 5 mM lithium chloride in 40-/zl aliquots in an oxygenated chamber. Agonists were added after 25 min and the reaction was stopped with a methanol:chloroform solution 45 min later. In appropriate experiments, cocaine was added 15 min prior to agonists and CBZ was added 15 min prior to cocaine. [3H]inositol-monophosphate (IP l) was isolated by adding water, separating aqueous and organic phases by centrifugation at 3000 rpm for 5 min and applying the aqueous layer to a Bio-Rad 1 × 8 formate phase column. After two rinses with water and three rinses with 5 mM disodium tetraborate/60 mM sodium formate, IP l was eluted with 0.2 M ammonium formate and quantified by standard liquid scintilation techniques. The incorporation of [3H]inositol into total inositol containing lipids was measured by evaporating the organic phase and counting the residual radioactivity. Results are expressed as the ratio of IP 1 to total inositol incorporated into inositol lipids.
RESULTS MATERIALS
AND METHODS
Materials
BTX was a generous gift of J.W. Daly, National Institute of Health (Bethesda, MD). Ibotenic acid (IBO) was obtained from ICN Biochemicals (Irvine, CA), lithium chloride and cocaine hydrochloride from Sigma (St Louis, MO), CBZ for in vitro use from Research Biochemical (Natick, MA) and [3H]myoinositol (16.5 mCi/mmol) from New England Nuclear (Boston, MA). Dietary CBZ was formulated by Bioserve (Frenchtown, NJ). Lidocaine and cocaine treatment
All experiments used adult male Sprague-Dawley rats, weighing 250-300 g at the beginning of the experiment. The rats were grouphoused with a 12-h alternating light-dark cycle and free access to food and water. Cocaine hydrochloride (40 mg/kg) or saline were injected i.p. once daily. Previous work in our laboratory has demonstrated that seizure-induced mortality is high with cocaine-kindled seizures ( ~ 70-100% after one or two injection-induced bouts of seizures). We, therefore, chose to sacrifice all animals 24 h after their sixth injection. Several rats sustained a single seizure during the course of injections; data from these rats were compared with rats which did not have seizures. Lidocaine hydrochloride (initially 70 mg/kg, increasing by 10 m g / k g every 10 injections) or saline was injected i.p. once daily 5 days/week. After a rat receiving lidocaine had at least two consecutive seizures (6-48 daily injections), it was sacrificed along with a saline-injected control rat and a rat that had received an identical number of lidocaine injections but had never developed seizures. CBZ treatment
Rats were fed a diet of rat chow containing CBZ or an inert binding agent for 7 days and then were sacrificed. The initial
Our initial experiment (Fig. 1) was an attempt to confirm, in slices of rat cerebral cortex, a previous report that in vitro cocaine could block BTX-induced PI hydrolysis. The concentration of BTX selected for this experiment, 0.05 tzM, provided maximal stimulation of PI hydrolysis under our assay conditions (unpubl. data; see also Fig. 2), similar to previously published results of BTX-induced PI hydrolysis in cortical slices lz. The use of higher concentrations of BTX to achieve maximal stimulation of PI hydrolysis in synaptoneurosome preparations 8 or cell culture systems 4 (1 /~ M) probably reflects the intrinsic differences between these preparations and the more interconnected, compartmentalized and heterogenous tissue slice preparation 12. The IC5o for cocaine in our experiment was ~ 100 /zM, almost identical to that previously identified in mouse cerebral cortex 12 while basal PI hydrolysis was unaffected by cocaine in the concentration range examined. We next examined the effect of repeated cocaine injections on BTX-induced PI hydrolysis; no change in BTX-induced PI hydrolysis was observed at any concentration of BTX (Fig. 2). Results from the rats which experienced a seizure did not significantly differ from the other rats (data not shown).
187
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Fig l. In vitro cocaine inhibits BTX-induced PI hydrolysis in rat cortical slices. Cortical slices from untreated rats were pooled and the effect of cocaine on basal (open circles) and 0.05 /xM BTXstimulated (closed circles) IP 1 formation was assessed. Results are means_+ S.E.M. of four independent experiments performed in triplicate.
In lidocaine-treated animals and matched controls, we assayed PI hydrolysis in the piriform cortex (PC) and hippocampus (HC) rather than frontal cortex (which included a portion of the PC) since these regions have been more specifically linked to the development of kindling. Lidocaine-kindled seizures or repeated lidocaine injections did not affect BTX-induced PI hydrolysis in either region (Fig. 3; A N O V A for HC: F = 0.302, P = 0.74; A N O V A for PC: F = 0.548, P = 0.58). No consistent effect of lidocaine kindling on BTX-induced PI hydrolysis was oberved at lower doses of BTX (data not shown). Since electrical kindling of the amygdala has been reported to increase IBO-induced PI hydrolysis t, we also examined the influence of lidocaine kindling on this measure in the H C and PC. Again, no effect was observed. However, similar to previous reports, a regional difference in IBO-induced PI hydrolysis was apparent; response in the H C was much greater than in the PC (Fig 4; A N O V A for HC: F = 0.245, P = 0.78; A N O V A for PC: F = 0.178, P =
0.84). Finally, we evaluated both the the direct impact of C B Z and the interaction between C B Z and cocaine on BTX-induced PI hydrolysis. Even a high concentration of CBZ (100 /zM) applied in vitro to slices of rat frontal cortex had no effect on BTX-induced PI hydro-
. . . . . .
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Fig 2. Repeated cocaine injections do not alter BTX-induced PI hydrolysis in rat cortical slices. Rats received 40 m g / k g cocaine (open circles; n = 8) or saline (closed circles; n = 4) i.p. daily for 6 days and cortical slices from each rat were assayed for BTX-induced IP l formation 24 h after the final injection. Results are m e a n s + S.E.M.; two independent assays were performed, each with unpooled slices from four treated and two control rats.
lysis and did not alter the ability of cocaine to decrease BTX-induced PI hydrolysis (Fig. 5). Cocaine and BTX, evaluated by separate A N O V A , each had a significant effect ( F = 12.05, P = 0.0032 and F = 9.80, P = 0.0064,
600
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Fig 3. Lidocaine kindling does not affect BTX-induced PI hydrolysis in rat PC or HC. PC and H C were sliced and assayed for 0.05 tzM BTX-induced PI hydrolysis 24 h after the last injection in lidocainekindled, lidocaine-injected and saline-injected rats matched for number of injections. Results are means_+ S.E.M. of nine separate assays each performed in triplicate. Values are calculated as percent of basal PI hydrolysis in each rat.
188 E1
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Fig 4. Lidocaine kindling does not affect IBO-induced PI hydrolysis in rat PC or HC. PC and HC were sliced and assayed for IBO (1 mM)-induced PI hydrolysis 24 h after the last injection in lidocainekindled, lidocaine-injected and saline-injected rats matched for number of injections. Results are m e a n s + S.E.M. of six separate assays each performed in triplicate. Values are calculated as percent of basal PI hydrolysis in each rat.
respectively) while CBZ did not ( F = 1.24, P = 0.28). No significant differences emerged when all comparison were combined in a three-factor A N O V A ( F = 0.037, P = 0.85). Similar results were obtained in slices of frontal cortex of rats which were treated with a CBZ diet for 7 days (Fig. 6). A N O V A results again indicate a significant effect of BTX ( F = 26.67, P = 0.0001), a trend toward a significant effect of cocaine ( F = 2.157, P = 0.1549) but no significant effect of CBZ ( F = 0.05,
eD o o. z O
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Fig 5, In vitro CBZ does not affect basal PI hydrolysis, BTX-induced PI hydrolysis or the attenuation by cocaine of BTX-induced PI hydrolysis. Frontal cortex from untreated rats was sliced and assayed for PI hydrolysis in the presence and absence of 100/zM CBZ, 300 ,ttM cocaine and 0.05 p.M BTX. Results are means + S.E.M. of three independent assays each performed with three to six replicates.
0/0
0/.05
COCAINE/BTX
300/0 CONCENTRATION
300/.05 (~M)
Fig 6. Chronic CBZ treatment in vivo does not affect basal PI hydrolysis, BTX-induced PI hydrolysis or the attenuation by cocaine of BTX-induced PI hydrolysis. Frontal cortex from rats treated for 7 days with CBZ (solid bars; n = 4) or a control diet (white bars; n = 4) was sliced and assayed for PI hydrolysis in the presence and absence of 300 p.M cocaine and 0.05 /~M BTX. Results are means+S.E.M. of four independent assays each performed with three to six replicates.
P = 0.8252; 0.9801).
three-factor
ANOVA:
F = 0.001,
P=
DISCUSSION The data demonstrate that, although in vitro administration of cocaine attenuates BTX-induced PI hydrolysis in rat frontal cortex, neither repeated cocaine injections nor lidocaine kindling alter BTX-induced PI hydrolysis in rat cortex or HC. Lidocaine kindling did not influence IBO-induced PI hydrolysis. The anticonvulsant drug CBZ had no effect when administered in vivo or in vitro on BTX-induced PI hydrolysis in frontal cortex and did not reverse the cocaine-induced attenuation of this phenomenon. These results suggest that neither repeated local anesthetic administration nor CBZ affected the coupling of the BTX-binding site to PI metabolism. We detected attenuation of BTX-induced PI hydrolysis by in vitro cocaine with an IC5o almost identical to that previously reported in slices of mouse cortex ]2. Others, using a dilute solution of guinea pig synaptoneurosomes and much higher concentrations of BTX, have reported an IC50 value > 10-fold higher t8, presumably reflecting the same difference in sensitivity between synapotoneurosome and tissue slice preparations observed in response to BTX. We observed a regional variation in the extent of IBO-induced PI hydrolysis similar to that previously reported 17, confirming the ability of our method to detect alterations in PI hydrolysis. The difference in BTX-induced PI hydrolysis be-
189
tween frontal cortex ( ~ 200% over control; Figs. 1, 2, 5, 6) and HC and PC ( ~ 400% over control; Fig. 3) may reflect an intrinsic regional difference in the coupling of PI hydrolysis to Na channels similar to that observed for IBO. Our results also imply that local anesthetics and CBZ do not alter the number or affinity of BTX-binding sites and therefore that Na channels are not up- or down-regulated by these agents though this possiblity will need confirmation by more direct methods. Overall, the data from this study suggest that Na channel-coupled PI hydrolysis is not altered by local anesthetic kindling. It remains possible that functional properties of Na channel conductance other than induction of PI hydrolysis may have some role in local anesthetic kindling and/or the anticonvulsant properties of CBZ, or that these phenomena alter Na channels in brain regions or at time points other than those assessed in the present study. Unlike electrical kindling 1, lidocaine kindling did not potentiate the PI response to IBO. This difference between the two types of kindling implies either that such a response is not central to the kindling process (i.e., that it is a unique by-product of electrical kindling) or that local anesthetic and electrical kindling are distinct processes produced by divergent mechansims. Acknowledgements. This work was supported in part by a PRAT fellowship from the National Institute of General Medical Sciences to R.L. Margolis. 1 Akiyama, K., Yamada, N. and Otsuki, S., Lasting increase in excitatory amino acid receptor-mediated polyphosphoinositide hydrolysis in the amygdala/pyriform cortex of amygdala-kindled rats, Brain Res., 485 (1989) 95-101. 2 Berridge, M.J., Downes, C.P. and Hanley, M.R., Lithium amplifies agonist-dependent phosphatidylinositol response in brain and salivary glands, Biochem. J., 206 (1982) 587-595. 3 Brown, G.B., Tieszen, S.C., Daly, J.W., Warnick, J.E. and Albuquerque, E.X., Batrachotoxinin-A 20-a-benzoate: a new radioactive ligand for voltage sensitive sodium channels, Cell Mol. Neuro: bioL, 1 (1981) 19-40. 4 Chuang, D.-M., Regulation by batrachotoxin, veratridine, and monensin of basal and carbachol-induced phosphoinositide hydrolysis in neurohybrid NCB-20 cells, Neurochem. Res., 15 (1990) 695-704. 5 Goddard, L.S., Mclntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 6 Gurwitz, D. and Sokolovsky, M., Dual pathways in muscarinic receptor stimulation of of phosphoinositide hydrolysis, Biochem/stry, 26 (1987) 633-638. 7 Gusovsky, F., Hollingsworth, E.B. and Daly, J.W., Regulation of phosphatidylinositol turnover in brain synatoneurosomes: stimulatory effects of agents that enhance influx of sodium ions, Proc. Natl. Acad. Sci. USA, 83 (1986) 3003-3007. 8 Gusovsky, F., McNeal, E.T. and Daly, J.W., Stimulation of phosphoinositide breakdown in brain synaptoneurosomes by agents that activate sodium influx: antagonism by tetrodotoxin, saxitoxin, and cadmium, Mol. Pharmacol., 32 (1987) 479-487. 9 Hondeghem L.M. and Katzung, B.G., Time- and voltage-depen-
dent interactions of antiarrhythmic drugs with cardiac sodium channels, Biochim. Biophys. Acta, 472 (1977) 373-398. 10 Kelly, P.H. and Iversen, S.D., Selective 6-OHDA-induced destruction of mesolimbic dopamine neurons: abolition of psychostimulant-induced locomotor activity in rats, Eur. J. Pharmacol., 40) (1975) 45-56. 11 Khodorov, B., Shishkovak, L., Peganov, E. and Revenko, S., Inhibition of sodium currents in frog Ranvier node treated with local anesthetics: role of slow sodium inactivation, Biochim. Biophys. Acta, 433 (1976) 409-435. 12 Kim, S.S. and Reith, M.E.A., Inhibition by cocaine of inositol phospholipid hydrolysis induced by the sodium channel activator batrachotoxin in mouse cerebral cortex, Biochem. Pharmacol., 37 (1988) 773-775. 13 Marangos, P.J., Weiss, S.R.B., Montgomery, P., Patel, J., Narang, P.K., Cappabianca, A.M. and Post, R.M., Chronic carbamazepine treatment increases brain adenosine receptors, Epilepsia, 26 (1985) 493-498. 14 McDonough, P.M., Goldstein, D. and Brown, J.H., Elevation of cytoplasmic calcium concentration stimulates hydrolysis of phosphatidylinositol bisphosphate in chick heart cells: effect of sodium channel activators, Mol. Pharmacol., 33 (1988) 310-315. 15 McLean, M.J. and Macdonald, R.L., Carbamazepine and 10,11epoxycarbamazepine produce use- and voltage-dependent limitation of rapidly firing action potentials of mouse central neurons in cell culture, J. Pharmacol. Exp. Ther., 238 (1986) 727-738. 16 Moneta, M.E., de la Fuente, M., Liberona, J.L. and Jaimovich, E., Sodium pathway markers in normal and kindled frog brains, Neurosci. Lett., 65 (1986) 331-335. i7 Nicoletti, F., Meek, J.L., ladarola, M.J., Chuang, D.-M., Roth, B.L. and Costa, E., Coupling of inositol phospholipid metabolism with excitatory amino acid recognition sites in rat hippocampus, J. Neurochem., 46 (1986) 40-46. 18 Nishizawa, Y., Gusovsky, F. and Daly, J.W., Local anesthetics: comparison of effects on batrachotoxin-elicited sodium flux and phosphoinositide breakdown in guinea pig cerebral cortical synaptoneurosomes, Mol. Pharmacol., 34 (1988) 707-713. 19 Post, R.M., Progressive changes in behavior and seizures following chronic cocaine administration: relationship of kindling and psychosis. In E.H. Ellinwood and M.M. Kilbey (Eds.), Advances in Behavioral Biology: Cocaine and Other Stimulants, Plenum Press, New York, 1977, pp. 352-353. 20 Post, R.M., Lidocaine kindled limbic seizures: behavioral implications. In J.A. Wada (Ed.), Kindling 2, Raven Press, New York, 1981, pp. 149-160. 21 Post, R.M. and Contel, N.R., Cocaine-induced behavioral sensitization: a model.for recurrent manic illness. In C. Perris et al. (Eds.), Biological Psychiatry, 1981, Elsevier, Amsterdam, 1981, pp. 746-749. 22 Post, R.M., Kopanda, R.T. and Lee, A., Progressive behavioral changes during chronic lidocaine administration: relationship to kindling, Life Sci., 17 (1975) 943-950. 23 Post, R.M., Uhde, T.W., Ballenger, J.C., Chatterji, D.C., Greene, R.K. and Bunney, W.E., Jr., Carbamazepine and its -10,11epoxide metabolite in plasma and CSF: relationship to antidepressant response, Arch. Gen. Psych., 40 (1983) 673-676. 24 Post, R.M., Weiss, S.R.B. and Clark, M., Amygdala versus local anesthetic kindling: differential anatomy, pharmacology and clinical implications. In J.A. Wada (Ed.), lO'ndling 4, Raven Press, New York, in press. 25 Scheel-Kruger, J., Braestrup, C., Nielson, M., Golembrowska, K. and Mogilnicka, F., Cocaine: discussion on the role of dopamine in the biochemical mechanism of action. In E.H. Ellinwood and M.M. Kilbey (Eds.), Advances in Behavioral Biology, Vol. 21, Cocaine and Other Stimulants, Plenum Press, New York, 1977, pp. 373-408. 26 Taouis M., Sheldon, R.S. and Duff, H.J., Upregulation of the rat cardiac sodium channel by in vivo treatment with a class I antiarrhythmic drug, J. Clin Invest., 88 (1991) 375-378. 27 Wang, G.K., Cocaine-induced closures of single batrachotoxinactivated Na + channels in planar lipid bilayers, J. Gen. Physiol., 92 (1988) 747-765.
190 28 Weiss, S.R.B., Post, R.M., Costello, M., Nutt, D.J. and Tandeciarz, S., Carbamazepine retards the development of cocaine-kindled seizures but not sensitization to cocaine-induced hyperactivity, Neuropsychopharmacology, 3 (1990) 273-281. 29 Willow, M., Kuenzel, E.A. and Catterall, W.A., Inhibition of voltage-sensitive sodium channels in neuroblastoma cells and synaptosomes by the anticonvulsant drugs diphenyl-hydantoin and carbamazepine, Mol. Pharmacol., 25 (1984) 228-234.
30 Willow, M., Taylor, S.M., Catterall, W.A, and Finnell, R.H., Down regulation of sodium channels in nerve terminals of spontaneously epileptic mice, Cell Mol. Neurobiol., 6 (1986) 213-220. 31 Zimanyi, I., Weiss, S.R.B., Lajtha, A., Post, R.M. and Reith, M.E.A.., Evidence for a common site of action of lidocaine and carbamazepine in voltage-dependent sodium channels, Eur. J. Pharrnacol., 167 (1989) 419-422.