Alteration in enzyme activities of de novo phosphatidylcholine biosynthesis in rat liver by treatment with typical inducers of microsomal drug-metabolizing system

49

Biochimico et Biophysics Acta, 620 (1980) 49-58 0 Elsevier/North-Holland Biomedical Press

BBA 5’7635

ALTERATION IN ENZYME ACTIVITIES OF DE NOVO PHOSPHATIDYLCHOLINE BIOSYNTHESIS IN RAT LIVER BY TREATMENT WITH TYPICAL INDUCERS OF MICROSOMAL DRUG-METABOLIZING SYSTEM KOZO ISHIDATE, MICHIKO TSURUOKA and YASUO NAKAZAWA Medical Research Institute, Tokyo Medical and Dental University, Z-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101 (Japan) (Received March 3rd, 1980)

Key words: Choline kinase; Cholinephosphate cytidylyltransferase; Cholinephosphotransferase; Drug metabolism; Phosphatidylcholine

synthesis; (Rat liver)

Summary

1. The activities of choline kinase, ethanolamine kinase, cholinephosphate cytidylyltransferase(s) and cholinephosphotransferase were compared in the liver subcellular fractions after the treatment of rats for two successive days with phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls. 2. The administration of phenobarbital resulted in a significant decrease in choline kinase activity while not affecting ethanolamine kinase activity. Both 3-methylcholanthrene and polychlorinated biphenyls caused considerable enhancement of choline kinase activity concomitantly with ethanolamine kinase activity. 3. The activity of cytosolic cytidylyltransferase was not affected by any of the inducers while the microsomal activity was significantly depressed by the administration of either phenobarbital or polychlorinated biphenyls. 4. The activity of microsomal cholinephosphotransferase decreased significantly after the treatment with both 3-methylcholanthrene and polychlorinated biphenyls and increased slightly after phenobarbital administration. 5. The observed opposite effects of phenobarbital and 3-methylcholanthrene on the enzymes in de novo phosphatidylcholine synthesis indicate that there exist a possible relation between induction of microsomal drug-metabolizing system and modulation of phosphatidylcholine biosynthesis in animal liver. .Introduction

The metabolism of phospholipid, particularly of phosphatidylcholine, in rat liver has been shown in in vivo experiments to be affected drastically by the

50

administration of phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls [ 121. The most remarkable change/was seen in the pool size of phosphorylcholine, a water-soluble intermediate in de novo synthesis of phosphatidylcholine. Both 3-methylcholanthrene and polychlorinated biphenyls increased the level of phosphorylcholine pool size in the liver by 2---3-fold, while phenobarbital significantly decreased the level, Severe inhibition of incorporation of intraperitoneally injected [ 32P]orthophosphate or [ *4C]choline into liver phosphatidylcholine was also observed in either 3-methylcholanthrene or polychlorinated biphenyls-treated rats [ 21. These chemicals are well-known inducers of microsomal drug-metabolizing enzymes although their mechanisms of the induction are different from each other; phenobarbital is a typical inducer of cytochrome P-450 (P-4501, P-450LM2, P-450b, P-450, (phenobarbital)), while 3-methylcholanthrene induces socalled cytochrome P448 (P-448,, P-450-LM4, P-450,, P-45Ou (methylcholanthrene)), and each cytochrome has a different molecular size and different catalytic activity [ 3-81. On the other hand, polychlorinated biphenyls have been shown to have the capability of induction of both types [6,9-111 because they are composed of several isomers of polychlorinated biphenyl molecules, some of which belong to the phenobarbital-type while others belong to the 3-methylcholanthrene-type of inducers, depending on the number and/or position of chlorine molecules in their structure [ 12-141. Thus the possible relationships between difference in induction mechanisms with respect to drug-metabolizing system and effect of these inducers on phosphatidylcholine metabolism, have led us to study the mechanism of their interaction with the enzymes involved in de novo synthesis of phosphatidylcholine in the liver. In the present paper, characteristic changes in the activity of choline kinase, ethanolamine kinase, cholinephosphate cytidylyltransferase(s) and cholinephosphotransferase were demonstrated in liver subcellular fractions after treatment of rats with the typical inducers. In addition, alteration of in vivo phosphatidylcholine metabolism by these inducers [ 21 was interpreted by the findings in the present in vitro experiment. Experimental Mu teriats

~~e~~~~-14C]Choline chloride, [ l-3~]eth~-l-ol-Z-amine hydrochloride, cytidine-5’diphospho-~~e~~y~-*4C]choline (ammonium salt) were obtained from the Radiochemical Centre, Amersham, U.K. Phosphoryl-[methyl-‘%]choline was purchased from New England Nuclear Corp. All radioactive chemicals were used without further purification. All other chemicals and solvents were of reagent grade. Inducer

treatment

of animals

Male Wistar rats (100-120 g) were used for all experiments. housed in one cage each, fed Clea CE-2 Laboratory Chow, and access to water. Phenobarbital and polychlorinated biphenyls biphenyl, tetra, from Wako Pure Chemical Industries Co. Ltd.,

2 rats were allowed free (polychloroJapan) were

51

given orally to experimental animals at dosages of 100 and 80 mg/kg per day, respectively. 3-Methylcholanthrene dissolved in arachis oil was intraperitoneally injected at a dosage of 50 mgfkg per day. All inducers were given once a day in the morning for two successive days while control animals were given equivalent volumes of the vehicle. Animals were killed in the morning on day 3 after 12 h starvation. Other details were as described in the previous paper [ 1,2].

Enzyme source For choline kinase, eth~olamine kinase and cholinephosphotransferase assays, a 20% homogenate of liver was prepared in 0.25 M sucrose using a motor-driven Teflon pestle homogenizer, For cholinephosphate cytidylyltransferase assay, saline was used instead of 0.25 M sucrose for the preparation of liver homogenate. The homogenate was centrifuged at 15 000 X g for 15 min and the supernatant was recentrifuged at the same speed for 15 min to minimize possible contamination of the microsomal fraction by light mitochond~a and lysosomes. The 15 000 X g supernatant was centrifuged at 105 000 X g for 60 min. The microsomal pellet and the supernatant were separated and the latter was used for choline kinase, ethanolamine kinase and soluble cytidylyltransferase assays. The microsomal pellet was homogenized in a proper amount of isotonic KC1 and recentrifuged at 105 000 X g for 45 min. The washed microsomes were resuspended in a small amount of isotonic KC1 using a Teflon pestle homogenizer and used for microsomal cytidylyltransferase and cholinephosphotransferase assays. Enzyme assay Choline kinase (ATP: choline phosphotr~sferase, EC 2.7.1.32) activity was determined by a modification of the method of Weinhold and Rethy f15]. Incubation mixtures contained 0.1 M Tris-HCl (pH 8.5), 10 mM MgCl,,, 10 mM disodium ATP (from equine muscle, Sigma), and 0.25 mM [methyl-i4C]choline chloride (spec. act. 0.7 Ci/mol) in a final volume of 0.5 ml. The reaction was started by addition of the enzyme preparation containing about 0.5 mg protein. Incubations were conducted at 37°C for 20 min and stopped by placing the tubes for 2 min in a boiling water bath. Blanks were done by stopping the reaction immediately after the addition of enzyme. An 0.25-ml portion of the mixture was applied to a column (0.5 X 2 cm) of Dowex 1 X-8(OH-), 200---400 mesh. The column was washed first with 2.5 ml 5 mM choline chloride then with 6 ml water and cholinephosphate was eluted with 0.5 ml 1 M NaOH followed by 1.5 ml 0.1 M NaOH. The radioactivity was determined with 10 ml Aquasol scintillation fluid (New England Nuclear Corp.). Ethanolamine kinase (ATP: ethanolamine 0-phosphotransferase, EC 2.7 .1.82) was determined by a slight modification of the method of Weinhold and Rethy [15]. Incubation mixtures contained 60 mM sodium glycylglycine (p&I 8.5), 3 mM MgC&, 3 mM disodium ATP 0.1 M KCl, 0.5 mM [l-3H]ethan-lolamine hydrochloride (spec. act. 2 Ci/mol) and enzyme preparation (0.2-0.3 mg protein) in a final volume of 0.2 ml. The reaction was started by the addition of enzyme and incubated at 37°C for 20 min. The reaction was stopped by placing the tubes for 2 min in a boiling water bath. Blank reactions were placed in the boiling water bath immediately after the addition of the enzyme. The

52

ethanolaminephosphate was separated from ethanolamine by applying a O.l-ml Sample to a column (0.5 X 2 cm) of Dowex 50W X-SjH’), IO@--ZOO mesh. The columns were washed three times with 1 ml water. The final wash was accomplished by a brief centrifugation. The water eluent was transferred into a scintillation vial and the radioactivity was measured with 10 ml Aquasol. Cholinephosphate cytidylyltransferase (CTP: cholinephosphate cytidylyltransferase, EC 2.7.7.15) was assayed by a modification of the method of Ansell and Chojnacki [16] _ The incubation mixtures contained 50 mM Trissuccinate (pH 7.51, 6.7 mM Mg(CH~COO~~, 2 mM trisodium CTP (from Kyowa Hakko Co. Ltd., 3apan), 1.7 mM phosphoryl-~~e~~~~-14C]~holine (spec. act, 0.2 CiJmof) and enzyme preparation of either 105 000 X g supernatant from 20% saline~homogenate or KCl-washed microsomes (each prep~ation contained l-3 mg protein per incubation) in a final volume of 0.3 ml. The assay was started by the addition of enzyme preparation and incubated at 37°C for 15 min. The reaction was stopped by the addition of 0.3 ml 10% trichloroa~etic acid. Blanks were done by stopping the reaction immediately after the addition of the enzyme. After brief centrifugation, a 0.45-ml portion of the supernatant was mixed with 1 ml aqueous suspension of charcoal (20 mg/ml, Norit A from ICN Pharmaceuticals Inc.). The mixture was shaken intermittently over a 15” min period to keep the charcoal in suspension, then centrifuged. The charcoal was washed five times with 2.5 ml unlabelled phosphorylcholine solution (2 mgfml) to remove unadsorbed phosphorylcholi~e, The labeled CDPcholine was then eluted from the charcoal by mixing ~gorously with 0.5 ml 87% (w/v) HCOOH for 5 min, centrifuged and a 0.3-ml portion of the supernatant was counted with 0.7 ml water and 10 ml Aquasol scintillation fluid. The reaction product was also checked by TLC on Avicel SF (microcrystalline cellulose, from Asahi Kasei Co. Ltd., 3apan) for trichloroaeetic acid-soluble fraction with a developing solvent system of n-C&H,OH/87% (w/v) HCOOH/HzO (60 : 24 : 16, v/v/v), Since the rate of CDPcholine production in the assay was linear for 30 min and proportional to the amount of enzyme preparation if no more than 3 mg protein were used with either analytical method of charcoal adsorption or TLC on Avicel SF, the former method was usually used for technical reasons. ~holinephospho~~sferase (~DPcholine:l,2d~cylglycerol cholinephosphotransferase, EC 2.7.8.2) was assayed as follows: the reaction mixtures contained 75 mM Tris-HCl (pH 8.0), 5 mM tetrasodium EDTA, 20 mM MgClZ* 8 mM dithiothreitol, 0.5 mM CDP-[~e~~~~-14~]choline (spec. act. 0.1 Ci/mol), 1 mM diacylglycerol in Tween dispersion and KCl-washed microsomes (O.l0.2 mg protein) in a final volume of 0.5 ml. Diacylglycerol was prepared from fresh egg lecithin through phospholipase C (CEostridium welchii, type I from Sigma) hydrolysis according to the method of Wood and Snyder [ 171 and purified by preparative TLC with a developing solvent system of C&H&HClJ GH@H (80 : 15 : 5, v/v/v). D~a~y~~lycer~l-Tween dispersion was prepared by the following procedure: 6 mg diacylglycerol were sonicated in 1 ml 0.2% Tween-20 solution using a sonic dismembrator {Artex Systems Corp.) at power setting 60 until no visible floating material was observed (usually 30 s). The reaction was started by the addition of microsomes and run at 37°C for 20 min. The reaction was stopped by the addition of 3.0 ml CH~l~/~H~OH (2 : 1, v/v)

53

followed by subsequent extraction of the reaction product. Blanks were stopped at zero time, while control incubations were carried out without added d~acy~~~ycero~for the correction of the con~~bu~~o~ of endu~en~~ d~cy~~~ycero1. The CXC& layer was separated by brief cent~~u~t~on and washed twice with 2 ml 50% C&OH. The washed CHCIB solution was transferred into a scintillation vial and evaporated. A toluene-based scintillation fluid containing 0.4% (w/v) 2,fidiphenyloxazole and 0.01% (w/v) 1,4-bis(5_phenyloxazolyl)2-Ibenzene was added and the radi~cti~ty counted, Protein was determined by the method of Lowry et al. [IS] with bovine serum albumin as standard. The radioactivity was determined in a Packard TriCarb liquid scintillation spectrometer (model 2002). The counting efficiency was c~culated by an external standard method. Results

Choline kinase Choline kinase is located in the soluble portion of the liver cell. The reaction rate was constant for 30 mm and proportions Wthe amount of enzyme within the range of Q.6 mg protein per incubation. As shown in Table I, the administration of phenobarbital for two successive days caused a significant decrease (80% of the cantrol) in enzyme activities when referred to either mg protein or g liver weight. Qn the other hand, the treatment of rats with ~-me~ylcholanthrene caused approx. ~100% increase in the activity, Po~ychl~r~ated b~p~euy~s also caused ~~~fi~nt elevation of the enzyme activity, though with a lower magnitude than ~-met~ylcho~n~

TABLE ‘I

PB (80 mg& per da~f, 3-MC (50 mglkg per day) or PCBs ilO mg/kg per day) was administered for two successive days and animals were killed on day 3, The assay conditions and other details BW described in Experimental. PB, phenobarbital; Q-MC, 3-methylcbalanthrene; PCBs, polychlorinated biphsnyls. Values are means !: S.E. of triplicate meaW9urementsfrom four animals. Treatmeat

Specific a&v&y

Activity

nmoljmin per mg protein

Control (96)

nmolfmin per g iiv+x

3.35 2.75 6.28 4-48

0.20 0.15 0.42 0.48

100 82 X87 134

23Q.Q ” 14.6 136.3” 8.8 413.6 * 28.9 293.8 ?. 34.5

Ethatlolamine kmase Control 1.24 + 0.08 PB 1.20 + 0.08 3-MC 2.50 ? 0.12 PCBs 1.76 f 0.20

100 97 202 142

$8.3 81,5 164.6 1lS.4

Choline kinasa Control PB 3-MC PCBs

? i i: r

?: 4.9 ?: 6.5 f 7.7. f 14.0

Activity nmd/min per whole liver

ControS (5%)

78 173 123

1263 1196 2807 2455

+ 119 It 154 +_239 ? 156

100 85 222 163

100 92 186 13X

466 517 1116 807

+ 25 f 44 t 51 + 72

100 111 240 173

GO&tP&

(WI

100

54

threne. When compared on the whole liver basis, the effects of 3-methylcholanthrene and polychlorinated biphenyls obviously were further exhibited because either of these inducers caused si~ificant enl~gement of the liver in experimental animals. Ethanolamine kinase The reaction rate of ethanolamine kinase in the present investigation was constant for 30 min and proportional to the amount of 105 000 X g supernatant below 0.5 mg protein. After the treatment of rats for two successive days, phenobarbital had no significant effect on ethanolamine kinase activity, while 3-methylcholanthrene caused approx. 100% increase in the activity when referred to the supernatant protein (Table I). The magnitude of stimulation by 3-methylcholanthrene was very close to that of choline kinase activity with either represen~tion of the activity. Polychlorinated biphenyls also had a stimulatory effect on ethanolamine kinase activity although the extent of the stimulation was smaller than that by 3-methylcholanthrene. As a result, activities of choline kinase and ethanolamine kinase in liver cytosol after treatment with the inducers were very similar to each other except that phenobarbi~l had a depressive effect on choline kinase activity, while it had no significant effect on ethanolamine kinase activity. Cholinephosphate cy tidylyltransferase Cholinephosphate cytidylyltransferase activity in liver is found in both 105 000 X g supernatant and microsomal fractions. Both activities were constant for 30 min and almost proportional to the amount of enzyme preparation in the range up to 3.0 mg protein per incubation. As shown in Table II, the activity in cytosol was not significantly affected by any of the inducers while microsomal activity was depressed to 60% of the control by both phenobarbital and polychlorinated biophenyls, when referred to the protein basis. Since microsomal protein content per g liver increased significantly by either phenobarbital or polychlorinated biphenyls treatment, the effect of these inducers on the activity per g liver weight would be diminished but still be significant. On the other hand, no appreciable change was found in cytidylyltransferase activity by 3-methylcholanthrene, both in cytosolic and microsomal fractions.

Cholinephosphotransferase activity is entirely located in the microsomal fraction in rat liver. The absolute reaction rate was definitely dependent on the preparation of diacylglycerol-Tween dispersion (Fig. 1). However, the linearity between reaction rate and incubation time or the amount of enzyme was not influenced by+exogenously added diacylglycerol preparation if the reaction was stopped within 30 min and the amount of enzyme was not more than 0.2 mg protein per incubation (Fig. IA and B). The activities of cholinephosphotransferase in liver microsomal fraction of rats pretreated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls are compared in Table III, in which the activity was represented on

55 TABLE II CHOLINEPHOSPHATE CYTIDYLYLTRANSFERASE ACTIVITIES IN LIVER 105 000 x B SUPERNATANT AND ~ICROSOMAL FRACTIONS FROM INDUCER-TR~ATRD RATS Treatments of animals were the same as in Table I. Each incubation contained 2-3 (supernatant) and 1-2 mg (microsomes) protein. The assay conditions are described in Experimental. Abbrevations used are as in Table I. Microsomsl activity

Supernatant activity

Treatment

nmoi/min per mg protein

nmol jmin per g liver

nmolfmin per mg protein

nrnolfmin per g liver

Expt. 1 Control PB % of control

0.11 + 0.09 * 0.71 + 0.03 92

80.3 f 10.6 66.5 f 1.9 88

1.30 t: 0.07 0.17 t 0.09 59

21.1 + 0.7 17.1 + 0.7 81

Expt. 2 Control 3-MC % of control

0.83 + 0.08 0.72 ** 87

85.8 + 5.6 75.2 88

1.19 + 0.14 1.10 + 0.10 92

17.9 ?r 2.3 16.3 _+0.9 91

Expt. 3 Control PCBs % of control

0.55 ?r 0.12 0.49 f 0.10 89

58.8 + 13.0 44.7 + 11.0 76

1.06 f: 0.09 0.62 f 0.03 58

18.3 + 1.5 13.5 + 0.7 74

* Values are means ? S.E. of trip&ate measurements from three animals. ** Values are means of triplicate measurements from two animals.

the basis of microsomal protein, g liver weight and whole liver, respectively. Phenobarbital and polychlor~ated biphenyls caused si~ificant.enl~gement of liver as well as an increase in the amount of microsomal protein, whereas 3-methylcholanthrene caused liver enlargement without appreciable increase in

10

20 30 40 INCUBATION TIME (min)

”

“.L

u.z

“.a

11.4

ENZYMECONTENT(mg protein)

Fig. 1. Cholinephosphotrapsferaae activity in rat liver microsomes as a function of incubation time (A) and protein content (B). The may conditions are described in Experimental. Open and closed circles represent results from separate experiments in which diacylglycerol-Tween dispersions were prepared separately.

56 TABLE III CHOLINEPHOSPHOTRANSFERASE TREATED RATS

ACTIVITIES

IN

LIVER

MICROSOMES

FROM

INDUCER-

Treatments of animals were the same as in Table I. Other details are described in Experimental. Values are means f S.E. of duplicate measurement from four animals. Treatment Specific activity Activity Activity nmol/min per mg protein Control Phenobarbital I-Methylcholanthrene Polychlorinated biphenyls

5.88 6.78 4.14 3.64

? r + +

Contro1

nmolfmin per g liver

(%) 0.16 0.43 0.68 0.18

100 115 70 62

Contro1 (%)

170.4 255.3 128.8 149.9

+ 2.8 + 15.1 ?: 21.7 + 9.9

100 150 75 88

nmoiimin &xr whole liver -~

Contro1

a24 + 87 1634 t 96 673f119 1007 c 129

100 198 82 122

(a)

microsomal protein content. The specific activity and the activity per g liver weight decreased considerably in rats treated with either 3-methylcholanthrene or polyehlorinated biphenyls. On the other hand, 50% increase in the activity per g liver weight was found in phenobarbit~-treated rats. In another experiment using a different preparation of diacyl~lycerol-seen dispersion as a substrate (cf. Fig. l), the specific activities (nmol) per mg protein/min obtained for microsomal cholinephosphotransferase were as follows: 15.36 + 0.59 for controls; 14.27 it 0.83 for phenobarbital-treated rats; 9.95 f. 1.94 for 3-methylcholanthreneand 7.67 + 1.47 for polychlorinated biphenyltreated rats. Discussion In the previous experiments [2], it was shown that the administration of either 3-methylcholanthrene or polychlorinated biphenyls to rats caused a severe inhibition of 13*PJo~hophosphate incorporation into liver mierosomal phospho~pids, especially phosphatidylcholine. At the same time, in the liver of these animals, a larger amount of radioacti~ty from intraperitoneally injected [methyl-14C]choline was detected in phosphorylcholine, a water-soluble intermediate in de novo phosphatidylcholine synthesis, when compared to the control animals. The calculation from in vivo studies showed that there occurred approx. l,S- and 2.3-fold large pool sizes of phosphorylcholine per g liver in 3_methylcholanthreneand polychlorinated biphenyl-treated animals, respectively. On the other hand, the phosphorylcholine pool size in the liver of phenobarbital-treated rats was less than 50% of the control although the apparent rate of precursor incorporation into liver microsomal phospholipids was not si~ificantly affected by phenob~bital [Z] . The effect of these inducers on phosphoryl~holine pool sizes in the liver could be’ well explained by the findings in the present investigation in which the activities of three kinds of enzyme involved in de novo phosphatidyl~holine synthesis were compared after two days of treatment of rats with the inducers. 3-Methylcholanthrene could elevate the level of phosphorylcholine by increasing cytosolic choline kinase activity (Table I). Folychlori~ated biphenyls

57

increased choline kinase activity while decreased microsomal cholinephosphate cytidylyltransferase activity (Table II), both of which could result in the accumulation of phospnorylcholine in the liver cells. Microsomal cholinephosphotransferase activity was depressed by either of the inducers (Table III), which also may influence the level of phosphorylcholine pool positively. On the other hand, phenobarbital would cause a decrease in phosphorylcholine pool size by decreasing cytosolic choline kinase activity. Phenobarbital also decreased microsomal cytidylyltransferase activity significantly. However, the decrease in membrane-bound activity may not be enough to maintain the pool size of phosphorylcholine at the control level, since the microsomal activity appeared to be only about 20% of total cytidylyltransferase activity in the liver cells (Table II). It is noteworthy that ethanolamine kinase activities showed behavior very similar to choline kinase activities after treatment by the inducers (Table I). The mechanism of increased activities of these kinase by either 3-methylcholanthrene or polychlorinated biphenyls administration is not established from the present studies. It might be a kind of adaptive change in the level of the enzymes or simply the result of the interaction between kinase proteins and the inducers (or metabolites) leading to the enzyme activation. There may be other indirect mechanisms leading to either induction or activation of the kinases by the administration of these inducers. The purification and complete separation of choline kinase from ethanolamine kinase in animal tissues have not yet been successful [15,19]. Recently, Brophy et al. [20] suggested from their electrophoretic studies that several isoenzymes of choline kinase and ethanolamine kinase exist in rat liver cytosolic fraction and that these are separate enzymes. If this were true, then it would be interesting to know which isoenzyme(s) is responsible for the inducer treatment, which may give an important suggestion in elucidating the control mechanism of phospholipid biosynthesis in animal liver. A number of different approaches have provided evidence that the reaction catalyzed by cholinephosphate cytidylyltransferase is the rate-limiting step in phosphatidylcholine biosynthesis in the liver [21,22] as well as other tissues [22-251. Recently, Choy et al. [26] reported that there are two forms of cholinephosphate cytidylyltransferase in rat liver cytosol, a high (H) and a low (L) molecular weight form. The L form requires some lipid component for the activity, but the activation of the enzyme is not accompanied by a simultaneous aggregation of the enzyme into the H form [27] ; this is in sharp contrast to the results obtained for lung enzyme [28]. In the subsequent paper [ 291, they reported that cytosolic and microsomal forms of cytidylyltransferase found in rat liver are immunologically identical, although the physiological distribution of the enzyme in liver cells has not yet been established. In the present investigation, the cytidylyltransferase activity in liver 105 000 X g supernatant was not significantly affected by the administration of the inducers while microsomal activity considerably decreased in either phenobarbitalor polychlorinated biphenyls-treated rats, suggesting that there exist a separate control mechanism between soluble and membrane-bound cytidylyltransferase activities in liver phosphatidylcholine synthesis. The last reaction in phosphatidylcholine biosynthesis is catalyzed by micro-

58

somal CDPcholine:l,2diacylglycerol cholinephosphotransferase. It is well understood that the activity of the cholinephosphotransferase does not seem to regulate the rate of phosphatidylcholine synthesis [22] although this enzyme acts at a branchpoint in the metabolism of diacylglycerol and has some specificities for diacylglycerol species [30,31]. In the present study, both 3-methylcholanthrene and polychlorinated biphenyls significantly decreased the activity of cholinephosphotransferase in rat liver, probably through their direct interactions with microsomal membranes because of their highly lipophilic characteristics. Consequently, these inducers may cause not only impaired phosphatidylcholine synthesis but also altered specificities of the enzyme for diacylglycerol species, which could result in concurrent changes in membrane integrity and permeability. In conclusion, phenobarbital and 3_methylcholanthrene, which are wellknown as typical inducers of the microsomal drug-metabolizing system in animal tissues with a different mechanism of induction, caused opposite effects on phosphatidylcholine synthesis in rat liver. Polychlorinated biphenyls, which have been known to be a mixed type of inducer of phenobarbital and a-methylcholanthrene on the drug-metabolizing system, also caused a mixed type of effect on liver enzymes in de nova phosphatidylcholine synthesis. The outstanding question raised by the present studies concerns the direct relationships between mechanism of induction of the drug-metabolizing system and phosphatidylcholine metabolism in animal liver. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

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4986 29 Choy, P.C., Schneider, W.J. and Vance, D.E. (1978) Eur. J. Biochem. 85.189-193 30 Holuh, B.J. (1978) J. Biol. Chem. 253,691-696 31 Morimoto, K. and Kanoh, H. (1978) J. Biol. Chem. 253.5056-5060