BIOCHEMICAL
Vol. 182, No. 3, 1992 February 14, 1992
OKADAIC ACID INHIBITS
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1226-1231
PHOSPHATIDYLETHANOLAMINE IN RAT HEPATOCYTES
BIOSYNTHESIS
Lilian B.M. Tijburg, Pieter S. Vermeulen, Marion G.J. Schmitz and Lambert van Golde’
M.G.
Laboratory of Veterinary Biochemistry, Utrecht University, P.O. Box 80.176,3508 TD Utrecht, The Netherlands Received
December
20,
1991
Okadaic acid, a specific inhibitor of protein phosphatase 1 and 2A, inhibited the synthesis of phosphatidylethanolamine via the CDPethanolamine pathway in isolated hepatocytes. Pulse-chase experiments and measurement of the enzyme activity demonstrated that the inhibition of phosphatidylethanolamine synthesis was not caused by an inhibition of ClP:phosphoethanolamine cytidylyltransferase, the putative regulatory enzyme. However, okadaic acid decreased the cellular diacylglycerol level to 30% of that in control cells. The data suggest that the availability of diacylglycerol limits phosphatidylethanolamine synthesis in okadaic acid-treated hepatocytes. o 1992 Academic ~~~~~~ W.
Okadaic acid is a long-chain polyether fatty acid (for review see ref. l), which was recently shown to be a potent and specific inhibitor
of protein phosphatases 1 and 2A
(2,3). These enzymes are two of the major protein phosphatases that dephosphorylate phosphoserine and phosphothreonine
residues in mammalian
cells (4,5). Okadaic acid
can enter into intact cells and can be used to identity reactions that are controlled reversible protein phosphorylation
in viva Haystead et aL (6) demonstrated
by
that in
isolated hepatocytes and adipocytes, okadaic acid increased the phosphorylation
of a
number of enzymes involved in glucose and lipid metabolism. Biosynthesis of the phospholipids amine
(PE)
can proceed
respectively. Recently,
phosphatidylcholine
via the CDPcholine
Hatch et al. (7) reported
(PC) and phosphatidylethanol-
and CDPethanolamine that okadaic
acid inhibited
To whom correspondence should be addressed. Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolanrine.
l
0006-291X/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1226
pathway, PC
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
biosynthesis in intact hepatocytes. The activity of CIFphosphocholine ferase, an important redistribution
cytidylyltrans-
regulatory enzyme of the pathway, was decreased as a result of a
of the enzyme from the endoplasmic reticulum
to the cytosol. These and
other (8) observations suggested that protein phosphatase 1 and/or 2A are involved in the regulation
of PC biosynthesis.
We previously showed that phorbol esters stimulate PE biosynthesis with a concomitant activation of the key-regulatory
enzyme CIRphosphoethanolamine
ferase (9). This implies that regulation occur via reversible phosphorylation
cytidylyltrans-
of PE synthesis, in analogy to that of PC, may
of mphosphoethanolamine
cytidylyltransferase.
In this study we examined the effect of okadaic acid on PE synthesis via the CDPethanolamine pathway in freshly isolated hepatocytes. METHODS
Ikdafion and ina&a&r of hepatocytes. Hepatocytes were isolated from male Wistar rats (10) and resuspended in Dulbecco’s modified Eagle’s medium supplemented with 10% delipidated (11) fetal calf serum, 2% defatted (12) bovine serum albumin and 0.05 mM ethanolamine. Cells were incubated in 25ml Erlenmeyer flasks (approx. 2.5 mg cell protein/ml) with 2.5 PCi [3H]ethanolamine (Amersham, U.K.) for O-90 min. In pulse-chase experiments hepatocytes were prelabelled with radioactive ethanolamine (125.000 dpm/nmol) for 30 min prior to chase with rmlabelled ethanolamine. Okadaic acid (Boehringer, Mannheim, Germany) was dissolved in dimethylformamide and added at a final concentration of 0.5 ,uM. The concentration of dimethylformamide was 0.4% in control and okadaic acid incubations At the end of the incubation period the cells were harvested by centrifugation and the phospholipids and aqueous ethanolamine metabolites were extracted (10). Labelled ethanolamine, phosphoethanolamine, CDPethanolamine and glycerophosphoethanolamine were separated by thin-layer chromatography on cellulose plates (Merck, Darmstadt, Germany) with ethanol/0.9% NaCl/ammoniumhydroxide (80:10:26, v/v) as the eluent. Analysis of phospholipids has been described previously (13).
E 4~sqys Hepatocytes were incubated in the absence or presence of okadaic acid for 60 min. The cells were harvested by centrifugation and homogenized in 0.5 ml of 0.25 M sucrose, 20 mM Tris HCl (pH 7.4), 1 mM EDTA and 10 mM NaP (10). To isolate the cytosolic fraction the homogenate was centrifuged at 130,000 x g for 20 min in a Beckman airfuge. The activities of ethanolamine kinase and CTP:phosphoethanolamine cytidylyltransferase were determined in the resulting supematant as described previously (13,14). Microsomes were obtained from an aliquot of the homogenized cell suspension and isolated by ultracentrifugation (10). Ethanolaminephosphotransferase activity was assayed in the microsomal fraction with endogenous diacylglycerol as the second substrate (10). Other m&ho&. Diacylglycerol was determined enzymatically with diacylglycerol kinase (Lipidex Inc., Westfield NJ.) (15). Protein levels were estimated by the method of Lowry et aL (16). Student’s r-test was used for the determination of significance. 1227
Vol.
182,
No.
3, 1992
RESULXS
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND DISCUSSION
Okxadaic add inhibits
PE synhesir in hepatocyttx The effects of okadaic acid on PE
synthesis were studied in rat hepatocytes in suspension. The incorporation glycerol into PE was inhibited
of [1(3)-3H]-
by 50% in okadaic acid-treated cells (not shown). While
PE biosynthesis from [3H]ethanolamine
was linear for almost 90 min in control cells, the
formation of PE in cells incubated with okadaic acid levelled off after 30 min (Fig. 1A). After 90 min the radioactivity
associated with PE in okadaic acid-treated
cells was
decreased by 52.5 -c 3.6% (n=3) as compared to controls. The biosynthesis of rH]PC was also inhibited,
probably as a result of the diminished
(Fig. 1A). Labelling
of ethanolamine,
(not shown) and phosphoethanolamine
CDPethanolamine,
labelling
of its precursor PE
glycerophosphoethanolamine
(Fig. 1B) was comparable
between control and
okadaic acid incubations. The observed changes in ethanolamine-labelled uptake
of ethanolamine
PE may have been due to an altered
or to isotope dilution
in okadaic acid-treated
cells. Thus, the
effect of okadaic acid was further evaluated in pulse-chase experiments. prelabelled
with [3H]ethanolamine
Cells were
and subsequently, chased in the absence or presence
of okadaic acid. Fig. 2 shows that the appearance of label in ethanolamine-labelled phospholipids Although
(PE + PC) was significantly
it is noteworthy
okadaic acid-treated
delayed in the presence of okadaic acid.
that the amount
of radioactive
phosphoethanolamine
cells was slightly higher than in control incubations
disappearance of labelled phosphoethanolaririne
in okadaic acid-treated
in
(Fig. 2), the cells was not
6
. B 5 -
0
20
40
60
so
100
Time
0
20
40
60
80
(mid
F&J. Effect of okadaic acid on the incorporation of [3H]ethanolamine into PE, PC (A) and phosphoethanolamine (B). Hepatocytes were incubated for up to 90 min in the absence (open symbols) or presence (filled symbols) of okadaic acid. Values represent the mean of duplicate incubations of one representative experiment out of three. (0 q, PE, (A& PC, (0 l ), phosphoethanolamine. 1228
100
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
.5 8 g if & a z
LO -
*
0,5-
E 0.0
0
2
‘0
20
40
60
80
time (mid
0
3
E&J. Pulse-chase study of the effects of okadaic acid on the metabolism of [‘H]ethanolamine. Cells were pulsed with [3H]ethanolamine and, subsequently, chased in the absence (open symbols) or presence (closed symbols) of okadaic acid. Values are the mean of duplicate incubations of one representative experiment out of three. (0 W), PE + PC; (0 l ), phosphoethanolamine. &J. Effect of okadaic acid on diacylglycerol levels in hepatocytes. Hepatocytes were incubated for 60 min in the absence (open bar) or presence of 0.5 PM okadaic acid (hatched bar). Diacylglycerol was determined as described in Materials and Methods. Data are the mean (f S.E.M.) of 4 independent cell experiments. * P < 0.05.
statistically significant from control incubations. acid does not significantly
These observations
indicate that okadaic
to CDPethanol-
affect the conversion of phosphoethanolamine
amine.
Effzct of okxuiai? acid on the enzymes trvolved in PE synthesis hepatocytes
with
of
okadaic acid for 60 rnin did not affect the activity of ethanolamine
kinase, nor that of etbartolaminephosphotransferase
did not alter the activity of phosphoethanolamine line with the observation
Table 1.
Treatment
(Table 1). Surprisingly,
cytidylyltransferase
that in pulse-chase experiments
okadaic acid
either. This is in
the disappearance
Effect of okadaic acid on the activaties of the enzymes of the CDPethanolamine path
Enzyme
control
okadaic acid nmol/min per mg protein
Ethanolamine kinase Phosphoethanolamine cytidylyltransferase Ethanolaminephosphotransferase
0.91 * 0.25 3.51 2 0.33
0.95 + 0.35 3.35 f 0.34
0.32 + 0.05
0.38 f 0.16
Data are presented as the mean r S.D. of 3 independent cell experiments. 1229
of
Vol.
182,
No.
3, 1992
BIOCHEMICAL
phosphoethanolamine
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
was similar in okadaic acid-treated cells and in controls (Fig. 2).
Hatch et aL (17) reported that okadaic acid inhibited of rat-liver phosphocholine
cytidylyltransferase
the time-dependent
activation
(18) in cytosol. In comparable
experi-
ments we examined the effect of okadaic acid on the activity of phosphoethanolamine cytidylyltransferase.
Although
incubation
okadaic acid resulted in an inhibition
of postmitochondrial
supematant
with 5 PM
of the enzyme activity by 17 f 11% (n=3), this
effect was not significant. Taken collectively, our data suggest that phosphoethanohun.ine cytidylyltransferase,
in contrast to phosphocholine
cytidylyltransferase
(7,17), is not
regulated by reversible phosphorylation. We previously showed (13) that the inhibitory
effect of glucagon on de nova PE
synthesis was due to a diminished supply of diacylglycerol, causing a decreased formation of PE from CDPethanolamine
and diaqlglycerol.
okadaic acid did not affect the formation
As our present studies suggested that
of CDPethanolamine,
we examined the effect
of okadaic acid on the cellular concentration of diacylglycerol. Treatment with okadaic acid for 60 min significantly decreased the diacylglycerol
of hepatocytes level by a factor
of 3.5 (Fig. 3). It has been reported that okadaic acid greatly inhibits fatty acid synthesis and stimulates lipolysis in adipocytes (6). It is plausible that this results in a significant decrease of the amount
of cellular
diacylglycerol,
which may in turn limit
PE
biosynthesis. The role of diacylglycerol was further explored in pulse-label experiments with a low concentration
of [3H]ethanolamine
(0.005 mM). Under these conditions the rate of PE
synthesis from ethanolamine
is relatively
incorporation
into PE at an ethanolamine
of ethanolamine
(data not shown). Apparently,
low (9). Okadaic
at low ethanolamine
acid did not affect the
concentration
concentration
of 0.005 mM
the amount
of
diacylglycerol in okadaic acid-treated cells was sufficient to maintain PE synthesis at the same level as in control cells. These data and those presented in Figs. 1 and 2, indicate that the concentration
of diacylglycerol
in okadaic acid-treated cells seems to limit PE
biosynthesis. In conclusion, we demonstrated that okadaic acid inhibits de nova PE synthesis. This inhibition
is not caused by a concomitant
cytidylyltransferase,
inhibition
but is probably due to a diminished
of CTP:phosphoethanolamine supply of diacylglycerol.
Acknowledgments These investigations were supported in part by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO). The research by LT. was made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences. 1230
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Cohen, P., Holmes, C.F.B. and Tsukitani, Y. (1990) Trends B&hem. Sci. 15,98102 Bialojan, C. and Takai, A. (1988) Biochem. J. 256, 283-290 Herchler, J., Miesler, G., Ruegg, J.C., Takai, A. and Trautwein, W. (1988) Weugers Arch. Ges. Physiol. 412, 248-252 Cohen, P. (1989) Armu. Rev. Biochem. 58,453-508 Cohen, P. and Cohen, P.T.W. (1989) J. Biol. Chem. 264,21435-21438 Haystead, T&J., Sim, A.T.R., Carling, D., Honnor, R.C., Tsukitani, Y., Cohen, P. and Hardie, D.G. (1989) Nature 337, 78-81 Hatch, G.M., Tsukitani, Y. and Vance, D.E. (1991) Biochim. Biophys. Acta 1081, 25-32 Sanghera, J.S. and Vance, D.E. (1989) J. Biol. Chem. 264, 1215-1223 Tijburg, L.B.M., Houweling, M., Geelen, MJ.H. and Van Golde, LM.G. (1987) Biochim. Biophys. Acta 922, 184-190 Tijburg, LB.M., Schuurmans, E.A.J.M., Geelen, M J.H. and Van Golde, LM.G. (1987) Biochim. Biophys. Acta 919, 49-57 Cham, B.E. and Knowles, B.R. (1976) J. Lipid. Res. 17, 176-181 Chen, R.F. (1967) J. Biol. Chem. 242, 173-181 Tijburg, L.B.M., Houweling, M., Geelen, MJ.H. and Van Golde, LM.G. (1989) Biochem. J. 257, 645-650 Weinhold, PA. and Rethy, V.B. (1974) Biochemistry 13, 5135-5141 Preiss, J., Loomis, C.R., Bishop, W.R., Stein, R., Niedel, J.E. and Bell, R.M. (1986) J. Biol. Chem. 261, 8597-8600 Lowry, O.H., Rosebrough, NJ., Farr, AL and Randall, RJ. (1951) J. Biol. Chem. 193, 265-275 Hatch, G.M., Lam, T-S., Tsukitani, Y. and Vance, D.E. (1990) Biochim. Biophys. Acta 1042,374-379 Pelech, S.L. and Vance, D.E. (1982) J. Biol. Chem. 257, 14198-14202
1231
Vol. 182, No. 3, 1992 February 14, 1992
OKADAIC ACID INHIBITS
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1226-1231
PHOSPHATIDYLETHANOLAMINE IN RAT HEPATOCYTES
BIOSYNTHESIS
Lilian B.M. Tijburg, Pieter S. Vermeulen, Marion G.J. Schmitz and Lambert van Golde’
M.G.
Laboratory of Veterinary Biochemistry, Utrecht University, P.O. Box 80.176,3508 TD Utrecht, The Netherlands Received
December
20,
1991
Okadaic acid, a specific inhibitor of protein phosphatase 1 and 2A, inhibited the synthesis of phosphatidylethanolamine via the CDPethanolamine pathway in isolated hepatocytes. Pulse-chase experiments and measurement of the enzyme activity demonstrated that the inhibition of phosphatidylethanolamine synthesis was not caused by an inhibition of ClP:phosphoethanolamine cytidylyltransferase, the putative regulatory enzyme. However, okadaic acid decreased the cellular diacylglycerol level to 30% of that in control cells. The data suggest that the availability of diacylglycerol limits phosphatidylethanolamine synthesis in okadaic acid-treated hepatocytes. o 1992 Academic ~~~~~~ W.
Okadaic acid is a long-chain polyether fatty acid (for review see ref. l), which was recently shown to be a potent and specific inhibitor
of protein phosphatases 1 and 2A
(2,3). These enzymes are two of the major protein phosphatases that dephosphorylate phosphoserine and phosphothreonine
residues in mammalian
cells (4,5). Okadaic acid
can enter into intact cells and can be used to identity reactions that are controlled reversible protein phosphorylation
in viva Haystead et aL (6) demonstrated
by
that in
isolated hepatocytes and adipocytes, okadaic acid increased the phosphorylation
of a
number of enzymes involved in glucose and lipid metabolism. Biosynthesis of the phospholipids amine
(PE)
can proceed
respectively. Recently,
phosphatidylcholine
via the CDPcholine
Hatch et al. (7) reported
(PC) and phosphatidylethanol-
and CDPethanolamine that okadaic
acid inhibited
To whom correspondence should be addressed. Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolanrine.
l
0006-291X/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1226
pathway, PC
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
biosynthesis in intact hepatocytes. The activity of CIFphosphocholine ferase, an important redistribution
cytidylyltrans-
regulatory enzyme of the pathway, was decreased as a result of a
of the enzyme from the endoplasmic reticulum
to the cytosol. These and
other (8) observations suggested that protein phosphatase 1 and/or 2A are involved in the regulation
of PC biosynthesis.
We previously showed that phorbol esters stimulate PE biosynthesis with a concomitant activation of the key-regulatory
enzyme CIRphosphoethanolamine
ferase (9). This implies that regulation occur via reversible phosphorylation
cytidylyltrans-
of PE synthesis, in analogy to that of PC, may
of mphosphoethanolamine
cytidylyltransferase.
In this study we examined the effect of okadaic acid on PE synthesis via the CDPethanolamine pathway in freshly isolated hepatocytes. METHODS
Ikdafion and ina&a&r of hepatocytes. Hepatocytes were isolated from male Wistar rats (10) and resuspended in Dulbecco’s modified Eagle’s medium supplemented with 10% delipidated (11) fetal calf serum, 2% defatted (12) bovine serum albumin and 0.05 mM ethanolamine. Cells were incubated in 25ml Erlenmeyer flasks (approx. 2.5 mg cell protein/ml) with 2.5 PCi [3H]ethanolamine (Amersham, U.K.) for O-90 min. In pulse-chase experiments hepatocytes were prelabelled with radioactive ethanolamine (125.000 dpm/nmol) for 30 min prior to chase with rmlabelled ethanolamine. Okadaic acid (Boehringer, Mannheim, Germany) was dissolved in dimethylformamide and added at a final concentration of 0.5 ,uM. The concentration of dimethylformamide was 0.4% in control and okadaic acid incubations At the end of the incubation period the cells were harvested by centrifugation and the phospholipids and aqueous ethanolamine metabolites were extracted (10). Labelled ethanolamine, phosphoethanolamine, CDPethanolamine and glycerophosphoethanolamine were separated by thin-layer chromatography on cellulose plates (Merck, Darmstadt, Germany) with ethanol/0.9% NaCl/ammoniumhydroxide (80:10:26, v/v) as the eluent. Analysis of phospholipids has been described previously (13).
E 4~sqys Hepatocytes were incubated in the absence or presence of okadaic acid for 60 min. The cells were harvested by centrifugation and homogenized in 0.5 ml of 0.25 M sucrose, 20 mM Tris HCl (pH 7.4), 1 mM EDTA and 10 mM NaP (10). To isolate the cytosolic fraction the homogenate was centrifuged at 130,000 x g for 20 min in a Beckman airfuge. The activities of ethanolamine kinase and CTP:phosphoethanolamine cytidylyltransferase were determined in the resulting supematant as described previously (13,14). Microsomes were obtained from an aliquot of the homogenized cell suspension and isolated by ultracentrifugation (10). Ethanolaminephosphotransferase activity was assayed in the microsomal fraction with endogenous diacylglycerol as the second substrate (10). Other m&ho&. Diacylglycerol was determined enzymatically with diacylglycerol kinase (Lipidex Inc., Westfield NJ.) (15). Protein levels were estimated by the method of Lowry et aL (16). Student’s r-test was used for the determination of significance. 1227
Vol.
182,
No.
3, 1992
RESULXS
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND DISCUSSION
Okxadaic add inhibits
PE synhesir in hepatocyttx The effects of okadaic acid on PE
synthesis were studied in rat hepatocytes in suspension. The incorporation glycerol into PE was inhibited
of [1(3)-3H]-
by 50% in okadaic acid-treated cells (not shown). While
PE biosynthesis from [3H]ethanolamine
was linear for almost 90 min in control cells, the
formation of PE in cells incubated with okadaic acid levelled off after 30 min (Fig. 1A). After 90 min the radioactivity
associated with PE in okadaic acid-treated
cells was
decreased by 52.5 -c 3.6% (n=3) as compared to controls. The biosynthesis of rH]PC was also inhibited,
probably as a result of the diminished
(Fig. 1A). Labelling
of ethanolamine,
(not shown) and phosphoethanolamine
CDPethanolamine,
labelling
of its precursor PE
glycerophosphoethanolamine
(Fig. 1B) was comparable
between control and
okadaic acid incubations. The observed changes in ethanolamine-labelled uptake
of ethanolamine
PE may have been due to an altered
or to isotope dilution
in okadaic acid-treated
cells. Thus, the
effect of okadaic acid was further evaluated in pulse-chase experiments. prelabelled
with [3H]ethanolamine
Cells were
and subsequently, chased in the absence or presence
of okadaic acid. Fig. 2 shows that the appearance of label in ethanolamine-labelled phospholipids Although
(PE + PC) was significantly
it is noteworthy
okadaic acid-treated
delayed in the presence of okadaic acid.
that the amount
of radioactive
phosphoethanolamine
cells was slightly higher than in control incubations
disappearance of labelled phosphoethanolaririne
in okadaic acid-treated
in
(Fig. 2), the cells was not
6
. B 5 -
0
20
40
60
so
100
Time
0
20
40
60
80
(mid
F&J. Effect of okadaic acid on the incorporation of [3H]ethanolamine into PE, PC (A) and phosphoethanolamine (B). Hepatocytes were incubated for up to 90 min in the absence (open symbols) or presence (filled symbols) of okadaic acid. Values represent the mean of duplicate incubations of one representative experiment out of three. (0 q, PE, (A& PC, (0 l ), phosphoethanolamine. 1228
100
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
.5 8 g if & a z
LO -
*
0,5-
E 0.0
0
2
‘0
20
40
60
80
time (mid
0
3
E&J. Pulse-chase study of the effects of okadaic acid on the metabolism of [‘H]ethanolamine. Cells were pulsed with [3H]ethanolamine and, subsequently, chased in the absence (open symbols) or presence (closed symbols) of okadaic acid. Values are the mean of duplicate incubations of one representative experiment out of three. (0 W), PE + PC; (0 l ), phosphoethanolamine. &J. Effect of okadaic acid on diacylglycerol levels in hepatocytes. Hepatocytes were incubated for 60 min in the absence (open bar) or presence of 0.5 PM okadaic acid (hatched bar). Diacylglycerol was determined as described in Materials and Methods. Data are the mean (f S.E.M.) of 4 independent cell experiments. * P < 0.05.
statistically significant from control incubations. acid does not significantly
These observations
indicate that okadaic
to CDPethanol-
affect the conversion of phosphoethanolamine
amine.
Effzct of okxuiai? acid on the enzymes trvolved in PE synthesis hepatocytes
with
of
okadaic acid for 60 rnin did not affect the activity of ethanolamine
kinase, nor that of etbartolaminephosphotransferase
did not alter the activity of phosphoethanolamine line with the observation
Table 1.
Treatment
(Table 1). Surprisingly,
cytidylyltransferase
that in pulse-chase experiments
okadaic acid
either. This is in
the disappearance
Effect of okadaic acid on the activaties of the enzymes of the CDPethanolamine path
Enzyme
control
okadaic acid nmol/min per mg protein
Ethanolamine kinase Phosphoethanolamine cytidylyltransferase Ethanolaminephosphotransferase
0.91 * 0.25 3.51 2 0.33
0.95 + 0.35 3.35 f 0.34
0.32 + 0.05
0.38 f 0.16
Data are presented as the mean r S.D. of 3 independent cell experiments. 1229
of
Vol.
182,
No.
3, 1992
BIOCHEMICAL
phosphoethanolamine
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
was similar in okadaic acid-treated cells and in controls (Fig. 2).
Hatch et aL (17) reported that okadaic acid inhibited of rat-liver phosphocholine
cytidylyltransferase
the time-dependent
activation
(18) in cytosol. In comparable
experi-
ments we examined the effect of okadaic acid on the activity of phosphoethanolamine cytidylyltransferase.
Although
incubation
okadaic acid resulted in an inhibition
of postmitochondrial
supematant
with 5 PM
of the enzyme activity by 17 f 11% (n=3), this
effect was not significant. Taken collectively, our data suggest that phosphoethanohun.ine cytidylyltransferase,
in contrast to phosphocholine
cytidylyltransferase
(7,17), is not
regulated by reversible phosphorylation. We previously showed (13) that the inhibitory
effect of glucagon on de nova PE
synthesis was due to a diminished supply of diacylglycerol, causing a decreased formation of PE from CDPethanolamine
and diaqlglycerol.
okadaic acid did not affect the formation
As our present studies suggested that
of CDPethanolamine,
we examined the effect
of okadaic acid on the cellular concentration of diacylglycerol. Treatment with okadaic acid for 60 min significantly decreased the diacylglycerol
of hepatocytes level by a factor
of 3.5 (Fig. 3). It has been reported that okadaic acid greatly inhibits fatty acid synthesis and stimulates lipolysis in adipocytes (6). It is plausible that this results in a significant decrease of the amount
of cellular
diacylglycerol,
which may in turn limit
PE
biosynthesis. The role of diacylglycerol was further explored in pulse-label experiments with a low concentration
of [3H]ethanolamine
(0.005 mM). Under these conditions the rate of PE
synthesis from ethanolamine
is relatively
incorporation
into PE at an ethanolamine
of ethanolamine
(data not shown). Apparently,
low (9). Okadaic
at low ethanolamine
acid did not affect the
concentration
concentration
of 0.005 mM
the amount
of
diacylglycerol in okadaic acid-treated cells was sufficient to maintain PE synthesis at the same level as in control cells. These data and those presented in Figs. 1 and 2, indicate that the concentration
of diacylglycerol
in okadaic acid-treated cells seems to limit PE
biosynthesis. In conclusion, we demonstrated that okadaic acid inhibits de nova PE synthesis. This inhibition
is not caused by a concomitant
cytidylyltransferase,
inhibition
but is probably due to a diminished
of CTP:phosphoethanolamine supply of diacylglycerol.
Acknowledgments These investigations were supported in part by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO). The research by LT. was made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences. 1230
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
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