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.     

Glycerolipid biosynthesis in rat adipose tissue. 11. Effects of polyamines on Mg2+-dependent phosphatidate phosphohydrolase

The effect of polyamines (spermine, spermidine and putrescine) on the Mg2+-dependent phosphatidate phosphohydrolase was investigated. Phosphatidate phosphohydrolase activity was measured in the presence of aqueous dispersed phosphatidate as substrate, and the release of inorganic phosphate was taken as a measure of phosphatidate phosphohydrolase activity. In the presence of various polyamines there was activation of the Mg2+-dependent phosphatidate phosphohydrolase activity. Under this condition, the Km of enzyme towards phosphatidase decreased from 1.6 x 10(-4) to 9.8 x 10(-5) M and the Mg2+ requirement decreased from 5 to 0.5 mM. These polyvalent cations did not replace Mg2+, but potentiate the phosphohydrolase activity in the presence of Mg2+. The activation of Mg2+-dependent phosphatidate phosphohydrolase activity by polyamines was observed in the presence of 3-sn-phosphatidylcholine, suggesting that these modulators of phosphatidate phosphohydrolase activity may be acting through different mechanisms. These studies demonstrate that polyamines may be important regulators of Mg2+-dependent phosphatidate phosphohydrolase activity in adipose tissue.     

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.     

Pulmonary surfactant synthesis. A highly active microsomal phosphatidate phosphohydrolase in the lung.

Lung cell-free homogenate, which contains about twice the units of phosphatidate phosphohydrolase per mg of protein compared to liver, was fractionated by differential centrifugation and the fractions were assayed for phosphatidate phosphohydrolase and marker enzymes of endoplasmic reticulum, mitochondria, and lysosomes. Over 60% of the lung phosphatidate phosphohydrolase was associated with the endoplasmic reticulum, compared to 50% of the total liver enzyme. Thus a major portion of the more active lung enzyme is potentially involved in lipid biosynthesis by the endoplasmic reticulum. Less than 0.2% of the total lung enzyme was found in a lamellar body fraction, consistent with previous findings. The lung microsomal phosphohydrolase was specific for lipid substrates, showing equal activity towards phosphatidic acid or lysophosphatidic acid and relatively low activities towards glycerophosphates. It had a neutral pH optimum, similar to the liver enzyme, but differed somewhat in its relative activity at extremes of pH. Stability at 65 degrees C was greater for the lung enzyme. Fluroide inhibited lung (or liver) microsomal phosphatidate phosphohydrolase, while tartrate, MgCl2, or EDTA had no effect. The presence of a high activity of phosphatidate phosphohydrolase in lung endoplasmic reticulum is consistent with the rapid synthesis of pulmonary surfactant phosphatidylcholine.     

Glycerolipid biosynthesis in rat adipose tissue 12. Properties of Mg2+-dependent and -independent phosphatidate phosphohydrolase

The properties of Mg²⁺-dependent and Mg²⁺-independent phosphatidate phosphohydrolase activities were investigated in different subcellular fractions in rat adipose tissue. Phosphatidate phosphohydrolase activity was measured in the presence of aqueous dispersed phosphatidate as substrate, and the release of inorganic phosphate was taken as a measure of phosphatidate phosphohydrolase activity. The Mg²⁺-dependent phosphatidate phosphohydrolase was inhibited in the presence of N-methyl- or N-ethylmaleimide, whereas the Mg²⁺-independent activity was unaffected by these agents. The Mg²⁺-dependent phosphatidate phosphohydrolase was more sensitive to proteolysis and to high temperature (55 °C) compared to the Mg²⁺-independent enzyme. The Mg²⁺-dependent phosphatidate phosphohydrolase activity was reduced significantly during aging without any appreciable effects on the Mg²⁺-independent phosphatidate phosphohydrolase activity. These studies demonstrate that, in addition to Mg²⁺-dependency, these two forms of phosphatidate phosphohydrolases differ in several respects irrespective of their location in the adipose cell.     

Effects of cyclic AMP, glucocorticoids and insulin on the activities of phosphatidate phosphohydrolase, tyrosine aminotransferase and glycerol kinase in isolated rat hepatocytes in relation to the control of triacylglycerol synthesis and gluconeogenesis.

Rat hepatocytes were incubated in monolayer culture in modified Leibovitz L-15 medium containing either 10% (v/v) newborn-calf serum or 0.2% (w/v) fatty-acid-poor bovine serum albumin. The addition of 100 nM-dexamethasone increased the activities of both phosphatidate phosphohydrolase and tyrosine aminotransferase by about 3.5-fold after 8h, and these activities continued to rise until at least 24h. Incubating the hepatocytes in the albumin-containing medium with 10 microM- or 100 microM-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate increased the activities of the phosphohydrolase and aminotransferase by 2.6- and 3.4-fold respectively after 8h. These increases were blocked by actinomycin D. The increases in the activities that were produced by the cyclic AMP analogue and dexamethasone were independent and approximately additive. Insulin when added alone did not alter the phosphohydrolase activity, but it increased the aminotransferase activity by 34%. The dexamethasone-induced increase in the phosphohydrolase activity was completely blocked by 7-144 microM-insulin, whereas that of the aminotransferase was only partly suppressed. Insulin had no significant Effects on the increases in the activities of phosphatidate phosphohydrolase and tyrosine aminotransferase that were produced by the cyclic AMP analogue, but this may be because the analogue is fairly resistant to degradation by the phosphodiesterase. The activity of glycerol kinase was not significantly changed by incubating the hepatocytes with insulin, dexamethasone and the cyclic AMP analogue alone or in combinations. It is proposed that high concentrations of cyclic AMP and glucocorticoids increase the total activity of phosphatidate phosphohydrolase in the liver and provide it with an increased capacity for synthesizing triacylglycerols and very-low-density lipoproteins, which is expressed when the availability of fatty acids is high. There appears to be a co-ordinated hormonal control of triacyglycerol synthesis and gluconeogenesis in diabetes and in metabolic stress to enable the liver to supply other organs with energy.     

The measurement of phosphatidate phosphohydrolase in human amniotic fluid.

Phosphatidate phosphohydrolase (EC 3.1.3.4) activity can be found in late gestational human amniotic fluid and is thought to originate in type II alveolar cells of the fetal lungs where it plays an important role in lung surfactant synthesis. In the present study, phosphatidate phosphohydrolase activity was detected and characterized in a 105 000 X g pellet of amniotic fluid using either [32P]phosphatidate or a water-soluble analog, 1-O-hexadecyl-rac-[2-(3)H]glycerol 3-phosphate as substrate. With either substrate, enzyme activity was optimal at pH 6.0. The soluble analog was hydrolyzed with a Km value of 163 micrometer and a V of 30 nmole/min per mg of protein, and offered several advantages over phosphatidate as a substrate for assaying phosphatidate phosphohydrolase in amniotic fluid. Using the synthetic analog, phosphatidate phosphohydrolase activity was measured in the 700 X g supernatant fraction of 30 human amniocentesis samples and compared with another index of fetal lung maturity, the phosphatidylcholine/sphingomyelin ratio. The results suggest that the new phosphohydrolase assay may be clinically useful in the assessment of fetal lung development.     

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.     

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.     

Mg2-dependent phosphatidate phosphohydrolase of rat lung: development of an assay employing a defined chemical substrate which reflects the phosphohydrolase activity measured using membrane-bound substrate

An assay of pulmonary phosphatidate phosphohydrolase activity has been developed that employs a chemically defined liposome substrate of equimolar phosphatidate and phosphatidylcholine. Enzyme assays employing this substrate resolved two distinct activities based upon their requirements for Mg2+. Assays were performed in the presence and absence of 2 mM MgCl2 and the Mg2+-dependent phosphatidate phosphohydrolase activity calculated by difference. The Mg2+-independent phosphatase activity resembled that found using aqueous dispersions of phosphatidate (PAaq). Approximately 90% of the Mg2+-dependent phosphatidate phosphohydrolase activity was recovered in the cytosol and the remainder was associated with the microsomal fraction. The Mg2+-dependent phosphatidate phosphohydrolase activity has kinetic parameters of Km = 55 microM, Vmax = 1.6 nmol/min/mg protein for the microsomal fraction, and Km = 215 microM, Vmax = 6.8 nmol/min/mg protein for the cytosolic fraction. These parameters resembled those found using the microsomal membrane-bound (PAmb) substrate. In addition, the pH optima and sensitivity to detergents and thermal inactivation are equal to those for the PAmb-dependent phosphatidate phosphohydrolase activity. In the course of these studies the microsomal and cytosolic activities were qualitatively equal, indicative of a single enzyme in two subcellular locations. In conclusion, the assay of Mg2+-dependent phosphatidate phosphohydrolase activity measured using equimolar phosphatidate and phosphatidylcholine liposomes is equivalent to that activity previously described using microsomal membrane-bound substrate. However, the chemically-defined system provides a more simplified starting point for further studies on this important enzyme.     

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.     

The effect of chronic diabetes, induced by streptozotocin, on the activities of some enzymes of glycerolipid synthesis in rat liver.

1. Rats were injected with a single dose of 35mg of streptozotocin/kg body wt. They exhibited a diabetes that was characterized by glycosuria, polyuria, polydipsia, hyperphagia, hyperglycaemia, increased concentrations of unesterified fatty acids, glycerol and triacylglycerols in the serum and an increased activity of glucose 6-phosphatase in the liver. 2. After 10 weeks the hepatic activities of the microsomal glycerol phosphate acyltransferase, phosphatidate phosphohydrolase, phosphatidate cytidylyltransferase, diacylglycerol acyltransferase, choline phosphotransferase, CDP-diacylglycerol--inositol phosphatidyltransferase and the soluble phosphatidate phosphohydrolase were measured. 3. The only significant changes were an increase in the activity of the soluble phosphatidate phosphohydrolase and a decrease in that of the CDP-diacylglycerol--inositol phosphatidyltransferase in the diabetic rats. 4. These results are discussed in relation to the control of glycerolipid synthesis.     

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.     

Problems encountered in measuring the activity of phosphatidate phosphohydrolase.

The measurement of phosphate release from phosphatidate overestimates the microsomal activity of phosphatidate phosphohydrolase from rat liver, since phosphate is also produced via the glycerol phosphate that results from the deacylation of phosphatidate. The determination of phosphate production can be a reliable assay for the soluble phosphatidate phosphohydrolase in rat liver, because the glycerol phosphate formed is not hydrolysed under the conditions used.     

Insulin antagonises the growth hormone-mediated increase in the activity of phosphatidate phosphohydrolase in isolated rat hepatocytes

Rat hepatocytes were incubated in monolayer culture, under serum-free conditions for 8 h. Rat growth hormone (up to 100 nM) increased the activity of phosphatidate phosphohydrolase by up to 47%. Insulin (500 pM or 35 nM), cycloheximide or actinomycin D reversed this effect. The ability of growth hormone to modify the effects of insulin is discussed in relation to the control of the phosphohydrolase activity and glycerolipid synthesis.     

Vanadate-sensitive phosphatidate phosphohydrolase activity in a purified rabbit kidney Na,K-ATPase preparation.

Reconstitution of purified rabbit kidney Na,K-ATPase in phosphatidylcholine/phosphatidic acid liposomes resulted in the absence of ATP in a time-, temperature- and protein-dependent formation of inorganic phosphate. This formation of inorganic phosphate could be attributed to a phosphatidate phosphohydrolase activity present in the Na,K-ATPase preparation. A close interaction of the enzyme with the substrate phosphatidic acid was important, since no or little Pi production was observed under any of the following conditions: without reconstitution, after reconstitution in the absence of phosphatidic acid, with low concentrations of detergent or at low lipid/protein ratios. The hydrolysis of phosphatidic acid was not influenced by the Na,K-ATPase inhibitor ouabain but was completely inhibited by the P-type ATPase inhibitor vanadate. Besides Pi diacylglycerol was also formed, confirming that a phosphatidate hydrolase activity was involved. Since the phosphatidate phosphohydrolase activity was rather heat- and N-ethylmaleimide-insensitive, we conclude that the phosphatidic acid hydrolysis was not due to Na,K-ATPase itself but to a membrane-bound phosphatidate phosphohydrolase, present as an impurity in the purified rabbit kidney Na,K-ATPase preparations.     

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.     

Induction of endothelial monolayer permeability by phosphatidate.

Released into the vasculature from disrupted cells or transported to the surface of adjacent effectors, phosphatidate and related lipids may potentiate endothelial cell activation. However, the effect of these lipids on endothelial monolayer barrier integrity has not been reported. The present study documents the induction of endothelial monolayer permeability by phosphatidate. Both long (di-C18:1) and medium (di-C10; di-C8) chain length phosphatidates increased permeability of bovine pulmonary artery endothelial cell monolayers assessed using a well characterized assay system in vitro. Barrier disruption effected by dioctanoyl (di-C8) phosphatidate was markedly potentiated by the addition of propranolol, an inhibitor of endothelial cell "ecto"-phosphatidate phosphohydrolase (PAP), a lipid phosphate phosphohydrolase (LPP) that efficiently hydrolyzes extracellular substrate. Disruption of barrier function by phosphatidate did not result from its non-specific detergent characteristics, since a non-hydrolyzable but biologically inactive phosphonate analog of dioctanoyl phosphatidate, which retains the detergent characteristics of phosphatidate, did not induce permeability changes. Furthermore, neither diacylglycerol nor lyso-PA effected significant increases in monolayer permeability, indicating the observed response was due to phosphatidate rather than one of its metabolites. Phosphatidate-induced permeability was attenuated by preincubation of endothelial cells with the tyrosine kinase inhibitor, herbimycin A (10 microg/ml), and enhanced by the tyrosine phosphatase inhibitor, vanadate (100 microM), implicating a role for activation of intracellular tyrosine kinases in the response. In addition, phosphatidate increased the levels of intracellular free Ca(2+) in endothelial cells and ligated specific binding sites on endothelial cell plasma membranes, consistent with the presence of a phosphatidate receptor. Since phosphatidate generated within the plasma membrane of adherent effectors potentially interacts with endothelial membranes, we evaluated the influence of phosphatidate-enriched neutrophil plasma membranes on endothelial monolayer integrity. The effects of ectopic phosphatidate on endothelial monolayer permeability were mimicked by phosphatidate confined to neutrophil plasma membranes. We conclude that phosphatidate may be a physiologic modulator of endothelial monolayer permeability that exerts its effects by activating a receptor-linked, tyrosine kinase-dependent process which results in mobilization of intracellular stored Ca(2+)and consequent metabolic activation.     

Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol

Assays for two distinct phosphatidate phosphohydrolase activities were established based upon a differential inhibition by N-ethylmaleimide (NEM). The activity that is insensitive to this reagent in rat liver is predominantly in the plasma membrane fraction, whereas the NEM-sensitive activity is in the cytosolic and microsomal fractions. The NEM-insensitive activity is further distinguished from the NEM-sensitive phosphohydrolase by: (a) being relatively stable to heat; (b) not being inhibited by phenylglyoxal, butane-2,3-dione, cyclohexane-1,2-dione, 2,4-dinitrofluorobenzene, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and diethyl pyrocarbonate; (c) being inhibited by NaF and phosphatidylcholine; and (d) not being stimulated by Mg2+. The NEM-insensitive activity was specific for phosphatidate. Both phosphohydrolase activities could be inhibited by chlorpromazine, propranolol, sphingosine, and spermine. The NEM-sensitive phosphatidate phosphohydrolase activity was increased by incubating hepatocytes for 12 h with glucagon and dexamethasone, and this effect was antagonized by insulin. The NEM-sensitive phosphohydrolase is concluded to be involved in glycerolipid synthesis. The activity of the NEM-insensitive phosphohydrolase was not altered by preincubation of rat hepatocytes in the short or long term with vasopressin, glucagon, insulin, triiodothyronine, or dexamethasone, but it might be modulated indirectly by sphingosine. The NEM-insensitive enzyme of the plasma membranes could be involved in signal transduction via the agonist-stimulated degradation of phosphatidylcholine through the phospholipase D pathway.     

Can phosphorylation of phosphatidate phosphohydrolase by a cyclic AMP-dependent mechanism regulate its activity and subcellular distribution and control hepatic glycerolipid synthesis?

Incubating the particle-free supernatant of rat liver with alkaline phosphatase decreased the activity of phosphatidate phosphohydrolase by 21-29%. When the particle-free supernatant was incubated with various combinations of Mg2+, ATP, cyclic AMP and cyclic AMP-dependent protein kinase this failed to alter significantly phosphatidate phosphohydrolase activity under the conditions employed. The incubation of hepatocytes in monolayer culture with 0.5 mM-8-(4-chlorophenylthio)adenosine 3',5'-monophosphate increased the total activity of phosphatidate phosphohydrolase as measured in vitro. This also decreased the proportion of the phosphohydrolase that was associated with the membrane fraction of the cells and increased that in the cytosolic fraction. Adding 1 mM-oleate to the hepatocytes promoted the translocation of phosphatidate phosphohydrolase from the cytosol to the membrane-associated compartment. Oleate overcame the effect of the cyclic AMP analogue in favouring the cytosolic distribution of the phosphohydrolase. These results are discussed in relation to the interaction of hormonal balance and substrate supply in controlling the synthesis of phosphatidylcholine and triacylglycerol in the liver in stress and in diabetes. It is proposed that the cytosolic phosphatidate phosphohydrolase activity represents a reservoir of potential activity that becomes expressed when the enzyme translocates to the membranes on which the synthesis of glycerolipids occurs.