Biochimica et Bioplo'sica Acta, 1081 (1991) 25-32
25
' 1991 ElsevierScience PublishersB.V.(Biomedical Division)0005-2760/91/503.50 ADONIS 000527609100052Y
The protein phosphatase inhibitor, okadaic acid, inhibits phosphatidylcholine biosynthesis in isolated rat hepatocytes G r a n t M. H a t c h ' , Y. T s u k i t a n i ~- a n d D e n n i s E. V a n c e t Lipid and Lipoprotein Research Grol p and Department of Biochemistry, Unwersitv of Alberta Edmonton (Canada) attd " F~muwa Phart~:aceutical Company. Tokyo (Japan)
(Received 30 July 1990) Key words: Phosphatidylcholinebiosynthesis:Okadaic acid: IRat liver): Protein phosphatase There is evidence that pbosphatidylchollae (PC) biosynthesis in hepatueytes is regulated by a phosphoD'lation-deph~phorylation mechanism. The phosphatases involved have not been identified. We, therefore, investigated the effect of okadaic acid, a potent protein phosphatase inhibitor, on PC biosynthesis via the CDP-eheline pathway in suspension cultures of isolated rat hepatocytes. Okadaic acid caused a 15% decrease ( P < 0.05) in IMe-3H]cboline uptake in continuous-pulse labeling experiments. After 120 min of treatment, the labeling of PC was decreased 46% ( P < 0.05) with a corresponding 20'70 increase ( P < 0.05) in labeling of phosphucholine. Cells were pulsed with IMe-3H]choline for 30 min and subsequently chased for up to 120 min with choline in the absence or presence of okadaic acid. The labeling of phosphocholine was increased 86% ( P < 0.05) and labeling of PC decreased 29% ( P < 0.05) by 120 rain of chase in okadale acid-treated hepatucytes. The decrease of label in PC was quantitatively accounted for in the phosphocholine fraction. Incubation of bepatncytes with both okadaic acid and CPT-cAMP did not produce an additive inhibition in labeling of PC. Choline kinase and cholinepbospbotrausferase activities were unaltered by treatment ~ith okadaic acid. Hepatocytes were incubated with digitonin to cause release of cytusolic components. Cell ghost membrane cytidylyltransferase (CT) activity was decreased 37% ( P < 0.005) with a concomitant 33% increase ( P < 0.05) in released eytusolic cytidylyltransferase activity in okadaic acid-treated hepatucytes. We postulate that CT activity and PC biosynthesis are regulated by protein phosphatase activity, in isolated rat hepatucytes.
Introduction
Phosphatidylcholine (PC) is the principal phospholipid in the mammalian liver [1]. The major pathway for PC biosynthesis is the CDP-choline or Kennedy pathway (Fig. 1) [2]. In addition. PC may be synthesized by progressive methylation of phosphatidylethanolamine (Fig. 1). It is well established that CTP : phosphocholine cytidylyltransferase (CT) catalyzes the rate-limiting step of PC biosynthesis via the CDP-choline pathway in rat liver [3] and is located in both the cytosolic anu microsomal fractions from mammalian liver [3]. The mtcrosomal CT is regarded as the active form of the enzyme [3-5] and the cytosolic CT a storage form (Fig. l) [3].
The translocation of the enzyme between these two subcellular compartments represents a plausible mechanism for the regulation of its activity and thus PC biosynthesis [6-8]. Evidence for the translocation of CT has been obtained in rat liver [9] rat hepatocytes [10], Hela cells [11,12]. rat lung [13]. chick embryonic myoCK ChoI ~
)
PChoI
CPT CDP-ChoI'~'-~ PC
T PEMT
er ERlAttice)
Phosphatasels)
Klnasels)
CT-
Abbreviations: DMSO, dimethylsulfoxide; PBS, phosphate-buffered saline; PMSF. phenylmethylsulfonylfluoride, PC, phosphatidylcholine. CT. cytidylyhransferase. Correspondence: D.E. Vance, Lipid and Lipt,protein ResearchGroup, 3rd Floor Heritage Medical Research Center, Facuhy of Medicine, Universityof Alberta. Edmonton,Alberta Canada, T6G 2S2.
Cyto~,l(Inactive) Fig. 1. Postulated regulatory mechanismsfor control of phosphatidylcholine biosynthesis in rat liver. Chol. choline: PChol. phosphocholine; CDP-Chol, CDP-choline; PC, phosphatidylcholine: CK, choline kinase: CT. cytidylyltransfcrase; CPT, cholinep;osphotransferase, PE. phosphatidylethanolamine; PEMT, phosphatidylethanolamine methyltransferase;ER. endoplasmicreticulum.
blasts [14-16], Hamster heart [17] and in the liver of fetal rats [18]. Recently. ~,at liver CT was demonstrated to be phosphorylated on a serine residue(s) by cAMP-dependent protein kinase in vitro [19]. In this same study, incubation of rat liver post-mitochondrial supernatant with exogenous alkaline phosphatase promoted the translocation of CT activity from the cytosolic to the microsomal fraction. However. the subcellular distribution of alkaline phosphatase activity, on the external side of the plasma membrz.ae [20], would preclude the importance of this enzyme in the dephosphory!ation and tran:;Iocation of CT in vivo [21]. This implies that f.here must be intracellular phosphatases involved in the dephosphorylalion and translocation of CT, If CT were indeed regulated by a phosphorylation-dephosphorylation mechanism in vivo (Fig. 1), then the activities of cellular protein phosphatases should influence PC biosynthesis. Type 1 and 2A phosphoprotein phosphatases are the dominant protein phosphatases acting on a wide range of phosphoproteins in vivo which regulate several metabolic pathways and muscle contraction [22]. Okadaic acid is a specific and potent inbihitor of type 1 and 2A cellular phosphoprotein phosphatases [23,24] and was demonstrated to inhibit the time dependent association of cytosolic CT activity with microsomal membranes in rat liver post-mitochondrial supernatant [21]. In this study we provide the first evidence that phospb.oprotein phosphatases I a n d / o r 2A are involved in the regulation of PC biosynthesis in intact rat hepatocytes. Materials and Methods
[Me- 3H]Choline and CDP[ Me-t4C]choline were obtained from Amersham. t.-[3-14C]Serine was obtained from New England Nuclear. Phospho[Me-~H]choline was synthesized enzymatically from [Me-~H]choline [25]. Silica gel 60 plates were obtained from Merck. Albumin was obtained from Sigma :~A-7030. All other biochemicals were of analytical grade and were obtained from either Sigma Chemical Company (St. Louis, MO) or Fisher Scientific (Edmonton. Canada). Okadaic acid was a generous gift of Dr. Y. Tsukitani, Fujisawa Pharmaceutical, Tokyo (Japan). Isolation, incubation and harvesting of hepato(ytes Male Sprague-Dawley rats (175-250 g) were used throughout the study. Rats were maintained on Purina rat chow and tap water, ad libitium, in a temperatureand light-controlled room. Rats were rendered unconcious with diethyl ether and anaesthetized with 2.27 m g / k g phenobarbital. Hepatocytes were isolated by a collagenase-perfusion technique as described [26] and finally suspended in Dulbecco's Modified Eagle's Medium containing 10% delipidated fetal calf serum
[27] (which had been extensively dialyzed against 0.9% NaCl) supplemented with 25~c fatty acid free albumin. The isolated cells excluded 90~ Trypan blue. Cells (4-10~') were shaken, at 100 rpm on a Vibrax Shaker and incubated in 25 ml Erlenmeyer flasks with 2 ml of medium containing [Me-3H]choline for 0-120 rain in continuous-pulse experiments. In pulse-chase experiments cells were incubated for 30 rain with [Me3H]choline prior to chase with unlabeled choline. Okadaic acid was dissolved in 10% DMSO with final concentration in the medium of 0.5 p.M okadaic acid and 0.125% DMSO. Control hepatocytes were incubated with 0.125% DMSO. Subsequent to continuous-pulse or pulse-chase experiments, cells were harvested by the addition of 4 ml ice-cold PBS. The resulting suspension was centrifuged at 500 × g for 2 rain and the supernatant removed by suction. In some experiments, the cell suspension was placed in test tubes on ice prior to eentrifugation. The supernatant from this centrifugation was removed for the determination of radioactivity in the medium and radioactivity in the aqueous choline-containing metabolites in the medium.
Isolation of radiolabeled choline-containing metabolites Hepatocytes were ha, vested as described above and to the pelleted cells was added 4 ml C H C 1 3 / C H a O H (2:1, v/v). The suspension was vortexed and 2 ml water was added to separate phases. The aqueous phase was removed and the organic phase was washed with 2 ml CH3OH/0.5% N a C I / C H C I 3 (50: 50: 0.5, v/v). The combined aqueous fractions were evaporated and resuspended in 0.2 ml of water. A 5 0 / d aliquot was applied to a thin-layer plate (silica gel 60) and choline, phosphocholine, glycerophosphoeholine and betaine were separated in a solvent system containing CH3OH/1.2ff~ N a C I / N H 4 O H (50 : 50 : 5, v/v). The organic phase was evaporated under nitrogen gas and resuspended in 0.2 ml CHCI.~/CH3OH (2:1, v/v). A 50-txl aliquot was applied to a thin-layer plate and phosphatidylcholine was separated from other phospholipids in a solvent system containing C H C I 3 / C H 3 O H / C H 3 C O O H / H 2 0 (50:30: 8:3, v/v). The aqueous and organic metabolites were visualized on the plates by exposure to iodine vapor. The silica gel containing compounds of interest were removed from the plate and radioactivity in each sample determined by liquid scintillation counting. In another experiment, cells were incubated with L-[3-~4C]serine for 30 min and subsequently chased for 60 rain in the absence or presence of 0.5 FM okadaic acid and the incorporation of label into fatty acids in the lipid fraction determined [28].
Subcelhdar rractionation and digitonin permeabilization of hepatocytes Hepatocytes were incubated m the absence or presence of 0.5 FM okadaic acid for 120 rain as described
27 above in the absence of [Me-~H]choline. The cells were harvested and homogenized in 2 ml of 0.145 M NaCI, 10 mM Tris-HCI (pH 7.4) (buffer A) with 60 handdriven strokes of a Potter-Elvehjem homogenizer. The homogenate was centifuged at 12000 × g for 10 min and the resulting supernatant centrifuged at 105000 × g for 60 min. The supernatant obtained from this centrifugation was designated the cytosolic fraction. The pellet was resuspended in 5 ml of buffer A and homogenized with 15 strokes of a Dounce B homogenizer. This suspension was centrifuged at 105 000 × g for 60 min. The pellet was resuspended in 0.5 ml 0.25 M sucrose, 10 mM Tris-HC1 (pH 7.4) and homogenized with 15 hand driven strokes of a Dounce A homogenizer and was designated the microsomal fraction. In some experiments, cells were harvested as described and permeabilized with digitonin. Cells were incubated at 4°C for 4 rain with 0.5 ml of i0 mM Tris-HCI (pH 7.4), 0.25 M sucrose, 0.5 mM PMSF, 2.5 mM EDTA, 20 mM NaF and 0,5 m g / m l digitonin. EDTA and NaF were added to the digitonin buffer to inhibit protein kinase and protein phosphatases activities, respectively, which might affect the phosphorylation state of the cytidylyltransferase [29]. The suspension was centrifuged at 500 rpm for 2 rain in a bench top centrifuge and the resulting supernatant quickly removed with a pasteur pipette. The pellet from this centrifugation was resuspended in 0.5 ml of 10 mM Tris-HCI (pH 7.4) 0.145 M NaCI, 1 mM EDTA, 2 mM dithiothreitol, 0.U25% sodium azide and sonicated with a probe sonicator (seven 1 s pulses) and designated the cell ghost membrane fraction. The supernatant was further centrifuged at 99000 rpm for 15 min in a bench top ultracentrifuge to sediment cell ghost membranes that may have been transferred with the supernatant. The resulting supernatant was designated the released cytosol. Microsomes were obtained from rat liver homogenate as described [211.
Enzyme assays and other analyses Choline kinase in the cytosolic fraction was assayed with [Me-3Hlcholine as described [30]. Cholinephosphotransferase in the microsomal fraction was assayed with CDP[Me-14Clcholine as described [311 except that 1,2 diacyl-sn-glycerol was replaced by diolein (18:1, [cis]-9). CTP:phosphocholine cytidylyltransferase in both the cell ghost fraction and released cytosol was assayed with phospho[Me-~H]choline as described [32]. Phosphatidylcholine : oleate vesicles (0.2 mM, 1 : 1 molar ratio) were used as the lipid activator of the inactive cytosolic enzyme. Lactate dehydrogenase activity in the cell culture medium was assayed as described [33] and protein was estimated by the method of Bradford [34]. Students" t-test was used for the determination of significance. The level of significance was defined as P < 0.05 unless noted otherwise.
Rc~,ults
O~uduic acid inhibits PC bio.~Tnthesis in heputoc3"tex Our initial approach was to determine the effect of okadaic acid (1 #M) on PC biosynthesis in monolayer cultured rat hepatocytes plated on either plastic or collagen-coated dishes. Much to our sui'prise the okadaic acid caused the cells to be released from the plates within 15 min. We subsequently used okadaic acid concentrations as low as 10 nM but after 120 min of incubation cell to cell adhesion was reduced and the cells demonstrated an irregular rounded appearance on the plate. These findings suggest that okadaic acid affected cell-cell adhesion and attachment to a substratum. Since suspension cells had been used by other laboratories [35], as an alternative, we incubated suspension cultures of hepatocytes with various concentrations of okadaic acid. We used 0.5 /tM okadaic acid since at this concentration 90e~, of the cells in suspension excluded Trypan blue after 120 rain of incubation. in addition, the cells did not exhibit leakage as judged by assay of lactate dehydrogenase activity in the medium ~0.22 + 002 and 0.23 + 0.01 # m o i / m i n per ml for control and okadaic acid-treated hepatocytes, respectively). It is well documented that inhibition of phosphoprotein phosphatases 1 and 2A by okadaic acid will cause a reduction in fatty acid biosynthesis [35]. As a control. hepatocytes were pulse-labeled with t.-[3-JaClserine for 30 min followed by chase with unlabeled serine for 60 min. Subsequently, the cells were harvested and radioactivity in fatty acids determined. Fatty acid labeling from t.-[3-~Clserine was reduced by approx. 80% in okadaic acid treated hepatocytes. Thus, okadaic acid inhibits phosphoprotein phosphatases 1 and 2A in the isolated rat hepatocytes used in this study. To determine if okadaic acid would influence PC biosynthesis, cells were pulse-labeled with [Me3Hlcholine in the absence and presence of 0.5 ~tM okadaic acid. Subsequently, the cells were harvested and homogenized in CHCI3/CH~OH and an aliquot taken for the determination of total uptake of radioactivity. Total uptake of [Me-3H]choline from 0-30 min was substantially higher than the uptake from 30-120 min (F~g. 2). This was probably due to a rapid equil;hrium between the labeled choline in the medium and the unlabeled choline in the hepatocytes. Total uptake of radiolabeled choline was essentially linear between 30 and 120 rain of continuous pulse in both control and okadaic acid-treated cell suspensions. Total uptake was inhibite~ ~ 15% ( P < 0.05) in the presence of okadaic acid at 30 120 min of pulse compared to control. Thus, okadaic acid appears to affect the uptake of choline into hepatocytes. The homogenate was separated into organic and aqueous phases and radioactivity i.~ phosphocholine in the aqueous phase and PC in the organic phase were determined. Radioactivity in PC increased
1
L 3'0
6'0
9'0
120
(min) Fig. 2. The effect of okadaic acid on the incorporation of [MeSH]choline into hepatocytes. Rat hepatocytes were incubated for 0-120 rain with 10 #Ci [Me-~H]choline in the absence or presence of 0.5 aM okadaic acid. n control; Ill, okadaic acid.treated. Each point represents the mean of three flasks and standard deviation is indicated by bars. The experiment was repeated tw;ce with similar results. Pulse Time
with time in both c o n t r o l a n d o k a d a i c a c i d - t r e a t e d cells (Fig. 3). R a d i o a c t i v i t y in PC w a s d e c r e a s e d b y 46% at 120 rain of t r e a t m e n t with o k a d a i c acid c o m p a r e d to control. R a d i o a c t i v i t y in p h o s p h o c h o l i n e increased f r o m
10
E
0 Pulse hme (mm) Fig. 3. The effect of okadaic acid on the incorporation of [Me3H]choline into phosphatidylcholine and phosphocholine. Rat hepatocytes were incubated for O-120 rain with 10 ~tCi [ Me.3Hlcholine in the ,,bsence or presence of 0.5 pM okadaic acid. Incorporation of the label: D. I1, phosphocholine; o. e, phosphatidylcholine. Open symbol:, are controls and closed symbols okadaic acid-treated. Each pc,mr represents the mean of th:ee flasks and standard deviatio,t is imlicated by bars. The experintent was repeated twice with similar results.
Chase Time (min) Fig. 4. The effect of okadaic acid on the incorporation of [Me~Hlcholine into phosphocholine and phosphatidylcholine. Rat hepatocytes were incubated for 30 rain with 10 txCi [Me-3HIcholine and subsequently chased with choline for 0-120 rain in the absence or presence of 0.5 #M okadaic acid. Incorporation of label: o, O, phosphatidylcholine: D, I , phosphocholine. The open symbols are controls and closed symbols are okadaic acid-treated. Each point represents the mean of three flasks and standard deviation is indicated by bars. The experiment was repeated twice with similar results. 3 0 - 9 0 m i n in b o t h c o n t r o l a n d o k a d a i c acid t r e a t e d cells. R a d i o a c t i v i t y in p h o s p h o c h o l i n e w a s d e c r e a s e d 15% in o k a d a i c a c i d - t r e a t e d cells at 30 rain o f pulse c o m p a r e d to c o n t r o l . Interestingly, there w a s n o c h a n g e in labeled p h o s p h o c h o l i n e b e t w e e n the t w o g r o u p s at 60 a n d 90 m i n b u t at 120 m i n o f pulse a 20% increase ( P < 0.05) in r a d i o a c t i v i t y in p h o s p h o c h o l i n e w a s observed in o k a d a i c a c i d - t r e a t e d cells c o m p a r e d to c o n trol. T h i s c o u l d result if the c o n v e r s i o n of p h o s p h o c h o l i n e to P C w e r e i m p a i r e d in o k a d a i c a c i d - t r e a t e d cells. In the c o n t i n u o u s - p u l s e e x p e r i m e n t s , c h a n g e s in the labeling of phosphocholine and PC may have been due to isotope d i l u t i o n o r altered u p t a k e o f [ M e - 3 H ] c h o l i n e [36]. T h u s , we e m p l o y e d a p u l s e - c h a s e p r o t o c o l to eliminate these possibilities. Cells were pulse-labeled with 10 # C i o f [ M e - 3 H ] e h o l i n e for 30 min a n d subseq u e n t l y c h a s e d with m e d i u m in the a b s e n c e o r p r e s e n c e of 0,5 # M o k a d a i c acid for u p to 120 min. Total cellular r a d i o a c t i v i t y d e c r e a s e d with time o f c h a s e f r o m 7 . 9 . 1 0 6 to 5 . 1 . 1 0 ~ d p m / f l a s k a n d w a s c o m p a r a b l e b e t w e e n c o n t r o l a n d o k a d a i c acid t r e a t e d h e p a t o c y t e s . T h e radioactivity lost f r o m the cells d u r i n g the c h a s e w a s recovered in the m e d i u m (a c o m b i n a t i o n of choline, phosphocholine, betaine and glycerophosphocholine). A n a l y s i s of cellular a q u e o u s c h o l i n e - c o n t a i n i n g m e t a b o lites revealed t h a t r a d i o a c t i v i t y in choline w a s 4 . 5 . 1 0 4 d p m / 1 0 6 cells, r e m a i n e d c o n s t a n t with time o f c h a s e and was comparable between okadaic acid-treated and c o n t r o l h e p a t o c y t e s . R a d i o a c t i v i t y in g l y c e r o p h o s p h o -
choline was decreased from 14.9.104 to 7.0-104 dpm/106 cells until 60 min of chase and remained constant thereafter. In addition, there was no significant difference in the labeling of glycerophosphocholine after treatment with okadaic acid compared to control. Radioactivity in cellular betaine was decreased with chase time from 60.1 - 104 to 3.7 • 1 0 4 dpm/106 cells and was comparable between control and okadaic acid-treated hepatocytes. Radioactivity was lost from phosphocholine and accumulated in PC during the chase (Fig. 4). Radioactivity in the phosphocholine fraction was increased by 86% at 120 min of chase in the okadaic acid-treated hepatocytes compared to control. Radioactivity in PC wa~ decreased by 29"% at 120 min of chase in okadaic acid treated hepatocytes compared to control. The decrease in radioactivity in the PC fraction was quantitatively accounted for in the phosphocholine fraction. Thus, okadaic acid inhibited the conversion of phosphocholine to PC in isolated rat hepatocytes. Okadaic acid promotes C T translocation from membranes to cytosol
The accumulation of radioactivity in the phosphocholine fraction and decrease in radioactivity in the PC fraction in okadaic acid-treated hepatocytes might im-
TABLE I The activities of phosphatidylt~toline biosynthetic en'.ymesfrom isolated rat hepatocytes incubated with 0.5 I~M okadaic acid for 120 mm
Values represent the mean+standard deviation (number of experiments). Enzyme
Choline kinase (cytosol) Cholinephosphotransferase (microsomal) Cytidylyhransferase a (cell ghost) (cytosol h)
Enzymeactivity (nmol/minper mg protein) control okadaic acid 2.05± 0.17(4)
2.32+ 0.18(4)
1.06± 0.29(41
0.92 + 0.17(4)
0.19±0.03(51 1.08± 0.16(5)
C.12+ 0.02(5)" 1.44± 0.30(5) 'j
(nmol/min per flask) Cytidylyhransferane a (cell ghost) (cytosol b)
0.55 +0.11(51 1.61 +0.29(51 e
0.34±0.04(5)~ 2.09+0.29(5)~
Cell ghostsand cytosol were recovered after permeabilization with digitonin. b Assayed in the presence of 0.2 mM phosphatidylcholine:oleate vesicles. ¢ P < 0.005. d P < 0.05. e p < 0.025.
ply that the activity of one or more of the enzymes of the CDP-choline pathway was affected. Thus, the effect of okadaic acid on the activities of the de novo biosynthetic enzymes of the CDP-choline pathway was investigated. Hepatocytes were incubated in the absence or presence of 0.5 .ttM okadaic acid for 120 rain and subsequently the enzyme activities in the microsomal and cytosolic fractions assayed. Cytosolic choline kinase and microsomal cholinephosphotransferase activities were unchanged in okadaic acid treated hepatocytes compared to control (Table 1). In addition, cholinephosphotransferase activities, assayed in the absence of exogenous diacylglyceroL were 0.27 _+0.03 and 0.27 _+ 0.06 nmol/min per mg protein for control and okadaic acid-treated hepatocytes, respectively. The specific activity of microsomal CT was decreased 50% from 0.69 + 0.04, in control hepatocytes, to 0.34 + 0.02 nmol/min per mg protein in the okadaic acid-treated hepatocytes. This was accompanied by a corresponding 31% increase in cytosolic CT activity from 2.26 + 0.09 in control hepatocytes to 2.96 + 0.06 nmol/min per mg protein in okadaic acid-treated hepatocytes. However, we found that our homogenization procedure did not efficiently rupture the hepatocytes completely since many cells were pelleted in the 12000Xg centrifugation. In addition, hepatocytes treated with okadaic acid exhibited a 20% increase in release of protein from the mitochondrial pellet into the post-mitochondrial supernatant. To circumvent these subcellular fractionation problems, hepatocytes were incubated in the absence or presence of 0.5/~M okadaic acid for 120 rain and subsequently incubated with 0.5 m g / m l digitonin solution. Digitonin treatment of cells has been used as an established method of perforating the plasma membranes of suspension cultures of hepatocytes [37,38]. Subsequent to permeabilization and centrifugation the CT activity was assayed in the cell ghost membrane and released cytosol. Cell ghost membrane and released cytosol protein concentrations were comparable between control and okadaic acid-treated hepatocytes (data not shown). Cell ghost membrane CT specific activity was decreased 37% with a concomitant increase in released cytosolic CT activity of 33% in okadaic acid treated hepatocytes compared to control (Table 1). Total CT activity was calculated based upon the activity and volume of cell ghost membrane and released cytosol obtained. Total cell ghost membrane CT activity was decreased 38,% with a corresponding increase in total released cytosol CT activity of 30% in okadaic acid-treated hepatocytes compared to control (Table I). The sum of the total CT activities were 2.16_+0.38 and 2.43 +0.29 nmol/min per flask for control and okadaic acid-treated hepatocytes, respectively. Therefore, the lowered conversion of labeled phosphocholine to PC was consistent with a decrease in membrane CT activity.
30 TABLE 11 Total rtldioutltil'iQ" and inhibition of [Me-~H]phosphocholine #ltlorportltion into phosphatidvlcholine in isolated hepatocTtes incubated in the absence vr presence of okadaic acid or CPT-cA MP or both
Isolated rat hepatocytes were incubated for 30 min with 2 ~Ci of [Me-3H]choline and okadaic acid. CPT-cAMP or both were added to the chase medium. Subsequently.total radioactivity and radioactivity incorporated into phosphatidylcholine after 120 min of chase was determined. Valuesrepresent the mean_+standard deviation (number of experiments). Compoundadded
No atldition (controll Okadaie acid (0.5~ M) CPT-,:AMP (0.5 mM) Okadaic acid (0.5 ,aM) an,] CPT-cAMP (0.5 raM)
T'~,t,dradio~ctivity in cells (dpm.10 5/ flask) 6.35+_0.12(3) 6.47+_0.44(3) 6.34_.+0.47(3) 6.18_+0.20(3)
Radioactivityin phosphatidylcholine (dpm.10 4/106cells)
10.21+-0.41(3) 8.06 _+0.22(3) * 7.68+0.62(3)* 8.59_+0.09(3)*
* P < 0.05.
To determine if okadaic acid directly affected CT activity in microsomal membranes, we incubated rat liver microsomes at 37°C for 2 h in the absence or presence of 0.5 p,M okadaic acid. Microsomal CT activity was 0.63 + 0.04 and 0.60 +__0.06 nmol/min per mg protein for control and okadaic acid-treated microsomes, respectively. Thus, membrane CT activity was unaltered in the presence of okadaic acid. T h e effect o f o k a d a i c acid a n d C P T - c A M P thesis in hepato~ytes
on P C biosyn-
It was previously demonstrated that cAMP analogues inhibit PC biosynthesis in isolated cultured rat hepatocytes [39]. We speculated that cAMP analogues might have an additive effect on the okadaic acid induced inhibition of PC biosynthesis. Cells were pulselabeled with 2 #Ci [Me-3H]choline for 30 min and subsequently chased for 120 min in the absence or presence of 0.5 FM okadaic acid and 0.5 mM CPTcAMP. Incubation with either okadaic acid or CPTcAMP or both did not affect the total cellular radioactivity (Table It). Okadaic acid or CPT-cAMP alone in the incubation medium inhibited phosphatidylcholine biosynthesis in isolated hepatocytes. However, the addition of both okadaic acid and CPT-cAMP in :he incubation medium did not decrease PC biosynthesis further. Discussion We previously demonstated that the time-dependent association of CT activity with the microsomal fraction was inhibited by preincubation of rat liver post-mito-
chondrial supernatant with okadaic acid [21], a potent and specific inhibitor of phosphoprotein phosphatases l and 2A [23,24]. It was not known what, if any, effect cellular phosphoprotein phosphatase activities had on PC biosynthesis in any model system of PC biosynthesis described to date. Thus. the objective of this study was to examine the effect of inhibition of cellular phosphoprotein phosphatase activities on PC biosynthesis in isolated rat hepatocytes. Okadaic acid caused an inhibition of PC biosynthesis in isolated rat hepatocytes. In addition, okadaic acid caused a reduction in fatty acid synthesis which was consistent with the inhibition of phosphoprotein phosphatases 1 and 2A [35]. These findings suggest that the activity of cellular phosphoprotein phosphatases affect the biosynthesis of PC in isolated rat hepatocytes. We were most intrigued to find that okadaic acid, at such remarkably low concentrations ( > 10 nM), inhibited attachment of our isolated hepatocytes to plastic or collagen-coated dishes. Okadaic acid is a polyether derivative of a C-38 long chain fatty acid [40]. Treatment of monolayer cultured hepatocytes with concentrations of C-16 and C-18 long chain fatty acids as high as 4 mM was shown not to affect cell attachment to a substratum for culture periods up to 2 h [10]. Okadaic acid was shown to enhance the contraction of cardiac papillary muscle [41]. These data might suggest that the activity of phosphoprotein phosphatases could be involved in cell adhesion properties by the regulation of contractile elements. Chlorophenylthio-cAMP, a potent activator of cAMP-dependent protein kinase, was demonstrated to inhibit choline uptake in monolayer cultured rat hepatocytes [39]. In the present study, okadaic acid caused an inhibition in choline uptake in isolated rat hepatocytes. These studies might suggest that choline uptake is modulated by a phosphorylation-dephosphorylation mechanism. Greater than 50% of choline taken up by hepatocytes is oxidized to betaine [42]. Clearly, choline oxidation and release of betaine into the medium was not affected by okadaic acid since cellular and medium levels of betaine were unaltered in the presence of okadaic acid. The increased label associated with phosphocholine was not due to an increased conversion of choline to phosphocholine since the labeling of choline and choline kinase activity were unaltered in okadaic acid-treated hepatocytes. Indirect evidence that diacylglycerol might regulate cholinephosphotransferase activity has been documented [43]. The decrease in fatty acid synthesis from serine, observed when cells were treated with okadaic acid, might imply that diacylglycerol availability to cholinephosphotransferase could have been ratelimiting and have affected the labeling of PC during the chase. However, this is unlikely since both endogenous and diacylglyceride-stimulated cholinephosphotrans-
fcrase activities were u n a f f e c t e d by o k a d a i c acid treatm, n[. O k a d a i c acid itself did not directly affect m i c r o s o m a l m e m b r a n e C T activity n o r the activity of purified C T [21]. Thus. the inhibitory effect of o k a d a i c acid o n C T activity was p r o b a b l y m e d i a t e d t h r o u g h an inhibition of p h o s p h o p r o t e i n p h o s p h a t a s e s . O k a d a i c acid has a r a p i d effect on the p h o s p h o r y l a t i o n state of m a n y enzymes of cellular regulation [35]. Thus, it could be a r g u e d that the decrease in PC biosynthesis observed after 30 min of e x p o s u r e to o k a d a i c acid might be a n indirect result of altered cellular m e t a b o l i s m i n d u c e d b y this c o m p o u n d . However, a d d i t i o n of partially purified catalytic subunits of p h o s p h o p r o t e i n p h o s p h a t a s e 1 a n d / o r 2A prom o t e d the association of purified C T with w a s h e d mic r o s o m a l m e m b r a n e s in a c o n c e n t r a t i o n d e p e n d e n t m a n n e r ( H a t c h , G . a n d Vance, D., u n p u b l i s h e d data). T h u s . inhibition of these p h o s p h a t a s e s by o k a d a i c acid m i g h t be expected to affect the subcellular d i s t r i b u t i o n o f C T activities a n d alter PC biosynthesis. Clearly, i n c u b a t i o n o f h e p a t o c y t e s with o k a d a i c acid c a u s e d a decreased association o f C T activity with cell g h o s t m e m b r a n e s a n d a retention o f C T activity in the released cytosol. T h e r e d u c t i o n in PC labeling was, therefore, a result of the lowered C T activity associated with the cell g h o s t m e m b r a n e s . T h e s e d a t a c o u p l e d with o u r previous findings [18.31,38] suggest t h a t PC biosynthesis is r e g u l a t e d b y a m e c h a n i s m w h i c h involves the p h o s p h o r y l a t i o n / d e p h o s p h o r y l a t i o n o f the C T t h r o u g h c A M P - d e p e n d e n t protein k i n a s e a n d cellular phosp h o p r o t e i n p h o s p h a t a s e s 1 a n d / o r 2A. T h e relative i m p o r t a n c e o f these t w o p h o s p h a t a s e s in the r e g u l a t i o n o f C T activity a n d PC biosynthesis is u n k n o w n . O k a d a i c acid in c o m b i n a t i o n with C P T - c A M P inhibited PC b i o s y n t h e s i s t h o u g h n o t to a g r e a t e r extent t h a n either of these c o m p o u n d s individually. P e r h a p s p h o s p h o r y l a t i o n / d e p h o s p h o r y l a t i o n is a f i n e - t u n i n g m e c h a n i s m for c o n t r o l l i n g the cellular level o f PC since o n l y a p o r t i o n o f PC b i o s y n t h e s i s ( 2 0 - 3 0 % ) was inhibited b y o k a d a i c acid a n d C P T - c A M P . Liver cells c a n a f f o r d to decrease fatty acid synthesis m a r k e d l y , w h e n c A M P levels rise, since fatty a c i d s c a n be supplied f r o m a d i p o s e tissue via t r a n s p o r t to the liver b y a l b u m i n . However, e x o g e n o u s s u p p l y o f PC to liver is p r o b a b l y limited to t h a t delivered via lipoproteins. T h u s , a m a j o r decrease in PC b i o s y n t h e s i s m i g h t t h r e a t e n the integrity o f the m e m b r a n e s o f the cell with lethal c o n s e q u e n c e s . A 2 0 - 3 0 % decrease in PC biosynthesis might, however, be a c c e p t a b l e over a limited time p e r i o d d u r i n g e n e r g y d e p r i v a t i o n ( w h e n levels of c A M P increase). It is a p p a r e n t t h a t the level o f PC in the liver is also regulated b y the rate of PC c a t a b o l i s m . W e have evid e n c e t h a t c o n d i t i o n s f a v o r i n g p h o s p h o r y l a t i o n in h e p a t o c y t e s c a u s e the rate of PC c a t a b o l i s m to be enhanced (unpublished data).
Acknowledgements This w o r k w a s s u p p o r t e d by a g r a n t from the M.R.C. of C a n a d a . T h e a u t h o r s a p p r e c i a t e the technical assistance of Drs. H. Jamil a n d A. Merrill a n d helpful discussions of R. S a m b o r s k i a n d Drs. L.B.M. T i j b u r g a n d T. M o g a m i . G . M . H . is a P o s t d o c t o r a l Fellow of the H e a r t a n d Stroke F o u n d a t i o n of C a n a d a D.E.V. is a Medical Scientist of the A l b e r t a Heritage F o u n d a t i o n for Medical Research. References 1 White. I).A. (19731 In Form and Function of Phospholipids. {Ansell, (LB., Hawthorne, J.N. and Dawson, R.M.('., eds.I, pp. 441-482, Elsevier, Amsterdam. 2 Kennedy, E.P. (19891 in Phoshatidylcholine Metabolism (Vance, D.E.. ed.I. pp. 1-8, CRC Press, Boca Raton. 3 Vance. D.E. (19891 in Phosphatidylcholine Metabolism (Vance, D.E., ed.I, pp. 33-46, CRC Press, Boca Raton. 4 Sleight, R. and Kent. C. 11980) J. Biol. Chem. 255, 10644-10650. 5 Weinhold, P.A., FcIdman, I).&., Quad,:, M.M...Metier, J.C and Brooks. R.L. 119811Biochim. Biophys. Acta 665, 134-144. 6 Vance. D.E. and Pelech, S.L. (19841 Trends Bitvchem. Sci. 9, 17-20. 7 Vance, I).E. (19891 in Phosphalidylcholine Metabolism (Vance. D.E.. ed.). pp. 225-240 CRC Press, Boca Raton. 8 Tijburg. L.M.B., Geelen, M.J.H. and Van Golde, L.M.G. 11989) Bit.chim. Biophy~,.Acta. 11104,1-19. 9 Pritchard. P,H., Chiang, P.K., Cantoni, (i.L. amt Vance. D.E. (19831 J. Biol. Chem. 257, 6362-6367. 10 Peh:ch, S.L.. Pritchard. P.H., Brindley, D.N. and Vance, D.E. 119831J. Biol. Chem. 258, 6782-6788. 11 Cnrnell, R. and Vance, D.E. 119871 Biochim. Biophys. At", 919, 26-36. 12 Pelech, S.L., Cook, H.W., Paddon, H.B. and Vance, D.E. 119841 Biochim. Biophys. Acta 795. 433-4411. 13 Weinhold. PH.. Rounsifer, M., William. S., Brubaker, P. and Feldman, P.A. (1984) J. Biol. Chem. 259, 10315-10321. 14 Sleight. R. and Kent, C. 11983l J. Biol. Chem. 258. 824-830. 15 Sleight, R. and Kent, C. 119831J. Biol. Chem. 258. 831-835. 16 Sleight, R. and Kent, C. 11983) 3. Biol. Chem. 258, 836-839. 17 Hatch, (;,M. and Choy, P.C, (19901 Biochem. J. 268, 47-54. 18 Pelech, S,L.. Power, E. and Vance, D.E. (1983l Can. J. Biochent. 61, 1147-1152. 19 Sanghera, J.S. and Vance, D.E. (1989) J. Biol. Chem. 264. 12151223, 211 Tokumitou, S.1. (1984) in Human Alkaline Phosphatases (Stigkraud. T. and Fishman, W.H.. eds.), pp. 189-197, Alan Liss, New York. 21 Hatch, G.M., Lam. T-S., Tsukitani, Y. and Vance. D.E. (19901 Bit;chim. Biophys. Acla. 1042. 374-379. 22 Cohen, P. (1988) Prt~. Royal S~.'. B234, 115-144. 23 Bialojan, C. and Takai. A. 11988) Biochem. J. 256, 283-290. 24 Hcrchlcr, J., Mieskes, G., Ruegg, J.C.. Takai, A. and Trautwein. W. 119881 Pfleugers Arch. Ges. Physiol. 412, 248-252. 25 Paddon, H.B. and Vance. D.E. (1977) Biochim. Biophys. Aeta 448. 181-189. 26 Davis, R.A., Engelhorn, S.C.. Panghurn, S.H.. Weinstein, D.B. and Steinberg. D. (19791 J. Biol. Chem. 254. 2010-2016. 27 Baisted, D.J.. Robinson, B.S. and Vance. D.E. 119881253. 693-701. 28 Slifc. C.W.. Wang, E.. Hunter. R.. Wang. S.. Burgess, C. Llotta, D.C. and Merrill, Jr.. A.H. (19891J. Biol. ('hem. 264,10371-10377.
29 Beg. Z.. Stonik. J.A. and Brewer. H.B. (1980) J. Biol. Chem. 255, 8541-8545. 30 Weinhold. P.A. and Rethy. V.B. (1974) Biochemistry 13. 51355141. 31 Vance. D.E. and Burke. D.C. (1974) Eur. J. Biochem. 43. 327-336. 32 Pelech. S.L. and Vance. D.E. (1982) J. Biol. Chem. 257. 1419814202. 33 Saggerson. E.D. and Greenbaum. A.L. (1969) Biochem. J. 115, 405-417. 34 Bradford. M. (1976) Anal. Biochem. 72, 248-254. 35 Haystead. T.A.J.. Sim. A.T.R.. Cnrling. R.L., Honnor. R.. Tsukitani. Y., Cohen. P. and Hardie. D.G. (1989) Nature 337. 78 81. 36 Whitehead. F.W., Trip. E. and Vance. D.E. (1981) Can. J. Biochem. 59. 38-47.
37 Mackall, J.. Meredith. M. and Lane. M.D. (1979) Anal. Biochem. 95. 270-274. 38 Watkins. P.A, Tarlow. D.M. and Lane, M.D. (1977) Proc. Natl. Acad. Sci. USA 74. 1497-1501. 39 Pelech, S.L. Pritchard. P.H. and Vance. D.E. (1981) J. Biol. Chem. 256, 8283-8286. 40 Tachibana. K. and Scheucer, P.J. (1981) J. Am. Chem. Soc. 103. 2469- 2471. 41 Kodama, I.. Kondo, N. and Shibata, S. (1986) J. Physiol. 378. "~59-373. 42 Pritchard, P.H. and Vance. D.E. (1981) Bioehem. J. 196, 261-267. 43 Lira, P.. Cornell, R. and Vance. D.E. (1986) Biochem. Cell Biol. 64, 692-698.
25
' 1991 ElsevierScience PublishersB.V.(Biomedical Division)0005-2760/91/503.50 ADONIS 000527609100052Y
The protein phosphatase inhibitor, okadaic acid, inhibits phosphatidylcholine biosynthesis in isolated rat hepatocytes G r a n t M. H a t c h ' , Y. T s u k i t a n i ~- a n d D e n n i s E. V a n c e t Lipid and Lipoprotein Research Grol p and Department of Biochemistry, Unwersitv of Alberta Edmonton (Canada) attd " F~muwa Phart~:aceutical Company. Tokyo (Japan)
(Received 30 July 1990) Key words: Phosphatidylcholinebiosynthesis:Okadaic acid: IRat liver): Protein phosphatase There is evidence that pbosphatidylchollae (PC) biosynthesis in hepatueytes is regulated by a phosphoD'lation-deph~phorylation mechanism. The phosphatases involved have not been identified. We, therefore, investigated the effect of okadaic acid, a potent protein phosphatase inhibitor, on PC biosynthesis via the CDP-eheline pathway in suspension cultures of isolated rat hepatocytes. Okadaic acid caused a 15% decrease ( P < 0.05) in IMe-3H]cboline uptake in continuous-pulse labeling experiments. After 120 min of treatment, the labeling of PC was decreased 46% ( P < 0.05) with a corresponding 20'70 increase ( P < 0.05) in labeling of phosphucholine. Cells were pulsed with IMe-3H]choline for 30 min and subsequently chased for up to 120 min with choline in the absence or presence of okadaic acid. The labeling of phosphocholine was increased 86% ( P < 0.05) and labeling of PC decreased 29% ( P < 0.05) by 120 rain of chase in okadale acid-treated hepatucytes. The decrease of label in PC was quantitatively accounted for in the phosphocholine fraction. Incubation of bepatncytes with both okadaic acid and CPT-cAMP did not produce an additive inhibition in labeling of PC. Choline kinase and cholinepbospbotrausferase activities were unaltered by treatment ~ith okadaic acid. Hepatocytes were incubated with digitonin to cause release of cytusolic components. Cell ghost membrane cytidylyltransferase (CT) activity was decreased 37% ( P < 0.005) with a concomitant 33% increase ( P < 0.05) in released eytusolic cytidylyltransferase activity in okadaic acid-treated hepatucytes. We postulate that CT activity and PC biosynthesis are regulated by protein phosphatase activity, in isolated rat hepatucytes.
Introduction
Phosphatidylcholine (PC) is the principal phospholipid in the mammalian liver [1]. The major pathway for PC biosynthesis is the CDP-choline or Kennedy pathway (Fig. 1) [2]. In addition. PC may be synthesized by progressive methylation of phosphatidylethanolamine (Fig. 1). It is well established that CTP : phosphocholine cytidylyltransferase (CT) catalyzes the rate-limiting step of PC biosynthesis via the CDP-choline pathway in rat liver [3] and is located in both the cytosolic anu microsomal fractions from mammalian liver [3]. The mtcrosomal CT is regarded as the active form of the enzyme [3-5] and the cytosolic CT a storage form (Fig. l) [3].
The translocation of the enzyme between these two subcellular compartments represents a plausible mechanism for the regulation of its activity and thus PC biosynthesis [6-8]. Evidence for the translocation of CT has been obtained in rat liver [9] rat hepatocytes [10], Hela cells [11,12]. rat lung [13]. chick embryonic myoCK ChoI ~
)
PChoI
CPT CDP-ChoI'~'-~ PC
T PEMT
er ERlAttice)
Phosphatasels)
Klnasels)
CT-
Abbreviations: DMSO, dimethylsulfoxide; PBS, phosphate-buffered saline; PMSF. phenylmethylsulfonylfluoride, PC, phosphatidylcholine. CT. cytidylyhransferase. Correspondence: D.E. Vance, Lipid and Lipt,protein ResearchGroup, 3rd Floor Heritage Medical Research Center, Facuhy of Medicine, Universityof Alberta. Edmonton,Alberta Canada, T6G 2S2.
Cyto~,l(Inactive) Fig. 1. Postulated regulatory mechanismsfor control of phosphatidylcholine biosynthesis in rat liver. Chol. choline: PChol. phosphocholine; CDP-Chol, CDP-choline; PC, phosphatidylcholine: CK, choline kinase: CT. cytidylyltransfcrase; CPT, cholinep;osphotransferase, PE. phosphatidylethanolamine; PEMT, phosphatidylethanolamine methyltransferase;ER. endoplasmicreticulum.
blasts [14-16], Hamster heart [17] and in the liver of fetal rats [18]. Recently. ~,at liver CT was demonstrated to be phosphorylated on a serine residue(s) by cAMP-dependent protein kinase in vitro [19]. In this same study, incubation of rat liver post-mitochondrial supernatant with exogenous alkaline phosphatase promoted the translocation of CT activity from the cytosolic to the microsomal fraction. However. the subcellular distribution of alkaline phosphatase activity, on the external side of the plasma membrz.ae [20], would preclude the importance of this enzyme in the dephosphory!ation and tran:;Iocation of CT in vivo [21]. This implies that f.here must be intracellular phosphatases involved in the dephosphorylalion and translocation of CT, If CT were indeed regulated by a phosphorylation-dephosphorylation mechanism in vivo (Fig. 1), then the activities of cellular protein phosphatases should influence PC biosynthesis. Type 1 and 2A phosphoprotein phosphatases are the dominant protein phosphatases acting on a wide range of phosphoproteins in vivo which regulate several metabolic pathways and muscle contraction [22]. Okadaic acid is a specific and potent inbihitor of type 1 and 2A cellular phosphoprotein phosphatases [23,24] and was demonstrated to inhibit the time dependent association of cytosolic CT activity with microsomal membranes in rat liver post-mitochondrial supernatant [21]. In this study we provide the first evidence that phospb.oprotein phosphatases I a n d / o r 2A are involved in the regulation of PC biosynthesis in intact rat hepatocytes. Materials and Methods
[Me- 3H]Choline and CDP[ Me-t4C]choline were obtained from Amersham. t.-[3-14C]Serine was obtained from New England Nuclear. Phospho[Me-~H]choline was synthesized enzymatically from [Me-~H]choline [25]. Silica gel 60 plates were obtained from Merck. Albumin was obtained from Sigma :~A-7030. All other biochemicals were of analytical grade and were obtained from either Sigma Chemical Company (St. Louis, MO) or Fisher Scientific (Edmonton. Canada). Okadaic acid was a generous gift of Dr. Y. Tsukitani, Fujisawa Pharmaceutical, Tokyo (Japan). Isolation, incubation and harvesting of hepato(ytes Male Sprague-Dawley rats (175-250 g) were used throughout the study. Rats were maintained on Purina rat chow and tap water, ad libitium, in a temperatureand light-controlled room. Rats were rendered unconcious with diethyl ether and anaesthetized with 2.27 m g / k g phenobarbital. Hepatocytes were isolated by a collagenase-perfusion technique as described [26] and finally suspended in Dulbecco's Modified Eagle's Medium containing 10% delipidated fetal calf serum
[27] (which had been extensively dialyzed against 0.9% NaCl) supplemented with 25~c fatty acid free albumin. The isolated cells excluded 90~ Trypan blue. Cells (4-10~') were shaken, at 100 rpm on a Vibrax Shaker and incubated in 25 ml Erlenmeyer flasks with 2 ml of medium containing [Me-3H]choline for 0-120 rain in continuous-pulse experiments. In pulse-chase experiments cells were incubated for 30 rain with [Me3H]choline prior to chase with unlabeled choline. Okadaic acid was dissolved in 10% DMSO with final concentration in the medium of 0.5 p.M okadaic acid and 0.125% DMSO. Control hepatocytes were incubated with 0.125% DMSO. Subsequent to continuous-pulse or pulse-chase experiments, cells were harvested by the addition of 4 ml ice-cold PBS. The resulting suspension was centrifuged at 500 × g for 2 rain and the supernatant removed by suction. In some experiments, the cell suspension was placed in test tubes on ice prior to eentrifugation. The supernatant from this centrifugation was removed for the determination of radioactivity in the medium and radioactivity in the aqueous choline-containing metabolites in the medium.
Isolation of radiolabeled choline-containing metabolites Hepatocytes were ha, vested as described above and to the pelleted cells was added 4 ml C H C 1 3 / C H a O H (2:1, v/v). The suspension was vortexed and 2 ml water was added to separate phases. The aqueous phase was removed and the organic phase was washed with 2 ml CH3OH/0.5% N a C I / C H C I 3 (50: 50: 0.5, v/v). The combined aqueous fractions were evaporated and resuspended in 0.2 ml of water. A 5 0 / d aliquot was applied to a thin-layer plate (silica gel 60) and choline, phosphocholine, glycerophosphoeholine and betaine were separated in a solvent system containing CH3OH/1.2ff~ N a C I / N H 4 O H (50 : 50 : 5, v/v). The organic phase was evaporated under nitrogen gas and resuspended in 0.2 ml CHCI.~/CH3OH (2:1, v/v). A 50-txl aliquot was applied to a thin-layer plate and phosphatidylcholine was separated from other phospholipids in a solvent system containing C H C I 3 / C H 3 O H / C H 3 C O O H / H 2 0 (50:30: 8:3, v/v). The aqueous and organic metabolites were visualized on the plates by exposure to iodine vapor. The silica gel containing compounds of interest were removed from the plate and radioactivity in each sample determined by liquid scintillation counting. In another experiment, cells were incubated with L-[3-~4C]serine for 30 min and subsequently chased for 60 rain in the absence or presence of 0.5 FM okadaic acid and the incorporation of label into fatty acids in the lipid fraction determined [28].
Subcelhdar rractionation and digitonin permeabilization of hepatocytes Hepatocytes were incubated m the absence or presence of 0.5 FM okadaic acid for 120 rain as described
27 above in the absence of [Me-~H]choline. The cells were harvested and homogenized in 2 ml of 0.145 M NaCI, 10 mM Tris-HCI (pH 7.4) (buffer A) with 60 handdriven strokes of a Potter-Elvehjem homogenizer. The homogenate was centifuged at 12000 × g for 10 min and the resulting supernatant centrifuged at 105000 × g for 60 min. The supernatant obtained from this centrifugation was designated the cytosolic fraction. The pellet was resuspended in 5 ml of buffer A and homogenized with 15 strokes of a Dounce B homogenizer. This suspension was centrifuged at 105 000 × g for 60 min. The pellet was resuspended in 0.5 ml 0.25 M sucrose, 10 mM Tris-HC1 (pH 7.4) and homogenized with 15 hand driven strokes of a Dounce A homogenizer and was designated the microsomal fraction. In some experiments, cells were harvested as described and permeabilized with digitonin. Cells were incubated at 4°C for 4 rain with 0.5 ml of i0 mM Tris-HCI (pH 7.4), 0.25 M sucrose, 0.5 mM PMSF, 2.5 mM EDTA, 20 mM NaF and 0,5 m g / m l digitonin. EDTA and NaF were added to the digitonin buffer to inhibit protein kinase and protein phosphatases activities, respectively, which might affect the phosphorylation state of the cytidylyltransferase [29]. The suspension was centrifuged at 500 rpm for 2 rain in a bench top centrifuge and the resulting supernatant quickly removed with a pasteur pipette. The pellet from this centrifugation was resuspended in 0.5 ml of 10 mM Tris-HCI (pH 7.4) 0.145 M NaCI, 1 mM EDTA, 2 mM dithiothreitol, 0.U25% sodium azide and sonicated with a probe sonicator (seven 1 s pulses) and designated the cell ghost membrane fraction. The supernatant was further centrifuged at 99000 rpm for 15 min in a bench top ultracentrifuge to sediment cell ghost membranes that may have been transferred with the supernatant. The resulting supernatant was designated the released cytosol. Microsomes were obtained from rat liver homogenate as described [211.
Enzyme assays and other analyses Choline kinase in the cytosolic fraction was assayed with [Me-3Hlcholine as described [30]. Cholinephosphotransferase in the microsomal fraction was assayed with CDP[Me-14Clcholine as described [311 except that 1,2 diacyl-sn-glycerol was replaced by diolein (18:1, [cis]-9). CTP:phosphocholine cytidylyltransferase in both the cell ghost fraction and released cytosol was assayed with phospho[Me-~H]choline as described [32]. Phosphatidylcholine : oleate vesicles (0.2 mM, 1 : 1 molar ratio) were used as the lipid activator of the inactive cytosolic enzyme. Lactate dehydrogenase activity in the cell culture medium was assayed as described [33] and protein was estimated by the method of Bradford [34]. Students" t-test was used for the determination of significance. The level of significance was defined as P < 0.05 unless noted otherwise.
Rc~,ults
O~uduic acid inhibits PC bio.~Tnthesis in heputoc3"tex Our initial approach was to determine the effect of okadaic acid (1 #M) on PC biosynthesis in monolayer cultured rat hepatocytes plated on either plastic or collagen-coated dishes. Much to our sui'prise the okadaic acid caused the cells to be released from the plates within 15 min. We subsequently used okadaic acid concentrations as low as 10 nM but after 120 min of incubation cell to cell adhesion was reduced and the cells demonstrated an irregular rounded appearance on the plate. These findings suggest that okadaic acid affected cell-cell adhesion and attachment to a substratum. Since suspension cells had been used by other laboratories [35], as an alternative, we incubated suspension cultures of hepatocytes with various concentrations of okadaic acid. We used 0.5 /tM okadaic acid since at this concentration 90e~, of the cells in suspension excluded Trypan blue after 120 rain of incubation. in addition, the cells did not exhibit leakage as judged by assay of lactate dehydrogenase activity in the medium ~0.22 + 002 and 0.23 + 0.01 # m o i / m i n per ml for control and okadaic acid-treated hepatocytes, respectively). It is well documented that inhibition of phosphoprotein phosphatases 1 and 2A by okadaic acid will cause a reduction in fatty acid biosynthesis [35]. As a control. hepatocytes were pulse-labeled with t.-[3-JaClserine for 30 min followed by chase with unlabeled serine for 60 min. Subsequently, the cells were harvested and radioactivity in fatty acids determined. Fatty acid labeling from t.-[3-~Clserine was reduced by approx. 80% in okadaic acid treated hepatocytes. Thus, okadaic acid inhibits phosphoprotein phosphatases 1 and 2A in the isolated rat hepatocytes used in this study. To determine if okadaic acid would influence PC biosynthesis, cells were pulse-labeled with [Me3Hlcholine in the absence and presence of 0.5 ~tM okadaic acid. Subsequently, the cells were harvested and homogenized in CHCI3/CH~OH and an aliquot taken for the determination of total uptake of radioactivity. Total uptake of [Me-3H]choline from 0-30 min was substantially higher than the uptake from 30-120 min (F~g. 2). This was probably due to a rapid equil;hrium between the labeled choline in the medium and the unlabeled choline in the hepatocytes. Total uptake of radiolabeled choline was essentially linear between 30 and 120 rain of continuous pulse in both control and okadaic acid-treated cell suspensions. Total uptake was inhibite~ ~ 15% ( P < 0.05) in the presence of okadaic acid at 30 120 min of pulse compared to control. Thus, okadaic acid appears to affect the uptake of choline into hepatocytes. The homogenate was separated into organic and aqueous phases and radioactivity i.~ phosphocholine in the aqueous phase and PC in the organic phase were determined. Radioactivity in PC increased
1
L 3'0
6'0
9'0
120
(min) Fig. 2. The effect of okadaic acid on the incorporation of [MeSH]choline into hepatocytes. Rat hepatocytes were incubated for 0-120 rain with 10 #Ci [Me-~H]choline in the absence or presence of 0.5 aM okadaic acid. n control; Ill, okadaic acid.treated. Each point represents the mean of three flasks and standard deviation is indicated by bars. The experiment was repeated tw;ce with similar results. Pulse Time
with time in both c o n t r o l a n d o k a d a i c a c i d - t r e a t e d cells (Fig. 3). R a d i o a c t i v i t y in PC w a s d e c r e a s e d b y 46% at 120 rain of t r e a t m e n t with o k a d a i c acid c o m p a r e d to control. R a d i o a c t i v i t y in p h o s p h o c h o l i n e increased f r o m
10
E
0 Pulse hme (mm) Fig. 3. The effect of okadaic acid on the incorporation of [Me3H]choline into phosphatidylcholine and phosphocholine. Rat hepatocytes were incubated for O-120 rain with 10 ~tCi [ Me.3Hlcholine in the ,,bsence or presence of 0.5 pM okadaic acid. Incorporation of the label: D. I1, phosphocholine; o. e, phosphatidylcholine. Open symbol:, are controls and closed symbols okadaic acid-treated. Each pc,mr represents the mean of th:ee flasks and standard deviatio,t is imlicated by bars. The experintent was repeated twice with similar results.
Chase Time (min) Fig. 4. The effect of okadaic acid on the incorporation of [Me~Hlcholine into phosphocholine and phosphatidylcholine. Rat hepatocytes were incubated for 30 rain with 10 txCi [Me-3HIcholine and subsequently chased with choline for 0-120 rain in the absence or presence of 0.5 #M okadaic acid. Incorporation of label: o, O, phosphatidylcholine: D, I , phosphocholine. The open symbols are controls and closed symbols are okadaic acid-treated. Each point represents the mean of three flasks and standard deviation is indicated by bars. The experiment was repeated twice with similar results. 3 0 - 9 0 m i n in b o t h c o n t r o l a n d o k a d a i c acid t r e a t e d cells. R a d i o a c t i v i t y in p h o s p h o c h o l i n e w a s d e c r e a s e d 15% in o k a d a i c a c i d - t r e a t e d cells at 30 rain o f pulse c o m p a r e d to c o n t r o l . Interestingly, there w a s n o c h a n g e in labeled p h o s p h o c h o l i n e b e t w e e n the t w o g r o u p s at 60 a n d 90 m i n b u t at 120 m i n o f pulse a 20% increase ( P < 0.05) in r a d i o a c t i v i t y in p h o s p h o c h o l i n e w a s observed in o k a d a i c a c i d - t r e a t e d cells c o m p a r e d to c o n trol. T h i s c o u l d result if the c o n v e r s i o n of p h o s p h o c h o l i n e to P C w e r e i m p a i r e d in o k a d a i c a c i d - t r e a t e d cells. In the c o n t i n u o u s - p u l s e e x p e r i m e n t s , c h a n g e s in the labeling of phosphocholine and PC may have been due to isotope d i l u t i o n o r altered u p t a k e o f [ M e - 3 H ] c h o l i n e [36]. T h u s , we e m p l o y e d a p u l s e - c h a s e p r o t o c o l to eliminate these possibilities. Cells were pulse-labeled with 10 # C i o f [ M e - 3 H ] e h o l i n e for 30 min a n d subseq u e n t l y c h a s e d with m e d i u m in the a b s e n c e o r p r e s e n c e of 0,5 # M o k a d a i c acid for u p to 120 min. Total cellular r a d i o a c t i v i t y d e c r e a s e d with time o f c h a s e f r o m 7 . 9 . 1 0 6 to 5 . 1 . 1 0 ~ d p m / f l a s k a n d w a s c o m p a r a b l e b e t w e e n c o n t r o l a n d o k a d a i c acid t r e a t e d h e p a t o c y t e s . T h e radioactivity lost f r o m the cells d u r i n g the c h a s e w a s recovered in the m e d i u m (a c o m b i n a t i o n of choline, phosphocholine, betaine and glycerophosphocholine). A n a l y s i s of cellular a q u e o u s c h o l i n e - c o n t a i n i n g m e t a b o lites revealed t h a t r a d i o a c t i v i t y in choline w a s 4 . 5 . 1 0 4 d p m / 1 0 6 cells, r e m a i n e d c o n s t a n t with time o f c h a s e and was comparable between okadaic acid-treated and c o n t r o l h e p a t o c y t e s . R a d i o a c t i v i t y in g l y c e r o p h o s p h o -
choline was decreased from 14.9.104 to 7.0-104 dpm/106 cells until 60 min of chase and remained constant thereafter. In addition, there was no significant difference in the labeling of glycerophosphocholine after treatment with okadaic acid compared to control. Radioactivity in cellular betaine was decreased with chase time from 60.1 - 104 to 3.7 • 1 0 4 dpm/106 cells and was comparable between control and okadaic acid-treated hepatocytes. Radioactivity was lost from phosphocholine and accumulated in PC during the chase (Fig. 4). Radioactivity in the phosphocholine fraction was increased by 86% at 120 min of chase in the okadaic acid-treated hepatocytes compared to control. Radioactivity in PC wa~ decreased by 29"% at 120 min of chase in okadaic acid treated hepatocytes compared to control. The decrease in radioactivity in the PC fraction was quantitatively accounted for in the phosphocholine fraction. Thus, okadaic acid inhibited the conversion of phosphocholine to PC in isolated rat hepatocytes. Okadaic acid promotes C T translocation from membranes to cytosol
The accumulation of radioactivity in the phosphocholine fraction and decrease in radioactivity in the PC fraction in okadaic acid-treated hepatocytes might im-
TABLE I The activities of phosphatidylt~toline biosynthetic en'.ymesfrom isolated rat hepatocytes incubated with 0.5 I~M okadaic acid for 120 mm
Values represent the mean+standard deviation (number of experiments). Enzyme
Choline kinase (cytosol) Cholinephosphotransferase (microsomal) Cytidylyhransferase a (cell ghost) (cytosol h)
Enzymeactivity (nmol/minper mg protein) control okadaic acid 2.05± 0.17(4)
2.32+ 0.18(4)
1.06± 0.29(41
0.92 + 0.17(4)
0.19±0.03(51 1.08± 0.16(5)
C.12+ 0.02(5)" 1.44± 0.30(5) 'j
(nmol/min per flask) Cytidylyhransferane a (cell ghost) (cytosol b)
0.55 +0.11(51 1.61 +0.29(51 e
0.34±0.04(5)~ 2.09+0.29(5)~
Cell ghostsand cytosol were recovered after permeabilization with digitonin. b Assayed in the presence of 0.2 mM phosphatidylcholine:oleate vesicles. ¢ P < 0.005. d P < 0.05. e p < 0.025.
ply that the activity of one or more of the enzymes of the CDP-choline pathway was affected. Thus, the effect of okadaic acid on the activities of the de novo biosynthetic enzymes of the CDP-choline pathway was investigated. Hepatocytes were incubated in the absence or presence of 0.5 .ttM okadaic acid for 120 rain and subsequently the enzyme activities in the microsomal and cytosolic fractions assayed. Cytosolic choline kinase and microsomal cholinephosphotransferase activities were unchanged in okadaic acid treated hepatocytes compared to control (Table 1). In addition, cholinephosphotransferase activities, assayed in the absence of exogenous diacylglyceroL were 0.27 _+0.03 and 0.27 _+ 0.06 nmol/min per mg protein for control and okadaic acid-treated hepatocytes, respectively. The specific activity of microsomal CT was decreased 50% from 0.69 + 0.04, in control hepatocytes, to 0.34 + 0.02 nmol/min per mg protein in the okadaic acid-treated hepatocytes. This was accompanied by a corresponding 31% increase in cytosolic CT activity from 2.26 + 0.09 in control hepatocytes to 2.96 + 0.06 nmol/min per mg protein in okadaic acid-treated hepatocytes. However, we found that our homogenization procedure did not efficiently rupture the hepatocytes completely since many cells were pelleted in the 12000Xg centrifugation. In addition, hepatocytes treated with okadaic acid exhibited a 20% increase in release of protein from the mitochondrial pellet into the post-mitochondrial supernatant. To circumvent these subcellular fractionation problems, hepatocytes were incubated in the absence or presence of 0.5/~M okadaic acid for 120 rain and subsequently incubated with 0.5 m g / m l digitonin solution. Digitonin treatment of cells has been used as an established method of perforating the plasma membranes of suspension cultures of hepatocytes [37,38]. Subsequent to permeabilization and centrifugation the CT activity was assayed in the cell ghost membrane and released cytosol. Cell ghost membrane and released cytosol protein concentrations were comparable between control and okadaic acid-treated hepatocytes (data not shown). Cell ghost membrane CT specific activity was decreased 37% with a concomitant increase in released cytosolic CT activity of 33% in okadaic acid treated hepatocytes compared to control (Table 1). Total CT activity was calculated based upon the activity and volume of cell ghost membrane and released cytosol obtained. Total cell ghost membrane CT activity was decreased 38,% with a corresponding increase in total released cytosol CT activity of 30% in okadaic acid-treated hepatocytes compared to control (Table I). The sum of the total CT activities were 2.16_+0.38 and 2.43 +0.29 nmol/min per flask for control and okadaic acid-treated hepatocytes, respectively. Therefore, the lowered conversion of labeled phosphocholine to PC was consistent with a decrease in membrane CT activity.
30 TABLE 11 Total rtldioutltil'iQ" and inhibition of [Me-~H]phosphocholine #ltlorportltion into phosphatidvlcholine in isolated hepatocTtes incubated in the absence vr presence of okadaic acid or CPT-cA MP or both
Isolated rat hepatocytes were incubated for 30 min with 2 ~Ci of [Me-3H]choline and okadaic acid. CPT-cAMP or both were added to the chase medium. Subsequently.total radioactivity and radioactivity incorporated into phosphatidylcholine after 120 min of chase was determined. Valuesrepresent the mean_+standard deviation (number of experiments). Compoundadded
No atldition (controll Okadaie acid (0.5~ M) CPT-,:AMP (0.5 mM) Okadaic acid (0.5 ,aM) an,] CPT-cAMP (0.5 raM)
T'~,t,dradio~ctivity in cells (dpm.10 5/ flask) 6.35+_0.12(3) 6.47+_0.44(3) 6.34_.+0.47(3) 6.18_+0.20(3)
Radioactivityin phosphatidylcholine (dpm.10 4/106cells)
10.21+-0.41(3) 8.06 _+0.22(3) * 7.68+0.62(3)* 8.59_+0.09(3)*
* P < 0.05.
To determine if okadaic acid directly affected CT activity in microsomal membranes, we incubated rat liver microsomes at 37°C for 2 h in the absence or presence of 0.5 p,M okadaic acid. Microsomal CT activity was 0.63 + 0.04 and 0.60 +__0.06 nmol/min per mg protein for control and okadaic acid-treated microsomes, respectively. Thus, membrane CT activity was unaltered in the presence of okadaic acid. T h e effect o f o k a d a i c acid a n d C P T - c A M P thesis in hepato~ytes
on P C biosyn-
It was previously demonstrated that cAMP analogues inhibit PC biosynthesis in isolated cultured rat hepatocytes [39]. We speculated that cAMP analogues might have an additive effect on the okadaic acid induced inhibition of PC biosynthesis. Cells were pulselabeled with 2 #Ci [Me-3H]choline for 30 min and subsequently chased for 120 min in the absence or presence of 0.5 FM okadaic acid and 0.5 mM CPTcAMP. Incubation with either okadaic acid or CPTcAMP or both did not affect the total cellular radioactivity (Table It). Okadaic acid or CPT-cAMP alone in the incubation medium inhibited phosphatidylcholine biosynthesis in isolated hepatocytes. However, the addition of both okadaic acid and CPT-cAMP in :he incubation medium did not decrease PC biosynthesis further. Discussion We previously demonstated that the time-dependent association of CT activity with the microsomal fraction was inhibited by preincubation of rat liver post-mito-
chondrial supernatant with okadaic acid [21], a potent and specific inhibitor of phosphoprotein phosphatases l and 2A [23,24]. It was not known what, if any, effect cellular phosphoprotein phosphatase activities had on PC biosynthesis in any model system of PC biosynthesis described to date. Thus. the objective of this study was to examine the effect of inhibition of cellular phosphoprotein phosphatase activities on PC biosynthesis in isolated rat hepatocytes. Okadaic acid caused an inhibition of PC biosynthesis in isolated rat hepatocytes. In addition, okadaic acid caused a reduction in fatty acid synthesis which was consistent with the inhibition of phosphoprotein phosphatases 1 and 2A [35]. These findings suggest that the activity of cellular phosphoprotein phosphatases affect the biosynthesis of PC in isolated rat hepatocytes. We were most intrigued to find that okadaic acid, at such remarkably low concentrations ( > 10 nM), inhibited attachment of our isolated hepatocytes to plastic or collagen-coated dishes. Okadaic acid is a polyether derivative of a C-38 long chain fatty acid [40]. Treatment of monolayer cultured hepatocytes with concentrations of C-16 and C-18 long chain fatty acids as high as 4 mM was shown not to affect cell attachment to a substratum for culture periods up to 2 h [10]. Okadaic acid was shown to enhance the contraction of cardiac papillary muscle [41]. These data might suggest that the activity of phosphoprotein phosphatases could be involved in cell adhesion properties by the regulation of contractile elements. Chlorophenylthio-cAMP, a potent activator of cAMP-dependent protein kinase, was demonstrated to inhibit choline uptake in monolayer cultured rat hepatocytes [39]. In the present study, okadaic acid caused an inhibition in choline uptake in isolated rat hepatocytes. These studies might suggest that choline uptake is modulated by a phosphorylation-dephosphorylation mechanism. Greater than 50% of choline taken up by hepatocytes is oxidized to betaine [42]. Clearly, choline oxidation and release of betaine into the medium was not affected by okadaic acid since cellular and medium levels of betaine were unaltered in the presence of okadaic acid. The increased label associated with phosphocholine was not due to an increased conversion of choline to phosphocholine since the labeling of choline and choline kinase activity were unaltered in okadaic acid-treated hepatocytes. Indirect evidence that diacylglycerol might regulate cholinephosphotransferase activity has been documented [43]. The decrease in fatty acid synthesis from serine, observed when cells were treated with okadaic acid, might imply that diacylglycerol availability to cholinephosphotransferase could have been ratelimiting and have affected the labeling of PC during the chase. However, this is unlikely since both endogenous and diacylglyceride-stimulated cholinephosphotrans-
fcrase activities were u n a f f e c t e d by o k a d a i c acid treatm, n[. O k a d a i c acid itself did not directly affect m i c r o s o m a l m e m b r a n e C T activity n o r the activity of purified C T [21]. Thus. the inhibitory effect of o k a d a i c acid o n C T activity was p r o b a b l y m e d i a t e d t h r o u g h an inhibition of p h o s p h o p r o t e i n p h o s p h a t a s e s . O k a d a i c acid has a r a p i d effect on the p h o s p h o r y l a t i o n state of m a n y enzymes of cellular regulation [35]. Thus, it could be a r g u e d that the decrease in PC biosynthesis observed after 30 min of e x p o s u r e to o k a d a i c acid might be a n indirect result of altered cellular m e t a b o l i s m i n d u c e d b y this c o m p o u n d . However, a d d i t i o n of partially purified catalytic subunits of p h o s p h o p r o t e i n p h o s p h a t a s e 1 a n d / o r 2A prom o t e d the association of purified C T with w a s h e d mic r o s o m a l m e m b r a n e s in a c o n c e n t r a t i o n d e p e n d e n t m a n n e r ( H a t c h , G . a n d Vance, D., u n p u b l i s h e d data). T h u s . inhibition of these p h o s p h a t a s e s by o k a d a i c acid m i g h t be expected to affect the subcellular d i s t r i b u t i o n o f C T activities a n d alter PC biosynthesis. Clearly, i n c u b a t i o n o f h e p a t o c y t e s with o k a d a i c acid c a u s e d a decreased association o f C T activity with cell g h o s t m e m b r a n e s a n d a retention o f C T activity in the released cytosol. T h e r e d u c t i o n in PC labeling was, therefore, a result of the lowered C T activity associated with the cell g h o s t m e m b r a n e s . T h e s e d a t a c o u p l e d with o u r previous findings [18.31,38] suggest t h a t PC biosynthesis is r e g u l a t e d b y a m e c h a n i s m w h i c h involves the p h o s p h o r y l a t i o n / d e p h o s p h o r y l a t i o n o f the C T t h r o u g h c A M P - d e p e n d e n t protein k i n a s e a n d cellular phosp h o p r o t e i n p h o s p h a t a s e s 1 a n d / o r 2A. T h e relative i m p o r t a n c e o f these t w o p h o s p h a t a s e s in the r e g u l a t i o n o f C T activity a n d PC biosynthesis is u n k n o w n . O k a d a i c acid in c o m b i n a t i o n with C P T - c A M P inhibited PC b i o s y n t h e s i s t h o u g h n o t to a g r e a t e r extent t h a n either of these c o m p o u n d s individually. P e r h a p s p h o s p h o r y l a t i o n / d e p h o s p h o r y l a t i o n is a f i n e - t u n i n g m e c h a n i s m for c o n t r o l l i n g the cellular level o f PC since o n l y a p o r t i o n o f PC b i o s y n t h e s i s ( 2 0 - 3 0 % ) was inhibited b y o k a d a i c acid a n d C P T - c A M P . Liver cells c a n a f f o r d to decrease fatty acid synthesis m a r k e d l y , w h e n c A M P levels rise, since fatty a c i d s c a n be supplied f r o m a d i p o s e tissue via t r a n s p o r t to the liver b y a l b u m i n . However, e x o g e n o u s s u p p l y o f PC to liver is p r o b a b l y limited to t h a t delivered via lipoproteins. T h u s , a m a j o r decrease in PC b i o s y n t h e s i s m i g h t t h r e a t e n the integrity o f the m e m b r a n e s o f the cell with lethal c o n s e q u e n c e s . A 2 0 - 3 0 % decrease in PC biosynthesis might, however, be a c c e p t a b l e over a limited time p e r i o d d u r i n g e n e r g y d e p r i v a t i o n ( w h e n levels of c A M P increase). It is a p p a r e n t t h a t the level o f PC in the liver is also regulated b y the rate of PC c a t a b o l i s m . W e have evid e n c e t h a t c o n d i t i o n s f a v o r i n g p h o s p h o r y l a t i o n in h e p a t o c y t e s c a u s e the rate of PC c a t a b o l i s m to be enhanced (unpublished data).
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