Biochimica et Biophysics Acta 876 (1986) 581-591 Elsevier 581 BBA 52231 Relationship between the displacement of phosphatidate phosphohydrolase from the membrane-associated compartment by chlorpromazine and the inhibition of the synthesis of triacylglycerol and phosphatidylcholine in rat hepatocytes Ashley Martin, Roger Hopewell, Paloma Martin-Sanz *, Janette E. Morgan and David N. Brindley ** Department of Biochemistry, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham NG7 2 UH (U.K.) (Received December 12th, 1985) Key words: Chlorpromazine; t-a-Phosphatidate phosphohydrolase; Phosphatidylcholine synthesis; Enzyme translocation; Triacylglycerol synthesis; (Liver) 1. Glycerolipid synthesis was studied in isolated hepatocytes by using 177 PM [ 14C]oleate and 1 mM [3H]glycerol. Chlorpromazine (25-400 FM) inhibited the synthesis of phosphatidylcholine and tri- acylglycerol. This was accompanied by an average increase of 1Zfold in the accumulation of the labelled precursors in phosphatidate at 200 PM chlorpromazine and a decrease in the conversion of phosphatidate to diacylglycerol of 76%. These results indicate that part of the inhibition of the synthesis of phosphatidylcho- line and triacylglycerol occurs at the level of phosphatidate phosphohydrolase. 2. The relative rate of triacylglycerol synthesis at different concentrations of chlorpromazine was approximately proportional to the rate of conversion of phosphatidate to diacylglycerol. Phosphatidylcholine synthesis increased at higher rates of conversion of phosphatidate to diacylglycerol, but it was relatively independent of the latter rate when this was inhibited by more than about 30% with chlorpromazine. 3. The addition of oleate to the hepatocytes caused a translocation of phosphatidate phosphohydrolase from the cytosol to the membrane-associated compartment. Chlorpromazine had the opposite effect and displaced the phosphohydrolase from the membranes in the presence or absence of oleate. 4. There was a highly significant correlation between the activity of phosphatidate phosphohydrolase that was associated with the membranes of the hepatocytes and the calculated conversion of [3H]phosphatidate to diacylglycerol. 5. Chlorpromazine also antagonized the association of the phosphohydrolase with microsomal membranes when cell-free preparations were incubated with combinations of oleate and spennine. Furthermore, it inhibited the transfer of the soluble phos- phohydrolase to microsomal membranes that were labelled with [ “C]phosphatidate and thereby decreased diacylglycerol production. 6. It is concluded that part of the action of chlorpromazine in inhibiting the synthesis of triacylglycerol and phosphatidylcholine occurs because it prevents the interaction of the soluble phosphatidate phosphohydrolase with the membranes on which glycerolipid synthesis occurs. This in turn prevents the conversion of phosphatidate to diacylglycerol. * Permanent address: Instituto de Bioquimica, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 3, Spain. ** To whom correspondence should be addressed. Abbreviation: Hepes, 4-(2-hydroxyethyl)-l-piperazineethane- sulphonic acid. Introduction Phosphatidate phosphohydrolase participates in the control of hepatic glycerolipid synthesis and its activity is regulated by both acute and long-term 0005-2760/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)
Transcript
Biochimica et Biophysics Acta 876 (1986) 581-591
Elsevier
581
BBA 52231
Relationship between the displacement of phosphatidate phosphohydrolase from
the membrane-associated compartment by chlorpromazine and the inhibition of
the synthesis of triacylglycerol and phosphatidylcholine in rat hepatocytes
Ashley Martin, Roger Hopewell, Paloma Martin-Sanz *, Janette E. Morgan and David N. Brindley **
Department of Biochemistry, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham NG7 2 UH
1. Glycerolipid synthesis was studied in isolated hepatocytes by using 177 PM [ 14C]oleate and 1 mM [3H]glycerol. Chlorpromazine (25-400 FM) inhibited the synthesis of phosphatidylcholine and tri- acylglycerol. This was accompanied by an average increase of 1Zfold in the accumulation of the labelled precursors in phosphatidate at 200 PM chlorpromazine and a decrease in the conversion of phosphatidate to diacylglycerol of 76%. These results indicate that part of the inhibition of the synthesis of phosphatidylcho- line and triacylglycerol occurs at the level of phosphatidate phosphohydrolase. 2. The relative rate of triacylglycerol synthesis at different concentrations of chlorpromazine was approximately proportional to the rate of conversion of phosphatidate to diacylglycerol. Phosphatidylcholine synthesis increased at higher rates of conversion of phosphatidate to diacylglycerol, but it was relatively independent of the latter rate when this was inhibited by more than about 30% with chlorpromazine. 3. The addition of oleate to the hepatocytes caused a translocation of phosphatidate phosphohydrolase from the cytosol to the membrane-associated compartment. Chlorpromazine had the opposite effect and displaced the phosphohydrolase from the membranes in the presence or absence of oleate. 4. There was a highly significant correlation between the activity of phosphatidate phosphohydrolase that was associated with the membranes of the hepatocytes and the calculated conversion of [3H]phosphatidate to diacylglycerol. 5. Chlorpromazine also antagonized the association of the phosphohydrolase with microsomal membranes when cell-free preparations were incubated with combinations of oleate and spennine. Furthermore, it inhibited the transfer of the soluble phos- phohydrolase to microsomal membranes that were labelled with [ “C]phosphatidate and thereby decreased diacylglycerol production. 6. It is concluded that part of the action of chlorpromazine in inhibiting the synthesis of triacylglycerol and phosphatidylcholine occurs because it prevents the interaction of the soluble phosphatidate phosphohydrolase with the membranes on which glycerolipid synthesis occurs. This in turn prevents the conversion of phosphatidate to diacylglycerol.
* Permanent address: Instituto de Bioquimica, Facultad de
Farmacia, Universidad Complutense de Madrid, Madrid 3,
Spain. ** To whom correspondence should be addressed.
mechanisms [l]. The rate of synthesis of phos- phatidate phosphohydrolase is increased by gluco- corticoids, glucagon and cyclic AMP [2,3]. Insulin antagonizes the effects of glucocorticoids and glucagon in increasing phosphatidate phos- phohydrolase activity in hepatocytes [2,3]. Acutely, phosphatidate phosphohydrolase activity can be increased by vasopressin [4], and the presence of high concentrations of fatty acids [5]. However, the most dramatic effect of long-chain fatty acids and their acyl-CoA esters is to promote the trans- location of phosphatidate phosphohydrol~e from the cytosol to the membranes on which glyceroli- pid synthesis occurs [1,5-71. The action of fatty acids in this respect in liver appears to be aug- mented at least in vitro by sperrnine [S]. The effect could also explain why spermine increases the rnicrosomal phosphatidate phosphohydrolase ac- tivity in preparations of adipose tissue [9,10]. Cyclic AMP analogues are also able to modify acutely the subcellular distribution of phosphati- date phosphohydrolase in hepatocytes. When ad- ded to isolated hepatocytes in the absence of additional fatty acid, they displace phosphatidate phosphohydrolase from the membranes whereas 1 mM oleate reverses this effect [6]. These events are reflected in the rate of glycerolipid synthesis, since oleate also reverses the cyclic AMP-induced in- hibition of the synthesis of triacylglycerols and phosphatidylcholine [ll]. It is therefore believed that the cytosolic phosphatidate phosphohydro- lase represents a functionally inactive reservoir that can become metabolically active when trans- locatidn to the membranes on which glycerolipid synthesis occurs. This enables cells to respond to an increased fatty acid supply by increasing their rate of triacylglycerol synthesis [l].
The present work was undertaken to determine whether an amphiphilic amine, chlorpromazine, might interfere with the translocation process and thereby modify glycerolipid synthesis. It is known that amphiphilic amines redirect glycerolipid synthesis away from the formation of triacylglyc- erols, phosphatidylcholine and phosphatidyl- ethanolamine, and towards that of phosphati- dylinositol, phosphatidylglycerol and diphos- phatidylglycerol (1,121. Part of this results from changes in phosphatidate metabolism, since chlor- promazine can inhibit phosphatidate phosphohy-
drolase while simultaneously stimulating the activ- ity of phosphatidate cytidylyltransferase [1,13,14].
The present work examines whether chlor- promazine can decrease the rate of synthesis of phosphatidylcholine and triacylglycerol by block- ing the fatty acid-induced translocation of phos- phatidate phosphohydrolase from the cytosol to the endoplasmic reticulum.
Materials and Methods
~uteriu~~ and animals. Unless stated to the contrary the sources of the materials have been described previously [4,5,8]. [ 3H]Phosphatidate was synthesized [15] essentially as described previ- ously by using 0.2 mM ~3H]pal~tate (50 Ci/ mmol) and then diluting to 0.4 Ci/mol with non- radioactive potassium phosphatidate. Chlorprom- azine HCl (Largactil) was a gift from May and Baker Ltd, Dagenham, U.K.
Preparation and incubation of hepatocytes. Monolayer cultures of rat hepatocytes were pre- pared and cells were incubated for about 20 h with three changes of modified Leibovitz-L15 medium that contained 10% (v/v) newborn calf serum and 28.6 PM choline chloride [5]. Primaria tissue culture dishes (Falcon) from Becton Dickin- son U.K. Ltd, Cowley, Oxford, U.K., were used instead of collagen-coated tissue culture dishes [3]. In some experiments the rate of glycerolipid synthesis was measured by replacing the medium with fresh medium containing [ 3H]glycerol (1.42 Ci/mol) and (‘4C]oleate (1 Ci/mol) as indicated. After incubating at 37°C for a further 15 min the medium was removed and the cells were sus- pended in 1 ml of ice-cold 0.25 M sucrose that contained 0.5 mM dithiothreitol and 10 mM Hepes, and which was adjusted to pH 7.4 with KOH. In other experiments unlabelled oleate and glycerol were added and the distribution of phos- phatidate phosphohydrolase between the cytosol and the membrane-associated comp~tment was determined by lysing the cells for 4 min with 0.5 mg of digitonin/ml in 0.25 M sucrose containing 0.5 mM dithiothreitol and 10 mM Hepes adjusted to pH 7.4 with KOH [5]. Results are expressed relative to lactate dehydrogenase activity to com- pensate for the number of viable cells on each dish 1~~31.
583
Translocation of phosphatidate phosphohydrolase in cell-free systems from rat liver. The supematant obtained from rat liver homogenates after centri- fuging for 18000 X g (r,,,,, = 10.7 cm) for 10 min
at 4°C was incubated with various combinations
of chlorpromazine, oleate and spermine for 10 min
at 37°C [7,8]. The microsomal and soluble frac-
tions were collected after centrifuging at 90 000 X g
(raV = 6.3 cm) for 90 min at 4°C [7].
In other experiments the translocation of phos-
phatidate phosphohydrolase onto microsomal
membranes that had been labelled with [i4C]phos-
phatidate was determined. These membranes were
prepared by incubating a mixture containing 16.6
mM potassium phosphate buffer (pH 7.4) 1 mM
dithiothreitol, 50 mM NaF, 13.4 mM MgCl,, 8
mM ATP, 20 mM rat-glycerol 3 phosphate, 25
PM CoA, 50 PM potassium[i4C]oleate (0.5
Ci/mol; prepared by warming oleic acid with a
20% molar excess of KOH), 3 mg of fatty acid-poor
bovine serum albumin/ml and 4 mg of micro-
somal protein/ml. The incubation was for 30 min
at 37°C and was stopped by cooling to 4°C. The
mixture was then layered over 0.25 M sucrose
containing 0.2 mM dithiothreitol/(pH 7.4) and
centrifuged for 90 min at 4°C and 76000 X g
(raV = 7.62 cm). The pellets which contained the
membranes labelled with newly synthesized phos- phatidate were resuspended in 0.25 M sucrose
containing 0.2 mM dithiothreitol and stored at
- 20°C.
Analytical methods. The methods for de-
termining the synthesis of glycerolipids [16], and
the activity of phosphatidate phosphohydrolase in
hepatocytes [4] and in cell-free preparations [7]
were essentially those described previously. In the
phosphatidate phosphohydrolase assays shown in
Fig. 3 the Tris buffer was replaced by 100 mM
potassium phosphate (pH 7.4) since this was shown to increase phosphatidate phosphohydro- lase activities [17].
Results
Effects of chlorpromazine on the incorporation of [3H]glycerol and [‘4C]oleate into glycerolipids by rat hepatocytes
Monolayer cultures of hepatocytes were in- cubated with 1 mM [3H]glycerol and 177 PM
[i4C]oleate for 15 min in order to measure the
initial rate of glycerolipid synthesis. This enabled
us to determine simultaneously whether chlor-
promazine had a different effect on the incorpora-
tion of these two precursors into glycerolipids. In
order to illustrate results from different experi-
ments on the same graph, the incorporations have
been expressed in relative terms (Fig. 1). The
addition of chlorpromazine, even at 400 PM, did
not appear to affect the integrity of the hepato-
cytes over the 15 min incubation period as judged
by the retention of the cytosolic marker, lactate
dehydrogenase, by the cells.
Chlorpromazine progressively decreased the in-
corporation of [ 3H]glycerol and [i4C]oleate into
the total lipid fraction (Fig. lA, C). At 400 PM
chlorpromazine, these inhibitions were 31 f 5 and
29 + 4 (means f S.E. from five independent ex-
periments), respectively. However, these inhibi-
tions were less dramatic than the changes in the
incorporations of the precursors into the individ-
ual lipid fractions.
Chlorpromazine produced a dose-dependent
decrease in the synthesis of triacylglycerol from
[ 3H]glycerol and [‘4C]oleate which reached 99 and
88%, respectively, at 400 PM chlorpromazine (Fig.
lB, D). There was also marked inhibition of the
synthesis of phosphatidylcholine when the con- centration of chlorpromazine was increased to 50
PM but from 50-400 I_IM there was little further
change (Fig. lB, D). The accumulation of [3H]-
glycerol and [14C]oleate in diacylglycerol ap-
parently increased by 11 (0.2 > P > 0.1) and 15%
(P < 0.02), respectively, at 25 PM chlorpromazine
but there were progressive decreases in these accu-
mulations as these concentrations increased from
50 to 400 PM. By contrast, the accumulation of
[3H]glycerol and [i4C]oleate in phosphatidate in- creased as chlorpromazine was added. At 200-400
PM chlorpromazine 76-84% of the 3H and 64-74%
of the i4C that was incorporated into glycerolipids was present in phosphatidate (Fig. lA, C).
These results indicate that the inhibition in the
synthesis of triacylglycerol and phosphatidylcho- line that is produced by chlorpromazine could be explained by a decreased activity of phosphatidate phosphohydrolase. This decrease was estimated by calculating the flux from phosphatidate to di- acylglycerol. To achieve this, the incorporations of
Total lipid
B
I I I I I 0 100 200 300 4w
Fig. 1. Effect of chlorpromazine on the
synthesis of glycerolipids by isolated rat
hepatocytes. Hepatocytes were incubated for
15 min at 37°C with 1 mM [1,3-3H]glycerol
and 177 PM [‘4C]oleate in the presence or
absence of chlorpromazine (see Materials and
Methods section). The relative incorporations
of [ 3H]glycerol (Fig. A, B) and [t4C]oleate
(Fig. C, D) into the total esterified lipid
fraction (A), phosphatidate (0) diacylglycerol
(A), triacylglycerol (0) and phosphatidylcho-
line (0) are shown. The incorporations are
all expressed relative to the incorporation of
[ ‘HIglycerol or [‘4C]oleate into the total lipid
fraction in incubations that did not contain
chlorpromazine. These values were 6.02 f 0.02
nmol of glycerol and 10.85 k2.03 nmol of
oleate incorporated per U of lactate dehydro-
genase present in the cells. The incorpora-
tions into phosphatidylethanolamine were less
than 1% of these values and the results have
been omitted from the figure. The relative
rate of conversion of phosphatidate to di-
acylglycerol (m) was estimated by summing
the 3H recovered in diacylglycerol, phos-
phatidylethanolamine, phosphatidylchohne
and triacylglycerol and this is shown in A.
Each point shown in the figures is the mean
* S.E. from five independent experiments and
the values from the individual experiments
are the means from triplicate dishes.
[ 3H]glycerol into diacylglycerol, phosphatidyletha-
nolamine, phosphatidylcholine and triacylglycerol
were added together. Fig. 1A shows that conver- sion was in fact progressively inhibited by chlor-
promazine. Estimates of the rate of conversion of phosphatidate to diacylglycerol are not shown for
the incorporations of [‘4C]oleate, since assump- tions would have to be made about the stoichiom-
etry of the relative incorporation of oleate into the various lipids. However, the results in Fig. 1C and D also clearly demonstrate that the conversion of [‘4C]phosphatidate to diacylglycerol was markedly decreased.
The relative rate of triacylglycerol synthesis at different concentrations of chlorpromazine was approximately proportional to the rate of conver- sion of phosphatidate to diacylglycerol (Fig. 2). By contrast, phosphatidylcholine synthesis increased
at higher rates of conversion of phosphatidate to
diacylglycerol but it was relatively independent of
this latter rate when this was inhibited by more than about 30% with chlorpromazine.
Effects of chlorpromazine in displacing phosphati- date phosphohydrolase from the membrane-bound to the cytosolic compartment of rat hepatocytes
This was investigated in parallel incubations to those described in Fig. 1. Translocation of phos- phatidate phosphohydrolase to the membranes was promoted by 177 PM oleate. Increasing concentra- tions of chlorpromazine progressively displaced phosphatidate phosphohydrolase from the mem- brane compartment and simultaneously increased the cytosolic activity (Figs. 3 and 4). There was also a small decrease of 16 + 11% (mean _t S.E. from three independent experiments) in the total
585
phosphatidate phosphohydrolase activity at the higher concentrations of chlorpromazine.
The relationship between the relative activity of the membrane-associated phosphatidate phos- phohydrolase from Fig. 3 and the relative conver- sion of [ 3 H~phosphatidate to dia~ylglycerol from Fig. 1 is shown in Fig. 4. There was a highly significant correlation between these measure- ments (r = 0.98: P < 0.001). However, if it is valid to extrapolate to a zero conversion of phosphati- date then the intercept indicated that about 36% of the phosphatidate phosphobydrolase stilf re- mained associated with the membrane compart- ment.
Similar results to those shown in Fig. 3 were obtained in three other independent experiments in which the oleate ~n~ntration was also varied. As expected from previous work [5,6], the activity of the membrane-associated phosphatidate phos- phohydrolase activity increased with increasing oleate. For example, the percentage of the total
Fig. 2. Relationship between the effects of chlo~rom~ne on
the conversion of phosphatidate to diacylglycerol in hepato-
cytes and the synthesis of phosphatidylcholine and tri-
acylglycerol. Chlorpromazine (O-400 PM) was used to de-
crease the calculated rate of conversion of phosphatidate
labelled with [ 3H]glycero1 to diacylglycerol and the consequent
effect of this on the synthesis of [3H]t~acyl~ycerol (0) and
[3H]phosphatidylcholine (e) is shown. The rest&s are taken
from the experiments described in Fig. 1. They are expressed
as means f SE. from five independent experiments relative to
the incorporation of [3H]glycerol in the total lipid fraction
which was taken as 100%.
phosphatidate phosphohydrolase activity bound to the membranes at 0.2 and 1.0 mM oleate was 62 and 72%, respectively. Addition of 200 PM chlorpromazine decreased the relative proportion of membrane-bound phosphatidate phosphohy- drolase to 28 and 50% at 0.2 and 1.0 mM oleate, respectively. Similarly, with no added oleate the percentage of the phosphatidate phosphohydro- lase that was membrane associated was decreased from 40% to 20% by adding 100 PM chlor- promazine. These results show that the effects of oleate and chlo~rom~ine are mutually antagon- istic.
0.8 r
I I I I I I 1 0 100 m 300 4w
[Chlorprmazine] @W
Fig. 3. Effects of chlorpromazine on the subcellular distribu-
tion of phosphatidate phosphohydrolase in rat hepatocytes.
Monolayer cultures of rat hepatocytes were incubated for 15
nun at 37’C with 177 PM oleate and various concentrations of
chlorpromazine. Cytosolic enzymes were then released by in-
cubating for 4 min at 4°C with 0.5 mg of digitonin/ml of 0.25
M sucrose cont~ning 0.5 mM di~ot~eitol and 10 mM Hepes
adjusted to pH 7.4 with KOH. The graph shows the total
phosphatidate phosphohydrolase activity (0) and that which
was calculated to be associated with membranes in the cell
ghosts (m) and in the cytosol (A) after correcting for the
incomplete release of cytosolic enzymes by measuring lactate dehydrogenase (LDH) IS]. The results are means from tri-
plicate dishes and they are all expressed relative to the totai
lactate dehydrogenase activity recovered from the dishes in
order to compensate for slight variations in the numbers of
viable cells. Similar results were obtained in five further inde-
pendent experiments (see text and Fig. 4).
586
Effect of chlorpromazine on the translocation of phosphatidate phosphohydrolase in cell-free systems of rat liver
Experiments were also performed to determine
whether the effect of chlorpromazine on the
oleate-induced translocation was a direct action.
This entailed measuring the translocation in a
cell-free system by using a supernatant that was
obtained from the homogenates of rat liver after
centrifuging for 18000 X g for 10 min [7,8]. This
was incubated with 500 PM oleate to cause the
association of phosphatidate phosphohydrolase
r=O.98
p<O.ool
/I I I
40 60 80
Relative activity of membrane associated PAP (%)
J 100
Fig. 4. Relationship between the effects of chlorpromazine in
displacing phosphatidate phosphohydrolase (PAP) from the
membrane-associated compartment of rat hepatocytes and the
calculated conversion of phosphatidate to diacylglycerol. The graph shows the effects of chlorpromazine at 25 PM (A), 50
pM (0) 100 PM (v). 150 PM (0) 200 f.rM (A), 300 PM(~) and 400 PM (v) on the relative phosphatidate phosphohydrolase
activity associated with membranes which was taken as 100%
when no chlorpromazine was added (0). These results are
taken from three independent experiments and the values are
expressed as means + SE. The relationship of these values is
compared to the change in the relative rates of conversion of
[ ‘Hlphosphatidate to diacylglycerol in hepatocytes that were incubated in parallel in the same experiments. The latter
results were included in Fig. 1A.
with the microsomal membranes. The addition of
increasing concentrations of chlorpromazine pro-
gressively decreased the specific activity of phos-
phatidate phosphohydrolase that was recovered in
the microsomal fraction after centrifugation (Fig.
5). There was, however, no significant decrease in
the total phosphatidate phosphohydrolase activity
that was measured after adding chlorpromazine.
Chlorpromazine was also able to prevent the
increase in phosphatidate phosphohydrolase that
was associated with the microsomal fraction after
incubating the 180000 g. min supernatant with 1
mM spermine (Table I). When 500 PM oleate
were added together with 1 mM spermine, chlor-
promazine (400 PM) was able to partially prevent
the translocation of phosphatidate phosphohy-
drolase to the microsomal membranes.
The translocation of phosphatidate phos-
phohydrolase onto membranes that had been
I I I I
200 400
Chlorpromazine @Ml
Fig. 5. Effect of chlorpromazine on the distribution of phos-
phatidate phosphohydrolase between the soluble and micro-
somal fractions obtained form rat liver homogenates. The
18000 g .min supematant from rat liver homogenates was
incubated for 10 min at 37°C with 500 FM oleate to cause the
translocation of phosphatidate phosphohydrolase onto the mi-
crosomal membranes [7]. The effect of adding chlorpromazine
simultaneously on the activity of phosphatidate phos-
phohydrolase in the microsomal (A) and soluble fractions (0) is
shown. Similar results were found in four independent experi-
ments.
587
TABLE I
EFFECT OF CHLORPROMAZINE IN PREVENTING THE OLEATE- AND SPERMINE-INDUCED TRANSLOCATION OF
PHOSPHATIDATE PHOSPHOHYDROLASE ACTIVITY FROM THE CYTOSOLIC TO THE MICROSOMAL FRACTION
The 180000 g’min supematant from rat liver was incubated under various conditions for 10 min at 37°C and the microsomal and
cytosolic fractions were then separated by centrifugation. The distribution of phosphatidate phosphohydrolase (PAP) activity in these
fractions is shown from two independent experiments, the second experiment being shown in parenthesis.
Additions Relative distribution of PAP (S)
cytosol microsomal fraction
Total nmol of diacylglycerol
formed/mm
(A) None
(B) + 500 PM oleate
(C) + 500 pM oleate and
400 uM chlorpromazine
(D) + 1 mM spermine
(E) + 1 mM spermine and
400 PM chlorpromazine
(F) + 500 FM oleate and
1 mM spermine
(G) + 500 FM oleate, 1 mM
spermine and 400 pM
chlorpromazine
92 (87) 8 (13) 40.3 (27.9)
63 (67) 37 (33) 33.2 (21.5)
95 (89) 5 (11) 41.1 (27.7)
71 (77) 29 (23) 25.2 (16.5)
89 (94) II (6) 31.1 (21.0)
28 (22) 72 (78) 26.5 (15.31)
72 (57) 28 (43) 23.7 (14.1)
labelled with [i4C]phosphatidate was also de- membranes that were labelled with [‘4C]phos- termined. In these microsomal membranes more phatidate were also incubated for 10 min at 37°C
than 70% of the added i4C had been converted to to determine the effect of chlorpromazine on the phosphatidate and less than 5% remained as un- conversion of phosphatidate to diacylglycerol. The esterified oleate (Table II). When these mem- membranes that were incubated with the particle- branes were incubated with particle-free super- free supernatant showed about a 9-fold increase in natant containing soluble phosphatidate phos- the synthesis of diacylglycerol compared to the phohydrolase, about 42% of the enzyme bound to unincubated microsomal membranes (i.e. + 0.3 the membranes after centrifugation. However, less nmol compared with +2.7 nmol). This relatively than 5% was translocated to the membranes when low rate of conversion relative to Fig. 5 reflects 400 PM chlorpromazine were also present. The the much lower concentration of phosphatidate in
TABLE II
EFFECT OF CHLORPROMAZINE ON THE CONVERSION OF MEMBRANE-BOUND PHOSPHATIDATE TO DI-
ACYLGLYCEROL
Microsomal membranes were labelled with phosphatidate by incubating them with [‘4C]oleate (see Materials and Methods section).
Samples of the membranes equivalent to 750 pg of protein were then incubated for 10 min at 37“C with combinations of
chlorpromazine and the cytosolic proteins from rat liver which contained soluble phosphatidate phosphohydrolase activity. Results
are expressed in terms of nmol of [i4C]oleate present. These results were also confirmed in an independent experiment.
Phosphatidate
Diacylglycerol
Oleate
Microsomal
fraction
before
incubation
27.2
1.2
1.4
Microsomal fraction incubated after the following additions:
none soluble proteins mpM 400 pM chlorpromazine
(I .2 mg) chlorpromazine + soluble proteins
(1.2 mg)
25.7 21.9 25.9 26.8
1.5 3.9 1.0 0.9
1.6 1.5 1.2 1.0
588
the incubations. The addition of 400 PM chlor-
promazine completely suppressed the action of the
soluble phosphatidate phosphohydrolase activity
and the synthesis of the diacylglycerol from the
membrane-bound substrate (Table II). However, the 400 FM chlorpromazine did not significantly
alter the phosphatidate phosphohydrolase activity
in these preparations when the activity was mea-
sured in the normal way with an excess of phos-
phatidate in the form of a mixed micelle with
phosphatidylcholine.
In other experiments the combined microsomal
and soluble fractions were incubated for 10 min at
37°C with 500 PM oleate. The mixture was then
recentrifuged at 90000 X g ( rav = 6.3 cm) for 90
min and the microsomal pellet was resuspended in
0.25 M sucrose containing 0.2 mM dithiothreitol and 10 mM Hepes (PI-I 7.4). The microsomal
membranes were washed by repeating this process
two more times. The addition of 400 PM chlor-
promazine released 90% of the phosphatidate
phosphohydrolase activity from the microsomal
membranes, whereas only 10% of the activity was released in the absence of chlorpromazine.
Discussion
The work demonstrates that glycerolipid synthesis in isolated hepatocytes is sensitive to the
presence of chlorpromazine. As expected from previous work [1,12,18], this amphiphilic amine
markedly inhibited the synthesis of phosphati-
dylcholine and triacylglycerol, but it had relatively less effect on the incorporation of [3H]glycerol and [‘4C]oleate into the total glycerolipid fraction.
The latter decrease is probably explained by the known inhibition of glycerol phosphate acyltrans-
ferase by chlorpromazine [16]. However, this en-
zyme is much less sensitive to inhibition by amphiphilic amines than is phosphatidate phos- phohydrolase [16]. This is reflected in Fig. 1 where the major effect of chlorpromazine appeared to be at the level of phosphatidate phosphohydrolase as judged by the marked inhibition in the flux of phosphatidate to diacylglycerol and the accumula- tion of phosphatidate. Similar effects and conclu- sions were reached using slices of rat liver and higher concentrations of fenfluramine derivatives
[16]. These compounds can also be classified as amphiphilic amines.
The main objective of the present work was to
investigate whether chlorpromazine inhibited the
expression of phosphatidate phosphohydrolase ac- tivity in the hepatocytes by preventing the translo-
cation of the cytosolic enzyme to its site of action
on the membranes that synthesize glycerolipids.
The translocation is promoted by long-chain fatty
acids and their CoA esters and polyamines might
augment this action [S-8,19]. Evidence was ob-
tained that chlorpromazine did antagonize the
oleate-induced translocation in cell-free systems
(Fig. 5, Table I) and in intact hepatocytes (Fig. 3)
under the conditions where it inhibited the synthe-
sis of triacylglycerols and phosphatidylcholine
(Fig. 1). Furthermore, the activity of phosphati- date phosphohydrolase which was associated with
the membrane compartment in the presence or absence of chlorpromazine was highly correlated
with the flux of phosphatidate to diacylglycerol
that was calculated to occur in the hepatocytes
(Fig. 4).
The graph indicates that the displacement of
about 74% of the initial phosphatidate phos- phohydrolase activity that bound to the mem-
branes at 177 yM oleate might completely prevent the conversion of phosphatidate to diacylgly~erol.
This could mean that the apparent residual phos-
phatidate phosphohydrolase activity is not
involved in the metabolism of newly synthesized
phosphatidate to diacylglycerol that is destined for
glycerolipid synthesis. For instance, there is more
than one phosphatidate phosphohydrolase activity in microsomal fractions, and the enzyme has been reported to occur in a variety of different mem-
branes [B]. Alternatively, the ~hlo~romazine is likely to partition into the endoplasmic reticulum
and bind tightly to phosphatidate [14]. This could prevent its hydrolysis by phosphatidate phos- phohydrolase in the intact hepatocytes even though its activity can be expressed in the assay in the presence of saturating concentrations of exoge- nous phosphatidate.
Chlorpromazine also inhibited the translocating effects of oleate and spermine in a cell-free system when added separately, or in combination (Table I). Prevention of the oleate-induced translocation of the soluble phosphatidate phosphohydrolase in
589
the cell-free system was also accompanied by an impaired ability of the microsomal membranes to convert phosphatidate to diacylglycerol (Table II). In addition, the lack of apparent inhibition of phosphatidate phosphohydrolase activity in the cell-free system when it was assayed with the artificial dispersion of phosphatidate and phos- phatidylcholine rather than with phosphatidate bound to microsomal membranes could have resulted from the relatively high concentrations of phosphatidate used in the former assay. It is known that the inhibition of phosphatidate phos- phohydrolase in the presence of Mg*+ is of a competitive type relative to phosphatidate [14].
The inhibition is thought to result from the interaction of amphiphilic amines with the phos- phatidate in the membranes that constitute the substrate for the reaction. This alters the physical properties of the membranes including their surface charge [18]. In the presence of Mg2+, chlorpromazine increases the positive charge as it partitions into the membrane and thus prevents phosphatidate phosphohydrolase interacting with the phosphatidate [1,14]. Conversely, the adsorp- tion of amphiphilic anions such as clofenapate or oleoyl-CoA into the membranes makes them more negative and facilitates the binding of phosphati- date phosphohydrolase [18]. These same events could form the basis for the opposing effects of fatty acids or acyl-CoA esters and chlorpromazine on the translocation which can also be viewed in terms of the binding of phosphatidate phos- phohydrolase to the membranes that contain phosphatidate. The association of oleate with the endoplasmic reticulum is paralleled by the translo- cation of phosphatidate phosphohydrolase, and both of these events can be reversed by albumin [19]. By contrast, chlorpromazine causes slightly more oleate to associate with the membranes, but it displaces phosphatidate phosphohydrolase probably by its effects on membrane charge [19].
It is not yet completely certain how spermine facilitates the translocation of phosphatidate phosphohydrolase to the membranes in vitro, but it appears to promote the association of phos- phatidate phosphohydrolase with lipids and lipo- philic proteins [19]. It can also stimulate the hy- drolysis of potassium phosphatidate by cytosolic phosphatidate phosphohydrolase, but it is much less effective than Mg2+ [14].
Fatty acids are well known to stimulate the synthesis of triacylglycerols and phosphatidylcho- line by hepatocytes, and this is paralleled by the translocation of phosphatidate phosphohydrolase to the membrane-associated compartment [1,5]. The present work demonstrates a competitive antagonism of chlorpromazine against these fatty acid effects. Thus, as the oleate concentration is raised, the amount of chlorpromazine required to produce the same phosphatidate phosphohydro- lase concentrations on the membranes is in- creased.
Although the effects of chlorpromazine may be mainly brought about by altering the charge on membranes it is also known to bind to calmodulin and to antagonize some of its actions. The interac- tion of calmodulin with its target enzyme requires the exposure of a lipophilic domain through a Ca2+-induced conformational change [20,21]. Chlorpromazine can bind to the hydrophobic do- main of calmodulin [21,22], and block the activa- tion by Ca*+-calmodulin of target enzymes [19, 20,211. This action could also be relevant, since Ca2+ appears to be involved in controlling phos- phatidate phosphohydrolase activity and the synthesis of triacylglycerols and phosphatidylcho- line [4]. An interference with Ca2+ metabolism or an interaction with phospholipid could also mod- ify the activity of protein kinase C [23-251. This enzyme’s activity also seems to be partly regulated by translocation to membranes and interaction with phospholipids [26,27]. Furthermore, the product of the phosphatidate phosphohydrolase reaction, diacylglycerol, is also an activator of protein kinase C. This could indicate an interac- tion in the control of the activities of phosphati- date phosphohydrolase and protein kinase C. There is already evidence that phosphorylation of phosphatidate phosphohydrolase by a cyclic AMP dependent mechanism may change its ability to translocate to membranes [6] and Ca’+-dependent phosphorylations may also be effective. Such ef- fects could also modify the activity of phosphati- date phosphohydrolase in hepatocytes, but they are unlikely to do so in the cell-free system.
So far, the inhibition of the synthesis of tri- acylglycerol and phosphatidylcholine by chlor- promazine has been discussed at the level of phos- phatidate phosphohydrolase and the inhibition of
590
diacylglycerol formation. This was accompanied
by a decrease in the accumulation of [3H]glycerol
and [ I4 Cloleate in diacylglycerol at concentrations
of chlorpromazine between 50 and 400 PM. At 25
PM chlorpromazine there was a slight stimulation
of diacylglycerol accumulation even though the
calculated flux from [ 3H]phosphatidate to [ 3H]di-
acylglycerol was decreased by 8% (Fig. 1). Such an
increase in diacylglycerol accumulation has been observed in other work with chlorpromazine or
other amphiphilic amines [1,14,28,29] and this
probably indicates further inhibitions of di-
acylglycerol acyltransferase or choline phos-
photransferase. Although direct evidence for the
inhibition of diacylglycerol acyltransferase by
amphiphilic amines has not been obtained
[1,14,28], there are indications that these com-
pounds can block the action of choline phos- photransferase [28]. However, the major inhibition
of phosphatidylcholine synthesis occurred with 50
PM chlorpromazine and the synthesis was fairly resistant to further inhibition by higher concentra-
tions of the drug. By contrast, triacylglycerol
synthesis was susceptible to inhibition by these higher concentrations of chlorpromazine (Fig. lB,
D). Thus, this provides a further example that
there is a preferential use of diacylglycerol for the
synthesis of phosphatidylcholine rather than tri-
acylglycerol when the availability of diacylglycerol
becomes restricted. It is normally considered that the rate of phos-
phatidylcholine synthesis in cells is controlled by
the availability of CDPcholine rather than di-
acylglycerol [30,31]. However, there are likely to be many conditions where diacylglycerol availabil-
ity is also limiting and where phosphatidylcholine
synthesis is dependent upon the product of these concentrations. Diacylglycerol can also promote
the binding of cytosolic CTP : phosphocholine cytidylyltransferase to membranes of the endo- plasmic reticulum and thereby cause its activation and the production of more CDPcholine [30,31]. This translocation and activation is also facilitated by fatty acids [11,30-331 and it closely resembles that of phosphatidate phosphohydrolase [1,5-B]. Such an activation of the cytidylyltransferase by diacylglycerol and fatty acids could account for part of the increased synthesis of phosphati- dylcholine that was observed at high rates of diacylglycerol formation (Fig. 2). Although the cytosolic form of the cytidylyltransferase in
hepatocytes was sensitive to inhibition by a variety
of amphiphilic amines including chlorpromazine,
this inhibition was overcome by phospholipid [34].
Likewise, the microsomal enzyme was also rela-
tively resistant to inhibition by amphiphilic amines
which in these experiments with hepatocytes did not significantly inhibit phosphatidylcholine
synthesis from [methyl- 3H]choline. This also indi-
cated that the microsomal cytidylyltransferase was
not inhibited in the intact cells [34]. This relative
resistance of the microsomal cytidylyltransferase
to inhibition by chlorpromazine is compatible with
the finding that there was little change in phos-
phatidylcholine synthesis when chlorpromazine
concentrations were increased from 50 to 400 PM
(Fig. lB, D).
At present we cannot explain why phosphati- dylcholine synthesis was not inhibited at all in the
experiments of Pelech et al. [34], although the chlorpromazine concentration was relatively low
at 20 PM. In other work with amphiphilic cationic
drugs, decreases in the incorporation of radioac-
tive glycerol, fatty acids or choline into phos-
phatidylcholine have been reported (Fig. 1, Refs.
12, 17, 28 and 29). However, Pelech and Vance
[35] did observe an inhibition of phosphatidylcho- line synthesis in HeLa cells which in the case of
trifluorperazine was prevented by 0.4 mM oleate. The inhibition was associated with a decrease in
the cytidylyltransferase activity. Although this
probably resulted because trifluoperazine and
chlorpromazine interfered with the interaction of
the cytidylyltransferase with phospholipid, it did not alter the distribution of the enzyme between the cytosolic and membrane-associated compart-
ment at the concentrations used [35]. It also re-
mains to be seen whether the control of the trans-
location of phosphatidate phosphohydrolase and the cytidylyltransferase are different in some re- spects despite the similar effects of fatty acids and cyclic AMP analogues [1,5-8,11,30-331. For ex- ample, the negative charge donated by phos- phatidate should provide a very specific binding site for phosphatidate phosphohydrolase on the membranes, whereas it is relatively unlikely that the cytidylyltransferase has a high affinity for phosphatidate.
The present work with chlorpromazine provides further evidence that phosphatidate phosphohy- drolase is an ambiquitous enzyme [1,5-10,19,36, 371. This means that it exists in different cell
591
compartments and that the control of its subcellu- lar distribution helps to regulate metabolism [38]. The present results also provide further informa- tion concerning the importance of phosphatidate phosphohydrolase in regulating glycerolipid metabolism and on the mechanism whereby amphiphilic amines inhibit the expression of phos- phatidate phosphohydrolase activity in cells. This effect is implicated in redirecting the synthesis of glycerolipids away from triacylglycerol and phos- phatidylcholine and towards the production of acidic phospholipids. This could help to change the physiological responses of cells, and contribute to the phospholipidosis that can be observed after the chronic administration of the more hydro- phobic amphiphilic amines that have long biologi- cal half lives [1,12]. It is specifically hoped that the knowledge that amp~p~lic amines can inhibit the translocation of phosphatidate phosphohydrolase to membranes can provide a further level for which to develop therapeutic agents that can regu- late glycerolipid synthesis, membr~e turnover and lipoprotein secretion.
Acknowledgements
We thank Professor D.E. Vance, Dr. S.L. Pelech and Dr. A. Salter for useful suggestions and dis- cussions. Travel grants were provided to P.M.-S. by the Fondo de Investigations Sanitarias, Minis- terio de Sanidad y Secugidad Social and the British Council and to Professor Vance and D.N.B. by Nato (149.81). The work was supported by an equipment grant from the Humane Research Trust and a project grant and a research studentship to A.M. from the Medical Research Council.
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