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PubMed2006

Lipid phosphate phosphatase-1 regulates lysophosphatidate-induced fibroblast migration by controlling phospholipase D2-dependent phosphatidate generation.

Lysophosphatidate (LPA) stimulates cell migration and division through a family of G-protein-coupled receptors. Lipid phosphate phosphatase-1 (LPP1) regulates the degradation of extracellular LPA as well as the intracellular accumulation of lipid phosphates. Here we show that increasing the catalytic activity of LPP1 decreased the pertussis toxin-sensitive stimulation of fibroblast migration by LPA and an LPA-receptor agonist that could not be dephosphorylated. Conversely, knockdown of endogenous LPP1 activity increased LPA-induced migration. However, LPP1 did not affect PDGF- or endothelin-induced migration of fibroblasts in Transwell chamber and "wound healing" assays. Thus, in addition to degrading exogenous LPA, LPP1 controls signaling downstream of LPA receptors. Consistent with this conclusion, LPP1 expression decreased phospholipase D (PLD) stimulation by LPA and PDGF, and phosphatidate accumulation. This LPP1 effect was upstream of PLD activation in addition to the possible metabolism of phosphatidate to diacylglycerol. PLD(2) activation was necessary for LPA-, but not PDGF-induced migration. Increased LPP1 expression also decreased the LPA-, but not the PDGF-induced activation of important proteins involved in fibroblast migration. These included decreased LPA-induced activation of ERK and Rho, and the basal activities of Rac and Cdc42. However, ERK and Rho activation were not downstream targets of LPA-induced PLD(2) activity. We conclude that the intracellular actions of LPP1 play important functions in regulating LPA-induced fibroblast migration through PLD2. LPP1 also controls PDGF-induced phosphatidate formation. These results shed new light on the roles of LPP1 in controlling wound healing and the growth and metastasis of tumors.

Animals↗
PubMedDate not supplied

Characterization of the pulmonary N-ethylmaleimide-insensitive phosphatidate phosphohydrolase.

Phosphatidate phosphohydrolase (PAPase) is a key enzyme involved in glycerolipid synthesis where it converts phosphatidic acid to diacylglycerol. Previous studies performed in lung have demonstrated the existence of 2 different forms of PAPases, namely PAP-1 and PAP-2. The former pulmonary Mg+2-dependent enzyme is N-ethylmaleimide (NEM)-sensitive, heat labile, and is involved in phospholipid biosynthesis. However, the function of the latter lung isozyme is unknown. PAP-2 activity was selectively assayed using NEM in the absence of Mg+2. Studies employing this assay and adult rat lung microsomal preparations demonstrated that PAP-2 activity was inhibited by amphiphilic amines, sphingoid bases, products of the PAP-2 reaction (monoacylglycerol [MAG] and diacylglycerol [DAG]), and substrate analogs such as lysophosphatidic acid (lyso-PA), ceramide-1-phosphate, and to a lesser extent, sphingosine-1-phosphate. Purified lung plasma membranes, prepared using discontinuous sucrose and Percoll gradients, showed that PAP-2 activity was enriched 6.9 +/- 1.6-fold over the whole homogenate and was between the enrichment for plasma membrane markers, 5'-nucleotidase (14.7 +/- 0.3) and Na+, K(+)-ATPase (4.0 +/- 0.2). Both phosphatidic acid and lysophosphatidic acid were good substrates for PAP-2 activity in this purified plasma membrane fraction. In contrast, sphingosine-1-phosphate was a relatively poor substrate. PAP-2 activity was slightly enriched in isolated type II cells and low in isolated rat lung fibroblasts. This study shows lung contains PAP-2 activity in plasma membranes and type II cells where it could play a role in signal transduction.

Animals↗
PubMed1993

Liposomal hamycin in the control of experimental aspergillosis in mice: effect of phosphatidic acid with and without cholesterol.

Hamycin incorporated into liposomes containing phosphatidylcholine (SPC) and phosphatidic acid (PA) had reduced toxicity and an enhanced antifungal activity in experimental aspergillosis in balb/c mice. Incorporation of cholesterol into liposomes led to a dose dependent decrease in the toxicity of hamycin. The LD50 (mg/kg) of hamycin contained in SPC/cholesterol/PA (molar ratio 4:5:1) liposomes was 2.8 whereas that in SPC/PA liposomes (molar ratio 9:1) was 0.35. Although the free drug had little or no protective effect on the animals, those administered liposomal hamycin at an equivalent dose (0.1 mg/kg) in the absence of cholesterol (SPC/PA; molar ratio 9:1) showed 90% survival after seven days of therapy. On the other hand the presence of cholesterol in the carrier phosphatidic acid liposomes (SPC/cholesterol/PA; molar ratio 4:5:1) at a similar dose (0.1 mg/kg) led to a 60% survival over the same time period. Hamycin incorporation in phosphatidic acid liposomes both in the presence or absence of cholesterol was found to be effective in reducing the fungal load in lung, liver, spleen and kidney. Studies with distribution of hamycin in various tissues by HPLC showed a significant reduction in the concentration of the liposomal drug in circulation as compared to those seem after administration of free drug.

Animals↗
PubMed2004

Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid.

Cells regulate the biophysical properties of their membranes by coordinated synthesis of different classes of lipids. Here, we identified a highly dynamic feedback mechanism by which the budding yeast Saccharomyces cerevisiae can regulate phospholipid biosynthesis. Phosphatidic acid on the endoplasmic reticulum directly bound to the soluble transcriptional repressor Opi1p to maintain it as inactive outside the nucleus. After the addition of the lipid precursor inositol, this phosphatidic acid was rapidly consumed, releasing Opi1p from the endoplasmic reticulum and allowing its nuclear translocation and repression of target genes. Thus, phosphatidic acid appears to be both an essential ubiquitous metabolic intermediate and a signaling lipid.

Active Transport, Cell Nucleus↗
PubMed1999

Redundant systems of phosphatidic acid biosynthesis via acylation of glycerol-3-phosphate or dihydroxyacetone phosphate in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae lipid particles harbor two acyltransferases, Gat1p and Slc1p, which catalyze subsequent steps of acylation required for the formation of phosphatidic acid. Both enzymes are also components of the endoplasmic reticulum, but this compartment contains additional acyltransferase(s) involved in the biosynthesis of phosphatidic acid (K. Athenstaedt and G. Daum, J. Bacteriol. 179:7611-7616, 1997). Using the gat1 mutant strain TTA1, we show here that Gat1p present in both subcellular fractions accepts glycerol-3-phosphate and dihydroxyacetone phosphate as a substrate. Similarly, the additional acyltransferase(s) present in the endoplasmic reticulum can acylate both precursors. In contrast, yeast mitochondria harbor an enzyme(s) that significantly prefers dihydroxyacetone phosphate as a substrate for acylation, suggesting that at least one additional independent acyltransferase is present in this organelle. Surprisingly, enzymatic activity of 1-acyldihydroxyacetone phosphate reductase, which is required for the conversion of 1-acyldihydroxyacetone phosphate to 1-acylglycerol-3-phosphate (lysophosphatidic acid), is detectable only in lipid particles and the endoplasmic reticulum and not in mitochondria. In vivo labeling of wild-type cells with [2-3H, U-14C]glycerol revealed that both glycerol-3-phosphate and dihydroxyacetone phosphate can be incorporated as a backbone of glycerolipids. In the gat1 mutant and the 1-acylglycerol-3-phosphate acyltransferase slc1 mutant, the dihydroxyacetone phosphate pathway of phosphatidic acid biosynthesis is slightly preferred as compared to the wild type. Thus, mutations of the major acyltransferases Gat1p and Slc1p lead to an increased contribution of mitochondrial acyltransferase(s) to glycerolipid synthesis due to their substrate preference for dihydroxyacetone phosphate.

Acylation↗
PubMed1990

Antibodies to liposomal phosphatidylserine and phosphatidic acid.

Polyclonal antisera to phosphatidylserine or phosphatidic acid were induced in rabbits by injecting liposomes containing phosphatidylserine or phosphatidic acid and lipid A. Adsorption of antisera with liposomes containing different phospholipids revealed that some degree of reactivity with one or more phospholipids other than the immunizing phospholipid was often observed. However, cross-reactivity with other phospholipids was not a universal phenomenon, and one antiserum to phosphatidylserine failed to cross-react (i.e., was not adsorbed) with liposomes containing other phospholipids. All of the antisera were inhibited by soluble phosphorylated haptens (e.g., phosphocholine but not choline), but one of the antisera to phosphatidylserine was inhibited both by phosphoserine and by serine alone. Liposomal membrane composition influenced the activity of antiserum to phosphatidylserine. Regardless of whether unsaturated (beef brain) or saturated (dimyristoyl) phosphatidylserine was used in the immunizing liposomes, the antisera reacted more vigorously with liposomes containing unsaturated than saturated phosphatidylserine. We conclude that liposomes containing lipid A can serve as vehicles for stimulating polyclonal antisera to phosphatidylserine and phosphatidic acid. Although cross-reactivity with certain other phospholipids can be observed, sera from selected animals apparently can exhibit a high degree of specific activity to the immunizing phospholipid antigen.

Animals↗
PubMed1995

Stimulation and binding of myocardial phospholipase C by phosphatidic acid.

Exposure of adult ventricular myocytes to exogenous natural phosphatidic acid results in the production of inositol phosphates by unknown mechanism(s). We characterized stimulation of myocytic phosphoinositide-specific phospholipase C (PLC) by synthetic dioleoyl phosphatidic acid (PA) as a potential mechanism for modulation of inositol phosphate production. Our data demonstrate that exogenous PA, at 10(-8)-10(-5) M, caused a concentration-dependent increase in inositol 1,4,5-trisphosphate in adult rabbit ventricular myocytes. PA also caused a concentration-dependent increase in in vitro activity of myocytic PLC in the presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLC-delta 1, the predominant isozyme of PLC expressed in adult rabbit ventricular myocytes, bound to liposomes of PA with high affinity in the presence of EGTA. The phosphomonoester group of PA was critical to in vitro stimulation of myocytic PLC activity and high-affinity binding of PLC-delta 1. We propose that binding of PLC-delta 1 to phosphatidic acid may be a novel mechanism for dynamic membrane association and modulation of PLC in adult ventricular myocytes.

Animals↗
PubMed1997

Modulating phosphatidic acid metabolism decreases oxidative injury in rat lungs.

We determined that lisofylline, a potent inhibitor of oleate- and linoleate-containing phosphatidic acid formation (half-maximal inhibitory concentration = 40 nM), prevented oxidant-mediated capillary leak in isolated rat lungs given interleukin-8 (IL-8) intratracheally and perfused with human neutrophils. Lung leak was prevented by lung, but not neutrophil, lisofylline pretreatment. Furthermore, although lisofylline inhibited IL-8-stimulated neutrophil production of phosphatidic acid in vitro, it did not prevent IL-8-stimulated neutrophil adherence, chemotaxis, or intracellular calcium mobilization or N-formyl-Met-Leu-Phe (fMLP)-stimulated oxidant production in vitro. Lisofylline also prevented acute capillary leak in isolated rat lungs perfused only with the oxidant generator purine-xanthine oxidase but did not scavenge O2-(+) or H2O2 in vitro. Finally, lisofylline-mediated protection against lung leak in both models was associated with alterations in lung membrane free fatty acid acyl composition (as reflected by the decreased ratio [linoleate + oleate]/[palmitate]). We conclude that lisofylline prevented both neutrophil-dependent and neutrophil-independent oxidant-induced capillary leak in isolated rat lungs and that protection appears to be mediated by blocking intrinsic lung linoleoyl phosphatidic acid metabolism. We speculate that lisofylline, in addition to our previously reported effects on cytokine signaling by intrapulmonary mononuclear cells, alters intrinsic pulmonary capillary membrane composition and renders this barrier less vulnerable to oxidative damage.

Animals↗
PubMed1994

Relative contribution of phosphoinositides and phosphatidylcholine hydrolysis to the actions of carbamylcholine, thyrotropin, and phorbol esters on dog thyroid slices: regulation of cytidine monophosphate-phosphatidic acid accumulation and phospholipase-D activity. II. Actions of phorbol esters.

The effects of phorbol dibutyrate (PDBu) on phosphatidylbutanol (PtdBut) generation in [3H]palmitate- or [3H]myristate-prelabeled dog thyroid slices were measured to assess the activity of phospholipase-D (PLD) in the presence or absence of the two inhibitors of protein kinase-C (PKC), staurosporine (STSP) and calphostin-C. The actions of the same agents on [3H]cytidine monophosphate-phosphatidic acid accumulation were also determined to evaluate phosphatidic (PA) generation and inositol recycling to phosphatidylinositol. The effluxes of [3H]choline and [3H]ethanolamine induced by the phorbol ester from prelabeled slices were also evaluated. PDBu (5 x 10(-9) to 5 x 10(-6) M) potently stimulated PLD activity, with a concomitant increase in fatty acids incorporation in phosphatidylcholine (PtdCho). However, under no condition did the phorbol ester result in cytidine monophosphate-phosphatidic acid accumulation. It stimulated the efflux of choline and ethanolamine while decreasing choline and ethanolamine phosphates in the slices and incubation medium. Calphostin-C, inhibiting PKC, decreased PtdBut and PtdCho formation induced by the phorbol ester, as opposed to STSP (5 x 10(-6) M), which did not affect these actions of PDBu and, moreover, reproduced by itself the effects of the phorbol ester on choline efflux and PtdBut generation despite efficient inhibition of other effects of PKC. These data demonstrate the existence in thyroid tissue of a PLD-hydrolyzing PtdCho, which was stimulated by phorbol esters and STSP. They also suggest that the PA formed after PKC stimulation and subsequent PLD activation is channeled toward PtdCho resynthesis when intracellular Ca2+ is not increased, whereas the PA accumulated with a concomitant increase in intracellular Ca2+ is diverted toward phosphatidylinositol synthesis. The physiological relevance of this Ca-independent stimulation of a PKC-coupled PLD in thyroid metabolism could be related to the growth-inducing and dedifferentiating effects of the phorbol esters.

Animals↗
PubMed1982

Lung phosphatidate phosphatase: activity during altered physiologic states.

Phosphatidic acid phosphatase (PAPase) which catalyzes the conversion of phosphatidic acid to 1,2-diacyl-sn-glycerol was studied in fetal, neonatal, and adult rat lung microsomal fractions from whole lung under normal and altered physiological states. The maximal activity was obtained at pH 7.0 with 1.0 mM phosphatidic acid as the substrate. Twenty-one-day-old fetal rat lung averaged 20.3 +/- 0.6 SE nmol/min/mg microsomal protein compared to 9.9 +/- l.0 nmol/min/mg in liver. Following birth there was a dramatic 53% increase in the PAPase activity. Twenty-one-day-old fetal rat lungs from diabetic mothers (streptozotocin-induced) and from mothers fasted the last four days of gestation did not show altered PAPase activity. Premature breathing for 3-6 hr on day 21 of gestation also did not affect the PAPase activity. These data demonstrate that the microsomal PAPase activity (l) increases dramatically only after birth (2) is not responsive to altered physiologic state (maternal diabetes, maternal fasting, and premature breathing) and (3) may not be an important regulatory enzyme in lung surfactant phospholipid production.

Animals↗
PubMed1976

Parathyroid hormone-mediated incorporation of 32P-orthophosphate into phosphatidic acid and phosphatidylinositol in renal cortical slices.

Parathyroid hormone increased the incorporation of Na2H32PO4 into phosphatidic acid and phosphatidylinositol in cat renal cortical slices. Incorporation was not observed into any other phospholipid. The effects were seen as early as one minute for phosphatidic acid and ten minutes for phosphatidylinositol. 8-Bromoadnosine 3',5'-monophosphate did not mimic the effects of parathyroid hormone. Concentrations of parathyroid hormone, 1 x 10(-8)M to 1 x 10(-7)M, which increased the incorporation of 32p into phosphatidic acid and phosphatidylinositol maximally, did not alter tissue cyclic AMP levels suggesting that the incorporation of 32p was independent of cyclic AMP.

Animals↗
PubMed1989

Rat adipose tissue phosphatidic acid phosphatase: lack of effect of nucleotides on cytosolic enzyme activity.

The soluble phosphatidic acid phosphatase from rat adipose tissue was partially purified using ammonium sulfate fractionation and hydroxyapatite chromatography. Administration of ethanol has been found to increase phosphatidic acid phosphatase activity. The enzyme activity has been found to be dependent on magnesium ions with maximal activity at 2-5 mM magnesium. The enzyme displays an apparent pH optimum of 7.0. The activity of the enzyme is not affected by addition of ATP or ADP, in contrast with the results for hepatic phosphatidic acid phosphatase. The results suggest that these two enzymes may be regulated by different mechanisms and that they may thus represent two different types of isoenzyme.

Adenosine Diphosphate↗
PubMed1992

Propranolol, a phosphatidate phosphohydrolase inhibitor, also inhibits protein kinase C.

Propranolol, a beta-adrenergic receptor antagonist, also inhibits phosphatidate phosphohydrolase, the enzyme that converts phosphatidic acid into diacylglycerol. This latter effect has prompted recent use of propranolol in studies examining the importance of diacylglycerol and phosphatidic acid in cellular signalling events. Here, we show that propranolol is also an inhibitor of protein kinase C. At concentrations greater than or equal to 20 microM, propranolol reduced [3H]phorbol dibutyrate binding (IC50 = 200 microM) and phorbol myristate acetate-stimulated superoxide anion release (IC50 = 130 microM) in human neutrophils. Scatchard analysis showed that propranolol lowers the number of phorbol diester binding sites without significantly affecting their affinity. In vitro kinetic analysis, performed in a mixed micellar assay with protein kinase C purified from human neutrophils, suggested a competitive inhibition of propranolol with the cofactor phosphatidylserine. Complex kinetic patterns were observed with respect to diacylglycerol and ATP, approximating competitive and noncompetitive inhibition, respectively. Taken together, these results suggest that the drug interacts at the level of the regulatory domain of the enzyme. Fifty % inhibition occurred at approximately 150 microM propranolol. Similar levels of inhibition were obtained using exogenous (histone) and endogenous (p47-phox, a NADPH oxidase component) substrates. Protein kinase C-alpha and protein kinase C-beta, two protein kinase C isozymes present in human neutrophils, were inhibited by propranolol in a comparable manner. In the range of concentrations tested (30-1000 microM), neither cAMP-dependent protein kinase nor neutrophil protein tyrosine kinases were affected. The racemic form of propranolol and the (+) and the (-) stereoisomers were equally active, and other beta-adrenergic receptor antagonists (pindolol) and agonists (isoproterenol) were inactive. This suggests that the inhibitory action of propranolol on protein kinase C is related to the amphipathic nature of the drug rather than to its beta-adrenergic receptor blocking ability. Analogs of propranolol were synthesized and found to be more potent protein kinase C inhibitors, with IC50 values in the 10-20 microM range. We conclude that the ability of propranolol to inhibit both protein kinase C and PA phosphohydrolase complicates interpretation of results when this drug is used in signal transduction studies. In addition, propranolol may be a useful prototype for the synthesis of new protein kinase C inhibitors.

Cells, Cultured↗
PubMed1991

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.

Animals↗
PubMed1986

Monoclonal anti-DNA IgM kappa in neuropathy binds to myelin and to a conformational epitope formed by phosphatidic acid and gangliosides.

Anti-DNA antibodies that cross-react with phosphorylated epitopes of other cellular constituents may be involved in the pathogenesis of autoimmune disease. An IgM monoclonal antibody from a patient with chronic lymphocytic leukemia (CLL) and neuropathy bound to denatured DNA and immunostained myelin in peripheral nerve and spinal cord. The monoclonal IgM bound to ELISA microwells coated with a mixture of phosphatidic acid and gangliosides at serum dilutions of up to 1/100,000, but binding to phosphatidic acid alone was observed at dilutions of less than 1/100 only, and there was no binding to gangliosides alone. Incubation with micelles containing phosphatidic acid and gangliosides selectively absorbed the monoclonal IgM and inhibited its binding to denatured DNA and to myelin. These observations suggest that autoantibodies may bind to conformational epitopes formed by two separate molecules, and that autoantibodies that cross-react with phosphorylated epitopes in DNA and neural tissue could be involved in autoimmune neurologic diseases.

Antibodies, Antinuclear↗
PubMed1989

Exercise-induced translocation of protein kinase C and production of diacylglycerol and phosphatidic acid in rat skeletal muscle in vivo. Relationship to changes in glucose transport.

Contraction-induced translocation of protein kinase C (Richter E.A., Cleland, P.J.F., Rattigan, S., and Clark, M.G. (1987) FEBS Lett. 217, 232-236) implies a role for this enzyme in muscle contraction or the associated metabolic adjustments. In the present study, this role is further examined particularly in relation to changes in glucose transport. Electrical stimulation of the sciatic nerve of the anesthetized rat in vivo led to a time-dependent translocation of protein kinase C and a 2-fold increase in the concentrations of both diacylglycerol and phosphatidic acid. Maximum values for the latter were reached at 2 min and preceded the maximum translocation of protein kinase C (10 min). Stimulation of muscles in vitro increased the rate of glucose transport, but this required 20 min to reach maximum. There was no reversal of translocation or decrease in the concentrations of diacylglycerol and phosphatidic acid even after 30 min of rest following a 5-min period of stimulation in vivo. Translocation was not influenced by variations in applied load at maximal fiber recruitment but was dependent on the frequency of nontetanic stimuli, reaching a maximum at 4 Hz. The relationship between protein kinase C and glucose transport was also explored by varying the number of tetanic stimuli. Whereas only one train of stimuli (200 ms, 100 Hz) was required for maximal effects on protein kinase C, diacylglycerol, and phosphatidic acid, more than 35 trains of stimuli were required to activate glucose transport. It is concluded that the production of diacylglycerol and the translocation of protein kinase C may be causally related. However, if the translocated protein kinase C is involved in the activation of glucose transport during muscle contractions, an accumulated exposure to Ca2+, resulting from multiple contractions, would appear to be necessary.

Animals↗
PubMed1986

Cholinergic stimulation of phosphatidic acid formation by rat cornea in vitro.

The cornea has one of the highest acetylcholine (ACh) concentrations of any tissue but the function of the ACh has remained enigmatic. During studies on corneal arachidonic acid metabolism, we observed that ACh stimulates formation of labeled phosphatidic acid in rat corneas whose phospholipids were prelabeled with [14C]arachidonate. ACh did not affect the metabolism of free [14C]arachidonate. [14C]Arachidonyl-phosphatidic acid formation was doubled after 10 min of incubation in the presence of ACh concentrations of 10(-4) M or greater. The stimulation by ACh could be completely blocked by atropine and scopolamine and partially blocked by d-tubocurarine. These studies suggest that intact rat cornea has muscarinic cholinergic receptors and that the enzymes of the inositol phospholipids pathway are present since phosphatidic acid is an obligatory intermediate in that cycle of reactions.

Acetylcholine↗
PubMed1985

Phosphorylation, transbilayer movement, and facilitated intracellular transport of diacylglycerol are involved in the uptake of a fluorescent analog of phosphatidic acid by cultured fibroblasts.

We have previously shown that a fluorescent derivative of phosphatidic acid, 1-acyl-2-[N-(4-nitrobenzo-2-oxa-1,3-diazole) aminocaproyl]phosphatidic acid (C6-NBD-PA) is rapidly transferred from liposomes to Chinese hamster fibroblasts at 2 degrees C, resulting in intense labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope, but not the plasma membrane. During this labeling, C6-NBD-PA is metabolized predominantly to fluorescent diacylglycerol (Pagano, R. E., Longmuir, K. J., Martin, O. C., and Struck, D. K. (1981) J. Cell Biol. 91, 872-877). In the present study we investigated the mechanism by which C6-NBD-PA enters cells and is translocated to intracellular membranes at low temperature. (i) When hydrolysis of C6-NBD-PA to diacylglycerol was prevented by using a nonhydrolyzable fluorescent phosphonate analog, intense labeling of the plasma membrane occurred but fluorescent lipid did not enter the cytoplasm of cells. (ii) Experiments using C6-NBD-PA and cells prelabeled with 32Pi demonstrated that some of the fluorescent diacylglycerol was rephosphorylated at 2 degrees C. (iii) When cells were treated with 1,3-[palmitoyl, N-(4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl]-glycerophosphate, the lipid was dephosphorylated to 1,3-diacylglycerol but its rephosphorylation could not be detected. Nevertheless, rapid labeling of cytoplasmic membranes occurred. (iv) Formation of fluorescent diacylglycerol at the plasma membrane by treatment of cells with fluorescent phosphatidylcholine followed by phospholipase C at 2 degrees C resulted in strong labeling of intracellular membranes. Based on these results, a working model is presented for the uptake and intracellular translocation of phosphatidic acid involving formation of diacylglycerol at the plasma membrane followed by its transbilayer movement, facilitated translocation to intracellular membranes, and rephosphorylation.

Animals↗