the enhancement of phosphatidylcholine biosynthesis by angiotensin ii in h9c2 cells

ELSEVIER Biochimica et Biophysica Acta 1259 (1995) 283-290

Biochi~ic~a et Biophysica A~ta

The enhancement of phosphatidylcholine biosynthesis by angiotensin II in H9c2 cells

Khai Tran a, Ricky Y.K. Man b Patrick C. Choy a,* Department of Biochemisto' and Molecular Biology, Universi~" of Manitoba, Winnipeg, Manitoba R3E OW3, Canada

b Department of Pharmacology, University of Manitoba Winnipeg, Manitoba R3E OW3, Canada

Received 27 January 1995; revised 18 July 1995; accepted 3 August 1995

Abstract

The effect of angiotensin II on the biosynthesis of phosphatidylcholine in rat heart myoblastic (H9c2) cells was investigated. Cells were incubated with [methyl-3H]choline, and the labelling of phosphatidylcholine at different time intervals was examined. When cells were pretreated with angiotensin II, a significant increase in the labelling of phosphatidylcholine was observed. Analysis of the labelled phosphatidylcholine precursors indicated that the conversion of phosphocholine to CDP-choline was enhanced by angiotensin II treatment. Determination of enzyme activities in the CDP-choline pathway revealed that the activities of choline kinase or CDP-choline: diacylglycerol cholinephosphotransferase were not changed, but the activities of CTP:phosphocholine cytidylyltransferase were stimulated in both the particulate and soluble fractions. The stimulation of the cytidylyltransferase by angiotensin II was not abolished by okadaic acid, indicating that the activation of the enzyme was not mediated via the okadaic-sensitive dephosphorylation mechanism. Alternatively, the stimulation of the cytidylyltransferase activity was completely abolished by protein kinase C inhibitors. Immunoblotting studies revealed that levels of the cytidylyltransferase in the soluble and particulate fractions were not affected by angiotensin II treatment. We conclude that the increase in phosphatidylcholine biosynthesis by angiotensin II was a direct result of the enhancement of the cytidylyltransferase activity. The enhancement of enzyme activity was not mediated via enzyme translocation, but by a mechanism which was intimately associated with the protein kinase C cascade.

Keywords: Angiotensin; Phosphocholine cytidylyltransferase; Phosphatidylcholine; Biosynthesis; Cardiac cell

1. Introduction

Angiotensin II is a hormone and growth factor produced by the renin-angiotensin system in the kidney and other tissues [1]. In the heart, angiotensin II plays a vital role in the production of cardiac hypertrophy which is linked to the development of hypertension and congestive heart failure [2,3]. The hypertrophic effect of angiotensin II is manifested by its ability to stimulate protein synthesis and growth in vascular smooth muscle and heart cells [4,5]. Recently, angiotensin II has been shown to induce the expression of some early growth signals such as c-fos, c-myc and c-jun proto-oncogenes [6]. Angiotensin II also stimulates the phosphorylation of different proteins includ-

* Corresponding author. Fax: + 1 (204) 7830864.

0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 5 ) 0 0 1 75- 1

ing the initiation factor 4E [7], phospholipase C-y 1 [8] and the high molecular mass phospholipase A 2 [9]. These effects are mediated by the interaction of angiotensin II to its specific receptor subtypes on the cell membrane, with the subsequent generation of several lipid second messen- gers [10]. Specifically, angiotensin II causes the activation of various signal transduction processes [11], including phospholipase A 2 [9,12], phospoliipase C [8,12] and phospholipase D [13]. The hydrolysis of phosphatidylcholine by phospholipase D [14,15] is regarded as a mechanism to provide the cell with sustained levels of second messen- gers such as phosphatidic acid and diacylglycerol subsequent to agonist stimulation [ 16,17].

Although the angiotensin II-induced phosphatidylcholine hydrolysis has been studied extensively, its effect on the control of phosphatidylcholine biosynthesis is less clear. Since the cellular level of phosphatidylcholine does not normally change, the rates of its degradation and

284 K. Tran et al. / Biochimica et Biophysica Acta 1259 (1995) 283-290

synthesis should be well-coordinated [18]. The coordina- tion of the hydrolysis and biosynthesis of phosphatidylcholine is exemplified by the fact that the utilization of diacylglycerol for phosphatidylcholine formation appears to be an important mechanism for the termination diacylglycerol-induced cell signalling [19]. In cardiac cells, the principle pathway for phosphatidylcholine biosynthesis is the CDP-choline pathway. In this pathway, the choline taken up by the cell is rapidly phosphorylated to phosphocholine and then converts to CDP-choline by the action of the CTP:phosphocholine cytidylyltransferase. The CDP- choline formed condenses with diacylglycerol for the production of phosphatidylcholine. We have shown earlier that the rate-limiting step of this pathway is at the conversion of phosphocholine to CDP-choline catalyzed by the cytidylyltransferase [20]. This enzyme is located in both cytosolic and microsomal fractions, and the modulation of its activity is generally accepted as an important mechanism for the regulation of phosphatidylcholine biosynthesis in mammalian cells and tissues.

In the present study, the role of angiotensin II on phosphatidylcholine biosynthesis in cultured myoblastic (H9c2) cells was examined. Specifically, the study was undertaken to determine (1) the effect of angiotensin II on the uptake of [methyl-3H] choline and the subsequent biosynthesis of phosphatidylcholine, and (2) the mechanism involved in this process.

2. Materials and methods

2.1. Materials

[methyl-3H]Choline was obtained from the New Eng- land Nuclear Division of Dupont (Mississauga, Ontario, Canada). Cytidine 5'-diphospho[methyl-~4C]choline was purchased from Amersham Canada Ltd. (Oakville, On- tario, Canada). Phosphoryl[methyl-3H]choline was prepared from [methyl-3H]choline with yeast choline kinase as previously described [21]. Angiotensin II, phorbol 12- myristate 13-acetate, l-(5-isoquinolinyl-sulfonyl)-2- methylpiperazine (H7), cell-cultured media and reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Okadaic acid was obtained from LC Laboratories (Woburn, MA, USA). Ro31-8220, a selective inhibitor of protein kinase C, was a generous gift of Dr. G. Lawton, Roche Products Ltd. (Welwyn Garden City, Hertfordshire, UK). Rabbit sera containing anti-cytidylyltransferase antibodies was a gift of Dr. D. Vance (University of Alberta, Canada). All lipid standards were obtained from Serdary Research Laboratory (London, Ontario, Canada). Thin layer chromatographic plates (silica gel G) were products of Fisher Scientific (Edmonton, Alberta, Canada). All other chemicals and solvents (reagent or HPLC grade) as well as culture plasticware were obtained from Baxter (Edmonton, Alberta, Canada).

2.2. Rat myoblastic H9c2 cells

These cells were obtained from the American Type culture collection. They were cultured in Dulbecco's modified eagle medium supplemented with 10% (by volume) of heat-inactivated newborn calf serum, 100 units/ml of penicillin G, 10 /zg/ml of streptomycin and 0.25 /zg/ml of amphotericin B. Cell cultures were maintained at 37°C saturated with humidified air /5% CO 2. Each dish of cells was subcultured at 1:5 ratio and confluency was usually obtained after 4 days of incubation. In all experiments, cells at 60-70% confluence were made quiescent by incubation with a serum-free medium for 20 h prior to the addition of angiotensin II.

2.3. Angiotensin H treatment and phosphatidylcholine labelling

Angiotensin II was dissolved in a serum-free medium to obtain a stock solution of 0.8 mM. Aliquots of the stock were added to quiescent cells maintained in serum-free medium, and the cells were routinely incubated with angiotensin II for 2 h in 37°C incubator. Subsequently, [methyl-3H]choline in a serum-free medium was added into each dish to obtain a final concentration of 30 /xM (5 /zCi/ml). After incubation for 1 h or at indicated times, cells were rinsed three times with ice-cooled phosphate- buffered saline. The cells were harvested in methanol/HCl (100:1, v / v ) and the suspension was pooled in a silanized test tube. Total lipids were extracted from the cells by the modified method of Bligh and Dyer [22]. A monophasic extraction was first obtained with the addition of chloroform and ammonium acetate to the suspension in order to achieve the ratio of 1:2:0.5 (by volume) of chloroform/ methanol/0.1% ammonium acetate. Aliquots of the mixture were taken for total radioactivity determination. Sub- sequently, phase separation was achieved by the addition of chloroform and 0.1% ammonium acetate to a final ratio of 1:1:0.8 (by vo lume)o f chloroform/methanol/0.1% ammonium acetate. The choline, phosphocholine and CDP-choline fractions in the upper layer (aquous phase) were resolved by thin-layer chromatography with a solvent system consisting of methanol/0.6% NaCI/NH4OH (50:50:5, by volume). Phosphatidylcholine in the lower layer (organic phase) was resolved by thin-layer chromatography with a solvent system consisting of chloroform/methanol/acetic acid/water (70:30:2:4, by volume). The fractions were identified by a Bioscan System 200 Imaging Scanner, and radioactivities in each fraction was determined by liquid scintillation spectometry.

2.4. Subcellular fractionation

Cells grown to 60-70% confluence in 100-mm dishes were made quiescent by incubation for 20 h with a serum- free medium. After incubation with angiotensin II for the

K. Tran et al. / Biochimica et Biophysica Acta 1259 (1995) 283-290 285

prescribed period of time, each dish was rinsed three times with ice-cooled phosphate-buffered saline and the cells were removed from the dish in the same buffer. The cells were sedimented by centrifugation, and then resuspended in 0.5 ml of homogenizing buffer (20 mM Tris-HC1, 0.1 M NaC1, 1 mM dithiothreitol and 1 mM phenylmethylsulfo- nyl fluoride, pH 7.0). The cell suspension was placed under sonic oscillation for 10 s using a Fisher Sonic Dismembrator (model 300) equipped with a microtip at a setting of 30% energy output. The cell homogenate obtained after sonic treatment was centrifuged for 5 min at 800 × g, and the supernatant obtained was centrifuged at 100000 X g for 60 min. The supernatant was designated as the soluble fraction and the pellet, designated as the particulate fraction, was rinsed twice and resuspended in the homogenizing buffer.

2.5. Determination of enzymes activities

Choline kinase activity was assayed by determining the radioactivity in the phosphocholine fraction formed from [methyl-3H]choline [23]. The reaction mixture contained 100 mM Tris-HCl (pH 8.0), 10 mM MgCI 2, 10 mM ATP, 0.25 mM [methyl-3H]choline (45000 dpm/nmol) and 0.1 mg enzyme protein in a final volume of 0.2 ml. The reaction mixture was incubated at 37°C for 15 min, and the reaction was terminated by boiling the mixture for 2 min. CTP: phosphocholine cytidylyltransferase activity was assayed by determining the radioactivity in the CDP-choline fraction [21]. The reaction mixture contained 80 mM Tris- succinate (pH 6.5), 6 mM magnesium acetate, 0.5 mM CTP, 1 mM phospho[methyl-3H]choline (20 /zCi//xmol) and 0.1 mg enzyme protein in a final volume of 0.1 ml. Cytidylyltransferase activity in the soluble fraction was determined in the presence of 0.1 mg of total lipid from hamster liver. The reaction mixture was incubated at 37°C for 15 min, and the reaction was terminated by placing the mixture in boiling water for 2 min. CDP-choline:l,2- diacylglycerol cholinephosphotransferase activity was determined by the radioactivity in the phosphatidylcholine fraction [24]. The reaction mixture contained 40 mM Tris- HC1 (pH 8.0), 10 mM MgC12, 1 mM EDTA, 0.2 mM CDP[methyl-lZC]choline (5500 dpm/nmol) , 1 mM diacylglycerol (prepared in 0.15% Tween 20 by sonication) and 0.1 mg protein in a final volume of 0.2 ml. The reaction was mixture was incubated at 37°C for 15 min, and the reaction was terminated by the addition of 1 ml of chloroform/methanol (1:1; v / v ) to the assay mixture. Thin-layer chromatography was employed for the separation of the choline-containing compounds as described in the preced- ing section.

2.6. Quantitation of diacylglycerol

Diacylglycerol in the tissue was purified from the total lipid extract by thin-layer chromatography with a solvent

containing light petroleum (b.p. 37-56°C)/die thyl ether/acetic acid (60:40:1, by vol.). The acyl groups in diacylglycerol were methylated and the fatty acid methyl esters were quantitated by gas-liquid chromatography [25].

2.7. Immunoblotting of CTP:phosphocholine cytidylyltransferase

Cells were grown to 60% confluence in 150-mm dishes and kept in serum-free medium for 20 h. Subsequent to exposure to angiotensin II, cells in each dish were soni- cated in 2 ml of 20 mM Tris-HC1 (pH 8.0), 100 mM NaCI, 2 mM EDTA, 50 mM NaF, 30 mM sodium pyrophosphate, 0.2 mM sodium orthovanadate, 10 p ,g/ml leupeptin and 20 /~g/ml aprotinin. The mixture was centrifuged at 100000 × g for 60 min to obtain the particulate and soluble fractions. An aliquot of each fraction (containing 30 /zg protein) was analyzed by sodium dodecylsulfate- polyacrylamide gel (10%) electrophoresis. Subsequently, proteins in the gel were transferred to a nitrocellulose membrane by semi-dry blotting (Biorad), and the enzyme band was allowed to react with rabbit anti-cytidylyltransferase antibodies. The enzyme band in the nitrocellulose membrane was detected with a coupled horseradish peroxidase system [26].

2.8. Other analytical procedures

Protein was determined by the method of Lowry et al. [27] with bovine serum albumin as standard. Student's t-test was used for statistical analysis of data.

3. Results

3.1. The effect of angiotensin H on the biosynthesis of phosphatidylcholine

To examine the role of angiotensin II in the regulation of phosphatidylcholine biosynthesis, H9c2 cells were pretreated with angiotensin II for 2 h, and then incubated with

Table 1 Effect of angiotensin II on the uptake of [methyl-3H]choline by H9c2 cells

Control Angiotensin II

Total uptake 21.8 + 0.6 22.6 + 0.6 Aqueous 18.2 + 0.5 18.3 _+ 0.8 Organic 3.5 _+ 0.1 4.4 + 0.2 *

Data are dpm× 10 4/dish. Cells were incubated in the presence or absence of 40 /~M of angiotensin II for 2 h and then with 30 /xM of [methyl-3H]choline (5 /xmCi/ml) for 1 h. Radioactivities in the total cellular extract, the aqueous phase (upper layer) and the organic phase (lower layer) were determined. Each set of data represents the mean_ standard deviation of 9 separate experiments. * P < 0.05.

286 K. Tran et aL / Biochimica et Biophysica Acta 1259 (1995) 283-290

16 r 1 4

12

. - 1 0 -o

I ° 8

x

E 6 o.

-o

0 0 5 1 0 1.5 2.0

Time (h)

Fig. 1. The effect of angiotensin II on the incorporation of radioactivity into phosphatidylcholine. H9c2 cells were preincubated with (solid bars) or without (hatched bars) 40 /zM of angiotensin II for 2 h and then incubated with 30 /zM [methylPH]choline (5 k~Ci/ml) for 0.5-2.0 h. The labelling of phosphatidylcholine was expressed as dpm/dish. Each set of data represents the mean+standard deviation of four separate experiments. * indicates a P-value of < 0.05 when compared to the appropriate control.

[methyl-3H]choline for 1 h. The uptake of [methyl- 3H]choline by H9c2 cells and its conversion to different metabolites in the CDP-choline pathway were determined.

As depicted in Table 1, treatment of cells with angiotensin II resulted in an accumulation of radioactivity in the

organic phase without any detectable change in the total uptake or in the aqueous phase when compared with the

3.5

=5

T o

2.5 ×

E o_

13

2.0

0.001 0.01 0.1 1 I0

A n g i o t e n s i n II ( u M )

I00

Fig. 2. The enhancement of phosphatidylcholine biosynthesis at different angiotensin I1 concentrations. Cells were preincubated with 0.001-40 /zM angiotensin II for 2 h and then incubated with 30 /zM [methyl- 3H]choline (5 /xCi/ml) for 1 h. The labelling of phosphatidylcholine was expressed as dpm/dish. Each set of data represents the mean_ standard deviation of four separate experiments. * indicates a P-value of < 0.05 when compared to the control value (2.05_4-0.03 × 104/dish).

control. Analysis of the organic phase by thin-layer chromatography revealed that angiotensin II caused a significant increase (25%) in the radioactivity associated with the phosphatidylcholine fraction. The time course for the la-

belling of phosphatidylcholine in the presence and absence of angiotensin II was studied. As shown in Fig. 1, the

labelling of phosphatidylcholine was found to be signifi-

cantly elevated at 0 .5-2 .0 h of incubation. Alternatively,

the effect of angiotensin II concentration to phosphatidylcholine labelling was examined. Cells were treated with 0.001-40.0 /~M angiotensin II for 2 h and then incubated with [methyl-3H]choline for 1 h. The labelling of phospha-

tidylcholine was found to be increased by 0 .1-40 /zM angiotensin II in a dose-dependent manner (Fig. 2). Higher

doses of angiotensin II (up to 100 tzM) did not cause any additional increase in phosphatidylcholine labelling, and the pool size of phosphatidylcholine was not changed

between the control and the angiotensin II-stimulated cells (data not shown). Hence, 40 ~ M angiotensin II was used in all subsequent experiments in order to obtain the maxi-

mum effect on the labelling of phosphatidylcholine.

3.2. Analysis of choline-containing metabolites

The mechanism for the enhancement of phosphatidyl-

choline labelling by angiotensin II was studied by examin-

ing the labelling of the choline-containing metabolites. When cells were pretreated with angiotensin II, followed

by pulse-labelling with [methyl-3H]choline, there was a small increase in the labelling of choline (16%) and a consistent decrease (1 1%) in the labelling of the phospho-

choline fraction (Table 2). However, labellings in the CDP-choline fractions were not significantly altered. Since the labelled CDP-choline was transient in the biosynthesis

of phosphatidylcholine, the conversion of phosphocholine to CDP-choline and subsequently to phosphatidylcholine

was enhanced by angiotensin II treatment in H9c2 cells.

3.3. The effect of angiotensin II on enzyme activities in the CDP-choline pathway

Enzyme activities for the synthesis of phosphatidylcholine between the control and the angiotensin II treated

Table 2 Labelling of soluble choline-containing metabolites

Control Angiotensin II

Phosphocholine 13.7 _+ 0.5 12.0 _+ 0.6 a CDP-choline 1.12 + 0.05 1.15 4- 0.03 Choline 4.2 _+ 0.6 4.9 + 0.4 ~

Data are dpm× 10-4/dish. Cells were incubated in the presence or absence of 40 ,eM of angiotensin II for 2 h and then with 30 /zM of [methyl-3H]choline (5 /xCi/ml) for 1 h. The labelling of choline-containing metabolites in the aqueous phase was determined. Eact set of data represents the mean _+ standard deviation of 9 separate experiments.

P < 0.05.

K. Tran et a l . / Biochimica et Biophysica Acta 1259 (1995) 283-290 287

cells were determined. Cells were incubated with or without angiotensin II for 2 h, and cell homogenates were prepared after incubation. The soluble and particulate fractions were obtained from the cell homogenate by differen- tial centrifugation. Choline kinase activity was assayed in the soluble fraction and CDP-choline:l,2-diacylglycerol cholinephosphotransferase activity was determined in the particulate fraction. Since the CTP:phosphocholine cytidylyltransferase were shown to be present in the particulate as well as in the soluble fractions, its activities in both fractions were examined. No significant change in the activity of choline kinase (1.02 + 0.19 n m o l / m i n / m g ) or cholinephosphotransferase (0.50 + 0.03 n m o l / m i n / m g ) was detected between the control and the angiotensin II-treated cells. Altematively, treatment with angiotensin II caused a 50% increase in cytidylyltransferase activity in the particulate fraction and a 90% increase in the cytosolic fraction (Table 3). The enhancement of the cytidylyltransferase activities observed in this study concurs with the finding that the conversion of phosphocholine to CDP- choline was increased by angiotensin II treatment. In view of the fact that the cytidylyltransferase activities in both subcellular fractions were enhanced, the direct action of angiotensin II on the enzyme activity was examined. En- zyme activities in both subcellular fractions were assayed in the presence of 1-40 /.tM angiotensin II. Our results show that the presence of angiotensin II in the assay mixture had no effect on any of the enzyme activity (data not shown).

3.4. The effect of angiotensin H on the leL, els of diacylglycerol

In view of the fact that diacylglycerol may regulate phosphatidylcholine biosynthesis by acting as a substrate for the cholinephosphotransferase and an activator of the cytidylyltransferase, the pool size of diacylglycerol was

o 3 %

o E

o 2 < ICi

I I I I I I I

0 20 40 60 80 100 120

Time (min)

Fig. 3. Effect of angiotensin II on the levels of diacylglycerol. Cells were incubated without (O) or with ( 0 ) 40 /.tM of angiotensin II for 2 h. Diacylglycerol was separated from the total lipid extract by thin-layer chromatography. The acyl groups were esterified to fatty acid methyl esters which were quantitated by gas-liquid chromatography.

determined in the control and the angiotensin II-stimulated cells. As shown in Fig. 3, angiotensin II stimulation caused a biphasic modulation of the diacylglycerol level in these cells. The level of diacylglycerol was increased during the first 10 min of stimulation, but decreased below the control value after 30 min of stimulation. It is possible that the cytidylyltransferase in soluble and particulate fractions were activated by the elevated level of diacylglycerol during the first 10 min of angiotensin II stimulation.

3.5. The effect of okadaic acid on angiotensin H-induced actA~ation of cytidylyltransferase

Table 3 Effects of angiotensin II and okadaic acid on the cytidylyltransferase activity

CTP: phosphocholine cytidylyltransferase

Soluble Particulate

Control 0.45 + 0.02 0.30 + 0.03 Angiotensin II 1.04 __+_ 0.04 0.45 + 0.03 Okadaic acid 0.60 + 0.01 0.22 __. 0.02 Okadaic acid + 0.90 _+ 0.05 0.44 -F 0.02 Angiotensin II

Data are nmol/min/mg. Cells were incubated in the presence or absence of 40 p.M of angiotensin II and/or 0.5 /xM of okadaic acid for 2 h. Subsequent to incubation, soluble and particulate fractions were prepared and the activity of the cytidylyltransferase was determined in each fraction. Each set of data represents the mean +standard deviation of 4 determinations from two separate sets of experiments.

The phosphorylation-dephosphorylation of the enzyme in the cytosol has been shown to be an important mechanism for the regulation of the cytidylyltransferase activity. In order to explore this possibility, okadaic acid was employed for this study. H9c2 cells were preincubated with okadaic acid for 15 min prior to the incubation with angiotensin II, and subcellular fractions were obtained after the incubation. As shown in Table 3, preincubation with okadaic acid (without angiotensin II) caused a decrease in the activity of the enzyme in the particulate fraction with a corresponding increase of activity in the soluble fraction when compared with controls. A shift in the cytidylyltransferase activity by okadaic acid treatment was also observed in another study [28]. However, preincubation with okadaic acid did not significantly alter the stimulatory effect on the cytidylyltransferase activity produced by angiotensin II in both subcellular fractions. Our results suggest that the enhancement of enzyme activity by


angiotensin II was not mediated via the dephosphorylation mechanism.

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3.6. The effect of protein kinase inhibitors on the angiotensin II-induced activation of the cytidylyltransferase activity

In this study, the involvement of a protein kinase for the activation of the cytidylyltransferase by angiotensin II was examined. H9c2 cells were preincubated with a protein kinase inhibitor for 30 min and then incubated with angiotensin II for 2 h. Subsequently, cytidylyltransferase activities in the soluble and particulate fractions were determined. As indicated in Table 4, the activation of the cytidylyltransferase by angiotensin II was eliminated when cells were preincubated with H7. In view of the fact that H7 caused the inhibition of both protein kinase A and C, a specific inhibitor for protein kinase C, Ro31-8220 [29] was employed in this study. When cells were preincubation with Ro31-8220, the activation of the cytidylyltransferase by angiotensin II was eliminated. Our results indicate that protein kinase C might be involved in the activation of the cytidylyltransferase during the agonist stimulation. To fur- ther confirm the participation of protein kinase C in this process, its activity in the cells were down-regulated by prolonged phorbol myristate acetate incubation prior to angiotensin II treatment. The prolonged incubation of cells with the phorbol ester itself did not cause any significant change in the activities of the cytidylyltransferase. How- ever, the stimulatory effect of angiotensin II on the cytidylyltransferase was eliminated by this treatment. Taken together, our results clearly suggest the involvement of protein kinase C in the stimulation of cytidylyltransferase activities during angiotensin II treatment.

Table 4 Effect of protein kinase inhibitors on the angiotensin II-induced activation of cytidylyltransferase

CTP: phosphocholine cytidylyltransferase

Soluble Particulate

Control 0.57 ± 0.01 0.39 ± 0.02 Angiotensin II 1.02 _ 0.04 0.65 ± 0.02 H7 0.55 ± 0.05 0.45 ± 0.04 H7 + Angiotensin II 0.44 ± 0.04 0.38 ± 0.02 Ro 0.51 ± 0.02 0.42 _ 0.04 Ro + Angiotensin II 0.41 ± 0.01 0.37 ± 0.02 PMA 0.47 ± 0.01 0.40 _+ 0.01 PMA + Angiotensin I1 0.39 -t- 0.05 0.28 + 0.03

Data are n m o l / m i n / m g . Cells were incubated with protein kinase inhibitors (50 /zM of H7 or 5 tzM of Ro31-8220) for 30 min, and then incubated with 40 /xM of angiotensin II for 2 h. In other experiments, cells were incubated in the presence or absence of 100 nM of phorbol myristate acetate (PMA) for 20 h and then incubated with 40 /xM of angiotensin II for 2 h. The cytidylyltransferase activity was determined in soluble and particulate fractions. Each set of data represents the mean + standard deviation of 4 determinations from two separate sets of experiments.

Control Angiotensin II Control Angiotensin II

Soluble Particulate

Fig. 4. Immunoblotting analysis of cytidylyltransferase. Cells were incubated with 40 /zM of angiotensin II for 2 h. Aliquots (containing 30 /xg of protein) of the soluble and particulate fractions were analyzed by sodium dodecylsulfate-polyacrylamide (10%) gel electrophoresis. Protein bands in the gel were blotted into a nitrocellulose membrane and then treated with rabbit anti-cytidylyltransferase antibodies. A coupled peroxidase system was used for the colour development of the cytidylyltransferase band.

3.7. The quantitation of cytidylyltransferase by immunoblotting

The effect of angiotensin II treatment on the level of cytidylyltransferase in both the soluble and particulate fractions was studied by immunoblotting. Rabbit sera containing anti-rat liver CTP:phosphocholine cytidylyltransferase antibodies [30] were found to interact with the enzyme in H9c2 cells. Hence, relative levels of cytidylyltransferase in the control and angiotensin II-stimulated cells were determined by immunoblotting. Aliquots containing 30 #g of protein from soluble and particulate fractions were analyzed by sodium dodecylsulfate-polyacrylamide (10%) gel electrophoresis. Protein bands separated by the gel electrophoresis were transferred to a nitrocellulose membrane by semi-dry blotting. Subsequent to incubation with the antisera, the relative amount of enzyme in each band was quantitated by color formation in a horseradish peroxidase coupled reaction [26]. The relative concentration of the cytidylyltransferase band was quantitated by densitometry. As depicted in Fig. 4, the Westem blot shows that the cytidylyltransferase in both soluble and particulate fractions were not affected by angiotensin II treatment.

4. Discussion

It is clear from this study that angiotensin II had no effect on the uptake of choline but caused an enhancement of phosphatidylcholine biosynthesis in H9c2 cells. The enhancement of phosphatidylcholine biosynthesis was produced by increasing the activity of CTP:phosphocholine cytidylyltransferase. However, angiotensin II had no direct effect on the cyfidylyltransferase activity, but its action was mediated in part via the protein kinase C cascade.

In mammalian hearts, the biosynthesis of phosphatidylcholine via the CDP-choline pathway is regulated by several mechanisms, including the rate of choline uptake and the modulation of the cytidylyltransferase activity. In earlier studies, choline uptake in the hamster heart was found

K. Tran et al. / Biochimica et Biophysica Acta 1259 (I 995) 283-290 289

to be inhibited by ethanolamine and other choline analogs [31,32], but stimulated by neutral amino acids [33,34]. More recently, choline uptake in cardiac myocytes was found to be regulated by hormonal stimulation [35]. How- ever, the uptake of choline in H9c2 cells was not affected by angiotensin II treatment. This is not surprising since the control of choline uptake has been shown to be a transient process for the short-term regulation of phosphatidylcholine biosynthesis [32,33].

The conversion of phosphocholine to CDP-choline catalyzed by the cytidylyltransferase is generally accepted as the rate-determining step for controlling phosphatidylcholine biosynthesis [36,37]. The decrease in the labelling of phosphocholine by angiotensin II is a clear indication of the enhanced conversion of this metabolite to CDP-choline, and subsequently, phosphatidylcholine. However, the decrease in phosphocholine labelling was not detectable in the aqueous phase of the extract. It is possible that the modest change (11%) in phosphocholine labelling might have been masked by the presence of other labelled choline-containing metabolites. The enhancement of phosphocholine conversion is supported by the fact that the cytidylyltransferase activities in both subcellular fractions were stimulated by angiotensin II treatment.

The microsomal form of the cytidylyltransferase is regarded as the more active form of the enzyme and the translocation of the enzyme from the cytosolic to the microsomal compartment represents an important mechanism for the enhancement of the enzyme activity in vivo [37]. In this study, it is clear that angiotensin II did not promote the translocation of the enzyme, since the enzyme levels in both fractions were not changed and enzyme activities in both compartments were enhanced. Another mechanism for the modulation of the cytidylyltransferase is by phosphorylation-dephosphorylation, of which the de- phosphorylated enzyme is the more active form [37]. Okadaic acid is a potent inhibitor of types 1 and 2A protein phosphatases and has been shown to inhibit the dephosphorylation of cytidylyltransferase resulting in a reduction of the enzyme activity [28]. Hence, the inability to eliminate the activation of the cytidylyltransferase by okadaic acid in the present study suggests that the enzyme activation was not caused by dephosphorylation.

Diacylglycerol has been shown to be a potent activator of the cytidylyltransferase. A recent study by Utal et al. [38] suggested that the elevation of diacylglycerol plays a role in the stimulation of phosphatidylcholine biosynthesis. The elevated level of diacylglycerol during the first 10 min of angiotensin II stimulation might contribute to the initial activation of the enzyme. Alternatively, diacylglycerol is an immediate substrate of the cholinephosphotransferase reaction for the synthesis of phosphatidylcholine. How- ever, the K m of diacylglycerol for this reaction is several times lower than the diacylglycerol pool in the cardiac cells [39]. Hence, the change in diacylglycerol pool observed in this study might not be large enough to affect the

rate of phosphatidylcholine formation via the cholinephosphotransferase reaction.

The involvement of protein kinase C in the regulation of phosphatidylcholine hydrolysis during agonist stimulation has been well documented [40,41]. Intracellular phos- pholipases including phospholipase A 2, C and D are mod- ulated by protein kinase C [42], and their products, such as unsaturated fatty acids and diacylglycerol, have been shown to stimulate the cytidylyltransferase activity by direct activation or by promoting the translocation of the enzyme [43]. In view of the fact that the choline uptake was not stimulated by angiotensin II, the increase in the labelling of choline (Table 2) might have arisen from an increase in phospholipase D activity. The involvement of phorbol esters in the enhancement of phosphatidylcholine biosynthesis has also been reported [44]. In the present study, the participation of protein kinase C in the activation of the cytidylyltransferase during angiotensin II treatment has been clearly demonstrated. However, the exact mode on the activation of the cytidylyltransferase by protein kinase C remains unclear. The direct phosphorylation of the cytidylyltransferase by protein kinase C is unlikely since the enzyme has been shown to be less active in the phosphorylated state. We propose that exposure of the H9c2 cells to angiotensin II resulted in the elevation of diacylglycerol, which might be one of the factors to cause the activation of protein kinase C. Diacylglycerol also produced the initial enhancement of the cytidylyltransferase activity, whereas protein kinase C might initiate a cascade of activity for the prolonged enhancement of the enzyme activity.

Acknowledgements

The study was supported by MRC and HSFM. The technical assistance of Ms Carol Lemoine is appreciated. We thank Dr. G. Arthur for his advice, Dr. G. Lawton of Roche Products Ltd. for the gift of Ro31-8220, and Dr. D. Vance for the gift of the anti-cytidylyltransferase antibodies.

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the enhancement of phosphatidylcholine biosynthesis by angiotensin ii in h9c2 cells

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