The influence of exogenous and of membrane-bound phosphatidate concentration on the activity of CTP: phosphatidate cytidylyltransferase and phosphatidate phosphohydrolase.

Rat liver microsomes were treated with phospholipase D to obtain microsomal membranes with varying amounts of membrane-bound phosphatidate. This treatment did not impair the activity of two microsomal-bound enzymes acting with phosphatidate as substrate, i.e. CTP: phosphatidate cytidylyltransferase and phosphatidate phosphohydrolase. The dependency of the activity of these enzymes on the concentration of membrane-bound phosphatidate was determined. Both enzymes showed a linear increase in activity with membrane-bound phosphatidate concentrations up to at least 100 nmol phosphatidate/mg microsomal protein. These results indicate that both enzymes have a large reserve capacity and suggest that the enzymes are operating intracellularly, i.e. at phosphatidate concentrations of 5-10 nmol/mg endoplasmic reticulum protein, far below their maximal capacity. The ratio of phosphatidate conversion into CDP-diglyceride and 1,2-diglyceride seems to be constant for a large range of membrane-bound phosphatidate concentrations. The membrane-bound enzymes cannot utilize phosphatidate substrate present in heat-denatured membranes, but are active on phosphatidate incorporated into membranes of phospholipid vesicles.     

Drugs affecting the synthesis of glycerides and phospholipids in rat liver. The effects of clofibrate, halofenate, fenfluramine, amphetamine, cinchocaine, chlorpromazine, demethylimipramine, mepyramine and some of their derivatives.

The effects on glycerolipid synthesis of a series of compounds including many drugs were investigated in cell-free preparations and slices of rat liver. p-Chlorobenzoate, p-chlorophenoxyisobutyrate, halofenate, D-amphetamine, adrenaline, procaine and N-[2-(4-chloro-3-sulphamoylbenzoyloxy)ethyl]norfenfluramine had little inhibitory effect on any of the systems investigated. Two amphiphilic anions, clofenapate and 2-(p-chlorophenyl)-2-(m-trifluoromethylphenoxy)acetate, both inhibited glycerol phosphate acyltransferase and diacylglycerol acyltransferase at approx. 1.6 and 0.7 mm respectively. Clofenapate (1 mm) also inhibited the incorporation of glycerol into lipids by rat liver slices without altering the relative proportions of the different lipids synthesized. The amphilic amines, mepyramine, fenfluramine, norfenfluramine, hydroxyethylnorfenfluramine, N-(2-benzoyloxyethyl)norfenfluramine, cinchocaine, chlorpromazine and demethylimipramine inhibited phosphatidate phosphohydrolase by 50% at concentrations between 0.2 and 0.9 mm. The last four compounds inhibited glycerol phosphate acyltransferase by 50% at concentrations between 1 and 2.6 mm. None of the amines examined appeared to be an effective inhibitor of diacylglycerol acyltransferase. Norfenfluramine, hydroxyethylnorfenfluramine and N-(2-benzoyloxyethyl)norfenfluramine produced less inhibition of glycerol incorporation into total lipids than was observed with equimolar clofenapate. The major effect of these amines in liver slices was to inhibit triacylglycerol and phosphatidylcholine synthesis and to produce a marked accumulation of phosphatidate. The results are discussed in terms of the control of glycerolipid synthesis. They partly explain the observed effects of the various drugs on lipid metabolism. The possible use of these compounds as biochemical tools with which to investigate the reactions of glycerolipid synthesis is considered.     

The effects of Triton X-100 and chlorpromazine on the Mg2+-dependent and Mg2+-independent phosphatidate phosphohydrolase activities of rat lung.

Lung contains both Mg2+-dependent and Mg2+-independent phosphatidate phosphohydrolase activities. Addition of Triton X-100 (0.5%) or chlorpromazine (1 mM) leads to a marked increase in the total phosphatidate phosphohydrolase activity in rat lung microsomes (microsomal fractions), but a decrease in the Mg2+-dependent activity. These observations suggest that the Mg2+-independent activity is stimulated, whereas the Mg2+-dependent activity is inhibited. However, the possibility exists that Triton X-100 could stimulate the Mg2+-dependent enzymic activity in an Mg2+-independent manner. In addition, the positively charged amphiphilic drug could be replacing the enzyme's requirement for Mg2+. These two possibilities were examined by using subcellular fractions in which the Mg2+-dependent phosphatidate phosphohydrolase had been abolished by heat treatment at 55 degrees C for 15 min. Heat treatment does not affect the microsomal Mg2+-independent phosphohydrolase to any great extent. Since the 6-8-fold stimulations due to Triton X-100 and chlorpromazine are retained after heat treatment of this fraction, the Mg2+-independent activity must be involved. Addition of Triton X-100 and chlorpromazine to cytosol virtually abolishes the Mg2+-dependent phosphatidate phosphohydrolase activity and decreases the Mg2+-independent activity by half. Heat treatment also abolishes the Mg2+-dependent activity and decreases the Mg2+-independent activity by over half. The Mg2+-independent phosphatidate phosphohydrolase activity remaining after heat treatment was not affected by Triton X-100 or chlorpromazine. These studies demonstrate that Triton X-100 and chlorpromazine specifically stimulate the heat-stable Mg2+-independent phosphatidate phosphohydrolase activity in rat lung microsomes. In contrast, the heat-labile Mg2+-independent phosphatidate phosphohydrolase activities in cytosol are inhibited by these reagents. Triton X-100 and chlorpromazine inhibit the Mg2+-dependent phosphatidate phosphohydrolase activities in both rat lung microsomes and cytosol. These results are consistent with the view that a single Mg2+-dependent phosphatidate phosphohydrolase present in both microsomes and cytosol is specifically involved in glycerolipid metabolism.     

Mechanisms for the effects of ethanol on hepatic phosphatidate phosphohydrolase.

1. The effects of the intramuscular administration of glycerol and dihydroxyacetone (40mmol per kg body wt.), sorbitol and glucose (20mmol per kg body wt.) or NaCl (1.5mmol per kg body wt. in 10ml of water per kg body wt.) were investigated on soluble phosphatidate phosphohydrolase and certain metabolites in rat liver. 2. The effects of ethanol and glycerol on phosphatidate phosphohydrolase were also studied in isolated perfused livers. 3. The administration of glycerol, sorbitol and dihydroxyacetone in vivo increased hepatic phosphatidate phosphohydrolase activity by 137, 63 and 32% respectively in 4h. 4. A significant positive correlation was found between the hepatic sn-glycerol 3-phosphate concentration and phosphatidate phosphohydrolase after the administration of various substrates in vivo. 5. The soluble phosphatidate phosphohydrolase activity tended to increase during perfusions of isolated rat livers without added substrates, and neither ethanol nor glycerol produced additional effects. 6. The activity of soluble phosphatidate phosphohydrolase was 2.5 times higher in the livers of hyperthyroid rats than in normal rats. This activity was not influenced by intragastric ethanol or glycerol administration, nor was the concentration of sn-glycerol 3-phosphate changed by these compounds. 7. It is concluded that the ethanol-induced increase in hepatic phosphatidate phosphohydrolase may at least in part be mediated by the hepatic concentration of metabolites, probably by the concentration of sn-glycerol 3-phosphate.     

The effects of amphiphilic cationic drugs and inorganic cations on the activity of phosphatidate phosphohydrolase.

1. Phosphatidate phosphohydrolase from the particle-free supernatant of rat liver was assayed by using emulsions of phosphatidate as substrate. 2. The inhibition of the phosphohydrolase by chlorpromazine was of a competitive type with respect to phosphatidate. The potency of various amphiphilic cationic drugs as inhibitors of this reaction was related to their partition coefficients into a phosphatidate emulsion. 3. The effect of chlorpromazine on the phosphohydrolase activity was complementary rather than antagonistic towards Mg2+. Chlorpromazine stimulated the phosphohydrolase activity in the absence of added Mg2+ and was able to replace the requirement for Mg2+. However, at optimum concentrations of Mg2+, chlorpromazine inhibited the reaction, as did Ca2+. The phosphohydrolase activity was also stimulated by Co2+ and to a lesser extent by Mn2+, Fe2+, Fe3+, Ca2+, spermine and spermidine when Mg2+ was not added to the assays. 4. It is concluded that the inhibition of phosphatidate phosphohydrolase by amphiphilic cations can largely be explained by the interaction of these compounds with phosphatidate, which changes the physical properties of the lipid, making it less available for conversion into diacylglycerol. 5. The implications of these results to the effects of amphiphilic cations in redirecting glycerolipid synthesis at the level of phosphatidate are discussed.     

Interleukin-1 rapidly stimulates lysophosphatidate acyltransferase and phosphatidate phosphohydrolase activities in human mesangial cells.

Phosphatidic acid (PA) is a cytokine in a variety of cell types, and an intermediary in cell activation. It is produced from membrane phospholipids by either lysophosphatidate acyl-CoA:acyltransferase (lyso-PA AT) or phospholipase D. Interleukin-1 (IL-1) stimulation of human mesangial cells (HMC) induced activation of lyso-PA AT, and synthesis of new PA species with significant increase in PA mass. These PA species were enriched in long-chain unsaturated acyl side chains (C18:1, C18:2, C20:5, and C22:6) in both the sn-2 and sn-1 positions, and stimulated the action of the lyso-PA AT as a positive feedback mechanism. Gas-liquid chromatography and mass spectrometry demonstrate that the acyl composition of phosphatidic acid does not resemble that of the major phospholipid fractions of this preparation and therefore is not the product of phospholipase D. The PA species were rapidly converted to 1,2-sn-diacylglycerols by phosphatidate phosphohydrolase, which also was activated by IL-1 via a separate mechanism involving a pertussis-sensitive G-protein. The activities of lyso-PA AT and phosphatidate phosphohydrolase were associated with plasma membrane enriched and refined microsomal fractions. IL-1 stimulation of a murine T cell (thymoma) line, EL-4, also caused stimulation of lyso-PA AT, resulting in PA formation. EL-4 mutants with defective IL-1 receptors did not demonstrate stimulation of lyso-PA AT, showing the necessity of intact IL-1 receptors for activation of this enzyme. We conclude that PA is a significant signaling intermediary for IL-1 via activation of lyso-PA AT and a G-protein, which activates phosphatidate phosphohydrolase. This system suggests a novel mechanism whereby a low intensity signal may be translated into cellular activation.     

Properties of phosphatidate phosphohydrolase and diacylglycerol acyltransferase activities in the isolated rat heart. Effect of glucagon, ischaemia and diabetes.

Myocardial triacylglycerol hydrolysis is subject to product inhibition. After hydrolysis of endogenous triacylglycerols, the main proportion of the liberated fatty acids is re-esterified to triacylglycerol, indicating the importance of fatty acid re-esterification in the regulation of myocardial triacylglycerol homoeostasis. Therefore, we characterized phosphatidate phosphohydrolase (PAP) and diacylglycerol acyltransferase (DGAT) activities, enzymes catalysing the final steps in the re-esterification of fatty acids to triacylglycerols in the isolated rat heart. The PAP activity was mainly recovered in the microsomal and soluble cell fractions, with an apparent Km of 0.14 mM for both the microsomal and the soluble enzyme. PAP was stimulated by Mg2+ and oleic acid. Oleic acid, like a high concentration of KCl, stimulated the translocation of PAP activity from the soluble to the particulate (microsomal) fraction. Myocardial DGAT had an apparent Km of 3.8 microM and was predominantly recovered in the particulate (microsomal) fraction. Both enzyme activities were significantly increased after acute streptozotocin-induced diabetes, PAP from 15.6 +/- 1.1 to 28.1 +/- 3.6 m-units/g wet wt. (P less than 0.01) and DGAT from 2.23 +/- 0.11 to 3.01 +/- 0.11 m-units/g wet wt. (P less than 0.01). In contrast with diabetes, low-flow ischaemia during 30 min did not affect PAP and DGAT activity in rat hearts. Perfusion with glucagon (0.1 microM) during 30 min did not affect total PAP activity, but changed the subcellular distribution. More PAP activity was recovered in the particulate fraction. DGAT activity was lowered by glucagon treatment from 0.37 +/- 0.03 to 0.23 +/- 0.02 m-unit/mg of microsomal protein (P less than 0.05). The role of PAP and DGAT activity and PAP distribution in the myocardial glucose/fatty acid cycle is discussed.     

Comparative effects of dietary fish oil and carbohydrate on plasma lipids and hepatic activities of phosphatidate phosphohydrolase, diacylglycerol acyltransferase and neutral lipase activities in the rat

In rats fed a fish oil-enriched diet, plasma triacylglycerols were lowered 51%. At the same time there was a mean 45% reduction in Mg2+-dependent phosphatidate phosphohydrolase activity in liver microsomes and a mean 20% decrease in microsomal triacylglycerol (neutral) and diacylglycerol hydrolase activities, but not of diacylglycerol acyltransferase. These observations support the hypothesis that decreases in the activities of phosphatidate phosphohydrolase and of both lipases are involved in the expression of the inhibitory effects of fish oil feeding on hepatic lipoprotein triacylglycerol secretion. Conversely, the feeding of a sucrose-enriched diet resulted in a mean 39% rise in plasma triacylglycerols, a 19% increase in triacylglycerol hydrolase and a mean 45% increase in Mg2+-dependent microsomal phosphohydrolase activity. The effects of the two nutritional interventions on phosphatidate phosphohydrolase activity confirm a key function for this enzyme in triacylglycerol formation.     

Oleate stimulation of diacylglycerol formation from phosphatidylcholine through effects on phospholipase D and phosphatidate phosphohydrolase.

Hydrolysis of exogenous phosphatidylcholine (PtdCho) to 1,2-diacylglycerol by rat liver plasma membranes was stimulated by oleate concentrations as low as 0.1 mM. In the presence of 75 mM ethanol, the fatty acid also enhanced phosphatidylethanol (PtdEtOH) formation from PtdCho. These effects were also observed with linoleate and arachidonate, but not with saturated fatty acids or detergents, and were minimal in microsomes or mitochondria. Release of [3H]choline from exogenous Ptd[3H]Cho was stimulated by oleate, whereas phosphoryl[3H]choline formation was inhibited. Oleate and other unsaturated, but not saturated, fatty acids also stimulated the conversion of exogenous [14C]phosphatidic acid to [14C]diacylglycerol. These data are consistent with stimulatory effects of these fatty acids on both phospholipase D and phosphatidate phosphohydrolase in liver plasma membranes. The stimulatory effect of guanosine 5'-O-[3-thio]triphosphate) (20 microM) on PtdEtOH and diacylglycerol formation from PtdCho was enhanced by low concentrations of oleate. Phospholipase A2 also stimulated PtdEtOH and diacylglycerol formation from exogenous PtdCho. It is proposed that unsaturated fatty acids may play a physiological role in the regulation of diacylglycerol production through activation of phospholipase D and phosphatidate phosphohydrolase.     

Regulation of the translocation of phosphatidate phosphohydrolase between the cytosol and the endoplasmic reticulum of rat liver. Effects of unsaturated fatty acids, spermine, nucleotides, albumin and chlorpromazine.

The translocation of phosphatidate phosphohydrolase between the cytosol and the microsomal membranes was investigated by using a cell-free system from rat liver. Linoleate, alpha-linolenate, arachidonate and eicosapentenoate promoted the translocation to membranes with a similar potency to that of oleate. The phosphohydrolase that associated with the membranes in the presence of [14C]oleate or 1mM-spermine coincided on Percoll gradients with the peak of rotenone-insensitive NADH-cytochrome c reductase, and in the former case with a peak of 14C. Microsomal membranes were enriched with the phosphohydrolase activity by incubation with [14C]oleate or spermine and then incubated with albumin. The phosphohydrolase activity was displaced from the membranes by albumin, and this paralleled the removal of [14C]oleate from the membranes when this acid was present. Chlorpromazine also displaced phosphatidate phosphohydrolase from the membranes, but it did not displace [14C]oleate. The effects of spermine in promoting the association of the phosphohydrolase with the membranes was inhibited by ATP, GTP, CTP, AMP and phosphate. ATP at the same concentration did not antagonize the translocating effect of oleate. From these results and previous work, it was concluded that the binding of long-chain fatty acids and their CoA esters to the endoplasmic reticulum acts as a signal for more phosphatidate phosphohydrolase to associate with these membranes and thereby to enhance the synthesis of glycerolipids, especially triacylglycerol. The translocation of the phosphohydrolase probably depends on the increased negative charge on the membranes, which could also be donated by the accumulation of phosphatidate. Chlorpromazine could oppose the translocation by donating a positive charge to the membranes.     

Partial purification and characterization of the soluble phosphatidate phosphohydrolase of rat liver.

A method is described by which the Mg2+-stimulated phosphatidate phosphohydrolase can be purified from the soluble fraction of liver from ethanol-treated rats. The increase in specific activity was about 416-fold. This involved purification by adsorption on calcium phosphate, chromatography on DE-52 DEAE-cellulose, separation on Ultrogel AcA-34 and chromatography on CM-Sepharose 6B. The effects of phosphatidylcholine, phosphatidate and Mg2+, Mn2+ and Zn2+ on the activity are described. Inhibitor studies indicate that the phosphohydrolase contains functional thiol groups and arginine residues.     

Partial purification and properties of microsomal phosphatidate phosphohydrolase from rat liver.

Microsomal phosphatidate phosphohydrolase (phosphatidate phosphatase EC 3.1.3.4) was solubilized and fractionated to yield at least two distinct enzymatically active fractions. One, denoted FA, was non-specific, had a relatively high Km for phosphatidic acid and was insensitive to inhibition by diacylglycerol. The second fraction, FB, was specific for phosphatidates, had a low Km, and was inhibited, non-competitively, by diacylglycerol. FA exhibited a sigmoid substrate-activity curve. The isolated FB aggregated to particles of about 10(6) in the absence of salts and could be dissociated by the addition of monovalent cations at ionic strength 0.4-0.6 to about 2-10(5) daltons and thereby doubled its activity. Dissociation was time- and temperature-dependent. F- was inhibitory. Divalent ions were not required for the activity of FA or FB and inhibited at concentrations exceeding 1 mM.     

The effects of cortisol, corticotropin and thyroxine on the synthesis of glycerolipids and on the phosphatidate phosphohydrolase activity in rat liver

1. Male rats were injected daily for 5 days with 0.15m-NaCl, corticotropin, cortisol or l-thyroxine and the rates of glycerolipid synthesis were measured in the livers after intraportal injection of [(14)C]palmitate and [(3)H]glycerol. 2. Injection of all three hormones decreased the rates of body-weight gain. 3. Cortisol treatment increased the weight of the liver relative to body weight. 4. Thyroxine treatment increased the relative rate of triacylglycerol synthesis from [(3)H]glycerol and decreased the relative accumulation of (3)H and (14)C in diacylglycerol. It did not significantly alter the accumulation of these isotopes in phosphatidate nor the activity of the soluble phosphatidate phosphohydrolase in the total liver. However, this activity increased by 1.5-fold when expressed relative to the soluble protein of the liver. The increased triacylglycerol synthesis appears to be related to a general increase in the turnover of fatty acids in the liver. 5. Treatment with cortisol and corticotropin increased the relative rate of triacylglycerol synthesis from [(3)H]glycerol, decreased the accumulation of (3)H in phosphatidate and increased the flux of both isotopes from phosphatidate to diacylglycerol. This appeared to be caused by the increased activity of the soluble phosphatidate phosphohydrolase that was observed in the livers of the cortisol-treated rats. 6. It is proposed that cortisol could be directly or indirectly involved in increasing the activity of hepatic phosphatidate phosphohydrolase in starvation, diabetes, laparotomy, subtotal hepatectomy, liver damage, ethanol feeding and in obesity. This enzyme adaptation could contribute to the potential of the liver to increase its synthesis and accumulation of triacylglycerols or to secrete very-low-density lipoproteins.     

The inactivation of rat adipocyte Mg2+-dependent phosphatidate phosphohydrolase by noradrenaline.

The inactivation of rat adipocyte Mg2+-dependent phosphatidate phosphohydrolase by noradrenaline [Cheng & Saggerson (1978) FEBS Lett. 87, 65--68; Cheng & Saggerson (1978) FEBS Lett. 93, 120--124] persists for at least 40 min in crude defatted homogenates kept at 0 degrees C or 20 degrees C, but is diminished at 37 degrees C. The effect of noradrenaline persists through the isolation of post-105000 g supernatants and is then stable in these preparations at 0 degrees C and 37 degrees C. Inclusion of albumin (10--20 mg/ml) in homogenization buffers abolishes the effect of noradrenaline. The effect of noradrenaline is not removed by dialysis of extracts or by raising the concentrations of Mg2+ or phosphatidate in assays.     

The involvement of phosphatidate phosphohydrolase and phospholipase A activities in the control of hepatic glycerolipid synthesis. Effects of acute feeding with glucose, fructose, sorbitol, glycerol and ethanol.

Rats were fed by stomach tube with a single dose of glucose, sorbitol, fructose, glycerol or ethanol of equivalent energy contents or with 0.15 M-NaCl. They were killed 6 h later and the relative rates of phosphatidate deacylation and dephosphorylation measured in the microsomal and supernatant fractions of the livers. Treatment with sorbitol, fructose, glycerol and ethanol increased phosphohydrolase activities in the microsomal and supernatant fractions. The only significant change in deacylase activity was an increase in the microsomal fraction produced by ethanol. It is proposed that hepatic triacylglycerol synthesis is partly controlled by the balance between phosphatidate phosphohydrolase and phospholipase A-type activities.     

Stimulation and inhibition of the activity of rat liver cytosolic phosphatidate phosphohydrolase by various phospholipids.

The influence of phospholipids on the activity of the soluble phosphatidate phosphohydrolase from rat liver was studied. Phosphatidylethanolamine stimulated the enzyme activity whereas phosphatidylglycerol, phosphatidylserine, and phosphatidylinositol were inhibitory. At a phospholipid concentration of 0.7 mg/ml, phosphatidylglycerol inhibited phosphatidate phosphohydrolase activity by 75%, while the enzyme activity was stimulated twofold in the presence of phosphatidylethanolamine. Both lysophosphatidylglycerol and lysophosphatidylethanolamine inhibited phosphatidate phosphohydrolase activity as did octylglucoside, sodium cholate, and Tween 20. The finding that phospholipids influence hepatic phosphatidate phosphohydrolase activity indicates that changes in the lipid environment may modulate the enzyme activity.     

Interactions of insulin, glucagon and dexamethasone in controlling the activity of glycerol phosphate acyltransferase and the activity and subcellular distribution of phosphatidate phosphohydrolase in cultured rat hepatocytes.

Rat hepatocytes were incubated in monolayer culture for 8 h. Glucagon (10nM) increased the total phosphatidate phosphohydrolase activity by 1.7-fold. This effect was abolished by adding cycloheximide, actinomycin D or 500 pM-insulin to the incubations. The glucagon-induced increase was synergistic with that produced by an optimum concentration of 100 nM-dexamethasone. Theophylline (1mM) potentiated the effect of glucagon, but it did not affect the dexamethasone-induced increase in the phosphohydrolase activity. The relative proportion of the phosphohydrolase activity associated with membranes was decreased by glucagon when 0.15 mM-oleate was added 15 min before the end of the incubations to translocate the phosphohydrolase from the cytosol. This glucagon effect was not seen at 0.5 mM-oleate. Since glucagon also increased the total phosphohydrolase activity, the membrane-associated activity was maintained at 0.15 mM-oleate and was increased at 0.5 mM-oleate. This activity at both oleate concentrations was also increased in incubations that contained dexamethasone, particularly in the presence of glucagon. Insulin increased the relative proportion of phosphatidate phosphohydrolase that was associated with membranes at 0.15 mM-oleate, but not at 0.5 mM-oleate. It also decreased the absolute phosphohydrolase activity on the membranes at both oleate concentrations in incubations that also contained glucagon and dexamethasone. None of the hormonal combinations significantly altered the total glycerol phosphate acyltransferase activity. However, glucagon significantly increased the microsomal activities, and insulin had the opposite effect. Glucagon also decreased the mitochondrial acyltransferase activity. There was a highly significant correlation between the total phosphatidate phosphohydrolase activity and the synthesis of neutral lipids from glycerol phosphate and 0.5 mM-oleate in homogenates of cells from all of the hormonal combinations. Phosphatidate phosphohydrolase activity is increased in the long term by glucocorticoids and also by glucagon through cyclic AMP. In the short term, glucagon increases the concentration of fatty acid required to translocate the cytosolic reservoir of activity to the membranes on which phosphatidate is synthesized. Insulin opposes the combined actions of glucagon and glucocorticoids. The long-term events explain the large increases in the phosphohydrolase activity that occur in vivo in a variety of stress conditions. The expression of this activity depends on increases in the net availability of fatty acids and their CoA esters in the liver.     

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

Glycerolipid synthesis was studied in isolated hepatocytes by using 177 microM [14C]oleate and 1 mM [3H]glycerol. Chlorpromazine (25-400 microM) inhibited the synthesis of phosphatidylcholine and triacylglycerol. This was accompanied by an average increase of 12-fold in the accumulation of the labelled precursors in phosphatidate at 200 microM 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 phosphatidylcholine and triacylglycerol occurs at the level of phosphatidate phosphohydrolase. 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. 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. 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. Chlorpromazine also antagonized the association of the phosphohydrolase with microsomal membranes when cell-free preparations were incubated with combinations of oleate and spermine. Furthermore, it inhibited the transfer of the soluble phosphohydrolase to microsomal membranes that were labelled with [14C]phosphatidate and thereby decreased diacylglycerol production. 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.