enhancement of phospholipid hydrolysis in vasopressin-stimulated bhk-21 and h9c2 cells

Molecular and Cellular Biochemistry 151: 69-76, 1995. �9 1995 Kluwer Academic Publishers. Printed in the Netherlands.

Enhancement of phospholipid hydrolysis in vasopressin-stimulated BHK-21 and H9c2 cells

Khai Tran, Xiliang Zha, Monroe Chan and Patrick C. Choy Department o f Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba, R3E OW3 Canada

Received 27 February 1995; accepted 11 May 1995

Abstract

The hydrolysis of phospholipids in vasopressin-stimulated baby hamster kidney (BHK)-21 and H9c2 myoblastic cells was investigated. Phosphatidylcholine and phosphatidylethanolamine in these cells were pulse labelled with [3H]glycerol, [3H]myristate, [3H]choline or [3H]ethanolamine, and chased with the non-labelled precursor until linear turnover rates were obtained. When cells labelled with [3H]glycerol or [3H]myristate were stimulated by vasopressin, no significant decrease in the labelling ofphosphatidylcholine was detected, but the labelling ofphosphatidic acid was elevated. However, the labeltings of phosphatidylethanolamine and its hydrolytic product were not affected by vasopressin stimulation. When the cells were pulse labelled with [3H]-choline, vasopressin stimulation caused a decrease in the labelled phosphatidylcholine with a corresponding increase in the labelled choline. The apparent discrepancy between the two types of labelling might be explained by the recycling of labelled phosphatidic acid back into phosphatidylcholine, thus masking the reduction in the labelled phospholipid during vasopressin stimulation. Alternatively, the labelled choline produced by vasopressin stimulation was released into the medium, thus reducing the recycling of label precursor back into the phospholipid and making the decrease in the labelling of phosphatidylcholine readily detectable. Further studies revealed that vasopressin treatment caused an enhancement of phospholipase D activity in these cells. The presence of substrate-specific phospholipase D isoforms in mammalian tissues led us to postulate that the differential stimulation of phospholipid hydrolysis by vasopressin was caused by the enhancement of a phosphatidylcholine-specific phospholipase D in both BHK-21 and the H9c2 cells. (Mol Cell Biochem 151: 69-76, 1995)

Key words: vasopressin, phospholipids, phosphatidylcholine, hydrolysis, phospholipase D

Abbreviations: BHK-21 cel ls- baby hamster kidney-21 cells

Introduction

Phosphatidylcholine and phosphatidylethanolamine are major lipid components of the biological membrane. Beyond their structural role as membrane-building blocks, phospholipids in the membrane are actively involved in the production of second messengers during signal transduction [ 1]. The rote ofphosphatidylinositol as a precursor for the rapid production ofinositol trisphosphate and diacylglycerol has been well-established [2, 3]. In the last several years, the hydrolysis ofphosphatidylcholine has been regarded as an important mechanism for the prolonged production of second messen-

ger during agonist stimulation [4]. Phosphatidylcholine has been shown to be hydrolyzed by the action ofphospholipase D for the production of choline and phosphatidic acid [5]. Alternatively, it can be hydrolyzed by the action ofphospholipase C for the production of phosphocholine and diacylglycerol [6, 7]. The involvement of phosphatidylethanolamine for the production of second messengers by the hydrolytic action of phospholipase D has also been demonstrated [8-10].

A number of agonists have been shown to modulate the breakdown ofphosphatidylcholine. For example, intefleukin- I and the ras oncogene have been shown to increase diacyl-

Address for offprints: P.C. Choy, Department of Biochemistry and Molecular Biology, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Mani- toba, R3E OW3 Canada

70

glycerol production by the enhancement of phosphatidylcholine hydrolysis [11-13]. Other agents which cause the elevated hydrolysis of phosphatidylcholine include adrena- line [14], platelet-derived growth factor [6] and bombesin [ 15]. Interestingly, phorbol 12-myristate 13-acetate has the ability to stimulate the hydrolysis ofphosphatidylcholine in a number of cell types [ 16-21 ]. The ability of the phorbol ester to stimulate the hydrolysis ofphosphatidylethanolamine has been demonstrated in leukemic HL-60, NIH 3T3 and baby hamster kidney -21 cells [8].

Vasopressin has been shown to display profound effects on the biosynthesis and hydrolysis ofphosphatidylcholine in mammalian cells. In cardiac myocytes, phosphatidylcholine biosynthesis is modulated by vasopressin in a biphasic manner [22], whereas in rat hepatocytes, phosphatidylcholine biosynthesis is inhibited by vasopressin at all concentrations [23]. Alternatively, vasopressin enhances the incorporation of labelled choline into phosphatidylcholine in glomerular mesangial cells [24]. The effect of vasopressin on phosphatidylcholine hydrolysis was studied in several cell lines [24-26]. In these studies, the cells were prelabelled with a phosphatidylcholine precursors, and the effect ofvasopressin on the production of the hydrolytic products were determined. Vasopressin was found to enhance the production of choline or phosphatidic acid in fibroblasts [25] and mesangial cells [24] which implied that phosphatidylcholine hydrolysis was stimulated in these cells. However, the vasopressin-induced accumulation of diradylglycerol in vascular smooth muscle cells was abolished when the cells were incubated with eicosapentaenoic acid [27]. In addition, the vasopressin-induced phosphatidic acid formation in smooth muscle cells was inhibited by incubation with linoleic acid [27]. At present, the reason for the attenuation of the vasopressin-induced phosphatidylcholine hydrolysis by these fatty acids is not entirely clear. The effect of vasopressin on phosphatidylethanolamine metabolism remains largely unknown.

In view of the significance of agonist-induced production of second messengers from the major phospholipids, it is important to study the regulation ofphosphatidylcholine and phosphatidylethanolamine hydrolysis by vasopressin in key mammalian organs. BHK-21 cells and rat heart H9c2(2-1) myoblasts were used for this study since the advantage of using homogenous cell types to study phospholipid metabolism has been well-documented [28]. In this study, labelling of phosphatidylcholine and phosphatidylethanolamine in these cells were achieved by incubating them with [1,3- 3H]glycerol, [9,10-3H]myristate and [methyl-3H]choline or [1,2-3H]ethanolamine. Subsequently, the stimulation ofphos- pholipid turnover by vasopressin in these cells were determined.

Materials and methods

Materials

[methyl-3H]Choline, [1-3H]ethanolamine, [1,3-3H]glycerol and [9,10-3H]myristic acid were obtained from New England Nuclear (Mississauga, Ontario, Canada). All cell culture media were purchased from Sigma Chemical Company (St. Louis, Mo., USA).All the lipid standards were obtained from Serdary (London, Ontario, Canada). Thin layer chromato- graphic plates (sil-G25) were the product of Macherey-Nagel (Duren, Germany) and obtained through Brinkman (Rexdale, Ontario, Canada). All other chemicals and solvents (reagent or HPLC grade) and all the culture plastioware were obtained from the Canlab division of Baxter Company (Mississauga, Ontario, Canada).

Cultured of BHK-21 and H9c2 Cells

BHK-21 and H9c2(2-1) cells were obtained from theAmeri- can Type Culture Collection and cultured as previously described [28]. The cells were seeded in 60 mm diameter dishes and incubated with a complete growth medium containing Joklik modified minimum essential medium supplemented with 1.8 mM calcium, 10% fetal calf serum, 100 units/ml of penicillin G, 10 lag/ml of streptomycin and 0.25 ,ug/ml am- photericin B. The cells were allowed to grow to 70% conflu- ence in a 37~ incubator saturated with 95% air/5%CO 2.

Labelling of phosphatidyleholine and phosphatidylethanolamine

Both BHK-21 and H9c2 cells were used for this study. The labelling of phosphatidylcholine and phosphatidylethanolamine were achieved by incubating the cells for 24 h with 2 ml of 0.1 mM of [3H]glycerol (7.5 laCi/ml) in the growth medium. Subsequent to pulse labelling, cells were rinsed three times with the serum-free growth medium and reincubated with the complete growth medium containing 1.0 mM ofunlabelled glycerol for 20 h prior to use.

Labelling of the base groups in phosphatidylcholine and phosphatidylethanolamine were achieved by incubating the cells with [3H]choline or [3H]ethanolamine. In a typical ex- periment, cells were incubated with 2 ml of 1.0 ~tM of [3H]choline (10.0 IxCi/ml) or [3H]ethanolamine (10.0 ~tCi/ml) in the complete growth medium for 2 h. The cells were then rinsed three times in the serum free growth medium and reincubated with the complete growth medium supplemented with 10.0 ~tM ofunlabelled choline or ethanolamine for 20 h prior to use.

The labelling of phosphatidylcholine containing a myristate group was achieved by incubating the cells with 2 ml of 0.3 I.tM of [3H]myristate (4.5 gCi/ml) in the complete growth medium for 4 h. The cells were rinsed three times with the serum free growth medium and reincubated in the complete growth medium containing 3 gM ofunlabelled myristate for 20 h prior to use.

71

system containing hexane/diethylether/acetic acid (70:30:1, by volume). The water soluble choline containing compounds (choline and phosphorylcholine) were separated by thin-layer chromatography. The plate was developed in a solvent system containing methanol/0.6% NaCI/NH4OH (50:50:5, by volume). The radioactivity in each fraction was determined by liquid scintillation spectrometry.

Vasopressin stimulation

Subsequent to the labelling of the phospholipid(s), the cells were incubated with the complete growth medium containing 0.5 p.M vasopressin at 37~ for the prescribed period of time. After incubation with the hormone, the medium was quickly removed and 1.0 ml of ice-cooled methanol/HC1 (100:1, v/v) was added to the dish. The cells in the dishes were removed and placed in a silanized test tube, and 0.8 ml of water, t.0 ml of chloroform and 0.05 mt of saturated NaC1 were added in order to cause phase separation. Total lipids in the cells were extracted by the method of Bligh and Dyer [29].

Phospholipase D assay

The determination ofphospholipase D activity in the agonist- stimulated and control cells were carried out by the proce- dure of Bocokino et al. [30]. The cells were incubated with 2 ml of 0.3 pM of [3H]myristate (4.5 laCi/ml) in the complete growth medium for 20 h. The cells were rinsed three times with the serum-free growth medium and reincubated with the complete growth medium for 1 h prior to the addition of vasopressin in the presence of 2% ethanol. Phospholipase D activity was determined by its ability to transfer the phosphatidyl moiety of a phospholipid to a primary alcohol to form phosphatidylethanol.

Analysis of lipid fractions by thin layer chromatography

Lipids in the organic extract were quantitated by thin layer chromatography. The ,volume of the organic phase was re- duced by a stream of nitrogen gas and aliquots of the con- centrated lipid extract were analyzed by thin layer chromatography. For the separation of phosphatidytcholine, phosphatidylethanolamine and phosphatidic acid, the plate was developed in a solvent system containing chloroform/methanol/ acetic acid/water (85:15:10:3, by volume). To simulta- neously separate phosphatidic acid, phosphatidylethanol and diacylglycerol, a double development protocol [31] was employed. The plate was first developed half-way in the upper phase of ethylacetate/isooctane/acetic acid/water (110:50:20:100, by v o l u m e ) and redeveloped in a solvent

Results

The labelling of the major phospholipids by [3HI-glycerol in BHK-21 and H9c2 myoblastic cells

Cells were grown to 70% confluency in 60 mm dishes. La- belling of the cellular tipids was achieved by incubating each dish for 24 h with 2 ml complete growth medium containing 0.1 mM [3H]glycerol (7.5 laCi/ml). Subsequent to pulse-labelling, cells in each dish were rinsed and reincubated (chased) with the complete growth medium containing 1 mM glycerol. The labellings of the total lipid extract as well as phosphatidylcholine and phosphatidylethanoIamine in BHK- 21 cells at different time points of the chase are depicted in Fig. 1. Maximum labellings of the lipid fractions were achieved at 2-4 h of the chase followed by rapid decreases in their tabellings within the first 12 h of the chase. Subse- quently, a linear rate of turnover was detected in both phos-

2.5

2.0

=6

I o

x

E 1 . 0 o_ " o

0.5

\ ~

To ta l l i p i d

PC

PE :

0 . 0 I 1 P I 1 t

0 4 8 ! 2 16 20 24

Chose t ime (h)

Fig. 1. Labelling of phosphatidylcholine and phosphatidylethanolamine with [3HI-glycerol in BHK-21 cells. Cells were incubated with medium containing 0.1 mM of PHI-glycerol (7,5 gCi/ml) for 24 h and then with medium containing 1.0 mM of unlabelled glycerol for 0-24 h. Cellular lipids were extracted and phosphatidylcholine (PC) and phosphatidylethanolamine (PE) fractions were isolated by thin-layer chromatography. Each point represents the mean of 4 ~ dishes and the standard deviation for each point is less than 10% of the mean values.

72

pholipid fractions between 12 and 24 h of chase. The same pattern of labelling was observed in H9c2 myoblastic cells when the cells were labelled with [3H]glycerol under identical experimental conditions (Fig. 2).

Effect of vasopressin on the turnover of [3H]glycerol labelled phosphatidylcholine and phosphatidylethanolamine

The effect of vasopressin on the hydrolysis of phosphatidylcholine and phosphatidylethanolamine in the BHK-21 and the H9c2 myoblastic cells were investigated. Lipids in BHK-21 cells were labelled with [3H]glycerol as described in the preceding section. At 20 h of the chase period, the medium was removed from the dish and replaced with the complete growth medium containing 0.5 taM ofvasopressin. The cells were incubated at 37~ for the prescribed period of time as depicted in Fig. 3. A small decrease in the labelling of phosphatidylcholine was detected after 2 and 4 min of incubation with vasopressin with a corresponding increase in the labelling of phosphatidic acid. Elevations in the labellings ofphosphatidic acid at 2 and 4 min were found to be statistically significant when compared with the control (at 0 min), but decreases in the labellings of phosphatidylcholine at the same time points were not statistically significant (p > 0.05). Vasopressin stimulation did not cause any additional release of radioactivity into the medium. No detectable changes in the labelling ofphosphatidylethanolamine or diacylglycerol was observed.

2.0

1.5

7~

I o 1.0

*\

•

"o

0.5

- ~ =PE =

0 . 0 I I I I I 1

0 4 8 12 16 20 24

Chose t ime (h)

Fig. 2. Labelling of phosphatidylcholine and phosphatidylethanolamine with [3H]-glycerol in H9c2 cells. Experimental conditions are identical to that described in Fig. 1.

0.75

0 �9 0.60

I o

x

E o.

"o

0.45

0 .50

0 .15

0 .00

,•'-•A-.....,.• P A

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DG = I - - - - - - - - . . R �9 = - - �9

I I I I I I

2 4 6 8 10 12

Time ( ra in )

5: ,< v

4 ~

b 3 -

x

E o_

2 "o

Fig. 3. Time course of vasopressin-induced turnover of [3HI-glycerol labelled phosphatidylcholine and phosphatidylethanolamine in BHK-21 cells. Cells were incubated with medium containing 0.1 mM of [3H]- glycerol (7.5 laCi/ml) for 24 h and incubated with medium containing 1.0 mM of unlabelled glycerol for 20 h. Subsequently, cells were treated with (solid symbols) and without (open symbols) 0.5 ~M of vasopressin for the indicated time points. The radioactivity in phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) and diacylglycerol (DG) fractions were determined. Each point represents the mean of 4-6 dishes with a standard deviation less than 10% of the mean value.

Lipids in H9c2 myoblastic cells were labelled with [3H]- glycerol, and the cells were stimulated with vasopressin in the same manner as described for the BHK-21 cells. The labelling of the phospholipids and diacylglycerol in these cells followed the same pattem as that found in the BHK-21 cells (Table 1). An increase in the labelling of phosphatidic acid was observed at 4 min of vasopressin incubation. Similarly, a small decrease in the labelling ofphosphatidylcholine was detected at 4 min ofvasopressin stimulation, but the decrease was not large enough to become statistically significant.

Table 1. The turnover of [3H]-glycerol labelled phosphatidylcholine and phosphatidylethanolamine in H9c2 cells in the presence of 0.5 laM vasopressin a

Time of stimulation (min) 0 4 10

(dpm X 106/dish) Phosphatidylcholine 0.52 • 0.07 0.45 • 0.04 0.50 • 0.06 Phosphatidyl- 0.26 • 0.05 0.27 • 0.06 0.27 • 0.08 ethanolamine (dpm x 10 -3/dish) Phosphatidic acid 3.41 • 0.46 5.19 • 0.54* 3.30 • 0.72 Diacylglycerol 0.67 • 0.10 0.73 • 0.09 0.71 + 0.12

"The experimental conditions were similar to that described in Fig. 3. *p < 0.05

73

Effect of vasopressin on the turnover of phosphatidylcholine and pho~phatidylethanolamine in cells labelled with [3H]choline or [3H]ethanolamine

In this study, the effect of vasopressin on the hydrolysis of [3H]choline labelled phosphatidylcholine or [3H]ethanolamine labelled phosphatidylethanolamine was investigated. In a set of preliminary experiments, BHK-21 cells were incubated with the complete growth medium containing 1.0 ktM of [3H]choline (10 ~C~/ml) for 2 h and chased with growth medium containing 10 ~tM unlabelled choline for up to 24 h. The labelling ofphosphatidyleholine reached a maximum at 4-6 h of the chase time. Similar to the previous set of experi- ment, a slow but linear tumover in the labelling ofphosphatidylcholine was observed between 16-24 h chase period (data not shown). At 20 h of chase, more than 90% of total radioactivity was found in the phosphatidylcholine fraction. A similar profile of labelling in phosphatidylethanolamine was observed when the cells were labelled with [3H]ethanolamine.

The effect of vasopressin on the hydrolysis of phosphatidylcholine was examined in BHK-21 cells at 20 h of chase. The complete growth medium was removed from the dish and replaced with the complete growth medium containing 0.5 l-tM ofvasopressin. The labelling ofphosphatidylcholine was significantly decreased after the first 2 min ofvasopressin incubation, and reached a minimum value at 4 min of incubation. However, no significant change in the labelling of phosphatidylethanolamine was detected throughout the 10 min incubation time (Fig. 4). Analysis of choline-containing compounds in the cellular extract revealed that the amount of labelled choline was increased by vasopressin stimulation. The increase in the labelled choline fraction appeared to have a temporal relationship with the decrease in the labelling of phosphatidylcholine. No significant change in the amount of labelled phosphocholine was detected throughout the stimulation period (Fig. 5). A small amount of labelled choline (1.2 + 0.2 x 103 dpm/ml) was found in the growth medium at 4 min of incubation, and the amount of labelled choline was significantly increased in the medium of the vasopressin- stimulated cells (3.4 • 0.4 x 103 dpm/ml).

The labelling of phosphatidyleholine by [3H]choline and the stimulation of phosphatidylcholine hydrolysis by vasopressin was also carried out in H9c2 myoblastic cells under identical conditions. The hydrolysis of phosphatidylcholine was similar to the pattern observed in the BHK-21 cells and maximum hydrolysis was detected at 5 min of stimulation (Table 2). The amount of labelled choline was elevated by vasopressin stimulation but no significant change in the amount of labelled phosphocholine was detected. When the cells were labelled with [3H]ethanolamine, there was no significant change in the hydrolysis of labelled phosphatidylethanolamine (data not shown).

T 0

x

E

4q

PE

0 I I I l _ A

0 2 4 G 8 10

Time (rain)

Fig. 4. Time course of vasopressin-induced turnover of [3H]-choline labelled phosphatidylcholine and [3H]-ethanolamine labelled phosphatidylethanolamine in BHK-21 cells. Cells were incubated with 1.0 laM of [3H]-choline (10 ~tCi/ml) or 1.0 gM of [3H]-ethanolamine (10 ~aCi/ml) for 2 h and chased with 10 ItM of unlabelled choline or ethanolamine for 20 h. The cells were then treated with (solid symbols) and without (open symbols) 0.5 gM of vasopressin for the indicated time points. The radioactivity in the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) fractions were determined. Each point represents the mean of six dishes with a standard deviation less than 10% of the mean value.

O

6

I O

4 x

E Q-

"O

I I I I

2 4 6 8

Time (min)

8

6 v

I O

4 x

E t X

"t3

2

t 0 10

Fig. 5. Time course of vasopressin-induced formation of choline- containing metabolites in BHK-21 cells. Cells were prelabelled with 1.0 IxM of [~H]-choline (10 laCi/ml) for 2 h, and chased with 10 ~tM of unlabelled choline for 24 h. The incubated cells were treated with (solid symbol) or without (open symbol) 0.5 I~M of vasopressin for the times indicated. The radioactivity in the choline (O, O) and phosphorylcholine (A, A) fractions were determined. Each point represents the mean of six dishes. The vertical bar is the standard deviation for each set of value.

74

Table 2. The turnover ofphosphatidy|choline in [3HI-choline labelled H9c2

cells ~

Control Vasopressm

(dpm x 104/dish)

Phosphatidylcholine 5.18 • 0.47 4. l 1 • 0,30* (dpm x 10-S/dish)

Choline 0.46 • 0.05 0.62 • 0.06* Phosphocholine 2.37 + 0.35 2.53 + 0.29

aCells were incubated with medium containing 1.0 ~tM of [3H]choline (10 laCi/ml) for 2 h and subsequently chased with medium containing 10.0/aM of unlabelled choline for 20 h prior to stimulation with 0.5/aM vasopressin. Each set of data represents the mean + slandard deviation from six dishes.

*p < 0.05.

Effect of vasopressin on the hydrolysis of [3H]myristate labelled phosphatidylcholine

When cells were labelled with [3H]myristate, a high propor- tion of the labelled fatty acid was shown to incorporate into phosphatidylcholine [32, 33]. Hence, the effect ofvasopressin on the hydrolysis of [3H]myristate labelled phosphatidylcholine was examined. BHK-21 ceils were incubated with the complete growth medium containing 0.3 ~tM of labelled myristate (4.5 laCi/ml) for4 h and chased with 3 ~tM ofunlabelled myristate in the complete growth medium for 20 h. The medium was removed and replaced with the complete growth medium containing 0.5 ~tM ofvasopressin for 5 min. No significant change in the labelling of phosphatidylcholine, phosphatidylethanolamine and diacylglycerol was detected by vasopressin stimulation. However, incubation with vasopressin caused a significant increase in the labelling of phosphatidic acid (Table 3).

Effect of vasopressin on phospholipase D activity

The data obtained from preceding sections indicate that vasopressin caused the stimulation ofphosphatidylcholine hydrolysis in both BHK-21 and H9c2 myoblastic cells for the

Table 3. The turnover of [3H]-myristate labelled phosphatidylcholine and phosphatidylethanolamine in BHK-21 ceils ~

Control Vasopressin

(dpm x 10 -6/dish) Phosphatidylcholine 2.25 • 0.08 2.20 • 0.06 Phosphatidyl- 0.70 • 0.04 0.71 • 0.04 ethanolamine (dpm x 10-Vdish) Phosphatidic acid 6.35 + 0.25 I 0.10 • 0.90* Diacylglycerol 7.58 + 0.38 7.24 • 0.44

~Cells were incubated with medium containing 0.3/aM [3H]myristate (4,5 /aCi/ml) for 4 h and subsequently chased with medium containing 3/aM of unlabelled myristate for 20 h prior to stimulation with 0.5 rtM vasopressin for 5 min. Each set of data represents the mean • standard deviation from four dishes. *p < 0.05

formation ofphosphatidic acid and choline. Hence the effect ofvasopressin on the activity ofphospholipase D in these two cell lines were determined. The cells were prelabelled by 0.3 ~tM [3H]myristate (4.5 ~tCi/ml) for 20 h., and then incubated with the complete growth medium containing 0.5 ~tM of vasopressin in the presence of 2% ethanol for 5 min. Phos- pholipase D activity was expressed by the amount of radioactivity in the phosphatidylethanol fraction [30]. A substantial increase (5~5 fold) in phosphatidylethanol formation was detected in the BHK-21 or the H9c2 cells within 5 min of vasopressin stimulation (Table 4).

Discussion

The present study was designed to investigate the effect of vasopressin on the hydrolysis of the major phospholipids in the BHK-21 and H9c2 cells. In order to study the phospholipid turnover, the cells were incubated with its labelled precursor and then chased for a prolonged period until the new synthesis of labelled phospholipids was no longer detectable. Our results show that the production ofphosphatidic acid was enhanced by vasopressin in both BHK-21 and H9c2 cells. The time course study in Fig. 3 suggests that increase in phosphatidylcholine hydrolysis is largely responsible for the elevated labelling in the phosphatidic acid fraction. Part of the accu- mulated phosphatidic acid produced may be converted into diacylglycerol which provides the cell with a sustained messenger following the initial hydrolysis of the phosphatidylinositol bisphosphate during agonist stimulation [4].

When the ceils were incubated with [3H]glycerol or [3H]myristate, the enhancement of phosphatidylcholine hydrolysis by vasopressin was detected in the phosphatidic acid fraction in the cellular lipid extract. When the cells were incubated with [3H]choline, vasopressin caused the enhanced release of labelled choline which was detected in the cellular lipid extract as well as in the incubating medium. Our results demonstrate that the phospholipase D activity was stimulated by vasopressin in BHK-21 and H9c2 cells. The stimulation of phospholipase D activity by vasopressin has also been, shown in other cell types [24, 25]. In this study,

Table 4. Phosphotipase D activity characterized by phosphatidylethanol formation in BHK 21 and H9c2 cells a

Phosphatidylethanol (dpm x 10 -4/dish) Control Vasopressin

BHK-21 3.07 • 0.46 17.60 + 0.78* H9c2 2.57 + 0.32 11.33 + 0.54*

~Cells were incubated with medium containing 0,3/aM o f [3H]myristate (4,5/aCi/ml) for 20 h and then treated with 0.5/aM of vasopressin in the presence of ethanol (2%) for 5 min. Phospholipase D activity was calcu- lated from the amount of phosphatidylethanol formed. Each set of data represents the mean • standard deviation from four dishes. *p < 0,05.

a corresponding decrease in the labelling of phosphatidylcholine was detected when the phospholipid was labelled with [3H]choline, but not with [3H]glycerol or [3H]myristate. A facile explanation to this apparent discrepancy is that the label in phosphatidyl-[3H]choline after phospholipase D hydrolysis was released into the medium, and resulted in a decrease in the labelling ofphosphatidylcholine.Alternatively, the label in [3H]glycerol or [3H]myristate-labelled phosphatidylcholine would remain in the phosphatidic acid moiety after phospholipase D hydrolysis and this labelled product was not released into the medium. We postulate that a significant portion of the labelled phosphatidic acid would be recycled back into phosphatidylcholine via diacylglycerol through the action of CDP-choline: diacylglycerol choline- phosphotransferase. The ability to convert phosphatidic acid into diacylglycerol by phosphatidate phosphohydrolase during agonist stimulation has been reported in several cell types [4, 5]. Our study illustrates that the ability to remove labelled products from the intracellular compartment is the key factor to reduce isotope recycling, thus allowing the direct de- tection of labelling reduction in phosphatidylcholine during agonist stimulation.

At present, the mechanism for the enhancement of phospholipase D activity by vasopressin is not clear. A number of hormones, including vasopressin, have been shown to cause an increase in intracellular Ca ++ level through the phosphotidylinositol bisphosphate-specific phospholipase C pathway in mammalian tissues and cell types [25, 34-36]. The elevation of intracellular Ca ++ is an important prerequi- site for the activation ofphospholipase D activity, thus pro- viding the cell with a prolonged second messenger via the generation of phosphatidic acid, and subsequently, diacylglycerol [4].

It is interesting to note that the activation ofphospholipase D by vasopressin caused the hydrolysis of phosphatidylcholine but not phosphatidylethanolamine in both cell types. The differential hydrolysis of the two major phospholipids exemplifies that the control of degradation of these two phospholipids are not identical. One plausible explanation to the preferred hydrolysis of phosphatidylcholine is that the activated pbospholipase D is highly specific for this phospholipid. The existence of multiple molecular forms ofphospholipase D with different specificities for various phospholipids have been documented. For example, a phospholipase D with a high degree of specificity on phosphatidylcholine has been demonstrated in several cell types [25, 37-40]. Another form of phospholipase D which is activated by phorbol esters has been shown to display a high specificity towards the hydrolysis ofphosphatidylethanolamine in HL- 60, NIH 3T3 and baby hamster kidney-21 cells [8-10]. Al- ternatively, a partially purified phospholipase D from rat brain has the ability to hydrolyze both phosphatidylcholine and phosphatidylethanolamine [41]. Recently, a cytosolic phos-

75

pholipase D has been identified in various bovine tissues [42] which acts on both phosphatidylcholine and phosphatidylethanolamine but is distinct from the membrane-bound enzyme. The presence of different forms of phospholipase D leads us to conclude that only the phosphatidylcholine- specific form ofphospholipase D was activated in the BHK- 21 and H9c2 cells during vasopressin stimulation.

Acknowledgement

This study was supported by the Heart and Stroke Founda- tion of Manitoba.

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enhancement of phospholipid hydrolysis in vasopressin-stimulated bhk-21 and h9c2 cells

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