THE.JOURNAL OF BIOI.OGICAI. CHEMISTRY Vol. 255, N o . 22, Issue of November 25, pp. 10896-10901, 1980 Printed tn U S A .

The Effects of Lithium Ion and Other Agents on the Activitymyoof Inositol-1-phosphatasefrom Bovine Brain*
(Received for publication, June 9, 1980)

Loretta M. HallcherS and WilliamR. Sherman8

From the Departmentsof Psychiatry and Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110

myo-Inositol-1-phosphatase has been partially puri- in rat testis and reported that it hydrolyzed both D- and Lfied from bovine brain. The enzyme has a molecular myo-inositol-1-P. The D-enantiomer of myo-inositol-1-P is weightofabout 58,000. BothL-myo-inositol l-phos- formed by the action of a phospholipase C on phosphatidyl phate and D-myo-inositol 1-phosphate are hydrolyzed inositol and may also be a degradative product in the metabby the enzymeas well as (-)-chiro-inositol3-phosphate olism of the polyphosphoinositides. It is thus probable that The myo-inositol-1-phosphatase, hydrolyzing both enantiomers and 2-AMP. Triphosphoinositideis not a substrate. by phosphatase is completely dependent on M 8 + , which of the inositol phosphates, functions as a part of the process . has a K, of 1 m ~ Calcium and manganese ions are by which the cell maintains its myo-inositol levels. competitive inhibitors of M&+ binding with Ki values In 1971, Allison and Stewart reported that the acute adminof 18 @ and 2 p ~ respectively. Lithium chloride inI , istration of lithium, adrug widely used to treatmanic-depreshibits the hydrolysis of both L- and D-myO-inOSitOl 1- sive illness, results in a 30% decrease in the level of myophosphate to the extent of 50%at a concentrationof 0.8 inositol cortex. Allison et aL(1976) showed that m ~ The phosphatase from testis is similarly inhibited lithium in rat cerebral causes the level of myo-inositol-1-P to . chloride also of by lithium. Lithium ion a noncompetitive inhibitor is M&+ binding and an uncompetitive inhibitor of myo- increase in the same tissue. We have recently found that, inositol 1-phosphate binding. Because lithium chloride during chronic LiC1-administration, the cortical levels of both administration elicits both an increase in the levels of D- and L-myo-inositol-1-P increase, with the D-enantiomer myo-inositol 1-phosphate and a decrease in the levels accounting for most of the effect. It t l, of myo-inositol in rat brain (Allison, 1978), and because was found in 1974, by Naccarato e a . that 250 mMLi, completely inhibits myo-inositol-1-phosphatase rat of these actions are blocked anticholinergicagents, we in vitro, by examined the effects cholinergic agonists and antag- mammary gland. Since inhibition of thisphosphatase by of onists on the enzyme and found none. The possibility lithium ion could account for at least part of the observed is effect in vivo, were led to study thei vitro effect of this thattheinhibition of this enzymebylithiumion we n related to the pharmacological actions oflithium is metal at pharmacologically effective concentrations (0.5 to 1.5 discussed. m).In this paper we report that lithium ion is a potent inhibitor of myo-inositol-1-phosphatase, we describe the and mode of the action of this inhibitor, and otheragents, on the myo-Inositol-1-phosphatase (EC 3.1.3.25) was first studied enzyme. in yeast by Chen and Charalampous (1966) who found that EXPERIMENTAL PROCEDURES this enzyme is a component of the pathway bywhich DPreparation of myo-Znositol-1-phosphatase-Wholebeef brains or glucose-6-P is converted to myo-inositol via the intermediate testes obtained from a local abattoir were either used fresh or kept L-myo-inositol-1-P.Eisenberg (1967) found the same enzyme frozen at -90C until used. All subsequent steps were performed at
* This work was supported by Grants NS-05159, NS-13781, and
AM-20579 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Present address, The Division of Biology and Biomedical Sciences, Washington University School of Medicine, 660 S. Euclid, St. Louis, Missouri 63110. 9 To whom correspondence should be addressed at Department of Psychiatry, Washington University School of Medicine, St. Louis, Missouri 63110. The recommended name for this enzyme (Nomenclature Committee of the International Union of Biochemistry) is 1L-myo-inositol1-phosphatase. Since from the work in this paper and in other studies it is evident that the natural substratesare both D- and L-myo-inositol 1-phosphate, we suggest that the name be simplified to myo-inositol1-phosphatase. Some of the early literature on inositol phosphates used absolute configuration notation that is the reverse of what is correct. The correct nomenclature is L-myo-inositol 1-phosphate for the product of the denovo synthetic pathway and D-myo-inOSitOl 1-phosphate for the phospholipase C product of phosphatidyl inositol hydrolysis. A ether)N, N, N, N-tetraacetic acid in a Waring Blendor. The homogenate was centrifuged at 15,000 X g for 30 min, and the supernatant was fractionated with ammonium sulfate. The 40 to 6 % precipitate 0 was dialyzed against 50 m~ Tris-HC1, pH 8.0,0.2 M KCl, and 0.1 m M ethylene glycol bis(B-aminoethyl ether)N, N, N, N-tetraacetic acid, applied to a column of Sephadex G-150 or Bio-Gel P-300,and eluted with the dialysis buffer containing 0.02%NaN3. There was a 90% recovery of activity from the Bio-Gel, as compared with 50%from the Sephadex. This preparation was used for most experiments reported here. Except as noted, the enzyme had a specific activity of 1.6 pnol of L-myo-inositol-1-Phydrolyzed per mg of protein per h. One unit of enzyme produces 1 nmol of P,/h at 37OC. Further purification of the enzyme was carried out by first applying the dialyzed (50 m Tris-HC1, pH 8.0) ammonium sulfate fraction M described above to a Whatman DE52 column which had been equilrecent discussion of cyclitol phosphate nomenclature clarifies errors in this area Agranoff, B.W. (1978)Trends Biochem. Sci. 3 1 ) N283(2, N285). W. R. Sherman, A.L. Leavitt, M. P. Honchar, L. M. Hallcher, and B. E. Phillips, manuscript in preparation.
4C. The tissue was homogenized in 2 volumes of 0.15 M KCl, 50 m M Tris-HC1, pH 8.0, and 0.1 m~ ethylene glycol bis(P-aminoethyl

10896

Effect of Li+, Ca2+, M n 2 +on myo-Inositol-1-phosphatase and

ibrated with the Tris buffer. The loaded column was washed with buffer, and the activity was then eluted with a IO-column-volume M linear gradient (0 to 0.3 M KCl) in 50 m Tris-HC1, pH 8.0. The DEAE-fraction was subsequently chromatographed on Bio-Gel P-300 followed by chromatography on Bio-Rad Affi-Gel Blue, which does not retain the phosphatase but which does remove some inactive protein. Columns were assayed using either 2'-AMP or DL-myo-inositol-I-P as substrate. Measurement of Activity-Assay mixtures usually contained 250 m~ KCl, 50 m Tris-HC1, pH 7.8, 3 m Mg *+, and 0.7 m L-myoM M M inositol-1-P, with changes or additions as noted, in a final volume of 300 pl. The reaction mixture was usually incubated for 5 min at 37'C for temperature equilibration, and the reaction initiated by the addition of enzyme, substrate, or Mg '+,depending on the experiment. The reaction was linear for at least 80 min.Incubations were normally run for 30 min and terminated with the addition of 50 p of 2 M LiCl. 1 Activity was measured as inorganic phosphate released using the method of Itaye and Ui (1966). Atvery low levels of Pi (0.5 to 5 nmol of phosphate released), a nonlinear standard curve was calculated with a computer program. Protein Estimation-Protein was measured using the Bio-Rad protein assay method using bovine IgG as the standard. Preparation of the myo-inositol 1-phosphates-L-myo-Inositol1phosphate was prepared by the conversion of D-glucose 6-phosphate to L-myo-inositol-1-Pby bovine testis synthase, according to the procedure of Burton andWells (1974), and was obtained from Elastin Products, St. Louis, MO. D-myo-Inositol 1-phosphate was prepared from phosphatidyl inositol purified from Asolectin (American Lecithin Co.) by extraction and chromatography on neutral a l ~ m i n aBase-catalyzed hydrolysis .~ of the phosphatidyl inositol (Pizer and Ballou, 1959),was followed by acidification with Dowex 50W-H+and extraction with diethyl ether. The solution was made basic with cyclohexylamine, washed with chloroform, and the D-myo-inositol-I-P was purified in three steps. The sample was fvst chromatographed on Bio-Rad AG I-X8 in the formate form according to the procedure of Burton and Wells (1974). The material obtained was then rechromatographed on the same resin using a linear 0 to 1.0 M formic acid gradient (Bartlett, 1958). The resulting mixture contained myo-inositol-1-P and -2-P in about a 4 l ratio. Pure D-?nyO-inoSitOl-l-P was finally obtained by high : pressure liquid chromatography on an aminopropyl bonded phase column 4 mm X 30 cm (Micropak-NH?, Varian Instruments) at pH 4.0 in 75 m~ ammonium formate. About 25mg could be separated per IO-min run, with myo-inositol-I-P eluting at 6 min and myoinositol-2-P a t 7.2 min. The purity of the myo-inositol-1-P,with respect to myo-inositol-2P, was established by packed column gas chromatography (Sherman et al.,1971). The enantiomeric purity of the D -myo-inositol-I-P was shown to be >97% by gas chromatography on a chiral glass capillary column (Chirasil-Val, Applied Science Labs).3 This is confiiation that the base hydrolysis, although capable of generating myo-inositol2-P, does not result in racemization (2.e. the migration of the phosphate moiety from the 1-position to the 3-position). The other substrates, L- and DL-myo-inositol-I-P,were also found to be enantiomerically pure. A l inositol phosphate substrates were prepared and l used as the dicyclohexylammoniumsalts. Triphosphoinositide (phosphatidyl inositol-4, 5-diphosphate) was the generous gift of J. N. Hawthorne, Department of Biochemistry, Queen's Medical Center, University of Nottingham, Nottingham, U. K. It was further purified by chromatography on Dowex 50W-H+and isolated as the ammonium salt (Dittmer and Dawson, 1961). EZectrophoresis-Polyacrylamide gel electrophoresis was carried out in a continuous Tris-glycine buffer at pH 8.2. The gels were 10 cm in length and were 7.5% or 10%cross-linked. To extract the phosphatase, following electrophoresis, the gels were cut into I-mm slices and incubated in 3 0 0 ~ of 0.1 M KCl, 50 m Tris-HC1, at pH 8.0 overnight 1 M at 4OC. Aliquots of the extracted enzyme were then incubated as described above.
RESULTS

10897

changed to 52,000 upon heating at 70C for 15 min (75% recovery of activity). The bovine brain enzyme, which has the same thermal stability with regard to activity, was chromatographed on a Sephadex G-100 column and compared with molecular weight standards (Andrews, 1964). Both heated (70C for 15 min) and unheated preparations of the phosphatase had apparent molecular weights of 57,000 as determined on Sephadex G-150. The molecular weight of the unheated enzyme was later determined on Bio-Gel P-300 and found to be 58,800. The heated and nonheated enzymes were also found to have the same Rf upon electrophoresis in a continuous Trisglycine system with 7.5% cross-linked polyacrylamide gels.

pH Effects
The enzyme was found to have a pH optimumbetween 7.5 and 8.0 This is similar to that for the rat testis (pH 7 to 8, Eisenberg, 1967), rat mammary gland phosphatase (pH 8, Naccarato et al., 1974), and the bovine lens enzyme (pH 7.7, Kabasawa et al., 1974). During an attempted purification step, the enzyme was dialyzed against 20 m citrate buffer at pH 6.0, and all of the M activity was recovered when the pH was adjusted to 8.0. Dialysis at pH 5.0, however, resulted in the complete loss of activity. Substrate Specificity Eisenberg (1967) found that myo-inositol-1-phosphatase from several organs of rat hydrolyzed D- and L-myo-inositol-lP, 2'-AMP, and P-glycerophosphate, but was inactive with myo-inositol-2-P.Enzyme from testis was studied more extensively and was found to hydrolyze (-)-chiro-inositol-3-P and a-glycerophosphate. Other phosphate esters such as ~-Glc-6P and ~ - G l c - l - P were not substrates. We have found similar specificity with the bovine brain enzyme, except that 2'-AMP is hydrolyzed at a lower rate; a- and P-glycerophosphate were not substrates. In an attempt toestablish that the activities of the bovine brain enzyme were al due to thesame phosphatase, electrol phoresis was performed on our most pure enzyme preparation, which had been purified through the Affi-Gel Blue stage, and which had a specific activity of 45 pmol/mg of protein/h. Following electrophoresis, the gelwas sliced, the enzyme extracted, and aliquots were then incubated with several substrates. As shown in Fig. 1, the hydrolytic rates versus the RF of the activity with L-myo-inositol-1-P,(-)-chiro-inositoll same 3-P, D-myo-inositol-1-P, and 2'-AMP al maximize at the location. The ratio of activities with these substrates is 1:O.B: 0.70.2. L-a-Glycerophosphate was not hydrolyzed. When gels run at the same time were stained with Coomassie brilliant blue R distinct protein bands were observed at the RFvalues of slices 48, 51, 54, 56, and 59, as well as at RF values more distant from the activity. Two-gel filtration-purified preparations of the phosphatase M were found to have a K,, for L-myo-inositol-1-P,of 0.10 m The (concentration range 0.15 to 2.5 m). D-enantiomer has the same K,,, value. No substrate inhibition was found to anLmyo-inositol-1-P concentration of 5 mM. The V,,, ratio of the L- to the D-enantiomer was 1.3. In several experiments with synthetic myo-inositol-1-P,the racemic mixture was hydrolyzed at a lower rate than the Lenantiomer. This was evidently due to an impurity since a mixture containing equal amounts of the pure D- and pure Lenantiomers had only the decrease in activity expected from the lower reactivity of the D form. Thus, the ratio of activity of L:DL:D was1:0.9:0.8 when the concentration of substrate was 0.7 m in each case. M Rat brain polyphosphoinositide phosphomonoesterase is a

Molecular Weight The molecular weight of myo-inositol-1-phosphatase from rat mammary gland was studied by Naccarato et al. (1974), who reported a native molecular weight of210,000 which
J. Eichberg, personal communication.

10898
0.6

Effect of Li+, Ca2+, Mn2+ myo-Inositol-1-phosphatase and on

04
a a Q
0

02

46

48

50 52 54 56 GEL SLICE NUMBER

58

60
005

FIG. 1. Polyacrylamide gel electrophoresis of myo-inositol1-phosphatase with a specific activity 'of 46 nmol/mg/h. L I I I I Twenty-six micrograms of protein was applied to the cylindrical gel. 0 5 I O 1 5 20 The gel was cut into 91 1-mm slices and assayed as described under [&++I(mM) "Experimental Procedures." Substrates were o " 0 , L-myo-inositolFIG. 2. The effect of magnesium on myo-inositol-l-phospha1-P; M(-)-inositol-3-P; MD-myo-inositol-I-P;A-A, , myo-inositol-I-P (0.8 m) was incubated in 50 m M 2'-AMP; and A-A, L-a-glycerophosphate. The apparent increase tase. W, , in activity at slice 52 is not reproducible. There is no other activity Tris-HCI, pH 7.8, with different concentrations of Mg2+.M using a stability constant for the magnesium complex with myothroughout the gel. inositol-I-P of M", an amount of L-myo-inositol-1-Pwas added at each point to give a calculated concentration of uncomplexed subsoluble enzyme which has been purified by Nijjar and Haw- strate equal to 0.57 m ~ The calculated concentration of the complex . thorne (1977). Although some of the reported properties of at 20 m Mg2+is 0.56 m. This shows that the inhibition by higher M is that enzyme differ from those of ours (e.g. inactivation at concentrations ofMg '+ not a result of substrate depletion. The 75C and metal requirements) we tested the bovine brain rates of each plot were arithmetically adjusted for graphical represenlower X 0.7). myo-inositol-1-phosphatase with triphosphoinositide as sub- tation (upper x 1.4, strate. As used by Nijjar and Hawthorne, the system con- An experiment was performed wherein the amount of free LM tained 1 m F- (which at thisconcentration is 50%inhibitory myo-inositol-1-P added initially was adjusted to maintain a M to our enzyme), 1 m triphosphoinositide, or 1 mhi L-myoconstant final concentration of the substance for each Mg2+ M inositol-1-P with 3 m cysteine and 1 m cetyltrimethylam- concentration, using the 50 M-' stability constant.As is shown M monium bromide. In thepresence of sufficient myo-inositol-l- in Fig. 2 (closed circles) the degree of inhibition is similar to phosphatase to hydrolyze 5.3 pmol of the inositol phosphate that found without consideration of the complex formation, per h, under standard conditions, we hydrolyzed 104 nmol of suggesting that magnesium inhibition is due to a direct effect L-myo-inositol-1-Pin 30 min (37OC) but less than 3 nmol of on the enzyme. triphosphoinositide. Calcium and Manganese-Fig. 3 shows that calcium and manganese are powerful inhibitors of the phosphatase. Fig. 4 Effects of Divalent Cations shows that Ca2+is a competitive inhibitor of M 2 + binding.' Magnesium-myo-Inositol-1-phosphatasehas no measura- The K, calculated from the datain Fig. 4 is 18 p ~Manganese . ble activity in the absence of Mg2+,and, as can be seen in Fig is also a competitive inhibitor of Mg2+(not shown) with a K, 2 (open circles), is maximally stimulated at a magnesium Of 2 pM. concentration of 3 to 4 mM. At higher concentrations, inhibiOur findings of potent calcium and manganese inhibition of tion occurs. Other metals that were tested in the place of the bovine brain myo-inositol-1-phosphatase, the inability and magnesium were Ca2+,Mn2+, Fez+, Zn2+,Cu2+,and Co2+.Of of either metal to substitute M F , is in contrast with other for these, Co2+gave 10% stimulation, while the rest gave ~ 3 %reports on this enzyme. Kabaswa et al. (1974) found that 3 activation at a concentration of 3.5 mM. m Mn2+ gave 30% of the activity of that concentration of M To the extent that a complex between magnesium and myo- Mg2+with the bovine lens myo-inositol-1-phosphatase. This inositol-1-P is formed it will reduce the concentrations of free may, however, have been due to a contaminating activity, myo-inositol-l-P2- and Mg2+available to the enzyme. With a since their enzyme preparation was about 30%as active toward stability constant of 50 M-', which is that given by O'Sullivan ~-Glc-6-P as was toward D-myo-inositol-1-P. Calcium was it and Smithers (1979) for hexose 6-phosphate, and with typical inhibitory to the bovine lens enzyme, although it was only incubation levels of Mg2+and myo-inositol-1-P (3 m and 0.7 studied at high (5 mM) concentration. Eisenberg (1967) also M mM), the calculated concentration of the magnesium complex found Mn2+to partially substitute for Mg+, as Naccarato did is 0.09 mM, an insignificant reduction in the amount of either et al. (1974).Both of the latter authors studied enzyme from Mg2+ or substrate. The calculated effect that this stability rat tissues; thus species differences could account for their constant has the K , value of myo-inositol-1-Pis a reduction results. on of 15%.Lineweaver-Burk plots, over a substrate concentration In some experiments a preincubation period was usedin the belief range of 0.03 to 2.6 mM, are linear. Therefore, over this concentration range the effect of complex formation on the that a slow equilibrium step occurred in the reaction. This was found to not be the case, and the step was not used in subsequent experireaction is neither inhibitory nor stimulatory. ments. Two of these experiments were in the study of the effect of It seemed possible that the apparent inhibition of the hy- Ca2+on Mg2+binding (30-min preincubation, the reaction initiated drolysis of myo-inositol-1-P by high levels of Mg2+might be with Mg") and in the study of the effect of Li' on Mgz+binding (45due to depletion of substrate by M$+ via complex formation. min preincubation, the reaction started with myo-inositol-1-P).

Effect of Li+, and Ca2+, Mn2+

on myo-Inositol-I-phosphatase

10899

the L-enantiomer and with (-)-chiro-inositol-3-P as well. Heated (7OoC, 15 min) and unheated enzyme are inhibited alike. Rubidium and cesium,like sodium, potassium, and ammonium are not inhibitory to concentrations of 4 m ~ In . the absence of M e , Li+ is not stimulatory. Thus itis evident that theeffect of lithium ion on this enzyme is unique among this group of cations and not a general (e.g. ionic strength) effect. The Mode of Action of Li+ To determine if lithium ion causes an irreversible change in the phosphatase, the enzyme was first incubated with 0, 0.5, 5, and 100 m~ LiCl at 37C for 30 min and then dialyzed against 50 m~ Tris-HC1, pH 8.0, for 2 days. The activities in all cases were comparable at the end of the treatment. In another experiment, we investigated whether Li+ effected a slow or a rapid change in the catalytic activity of the enzyme. The phosphatase was incubated in standard buffer with Lmyo-inositol-1-P, and aliquots were removed periodically for i, measurement of activity. After 30 mn LiCl wasadded to give a final concentration of 0.6 mM. The reaction immediately assumed a new rate which continued linearly for 80 min.Thus Li' is a rapid and reversible inhibitor of the phosphatase. It has beensuggested that part of the pharmacological action of lithium may be due to competition for magnesium sites in certain M8+-dependent enzymes (Birch 1974; Birch, 1978). In five separate experiments with rnyo-inositol-l-phosphatase, we found no evidence that Li+ is competitive with M F . Instead, the effect of Li' onMg2+binding is pure noncompetitive inhibition, i.e. there is no effect of lithium ion on the K,,, of Mg2+.In experiments5using 0, 0.1, 0.3, 0.5, and 1 m~ LiCl and analyzing the data by the Lineweaver-Burk obtained for Mg2+ was method, the mean K,,, value which was 0.9 0.1 m (S.D.). A direct linear plot (Eisenthal and M Cornish-Bowden, 1974), which very sensitive to differences is between competitive, uncompetitive, noncompetitive, and mixed inhibition, also indicated that Li+ was noncompetitive a inhibitor of M e . The direct linear plot, which is also less

FIG.3. Inhibition of myo-inositol-1-phosphatase by Cap+ and Mn2+. Incubations with 24 units of enzyme activity wereunder
standard conditions, as described under "Experimental Procedures."

241 2ot
22
18t

FIG.4. Lineweaver-Burk plot showing the competitive inhibition of magnesium binding by calcium. V, nanomoles of
phosphate released per min. Lines were obtained by linear regression analysis of the data. Standard incubation conditions were used with 23 units of enzyme activity.

The Effects of Li+ and Other monovalent Cations myo-Inositol-1-phosphatase stimulated 1.5-fold over low is salt media by 0.3 M K' and Na'. This stimulation persists to levels as high as 0.5 M. Ammonium ionstimulates 2-fold at 0.1 M, an effect that decreases at higher concentrations, falling to the basal level at 0.5 M. Fig. 5 shows the results of adding low concentrations of NH,' and Group l a metals to incubations of the phosphatase with L-myo-inositol-1-Ppresence of 250 m~ K'. Only lithium ion is inhibitory, giving 50% inhibition at about 0.8 mM. The inhibition by Li+ occurs to the same degree with the D- and

FIG.5. Effects of monovalent cations on the myo-inositol-lphosphatase catalyzed hydrolysis of substrate using 17 units of enzyme activity under standard incubation conditions (in the presence of 250 m~ K+). Ions used were: A-A, NH,+;
HRb'; o"--o, , Cs'; W,Na'; and V, The Li'. effect of lithium i unique among these ions. s

10900

Effect of L, Ca2+, i ' and M n 2 +on myo-Inositol-1-phosphatase

The Effect of Cholinergic Agonists and Antagonists on the Phosphatase With and Without Added Li' The elevation of myo-inositol-1-P levels by lithium ion in rat brain may, in part, be due to the direct action of Li+ on myo-inositol-1-phosphatase. Since the in vivo lithium effect is blocked by muscarinic anticholinergic agents and is mimicked by cholinergic agonists of the muscarinic type (Allison, 1978), we examined the effect of these agents on the phosphatase in vitro. Atropine, a cholinergic antagonist, when administered to rats (35 mmol/kg) along with LiCl (5 meq/kg), blocks the lithium-induced elevation of myo-inositol-1-Plevels in rat cerebral cortex, while the administration of atropine alone has the effect of reducing levelsof the inositol phosphate (Allison et al., 1976). We found atropine (lo-' to 1 0 - 8 ~to have no ) effect on the hydrolysis of myo-inositol-1-P by the phosphatase in the presence and absence of 0.4 m~ lithium chloride. Scopolamine hydrochloride and benztropine methanesulfonate, both of which are cholinergic receptor blocking agents, as well as physostigmine, a cholinesterase inhibitor, also had no effect on the phosphatase in experiments identical with that with atropine. Pilocarpine, a muscarinic agonist, choline chloride, and acetylcholine chloride were also without direct effecton the enzyme. Thusthe in vivo changes inmyoinositol-1-P due to the cholinergic drugs must result from receptor interactions, while the lithium effect appears to be due to both a receptor-mediated response and to the inhibition of the phosphatase.
DISCUSSION

sensitive to statistical bias (Cornish-Bowden,1979), gave K , a for M ' of 1.0 f 0.1. Analysis of this data by the s / V versus g s and the V uersus V / s methods both gave K, values of 1.1 f 0.2. Thus lithium ion reduces the rate of myo-inositol-1-P hydrolysis without altering the binding of magnesium ion. We also have found no evidence of competition by Li' for myo-inositol-1-P binding. Incubations with varied myo-inositol-1-P concentration andconstant [Mg"] with three Li' concentrations, when analyzed by the Lineweaver-Burk method, show lithium ion to be an uncompetitive inhibitor of myo-inositol-1-P (Fig. In uncompetitive inhibition the ratio 6). KJV is constant for all levels of the inhibitor, thus as Li+ reduces the rateof substrate hydrolysis it increases the binding of myo-inositol-1-P.The three KJV (slope) values forLi' in the Lineweaver-Burk plot (Fig. 6) are 0.61, 0.69, and 0.70. The 0 lithium concentration slope is 0.66. Analysis of the experimental data by direct linear plot as well as by the l / V versus i and s/V versus i methods all uniquely show the inhibition to be uncompetitive. An explanation of the mechanism of this inhibition is that lithium ion binds only to the enzyme-substrate complex and that this form of the enzyme is of low activity.

Other Inhibitory Agents myo-Inositol-2-P is a competitive inhibitor of bovine brain myo-inositol-1-phosphatase with a Ki of0.3 m ~ Rat mam. has mary myo-inositol-1-phosphatase also been reported to be inhibited by myo-inositol-2-P (Naccarato et al., 1974).Sulfate inhibits the activity by 40% at 17 m ~ Fluoride exhibits an . inhibition-concentration profiie almost identical with that of lithium chloride; however,fluoride inhibition is probably due to the removal of Mg2+.Neither myo-inositol, D-sorbitol,nor D-glucose is inhibitory to the phosphatase when the concentration of each is 30 mM.

Insofar as is known, myo-inositol can be generated in the central nervous system by four routes: myo-inositol-1-P synthase (Eisenberg, 1967; Mauck et al., 1980),the epimerization of scyllo-inositol (Hipps et al., 1977; Sherman et al., 1978),the metabolism of the phosphoinositides (e.g. Hawthorne and Pickard, 1979), and transport (Spector and Lorenzo, 1975; Spector, 1976).Three pathways are known to utilize free myoinositol: transport outward from brain to plasma and cerebrospinal fluid (Spector, 1978), epimerization to scyllo-inositol and neo-inositol, and uptake into the phosphoinositides. Of these pathways that contribute to the myo-inositol pool, two produce myo-inositol-1-P:L-myo-inositol-1-P synthase converts ~-Glc-6-P L-myo-inositol-1-P, and that portion of to phosphatidyl inositol metabolism which occurs by the phospholipase C pathway produces D-myo-inositol-1-P.Rat kidney cortex fractions are known to degrade polyphosphoinositides to myo-inositol-1-P (Lapetina et al., 1975); however,it is not yet known whether similar enzymesoccurin brain. With respect to whether the lipid or synthase pathway is more active in producing myo-inositol-1-P,we have found rat cerebral cortex to contain D- and L-myo-inositol-1-Pin a concenWhile it is uncertain to what degree tration ratio of3.5 to these two pathways feed the free myo-inositol pool, it has been estimated that there is sufficient phosphatidyl inositol I phospholipase C in brain to hydrolyze all of that tissue's phosphatidyl inositol in 30 s (Hawthorne and Pickard, 1979). Thus thepotential for a major source of D-myo-inositol-1-Pis constantly present in brain. In each of these cases the agent for the hydrolysis of the D- and L-enantiomers is probably the 2 4 6 8 1 0 enzyme we have studied in this paper. l/[MlP] ( m M ) The effect oflithium on myo-inositol-1-P levels in cererat FIG. 6. Lineweaver-Burk plot showing the uncompetitive bral cortex is large in magnitude. A 5 meq/kg dose of LiCl, inhibition of myo-inositol-1-P binding by lithium ion. The K,/ V,, (slope) valuesare 0 6 (no Li+), 0.61 (0.096 mhf Li+),0.69 (0.26 which results in brain tissue levels of 6.9 meq of Li+/kg of dry .6 from m~ E+), 0.70 (0.51 m~ Li+).V, nanomoles of phosphate released tissue, causes an increase in brain myo-inositol-1-P levels and per min. Lines were obtained by linear regression analysis. Standard 0.34 mmol/kg dry weight to 2.9 mmol/kg dry weight (Allison incubation conditionswere used with 5.5 units of enzyme activity. et al., 1976).It has been found that this increase is due to the

Effect of Li+, Ca2+, andMn2+on myo-Inositol-1-phosphatase

elevation of both enantiomers, with D-myo-inositol-1-P accounting for 90% of the ~ h a n g e . ~ T h e increase in the L-enantiomer could be attributed to Li+ stimulation of L-myo-inositol-1-P synthase; however, i vitro experiments with the synn thase have excluded that possibility.6Therefore, inhibition of myo-inositol-1-phosphatase Li' probably causes the inby crease in the L-enantiomer. The 10-foldgreater increase of the D-enantiomer in lithium-treated ratsis difficultto account for solely on the basis of phosphatase inhibition, since the L form is hydrolyzed by the enzyme only slightly faster than the Denantiomer ( V,,L/V,.D = 1.3). It is thus likely that lithium increases the rateof formation of D-myo-inositol-1-Pand also decreases the rate of its hydrolysis through inhibition of the phosphatase. Since D-myo-inositol-1-P is probably derived entirely from the phosphoinositides, we conclude that lithium in some way stimulates the metabolism of these lipids. The evidence that this stimulation involves a muscarinic cholinergic response is compelling. The in vivo effect of lithium on brain levels of myo-inositol-1-P (Allison et al., 1976) and of myo-inositol (Allison Blisner, 1976) is blocked byatropine, and atropine itself causes myo-inositol-1-P levels fall in cerebral to cortex of rat (Allison et al,. 1976), and muscarinic cholinergic agonists mimic the effect of lithium on inositol phosphate in the rat (Allison, 1978).The metabolism of both phosphatidyl inositol and of the polyphosphoinositides is known to be stimulated by cholinergic agonists in the central nervous system (e.g. Lunt and Pickard, 1975; Soukup et al., 1978). These facts suggest to us that lithium ion acts in vivo in a dual capacity: to alter the metabolism of one, or both families, of the phosphoinositides, generating D-myo-inosito~-~-P, and to inhibit myo-inositol-1-phosphatase, thus reducing the rate of hydrolysis of the inositol phosphate to free inositol. Lithium is a highlyeffective agent in thetreatment of manic-depressive illness. The involvement of a phosphoinositide, a class of lipids known to be active in synaptic function, in a significant effect of lithium in brain gives somehope that the therapeutic and the biochemical effect are related. Both calcium and manganese play significantroles in phosphatidyl-inositol metabolism. Calcium is required for the muscarinic activation of phosphatidyl inositol turnover in synaptosomes (Miller, 1977; Griffii et al., 1979). Although the , required levels of Ca2+in the medium are low (-0.1 p ~Griffin et al., 1979) when compared with the K, of Ca2+which we have measured with the phosphatase, it is not known what the actual cytosol Ca2+levels are during activation in vivo. Polyphosphoinositide metabolism in synaptosomes is directly stimulated by calcium, one of the products being an inositol diphosphate which appears to be derived mainly from phosphatidyl inositol 4-phosphate (Griffin and Hawthorne, 1978). Again, the intrasynaptosomal level of Ca" required for this stimulation occurs with effect is not known; however, maximal 0.4 m~ calcium in the medium. Manganese ion isrequired for phosphatidyl inositol synthesis (Paulus and Kennedy, 1960) and may play a regulatory role in the synthesis of phosphatidyl inositol in synaptosomes (Yandrasitz and Segal, 1979). Once again, the cytosol Mn2+levels are conjectural, but 0.1 to 1.0 IILM concentrations are typical in uitro. The low levels of Ca2+ and Mn2+which are inhibitory to myo-inositol-1-phosphatase make these metals potential regulators of that enzyme in vivo. What interrelationships exist between D-myo-inositol-1-Pand phosphoinositide metabolism are unknown except for the lipid-product-inositol-precursor role of D-myo-inositol-1-P. It is, however, tempting to speculate that there is a common and controlling basis in the stimulation-inhibition of the various parts of these highly active pathways.
R. W. Wise and W. R. Sherman unpublished data.

10901

We have found that myo-inositol-1-phosphatase from bovine testis is also inhibited by lithium ion. It is known that decreased myo-inositol synthesis in testis is accompanied by a decrease in the number of spermatids and spermatozoa, suggesting a role for myo-inositol in spermatogenesis (Morris and Collins, 1971). thus mustbe consideredwhat the effects It of lithium may be on testis and on other organs outside the central nervous system both from the standpoint of the effect on myo-inositol levels and on levels of myo-inositol-1-P.
Acknowledgement-We wish to acknowledge the work of S. L. Goodwin, who performed some of the initial experiments in this study.
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