Lithium and the phosphoinositide cycle: an example of uncompetitive inhibition and its pharmacological consequences

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Lithiumand the phosphoinositidecycle: an exampleof uncompetitive inhibitionand its pharmacologicalconsequences Stefan R. Nahorski, C. Ian Ragan and R. A. John Challiss The ability of lithium to rxert profound and selective psychopharmncolo~caI efftcts to ameliorate manic-depressioe psychosis has been the focus of considerable research effort. There is increasing evidence that lithium exerts its therapeutic action by inferfen’xg with pofyphospkoinositide metabolism in brain and prcwntion of inositof recycling by an uncompetitive inhibiffon of inositol monophosphutuse. Stefan Nahorski, Ian Ragan and John Challiss discuss this t.nusual stimulus-dependentfountof enzyme inhibition, emphnsizing that the sekctiuity exhibited by lithium depends upon the degree of inositol lipid hydrolysis and polyphosphoinositide dephosphorylafion. It is now well established that a variety of agonists acting at cell surface receptors can alter cellular function by activating phosphoinositidase C (via a guanine nude&de exchange protei$. The major (or, at least, the initial) target of this enzyme is phosphatidyhnositol I$-bisphosphate (PIP& and its immediate products - sn(l,Z)diacylglycerol (DAG) and inositoll,4%risphosphate (IPs) are important second messengers in many cells, including neurons. IPs, and perhaps its phosphorylated product inositoll,3,4~tetraki@osphate (IP,), have been established to control intracellular Ca** homeostasis and the activation of protein kinase C by DAG. However, despite the fundamental importance of this signalling system, from a pharmacologi5. R. Nuhorski is Professor of Pharmacobgv. and R. A. /. Chnlliss i3 a Wclfcome Lccfrrrr in Biochemicd Phemacd~ in tht Dqmrtment of Pharmacology md Theraprulics, Uniurrsity of kiccsfn, PO Box 138, Medical Scirnrrs Mding, Ueiwrsity Road, Leiresler lE1 9CIN. UK. C. I. Rqan is Director of Biorlwmietry at Mrrck. Sharpr 6 Dohw Research Labotafor~rs. Nrurosci.wcrs Rrsearch Cmtrr. Ter@s Pnrk, Herlow, Escx CM20 2QR. UK.

cal standpoint there have been few advances in attempting to perturb phosphoinositide (PI) metabolism other than via the traditional approach of targeting cell surface receptors selectiveiy. Almost certainly this reflects the reluctance of the pharmacologist to target drugs at apparently ubiquitous second messenger systems, although the development and utility of isoenzyme-selective inhibitors for cyclic nucleotide phosphodiesterases’ may encourage a similar approach for phosphoinositidase C and protein kinase C isoenzymes. This review highlights lithium as one of the few selective agents that can perturb PI signalling. Although this monovalent ion has been used for nearly 40 years in the treatment of manic-depressive illness, its ability to interfere with inositoi recycling, and convincing evidence that receptor-stimulated PI second messengers are sup pressed by lithium, are only just being recognized. A variety of hypotheses have been forwarded to explain its therapeutic success, that it including suggestions exerts its primary effect at the level of the receptor, G protein

and second messenger-. However, it is only recently that a hypothesis has been forwarded that contains adequate provision to explain the tissue anh cellular selectivity exhibited by lithium concentrations within the therapeutically effective range. Berridge and colleagues9 first suggested that lithium might reduce the supply of inositol required to sustain PI synthesis and have recently amplified this view using examples of lithium action in neuronal tissue and during embryonic developmentlo. This action of lithium probably results from its unusual uncompetitive inhibition of the enzyme inositol monophosphatase. Uncompetitive inhibition provides unique selectivity that depends upon the strength of the initial receptor stimulus and thus the eventual concentration of enzyme substrate. Although this type of inhibition (see Box) may have catastrophic consequences for cellular metabolism, it may also provide a selectivity of action directed at those populations of cells in which the susceptible pathway is abnormally activated. PI metabolism and inositol recycIb8g Receptor-G-protein activation results in the phosphodiesteratic cleavage of PIP, by a phosphoinositidase C. It is not certain whether more than one inositol phospholipid may be cleaved in response to receptor activation (or indeed in response to an increase in [CasQ because the inositol (poly)phosphates that would be formed are further metabolized by the same enzymes responsible for the inactivation of 1% (Fig. 1). However, a feature of such a mechanism is that it would allow for a disproportionate production of the intracellular signals of IPs and DAG with the latter arising directly from phosphatidyhnositol or phosphatidylinositol 4-phsphate (PIP,), perhaps temporally dissociated from the response that generates IP,. The complexity of the metabolic pathways for inositol phosphates (Fig. 1) appears to relate to the need to terminate the action of IPJ rapidly, in order to generate other messengers such as IP, (Reb 1.3) and to conserve efficiently the cellular supplies of inositol for

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Uncompetitiveinhibitors and their effects on met8bolk pathwrtlrs Pure uncompetitive inhibition is defined as that which results in ~arak\ Lineweaver-Burkplots as shown in the Figurb. Mechanistically, this m&e of inhibition arises when the inhibitor does not interact with the enzyme form that binds the substrate01 with any form of the enzyme upstream of substrate bindiig that is a pmr of the substrate-bound form. The simplest mechanism to assume is therefore one in which the inhibitor binds to the enzyme-substrate complex only, and not to the hpe enzyme. The rather unusual and counterintuitive eonsequences of uncompetitive inhibition can be simply demonslrakd from this form of graphical representation. Starting at substrate amcentrations defined by the points A, B or C, addition of a fixed concentration of inhibitor causes a decrease in velocity (increase in l/v) which is clearIy pmportionately greatest at point A, lesser at point B and ieast at point C, i.e. uncompetitive inhibitors work best at high sub&late concentrations. This does not mean, however, that these inhibitors necessarily produce an insurmountable blockade. At point C, forexample, a modest rise in substrate (to point C’) will restore the velocity in the presence of inhibitor to the same as the uninhibited rate. However, at point B,therisewouldhavetobeinfinitelyhighsincethe cormponding point B’ is at l/s = 0. Furthermore,at point A, increasing substrate can only partially overcome the inhibition. Thus, restoration of activity requires progressively greaterincreases in substrate as the staaing substrate cuncentration is raised, and eventually full restorationbecomes impossible.

degree of stimulus dependency is not so great with the Latter. The implications of this for celhdar metabolism were first pointed out by Comish-Bow&n’. If an inhibitor is inttuduced into a metabolic pathway, there will in generalbeadecreaseinthefluxthroughthatpaihway anddxuxgestotheconcentrationsdthepathway intermediates. The magnitude of the &crease in ftux depends on the flux control coefficient of the enzyme in question. If it is low, then inhibiting the amyme will

to restmefhax to some&gappnkhing the rumnal level and au other metabolite lewd5 (in a siaple world) woukl remain substantially unaltered. Some&&g like thismurrthspparwhenBthiumbntdedtoa~.?hm isaninaepscin inositolm~~on, but, at least at short eqosure -ii&, inositoI *ho+ phak levels are relativdv unrffsted. Theinmin ‘mositolmonophosphate -kquired to restore the status quodependsonthedegreeofstim&tionofthec4;the fastei that the inositol monopw enzyme is working prior to addition of iithiwn, the gzeater will be the increase in inositol monophosphate. Indcad, if the concentrationoflithiumishigh~itmaynotbe possible to mrercome theblockademdmab&atewould rise ind$fbIitdy. In pmctke, of anUse, baraw we ale dealiigivith ~multktep met&ok pathway, and one thatisrvdk,themam&uleofanvknxeeseuiUbe limitedhcetheto4masaofi&&drcnilrbkis constant.

Eventually there will be a &&ion

in the

concentration6of cycle iwrme&atea (e.g. lP3 and rP3 and a slowing of tha cycle Bux. tive In general, the concentfitiar of an uncem inhibitorthatkadrtostimtdusdcpcndclry 2 no&be muchlessthanthatwh&hgivesriaetomkurmountableblockade.Ontheotherhand,bww~&tkms 0ftheinhibitorwiUnotcausemachdbhuknato either flux or met&o& levels under auy celklu

111

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concentration. UnannpeHtivene~ is thereforea&active from the

Noncompetitive inhibition can also provide an insurmountabfe blockade at hi substrate and inhibitor concentrations. The diffcram is that pure noncom@tivefnhibftomareequaUyeffectiveathighorlow 3ubstrate cffsahn

concentcatioh, at low

substrate

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of the aBuiar effects of uncompetitive inhibition applies equally to noncompetitive (or irreversible) inhibitors with the proviso that the folbwing

pointofviewofselective&ugaUionbutit~a price thmugh potential mechmdam-hmed toxkity. P~hsprthisaCUWItsfOCthc~WWftldsnrsdunlrm inmehbotkregulatioiG.However,thaeisnothcgcticalmsonwhydNgrJesignrhwldnotbed&cted towards inhibitors of this type. Since unuqetitive

kinelica can arise from inhtbitton by subs&&e or product analogy in certain kinetic me&nkms, rational design should be possible in some in&axes.

discus&on

Refuence 1 Cornish-Dowden,A. (1985)FEES Lctt. 203.34

phosphatidylinositol resynthesis. The audal enzyme in this last process is inositol monophos-

in&to1 J-phosphate that arises by fiigny synthesis hrn glucose

phatase, since it can recover inositol from receptor-mediated

tie ibility of a cell to maintain sufficient supplies of inositol is crucial to the maintenance and efficiency of PI signalling. Al-

breakdown

of the PIs and alto

serves to recruit inositol from

though cells can take up inositol

from extrace&~lar sources, these cdn be absent UL h-&tirnt because of blood-tissue barriers”, and then the recycling of inositol from Pls and/or its de nouo syn thesis from glucose become the

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glucose B-phosphate

major routes for conserving cellular inositol pools. An inhibitor of this recycling would be expected to have profound effects on PI signaNn in those celLsthat have limited access to exogenous inositol. It is now clear that lithium posaKsyes unusual properties that allow it to act as an uncompetitive inhlbltor of inositol monophosphata# and to induce a stfmulus-dependent suppression of PI synthesis which may be particularly marked in cells with limited access to extracellular inositol. InhfbWonof in&o1 Phorphata=s The first indications that lithium may inhibit inositol monophosphatase in oivo came from AIlison, Sherman and colleagues (see Ref. 13 for a review). Plasma lithium in the therapeutic range (OS-1 nut) increased cerebral inositol l-phosphate and caused a proportionately smaller, but stoichiometrically similar, reduction in inositoW4. These effects were shown to be dependent on a pre-exlstlng tonic acetykholine receptor activation, since they were not observed after admlhWration of muscarlnic antagonists, but were enhanced following choline&erase inhibi!ionf3. Sub uent studies by H&her ark “4h S ennan’? which have been confinned by others

using the purified enzyme=, revealed that lithium is an uncompetitive inhibitor of inositol monophosphatase. This unusual mode of inhibition forms the basis of the stimulus dependence of the drug (i.e. lithium will exert anlnflwnceoncellspro onal to the lwel of PI hydra r;“” sis and the consequent generation of lnositol monophosphates)9**o. The details of this inhibition by lithlum and its influence on the flux of PI metabolism am provided in the Box. The pathways contain lithiumsensitive and -insensitive enzymes, and inhibition by lithium of both I(1,4)P2.!I(I,3,4)P3l-phosphatase and inositol monophosphatase has been studied. Roth enzymes have been purified and sequenced and the uncompetitive inhibition by lithium examined*6*x7. Although potent uncompetitive inhibition Ty the monovalent ion (4 05-l mw) can be observed for 1(1,3,4)Psl-phosphatase, more research has been directed at the monophosphatase because of the lilcelllood of hlgh substrate concentrations during cell stimulation and its crudal position in inositol recycling. Coaequencee of lithium action OnPIsynthesls Continued PI synthesis is dependent upon the maintenance

of concentrations of myo-inositol and cytidine monophospborylphosphatidate (CMP.PA) to sustain incorporation into phosphatidyhnositol (Pig. 1). The enzyme involved, Bositol synthase (mye-inositol 3 osphatldyl tmnsferase}, has a re E?tlvely high & for inositol (at least in liver and braW*‘~, so it would be antlcIpated that inhibition of phosphatldylinositol synthesis and accumulation of CMP.PA wouId be sensitive indicators of lnositol depletion. Indimct evidence that lithium might result in a reduction in the rate of synthesis of phosphatidylinositol is provided by reports that under conditions of incmased receptor stimulation, CMP.PA accumulates dramatically in parotid gland sllce8’ and rat cerebral cortex slices in the presence of llthiumzl~. As argued above, the uncompetitive nature-of -the inhibition of inositol monoPhosphatase by lithium pmvides a strong stimulus depe&ncy for this inhibitor on the avaiIabiIity of inositol for phosphatldylinositol synthesis. This ensures that with other pammemm (inositol uptake, K,,, for ph~phatidykositol syn thsis’ bemg equaL those cells * most actively stimulated willTe sekctlvelyvulnerable. In the absence of an efficient uptake system (see below) sup-

TiPS - August 1991 [Vol. 12]

300 pression of inositol via inhibition of recycling by lithium should reduce the synthesis of PIs. There is evidence for a lithium-induced fall in the labelling of phosphatidylinositol in a variety of cells and tissue preparations including GH3 cells, adrenal glomerulosa cells and rat parotid gland slicess,20,23. Perhaps surprisingly, labelling of PIP2 in each of these preparations appears to be unaffected by lithium, which questions the hypothesis that lithium results in a reduced PIP 2 pool in actively stimulated cells. However, even in single cells, PIs may be in compartments, so that only a proportion of the total PIP 2 is in an agonist-sensitive pool (see Ref. 10). In addition, in preparations of heterogeneous tissues such as the brain, in which stimulated cells may represent only a very small proportion of the total, the effects of lithium on PI synthesis may be entirely masked. The properties and distribution of the key enzyme phosphatidylinositol synthase are crucial to the interpretation of the results above and to the vulnerability of cells to lithium. There is increasing evidence for heterogeneity of phosphatidylinositol synthases, in both their tissue localization and subcellular distribution. In GH3 cells a plasma membrane phosphatidylinositol synthase has high affinity for inositol (Km 60 11M) compared with the enzyme located on the endoplasmic reticulum24 . In rat liver (Km 2.5 mM; Ref. 18) and brain (K m 4.6 mM; Ref. 19) the very low affinity of this enzyme for inositol could, in the absence of other sources of inositol, make these tissues particularly susceptible to any inositol depletion that results from the uncompetitive inhibition of inositol monophosphatase by lithium. More information on the properties and subcellular distribution of phosphatidylinositol synthase(s) in different tissues is required in order to understand the significance of compartmentation of PIs, their availability to receptor-stimulated phosphoinositidase C and their dependence on inositol recycling. Lithium reduces PI second messengers by inositol depletion The problems in interpretation caused by PI compartmentation in

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Fig. 2. Effects of lithium inhibition and myo-inositol supplementation on CMP.PA and inositol polyphosphate accumulation in cerebral cortex slices. a, b, c: Rat cerebral cortex slices were prepared and incubated 22 in the absence (0) and presence (0) of 1 mM LiCI for various times after addition of 1 mM carbachol. Where indicated, 10 mM myo-inositol was added to the incubation medium 1 h before agonist challenge. Statistically significant differences between LiCI plus agonist groups in the absence (0) and presence (-) of inositol supplementation are indicated as "p
cells have undoubtedly contributed to the lack of clear evidence for the inositol depletion theory of lithium action. However, assessment of agonist-stimulated IP3 and IP4 generation has recently provided compelling evidence that lithium-induced inositol depletion can result in depletion of a discrete agonist-sensitive pool of PIP2, at least in vitro. Consistent with the inhibitory effects that lithium exerts on inositol monophosphatase and I(I,4)P2/I(I,3,4)P3 I-phosphatase, agonist-induced accumulation of inositol monophosphates, inositol l,4-bisphosphate and, at least in some cells, I(I,3,4)P 3 is greatly enhanced by lithium in several tissues. However, studies in brain slices have revealed marked reductions in agonist-stimulated IP3 (Refs 22, 25) and IP4 (Refs 22, 25, 26) accumulation by relatively low concentrations of lithium. The results in all these studies have similar characteristics, indicating

that they are due to lithiuminduced inositol and PI depletion (Fig. 2) - i.e. they are time-dependent (a 5-10 min lag is generally observed before depletion of IP3 and IP 4 begins) and are induced by lithium (IC so 0.1-0.3 mM) in the same concentration range as that associated with inositol monophosphate accumulation21 .22 . The concentration of lithium that causes half-maximal CMP.PA accumulation in cerebral cortex slices in vitro is slightly higher than that necessary to cause halfmaximal inositol monophosphate accumulation. This can be explained by the fact that accumulation of CMP.PA occurs only when the tissue inositol concentration is so low that it is the rate-limiting step in phosphatidylinositol synthesis. The lithium-induced CMP.PA accumulation is also likely to result in an elevated level of phosphatidic acid and perhaps DAG in the tissue (Fig. 1); indeed a small accumulation of

13% - Augusf 7991[Vol. 121 this second messenger has been reported in lithium-treated GHs cells stimulated with thyrotropinreleasing hormones. Therefore, it is possible that lithium exerts an indirect effect upon the activity of protein kinase C. Most of these observationshave been made in cerebral cortex under muscarinic acetylcholine receptor stimulation. Although there have been suggestions that the actions of lithium may be agonist specific?‘, this could mfleet the strength of muscarinic stimulation relative to other types of receptor-mediated stimulation of PI hydrolysis and, therefore, the relative degree of uncompetitive inhibition by lithium of the inositol monophosphatase. The lag time observed before inositol polyphosphate depletion may relate to the time required to achieve a sufficient inhibition of inositol monophosphatase, so that inositol and phosphoinositide remea are below those needed to maintain normal signalling. In this context, it is of interest that CMP.PA accumulation in muscarlnic receptor-stimulated cerebral cortex slices is immediate upon addition of agonist plus litbium2’~, but only occurs after a 15 min lag period in the parotid glands”. Indeed, in GH:, cells 8timuIated with thyrotropinreleasing hormone, lithium causes only a very small accumulation of CMP.PA and does not influence IPs generatio@‘. Again, this may highlight differences in resting inoaitol monophosphates, inositol reserves, the affinity of inositol for different phosphatidylinositol synthases and/or the localization of isoenzymes and their association with the agonist-sensitive pool of PIs. Our prehminary data from a regional study of rat brain show that the corpus striatum (at least in oitro) is particularly vulnerable to the inhibitory effects of lithium, in that carbachol-stimulated IP, accumulation is reduced without an apparent lag (S. Jenkinson et al., unpublished). Recently, a method has been described to localize agoniststimulated PI metabolism in the CNS, based upon the autoradiographic detection of IsH]CMP.PA in ccmbral cor@x sectio#. Although qualitatively useful, such an approach will, as described above. not onlv dewnd

301

upon the extent of receptor stimulation, but will also critically depend upon regional variations in the supply of inositol, the properties of the phosphatidylinositol synthase and perhaps the accessibility of lithium to the inositol monophosphatase itself. The ShVrigf!St evidence that lithium impairs PI synthesis and the generation of inositol polyphosphate second messengers in brain slices by inositol depletion has come from experiments in which tissue has been preincubated with inositol. Accumulation of CMP.PA following stimulation of cerebral cortex slices with lithium plus agonist can be suppressed by preincubating slices with relatively high concentrations of myo-inositol but not scyllo-inosito12iJs (Fig. 2). These manoeuvres also significantly increase the lag period before the fall in IPs and IP, is observedzz (Fig. 2). This is consistent with the hypothesis that the lag represents the period required severely to deprive cerebral cortex cells of inositol and consequently reduce inositol polyphosphate synthesis. These observations suggest that, at least in oitro, brain tissue is particularly vulnerable to lithium. The requirement for high myoinositol concentrations to reverse only partially the actions of lithium suggests that slices are severely depleted of inositol during incubation and possess only low-affinity uptake mechanisms. This emphasizes the tight coupling between Pi hydrolysis and inositol recovery by dephospborylatlon of inositol phosphates that maintains the available inosito1 pool in stimulated slices. This in turn emphasizes that even moderate inositol depletion can lead to reduced inositol polyphosphate accumulation in cerebral slices. Extrapolationof these data to the situation in uiuo in which access to inositol may be limited by the blood-brain barrier” is discussed below. These effects of lithiun. are not exclusive to the brain, at least in vitro. Balla and colleagueslg have reported major inhibitory effects of lithium on angiotensin-stimulated accumulation of Il3 and II’4 in adrenal glomerulosa cells. As in the brain slice, a five minute lag was observed before inhibition

was manifest. Lithium also impairs

the ability of angiotensin (but not ACTH which activates adenylyl cyclase) to induce aldosterone secretion in glomerulosa cells”. Although it was not possible to reverse the action of lithium on secretion with myo-inositol, the inabiiity of this cyclitol even to overcome the inhibition of inositol lipid synthesis in glomerulosa cells suggests a very limited access of exogenous, inositol to these cells. By contrast, there are now several examples of functional effects of lithium that are sensitive to myo-inositol reversal. These include the late-phase, glucose-induced insulin secretion in rat isletsar and positive inotropic effects of a,-adrenoceptor agonisd’. These observations, as well as others made in embryonic development modelsss, support an action of lithium on PI signalling as outlined above. Two recent electrophysiological studies using hippocampal slices point to CNS effects of lithium mediated via inositol debletion -.and loss of PI signalling. In hippocamnal slices. the CA1 oonulation spil;! elicited by s&&ion of Schaeffer collaterals is inhibited by adenosine, and this action of adenosine can itself be blocked by muscarinic agonists. Worley et al. have shown that lithium, after a 10-15 minute lag, reverses the effects of muscarinic receptor agonists on the inhibitory actions of adenosi#. In another study it has been shown that muscarinic agonist-stimulated increases in neuronal firing rates in hippocampal slices decrease with agonist exposure time, and this desensitization is reversed by lithium; furthermore, exogenous application of myo-inositol can prevent (or reverse) the effect of lithium%. Thus preparations using both biochemical and electrophysiological approaches in vitro emphasize an effect of lithium on PI signalling that would be predicted from the detailed rrolecular information now available on the uucompetitive inhibition of inositol monophosphatase by this monovalent ion.

In&o1 homeostasisand lithium bna We are still largely ignorant of the cellular mechanisms that regu-

TiPS - August 1991[Vol. 121

302 late cellular myo-inositol concentration and those that determine the relationship between plasma lithium concentration and consequent tissue and cell levels of this ion. With respect to inositol homeostasis, cellular myo-inositol CORconsiderably centrations are greater in brain tissue than in cerebrospinal fluid, suggesting that concentrative mechanisms must be operative (see Ref. 35). ,4 Na+-dependent myo-inositol transport system of relatively low capacity has been demonstrated in rabbip and rap cerebral cortex slices in vitro. The system is also ctpable af transporting the stereoisomer scyllo-inosito136. The dependence of this transport on Na’ may explain the ability of the brain to maintain high intracellular concentrations of myoinositol, and suggests that the rate of transport may be inhibited during neuronal excitation when there is a decreased transmembrane Na+ gradienp7. Such a mechanism could exert a localized effect (e.g. within dendrites); however, it is not known whether such an inhibition of the inositol transporter makes any contribution to the depletion in cellular myo-inositol levels induced by agonist plus lithium. Another question is whether differences in inositol homeostasis exist between neuronal and glial cells. A recent report by Glanville et al.” presents data on the differences in the abilities of cultured cells of neuronal and glial origin to accumulate and metabolize myo-inositol. Striking differences were noted, with glioma cells apparently able to accumulate and maintain a 30-fold greater intracellular free inositol concentration than neuroblastoma cells. These data suggest a role for gIiaI cells as a reservoir for myo-inositol, which may be important in maintaining intracerebral inositol homeostasis. Although transmembrane influx and efflux mechanisms for lithium have been studied in some detail in simple model systems (e.g. erythrocytes), it is not clear whether the findings of such studies can be extrapolated to our understanding of lithium handling in the CNS. Early stucl;Ls suggested that Na+ channels may be of considerable importance for

access of lithium to excitable cells, and more recent studies using synaptosomal preparations also emphasize that lithium transport is dependent on the plasma membrane potential and maintenance of other cationic gradients (see Ref. 39). NMR spectroscopic and imaging techniques may prove useful for a non-invasive study of the concentration and distribution of lithium in the human brain. The report of Renshaw and Wicklunda describing ‘Li-NMR studies in human subjects suggests this approach will be possible. Lithium and PI responses in vivo Many of the data on which our present understanding of lithium action is based are founded upon experiments performed in viva. These experiments established that a near-stoichiometric relationship exists between inositol l-phosphate accumulation and myo-inositol depletion in brain following systemic lithium administration, and they also clearly demonstrated the potentiating effects of muscarinic receptor agonists or choline&erase inhibitors, and the ameliorating effects of muscarinic receptor antagonists (see Refs 13,35). Minimal data, however, are available on the effects of lithium on the iinmediate products of agonist-stimulated PIP, hydrolysis in vivo. There are two very recent reports on the effects of muscarinic agonists on 1% accumulation in vivo in lithiumtreated mice”’ and rats”. In the former study, where both in vivo [3H]inositol labelling and IPs mass assay strategies were employed, chronic lithium treatment surprisingly enhanced IP3 accumulation. By contrast, in the latter study, reductions in basal levels of IPs and decreased responsiveness to muscarinic stimulation were observed. These apparently contradictory studies highlight the major difficulties in designing suitable and comprehensive experiments in vivo, because it is difficult to choose appropriate sampling times and, in particular, to separate direct and indirect effects of administered agonists”. Despite these problems it is important to consider the chronic effects in vivo of lithium treatment in order to understand the therapeutic response in human sub-

jects. Particular issues for consideration are the compartmentation of myo-inositol pools with agonist-responsive PI systems, the accessibility of the CNS - and neurons in particular - to plasma myo-inositol, and the adaptive responses of the PI cycle. A combination of lithium treatment in viva and subsequent assessment in vitro of PI metabollmn provides some clues about this last issue. There is general agreement between the results of such studies that receptor-stimulated PI tumover in cerebral cortex slices in response to a variety of agonists is decreased by chronic (14-16 da& lithium treatment in viva; . There is some variation between agonists in the dependency on lithium concentration and the time of onset of these effects. Thus lithium administration for short (18-24 h) periods is sufficient for aneffecttobeobservedfor5-mand acetylchoIine-induced responses, while longer periods of lithium administration are required to see decreased PI tumover in response to noradrenaline”*“. Despite the consensus shown by these studies ex viuo, there is little evidence from experiments in vivo for the occurrence of such long-term adaptive changes*. multlpleUsing regression data analysis to overcome differences in final cerebral cortical lithium concentrations in acutely and chronically treated rats, Honchar et al. have provided convincing evidence that basal and agonist-stimulated inositol l-phosphate accumulation is unaffected b&the duration of lithium treatment . These workers have also provided evidence against the occurrence of any adaptive changes in global or regional brain inositol phospholipid levels or inositol monophoaphatase activity, in studies whele rats were treated with lithium administered intraperitoneally or in the diet for 58 days*. 0

cl

0

There is now compelling evidence that lithium can, through its uncompetitive inhibition of inositol monophoaphrtase, deprive cells of myo-inoaftol and reduce agonist-stimulated generation of inositol polyphosphate second

TIPS- August 1991/Vool. 121 messengers. Much of this evidence has come from biochemical experiments in vitro, generally under conditions of sustained and maximal agonist stimulation. It is not known how this relates to the situation in vim, where there may be variable receptor stimulation and accessibility to inositol between, and perhaps within, different tissues. This mechanism, however, does not prove that inhibition of incsitol monophosphatase is necessary and sufficient to explain the therapeutic effect of lithium. Lithium also inhibits other cellular processes at concentrations at, or close to, its therapeutic plasma level; these include adenylyl cyclase, guanylyi cyclase, receptor-G-protein interactions and Ca*+ influx via the NMDA recepto?*s35147. This lack of specificity, and particularly lithium’s ability to interfere with M#+dependent reactions, coupled with uncertainty about the intracellular concentration in viva, will always create difficulties in proving a single site of action. Different experimental approaches are needed, such as the use of selective inhibitors of inositoi monophosphatase. Some progress has already been made in the design of such agents’s. Whether competitive inhibitors of this type would exhibit the same effect in viuo on the PI cycle as lithium is uncertain, apart from the question of whether they would be efficacious in the treatment of man&depressive illness. A genetic approach is another alternative. The gene for inositol has been monophosphatase cloned@ and it is now theoretically possible to use transgenic technology to produce animals that possess a greater or lesser inositol monophosphatase activity, or that have a form of the enzyme with altered lithium sensitivity. These would be invaluable for investigating the action of lithium on the PI cycle, but in the absence of aniulal models of manic-depressive illness we would not necessarily gain any insight into the therapeutic mechanism of lithium. A better understanding may only be possible when new drugs of greater selectivity and potency than lithium are shown to be efficacious in the clinic.

303 Acknowledgements Much of the authors’ (S. R. N. and R. A. J. C.) own work described here has been generously supported by the Wellcome Trust, the MRC and the SERC. We also gratefully acknowledge the contributions made to this work by Ian Batty, Eleanor Kennedy and Stephen Jenkinson. We thank Mrs Lyn McCarthy for preparation of the manuscript. References 1 Berridge, hf. J. and 2 3 4

5 6

7 8

9

hvine, R. F. (1969) Nature 341,197-2(X Nishizuka, Y. (19B8)Notun 334,661-665 Nahorskf, S. R. (1988) TrendsNcumsci. 11,444-%% Nicholson. C. D.. Chalfiss. R. A. 1. and Shahid, M. (1991j TrendsP&&c&. Sri. 12.19-27 &mmond, A. H. (1987) Trends Pharmocof.Sri. 8,129-133 Bunney, E. and Gar&nd.Bunney, 8. L. (1987) in Psychopharmacology: the Third Generation of progress(Meltzer, H. Y., ed.), pp. 553-565, Raven Press Wood, A. J. and Goodwin, G. M. (1987) Psychol. Med. 17,5T%al Avissar, S., Srhreiber, G., Danon, A. and Belmaker, R. H. (1%) Nature 331, 44U42 Berridg. M. J., Dowries, P. F. and Hanley, M. R. (1982) Biochem.J. 206, Q77-545

__. ___ 10 Berridge, M. J., Downn, P. F. and Ha&v, M. R. (lm) Cell 59,411-419 11 Lewin; L. M., Yanna, Y.. Sulimovici, 5. and Kraicer, P. F. (1976) Biochcm.1.156, 375-380 12 Hallcher, L. M. and Sherman, W. R. (19BOJJ. Biof. Chum. 255,1~10901 13 Sherman, W. R., Gish, 8. G., Honchar, M. P. and Munseil, L. Y. (1986) Fed. Proc. 45.2639-2646 14 Sherman, W. R., Munsell. L. Y., Gish, B. C. and Honchar, M. P. (1985) J. Neurochen#.44, 798-607 15 Gee, N. S. et of. (19BB)Biochem.1. 249, B83-BB9 16 Gee, N. S., Reid, C. G., Jackson,R. G.. Bamaby, R. J. and bgan, C. 1. (19ga) Biochem.J.253,777-782 17 York, J. D. and MaJerus, P. W. (1990) Proc. NaH Acad. Sci. USA 87,954&9552 18 Takenawa, T. and Egrwa. K. (193 1. Biol. Chem. 252,5419-5423 19 Ghaiayini, A. and Eichberg, J. (19B5) J. Nrumchem. 44.175-182 20 Dowries. C. F. and Stone, M. A. (19%) Biochrm.J. 234,199-204 21 Godfrey, P. P. (19891 Biochrm.I. 25% 621-624 22 Kennedy, E. D.. Challirs, R. A. J., Raean, C. I. and Nahorski, 5. R. (1990) B&hem. J. 267,7Bl-786 23 Balla, T., Enyedi, P., Hunyady, L. and Spat, A. (1984) FEBS L&t. 17l,179-182 24 Imai, A. and Gershengom, M. C. (1987) Nature 32% 72&?28 25 Kennedy, E. D., Challirs, R. A. J. and Nahorski, ‘3. R. (19B9)J. Neomrhcm.53, 1652-1655 26 Whitworth, P. and Kendall, D. A. (19B8) J. Nelrmcfrrot.51,25&265 27 Hughes, P. 1. and Drummond, A. H. (1987) fliochem./. 248.463-470

28 Hwang, P. hi., Bredt, D. S. and Snyder, S. H. (1990) 5cienct 249,&&804 29 B& T., Baukd, A. 1.. Guillemette, G. and Catt. K. J. (19BB)I. Biol. Chrm. 263, 4oIwo91 30 Zawafich, W. S., twalich, K. C. and Rasmussen, H. (19B9) Biochcm./. 262, 557-561 31 Mantelli, L. et al. (1988) Eur. J. Phamtacol. 150, 12F129 32 Busa. W. B. and Gimlich, R. L (1989) Deu. Biol. 132,315-324 33 Wodey, P. F., Heller, W. A., Snyder, S. H. and Baraban, J. M. (1988) Sriencr 239,142&1429 34 Pontzer, N. J. and Crews, F. T. (1990) J. Plmmacol. Exp. Thcr. 25?,921-929 35 Sherman, W. R. (1%9) in lnosifolLipids in Cell SignaIling (Micbell, R. H., Drummond, A. H. and Dowries, C. P., eds), pp. 39-79, Academic Press 36 Spector, R. (1576) 1. Nrurochem. 27, 1273-1276 37 Howerton, 1. C. and Rutledge, C. 0. (1988 Bfochtnr.Pharmacol. 37,911-915 38 GlanviUe, N. T., Byers, D. M., Cook, H. W., Spence, M. W. and Palmer, F. B. St C. (1%) Biochim.Biophys.Acta 1004, X9-179 SchmalzinB,G. (1986) J. Viol. Chem.261, Renshaw, P. F. and Wicklund, S. (1988) Biol. Psychiatry23, w75 Whitworth, P., Heal, D. J. and Kendall, D. A. (lm) Br. 1. Phamwul. 101.39-U Jope, R. S., Smith, R. E. and MaQarrie, R. A. (1990) .9oc. Ncurosci. Abslr. 16, 377.17 43 Kendall, D. A. and Nahorski, 5. ?, (1987) J. Z~rtnnnro;. L’xp. Thor. 241, la?3-1027 44 Gmifrry. P. P., McCIue, S. J., White, A. M., Wood, A. J. and Grahame-Smith, D. G. (19B9)J. Neumchcm.52,49B-!i& 45 Honchar, M. P., Vo&r, G. P., Gish, B. G. and Sherman. W. R. (1990) 1. Neurachrm. 55,1521-1525 I 46 Honchar, M. P., Ackermann, K. E. and Sherman, W. R. (1989) J. Neurochem.53, 590-594 _._ _. 47 Port, R. M. (1991) in Ltlhmmand fileCrli: Pkarmacofo~yand Biochemistry(Birch, N. J.. ed.), pp. lp9-241, Audemic Press R., Carrick, C., Leeson, P. D., 48I Baker, Lennon, 1. C. and Liverton. N. J. (1991) 1. Chew. Sot. Ser. Chem. Commun. 491 Diehl, R. E. cl at. (1990) 1. Biof. Chcm. 265,5946-5949

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