Rat Hippocampus Elaine, E

Br. J. Pharmacol. (1989), 98, 581-589

Inhibition of agonist-stimulated inositol lipid metabolism by the anticonvulsant carbamazepine in rat hippocampus Elaine, E. McDermott & 'S.D. Logan

Department of Physiology, The Medical School, University of Birmingham, Birmingham B15 2TJ

1 The effect of the anticonvulsant, anti-manic drug carbamazepine was examined on inositol lipid signalling in rat hippocampus in vitro. 2 Hippocampal miniprisms were labelled with [3H]-inositol before stimulation with a variety of neuroactive agents that increase phosphoinositide turnover. 3 The presence of carbamazepine (0.1-100 yM) during labelling caused a dose-related reduction of basal and carbachol-evoked [3H]-inositol phosphate accumulations. The effect of the drug on basal inositol phosphate levels was lost when slices were labelled with [3H]-inositol before incubation with carbamazepine. 4 Incubation of slices with carbamazepine after labelling with [3H]-inositol and before stimulation showed the inhibitory effect of the drug to be selective according to the agonist used. Responses to carbachol, histamine and the sodium-channel agent veratrin were reduced by carbamazepine whilst the responses to 5-hydroxytryptamine, noradrenaline and substance P were unaffected. 5 Inhibition of carbachol, histamine and veratrin-induced stimulation by carbamazepine share a similar dependence on length of pre-incubation time with the drug. However, the effect of carbamazepine (100pM) on the respective dose-response curves suggests that the mechanism of inhibition of the carbachol response differs from the inhibition of the histamine and veratrin responses. These effects may be significant in the mechanism of action of carbamazepine as an anticonvulsant and in its effectiveness against manic depression.

Introduction Carbamazepine (5H-dibenz[b,f]azepine-5-carbo- drug (Morselli, 1976), Cbz has been found to affect xamide, Tegretol, Ciba-Geigy) is a first-line anti- binding (Skerritt et al., 1982; 1983; Marangos et al., convulsant used in the treatment of generalised 1983; 1985; 1987) and functionality (Lewin & Bleck, (tonic-clonic) and partial seizures. In addition, it is 1977; Phillis, 1984) of purinoceptors. At higher con- effective against trigeminal neuralgia (Bonduelle, centrations it has been shown to bind to the 'micro- 1976), arrhythmia and manic depression (Post et al., molar benzodiazepine receptor' (Weiss et al., 1985) 1983). Electrophysiological experiments (Willow et and inhibit Ca2 +-calmodulin-dependent protein al., 1983), receptor binding studies (Willow & phosphorylation (De Lorenzo, 1984). Catterall, 1985) and release studies (Crowder & The phosphoinositide hydrolysis signalling Bradford, 1987; E.E. McDermott, J.D. Turner & pathway involves the metabolism of the membrane S.D. Logan, unpublished results) have shown lipid phosphatidylinositol-4,5 bisphosphate (PtdIns4, carbamazepine (Cbz) to inhibit Na' and Ca2+ influx 5P2) to produce the second messengers inositol 1,4,5- by an interaction with voltage-dependent sodium trisphosphate (which mobilises intracellular calcium) channels (Willow, 1986). At therapeutic levels of the and diacylglycerol (which activates protein kinase C) (for reviews see Berridge & Irvine, 1984; Downes, 'Author for correspondence. 1986).

Several factors prompted the investigation into an buffer. This produced tissue densities of 1-1.5 mg per action of Cbz on this pathway. Firstly, Cbz shares its tube. Slices were incubated for 1 h with the label therapeutic action against depression with lithium before 10pl LiCl was added to give a final concen- which is established as having several effects on ino- tration of 10mM. After 10min the agonists were sitol lipid metabolism (Hallcher & Sherman, 1980; added in 10p1 to give a final volume of 300p1. Incu- Batty & Nahorski, 1987; Erneux et al., 1987; Acker- bations were re-gassed and stoppered every 20-30 mann et al., 1987; Kendall & Nahorski, 1987; min. Newman & Lerer, 1988; Ragan et al., 1988). Activa- Alternatively, for bulk labelling of tissue, the slices tion of protein kinase C appears to be involved in were suspended in 5 ml of buffer and preincubated the generation of hippocampal long term poten- for 10min at 37°C. They were then labelled by incu- tiation (Malenka et al., 1986), a phenomenon which bating with 0.4-0.8 pM [3H]-inositol for 1 h at 37°C has been compared to kindling (Baudry, 1986). in a shaking water bath under an atmosphere of Finally, increases in the products and metabolites of 95%02/5%CO2. Slices were washed with fresh inositol lipid turnover have been shown to be associ- buffer to remove excess label before incubation of ated with various seizure models (Bazan et al., 1986; 50.u1 aliquots in conditions as for continuous label- Iadorola et al., 1986) and with processes linked to ling. epilepsy, such as long term potentiation (Lynch et Incubations were terminated by addition of 250p1 al., 1988) and electroconvulsive shock (Vadnal & of ice-cold pefchloric acid (10%w/v) and put on ice Bazan, 1988). for 1Omin. These studies examined the effects of Cbz on the [3H]-inositol mono- and polyphosphates were inositol lipid signalling system in rat hippocampal extracted from the slices by a method based on that slices in vitro. Muscarinic agonists have been demon- of Sharps & McCarl (1982). After centrifugation at strated to stimulate hydrolysis of PtdInsP2 in this 2000g for 5min, the supernatant was removed and tissue (Stephens & Logan, 1989a) leading to rapid neutralised with 0.5 ml of a 1:1 (v/v) mixture of tri-n- formation of several inositol phosphates including octylamine and Freon. In later experiments maleic Insl,4,5P3 and Insl,3,4,5P4 (Stephens & Logan, acid was added (final concentration 75 mM) to mini- 1989b). The results indicate apparent inhibition of mise loss of inositol trisphosphate (InsP3) (see Wreg- both basal and cholinergically-stimulated [3H]-ino- gett et al., 1987). However, no significant differences sitol phosphate accumulation when the drug is were observed in recovery of total inositol phos- present during the labelling procedure, possibly by phates with this amendment. This may be due to affecting [3H]-inositol distribution in the inositol metabolism of InsP3 during long incubations. After lipids. In addition, the presence of Cbz after labelling being vortexed for 30s the phases were separated by caused selective reduction of particular agonist- centrifugation at 2000g for 5min (4°C). An aliquot stimulated [3H]-inositol phosphate accumulations. of the upper aqueous phase containing the [3H]-ino- Such effects may be of importance in the mecha- sitol phosphates was then removed for separation by nisms of action of the drug as an anticonvulsant or anion-exchange chromatography. antidepressant. Part of this work has been published In experiments in which total [3H]-inositol lipids previously in abstract form (McDermott & Logan, were measured, lipids were separated from water 1988). soluble products using chloroform: methanol (1:2) according to the method of Donaldson & Hill (1986).

Separation of[3H]-inositol phosphates Methods [3H]-inositol-labelled acid-soluble metabolites were Male Sprague-Dawley (300-400 g) rats were killed by separated by use of 0.6 x 2cm columns of BioRad cervical dislocation and brains removed rapidly. AG1 anion-exchange resin (1 x 8, 20040 mesh, Hippocampal tissue was dissected on ice and the for- formate form) made up in glass Pasteur pipettes. An mation chopped transversely at 350pum intervals in aliquot of water-soluble metabolites was applied to three directions (600 rotation between each) by the columns in IOml of 5 mm di-sodium tetraborate/ means of a McIlwain tissue chopper. The miniprisms 0.5 mM ethylenediaminetetraacetic acid (EDTA) after formed were then washed several times in gassed the resin had been equilibrated with 20 ml of distilled (95%02/5%CO2) Krebs-Ringer bicarbonate buffer water and lOml tetraborate/EDTA. (Stephens & Logan, 1986). Before collection of the inositol phosphates, free After the wash procedure, 50y1 aliquots of tissue [3H]-inositol was removed with 20ml of water; gly- were added to vials containing 3-7 uCi myo-2 [3H]- cerophosphoinositides (GroPIns) were then eluted inositol (specific activity 10-20 Ci mmol- ) in gassed with 15 ml of 50mM ammonium formate (AF). [3H]- CARBAMAZEPINE AND INOSITOL PHOSPHATES 583 inositol phosphates (InsPs) were eluted with 8 ml of Table 1 Effect of carbamazepine (Cbz) on basal 1.025M AF/0.1 M formic acid and 2 ml taken for and carbachol-induced inositol phosphate accu- liquid scintillation counting after the addition of mulations in rat hippocampus scintillant (Optiphase-X). Carbachol Protein values were estimated by the method of Basal (1 mM) Lowry et al. (1951) with bovine serum albumin (BSA) standards. (a) Cbz present during labelling Control (+ Li) 100+3 100 + 5 0.1 IM Cbz 88 + 15 97 + 4 Materials 1 M Cbz 100 +8 89 + 7 1OpM Cbz 82 + 4* 83 + 1* Carbamazepine was provided by Ciba-Geigy. 100pM Cbz 75 + 7** 72 + 5** 100pM Cbz (-Li) 77 + 8** ND Myo-2-[3H]-inositol (10-20Ci ml-1) was purchased Vehicle 0.4% DMSO 106 + 16 ND from Amersham U.K. Tri-n-octylamine and Freon (b) Cbz present after labelling were purchased from Aldrich Chemical Co; car- Control (+ Li) 100 + 5 100+9 bachol, 5-hydroxytrypamine, histamine dihydrochlo- 100pM Cbz (+Li) 99 + 14 66 + 10* ride, noradrenaline, veratrin and tetrodotoxin from the Sigma Chemical Co. Inorganic salts and organic Results are expressed as percentage of control (i.e. solvents were purchased from Fisons or Fluka. incubations without Cbz). Basal d.p.m. is approximately 1000d.p.m./50pl slices. (a) Slices were incubated with Cbz (0.1-100pM) for the total experiment including the 1 h labelling period with [3H]-inositol. (b) Slices were first prelabelled with Results [3H]-inositol then incubated with 100pM Cbz for 1 h before addition of 1 mm carbachol. Values rep- Incubation of hippocampal slices with Cbz during resent means ± s.e. mean (n = 4-12) with sta- labelling with [3H]-inositol resulted in decreased tistical analysis by Student's t test. *P < 0.05, basal and carbachol-stimulated total [3H]-inositol **P < 0.01. ND-not determined. phosphate levels, the maximum reduction with 100yM Cbz being 25% and 28%, respectively (Table la). This inhibition by the anticonvulsant appears to be dose-related and significant reductions of 17-18% phosphates is seen when Cbz is present after the were attained at approximately 10pM Cbz. Use of labelling process this suggests that there may be higher concentrations of the drug was limited by the some effect on the distribution of the label within the need for high levels of vehicle (dimethylsulphoxide) lipids (Table lb). It is unlikely that the general level to maintain its solubility. Such concentrations also of incorporation of [3H]-inositol into membrane lessen the relevance of the effects to the actions seen lipids is significantly decreased since measurements in vivo at therapeutic levels. Further investigation of of 3H-lipids (following a chloroform-methanol the action of the drug on inositol lipid hydrolysis extraction) are not significantly different in control was therefore carried out with 100 uM Cbz in 0.4% tissues (43,582 + 5,032 d.p.m.) compared with tissues dimethylsulphoxide. Although this concentration is labelled in the presence of 100 pM Cbz (47,334 + above the average therapeutic levels measured in 5,656 d.p.m.). However, when Cbz was present after plasma (10-50pM), Cbz is absorbed rapidly and is the labelling process the inhibition of the carbachol concentrated in brain and other tissues (Morselli et response remained, and was of a similar magnitude al., 1971) and may lead to levels of 100pM. (34%) to that observed when Cbz was present during The presence of LiCl (10mM) during incubation the labelling process. This suggests that there is more had no effect on the inhibition caused by Cbz. This than simply a general membrane stabilisation suggests that despite a common therapeutic action in involved in the reduction of the carbachol response. manic depression, Cbz does not interfere with the Further investigations used a variety of agents to activity of inositol monophosphatase as does stimulate phosphoinositide turnover and showed lithium. that the inhibitory action of Cbz appears to be selec- The lipophilic nature of Cbz suggests that its tive (Figure 1). Stimulation by carbachol (1 mM) was inhibitory effect on [3H]-inositol phosphate levels reduced by 25% from 4.5 fold to 3.4 fold, histamine may be related to membrane stabilisation, perhaps (100pM) stimulation by 60% from 4.3 fold to 1.7 fold altering the relative amounts of phosphatidylinositol and the veratrin (100pM) response by 41% from 2.7 (PtdIns), phosphatidylinositol phosphate (PtdInsP) fold to 1.6 fold. However, Cbz had no effect on and phosphatidylinositol bisphosphate (PtdInsP2). responses to noradrenaline (1 mM), 5-hydroxy- Moreover, since no reduction in basal [3H]-inositol tryptamine (1 mm) or substance P (10pM). 584 E.E. McDERMOTT & S.D. LOGAN

-0 300 0 C-) 0

Control Carbachol Histamine Veratrin Noradrenaline 5-HT Substance P Figure 1 Effect of carbamazepine on stimulation of total [3H]-inositol phosphate accumulation by various agents in rat hippocampus. Slices pre-labelled with [3H]-inositol were incubated with 100/OM carbamazepine (Cbz) for 1 h before stimulation with carbachol (1 mM), histamine (100yM), veratrin (1001iM), noradrenaline (1 mM), 5- hydroxytryptamine (5-HT, 1 mM) or substance P (10pM). Values are expressed as percentage of unstimulated. Columns represent the mean (n = 4-12) and vertical bars show s.e.mean. Open columns, control responses in absence of Cbz; stippled columns, responses in presence of Cbz. Statistical analysis was by Student's t test, *P < 0.05, **P < 0.005.

Time course experiments which involved increased pre-incubation time with 100pM Cbz before stimulation (for a constant time of 45 min) with carbachol, veratrin and histamine showed the inhibition by the 120 anticonvulsant to be dependent on the length of time the tissue was pre-incubated with Cbz (Figure 2). A pre-incubation of approximately 40min was needed for a statistically significant reduction of carbachol 100 stimulation, whilst approximately 60 min was needed for reductions of both veratrin and histamine stimu- lations. Comparison of the effects of Cbz (100pM) on carbachol, histamine and veratrin dose-response curves 80 showed differences in the apparent type of inhibition 0C 60 (Figure 3). The presence of Cbz caused a parallel shift to the right of the carbachol curve which was not observed with the dose-response curves of hista- 40 mine and veratrin, although in these cases there was a reduction in the maximum response. The effect of Cbz on the response to veratrin may 0 20 40 60 be explained by its established action at sodium Preincubation time with Cbz (min) channels, as the presence of the sodium channel blocking agent tetrodotoxin (5pM) in incubations Figure 2 Time-dependence of inhibition of stimulated blocked the veratrin response (Table 2). However, inositol phosphate accumulation by carbamazepine the toxin had no effect on stimulation of InsP accu- (Cbz). After pre-labelling with [3H]-inositol, hippocam- mulation by carbachol or histamine. Hence the pal slices were incubated with Cbz (100/M) for various lengths of time before incubation with (0) carbachol reduction of these responses by Cbz does not appear (1mM), (0) histamine (2mM) or (*) veratrin (100/AM) to be due to inhibition of a polysynaptic effect and for 45min. Values represent mean (n = 4-6); *P < 0.05, release of endogenous agonists of phosphoinositide **P < 0.02, ***P < 0.01 by Student's t test. turnover. CARBAMAZEPINE AND INOSITOL PHOSPHATES 585

a b c 500 *

400

°- 300- *** *** C 0 0 O 200

100 T

- + 6 5 4 3 8 7 6 5 4 3 7 6 5 4 Cbz -Iog [CarbacholJ (M) -log[Histaminel (M) -logWVeratrinI (M) Control Figure 3 Effect of carbamazepine (Cbz) on (a) carbachol, (b) histamine and (c) veratrin-stimulated total [3H]- inositol phosphate accumulation in rat hippocampal slices. Hippocampal slices were incubated with 100pM Cbz for h after prelabelling with [3H]-inositol and before addition of agonists. Responses in absence of Cbz (solid symbols), responses after preincubation with Cbz (open symbols). Values represent mean (n = 4-9); vertical lines show s.e.mean. *P < 0.05, **P < 0.025, ***P < 0.005 by Student's t test.

For comparison we investigated the effect of the Discussion anticonvulsant phenytoin on [3H]-inositol phosphate accumulation in response to carbachol (1 mM), This study demonstrates an inhibitory action of the histamine (100gM) and veratrin (100pM). Incubation anticonvulsant drug Cbz on inositol lipid metabo- of slices for one hour with 100puM phenytoin resulted lism in rat hippocampal slices. This is in common in a 38% decrease in carbachol stimulation and a with the in vivo effect of Cbz on electroconvulsive- 33% decrease in the veratrin response but had no shock-induced inositol phosphate accumulation in effect on that of histamine (Table 2). rat cortex and hippocampus (Vadnal & Bazan, 1988). When present during the labelling procedure Table 2 The effects of tetrodotoxin (TTX) and with [3H]-inositol both basal and carbachol- phenytoin on inositol phosphate accumulation stimulated inositol phosphate accumulations were stimulated by carbachol, histamine and veratrin. reduced. This possibly reflects a general membrane stabilisation, due to the lipophilic nature of Cbz. Basal Carbachol Histamine Veratrin Such an interaction could inhibit lipid turnover reducing incorporation of [3H]-inositol into phos- TTX 112+8 95+8 108+6 56+4 phoinositides or changing the distribution within (SpM) Phenytoin 106 + 13 62 + 3 100 + 23 67 + 9 PtdIns, PtdInsP and PtdInsP2. The latter explana- may more as no (100p M) tion be likely change in the levels of total labelled lipids was observed. Hippocampal slices were prelabelled with [3H]- A second effect, produced by incubation with Cbz inositol for one hour before incubation with TTX for one hour after prelabelling and before stimu- (5pM) for 10min or phenytoin (100pM in 0.4% lation, involved a selective reduction of carbachol, DMSO) for one hour before stimulation with car- veratrin and histamine-evoked [3H]-InsP accumula- bachol, histamine and veratrin (40 min). Results tion. The lack of effect of Cbz on phosphoinositide are expressed as percentage of basal or stimulated responses to noradrenaline, 5-hydroxytryptamine [3H]-inositol phosphate accumulation measured and substance P indicates that the drug does not in the absence of TTX or phenytoin. Values represent means + s.e.mean of a single experiment have a general action at the phosphoinositidase. conducted in quadruplicate, essentially identical This selectivity may indicate an interaction of Cbz results were obtained on two to five further with particular receptors or receptor-transduction occasions. mechanisms. 586 E.E. McDERMOTT & S.D. LOGAN

Time course experiments showed inhibition of of a polysynaptic response as inclusion of tetro- [3H]-InsP accumulation by Cbz (100pM) to increase dotoxin had no effect on these responses (Table 2). with time of incubation with the anticonvulsant However, studies on guinea-pig synaptoneurosomes (Figure 2). This time lag may suggest that some form (Gusovsky & Daly, 1988) suggest that sodium influx of equilibrium is reached between Cbz and a binding due to such agents as veratrin may increase intracel- site within the lipid bilayer. Alternatively it may lular calcium levels via the Na+/Ca2+ exchanger, reflect the need for a modulator substance to accu- resulting in a direct enhancement of phospholipase mulate and reach a threshold. activity. If, by its action at sodium channels, Cbz Although Cbz-induced inhibition of carbachol, produces a significant change in intracellular calcium veratrin and histamine responses had a similar time levels then this may provide a mechanism for its dependence, the apparent nature of the effect on car- effects on responses to other agonists. Selectivity bachol differs from that on veratrin and histamine could then arise from the variable calcium sensitivity responses. This can be seen by comparison of their in phosphoinositide turnover in response to particu- respective dose-response curves in the presence or lar agonists. For instance, the response to histamine absence of Cbz (Figure 3). The parallel shift to the in brain slices has been shown to require higher right of the carbachol dose-response curve is sugges- levels of Ca2 + than those of carbachol and 5- tive of competitive inhibition by Cbz of carbachol hydroxytryptamine (Kendall & Nahorski, 1984). stimulation. However, the effect of Cbz (100pM) on However, another possible explanation for inhibi- histamine and veratrin stimulation does not appear tion of the histamine-induced response arises from to be competitive but a consequence of a decreased the apparent interaction of histamine and adenosine maximal response. receptors. In addition to the potentiation of stimu- Explanation of the action of Cbz on carbachol- lated cyclic AMP levels by H1 agonists evoked [3H]-InsP accumulation is difficult as recep- (Hollingsworth & Daly, 1985, Hollingsworth et al., tor binding studies show no interaction with the 1986), there is now evidence of an alteration in muscarinic receptor at similar concentrations to histamine-induced phosphoinositide turnover by those used in inositol lipid studies (Skerritt et al., adenosine agonists (Hill & Kendall, 1986; 1987; 1983). This discrepancy may arise from differences in Kendall & Hill, 1988). An interaction of Cbz with time of incubation with Cbz, or perhaps reflects adenosine receptors (Marangos et al., 1983; 1985; competition with carbachol at a step beyond the 1987; Skerritt et al., 1982; 1983; Fujiwara et al., receptor-agonist interaction site. Such a selective 1986) may affect the H1-receptor in a manner similar action of Cbz on the muscarinic response is inter- to that of an A1-agonist. Interestingly, although in esting in view of induction of seizures by agonists guinea-pig and mouse cortical slices adenosine such as pilocarpine (Turski et al., 1983). (A )-receptor activation can modulate the histamine The reduction in the veratrin response can be response, no effect could be observed in rat cortical explained by the action of Cbz at the voltage- slices (Kendall & Hill, 1988). However, no such dependent sodium channel (Willow, 1986). Electro- experiment appears to have been performed in rat physiological and neurochemical studies show Cbz hippocampal slices, hence an effect on Cbz via aden- to inhibit binding of sodium channel agents such as osine receptor interaction cannot be discounted. batrachotoxin-B and veratrin to the neurotoxin In addition to the above putative mechanisms of receptor 2 (Schauf et al., 1974; Worley & Baraban, the effect of Cbz on histamine stimulation, there is 1987; Willow et al., 1985; Willow & Catterall, 1985). also the possibility of a direct interaction with the The model of this interaction proposed by Willow H -receptor. This is suggested by the binding studies (1986) explains the voltage and use-dependency of that show tricyclic antidepressants, that are structur- the interaction of Cbz with the channel. It proposes ally similar to Cbz, act as H,-antagonists in neuro- that Cbz binds with high affinity to the inactive state blastoma and guinea-pig ileum (Taylor & Richelson, of the channel, stabilising it, preventing its closure 1979). and subsequent activation. The need for the channel Preliminary studies have examined the effects of to pass through the activated state before binding of phenytoin, an anticonvulsant with a similar thera- Cbz may also explain the time lag seen in inositol peutic spectrum to Cbz, on phosphoinositide phosphate studies (Figure 2) the resulting decrease in responses to carbachol, histamine and veratrin. As inositol lipid turnover being due to a reduction in would be expected phenytoin, which has also been the depolarisation-induced release of endogenous established to act at sodium channels (Willow, 1986), agonists (Olpe et al., 1985; Crowder & Bradford, reduced veratrin-stimulated inositol phosphate accu- 1987; E.E. McDermott, J.D. Turner and S.D. Logan mulation to 68% of the normal response (Table 2). unpublished observations). It appears, however, that The response to carbachol (1mM) was reduced to the effect of Cbz on the histamine and carbachol 75%, but basal and histamine-stimulated levels were response is not due to a reduction in Na+ influx or unaffected. The two inhibitory actions of phenytoin CARBAMAZEPINE AND INOSITOL PHOSPHATES 587 observed in these studies were of similar magnitude lithium may be due to competition with the magne- to those of 100yM Cbz perhaps reflecting the sium needed for GTP binding, Cbz may take effect common action of the drugs as anticonvulsants. The by altering interactions of the receptor, G protein lack of effect on histamine stimulation by phenytoin and phospholipase C within the lipid bilayer. could reflect the closer structural similarity between Thus, we postulate that Cbz can affect the inositol Cbz and the tricyclic antidepressants and the action lipid signalling pathway in several complex and of Cbz as an antidepressant. perhaps interactive ways-(i) by altering the basal These results support the finding of Vadnal & turnover of the lipids, possibly by affecting the dis- Bazan (1988) that Cbz and Li' do not share an tribution of membrane inositol lipids, (ii) by affecting inhibitory action on the inositol-1-phosphatase. the receptor-G-protein-enzyme complex and (iii) by These authors found that Cbz inhibited the increase acting directly at the receptors for some ligands that in [3H]-InsP3 level, following electroconvulsive utilise this signalling pathway. shock treatment in rat cortex and hippocampus, sug- Such interactions of Cbz with this signalling gesting interference with signal transduction at the system may be important in its therapeutic action membrane. However, it is conceivable that as yet against manic depression. In addition these studies unclarified actions of lithium on phosphoinositide suggest that the inositol lipid signalling pathway metabolism (e.g. reduction of carbachol-induced ino- may be a site of action of anticonvulsant drugs and sitol tetrakisphosphate levels in brain, Batty & thus this mechanism may be important in epilep- Nahorski, 1987) may be common to both drugs. A togenesis. possible shared site of action is in the receptor/G protein/phospholipase transduction system where E.E.M. is an MRC research student. We are grateful lithium appears to reduce agonist-stimulated GTP to Ciba-Geigy for the gift of carbamazepine and binding (Avissar et al., 1988). Whereas the action of financial support.

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