Phosphatidic Acid Synthesis Research Articles

Enhanced de novo synthesis of phosphatidic acid and phosphatidylinositol in rat pancreatic islets exposed to nutrient or neurotransmitter stimuli

A stimulation of [ 3H]glycerol incorporation into phosphatidic acid and phosphatidylinositol was observed upon exposure of rat pancreatic islets to the nutrient secretagogues α-ketoisocaproate and glucose, or to the neurotransmitter stimuli carbamylcholine and cholecystokinin. These effects were associated with reduced labeling of phosphatidylcholine and, in some cases, phosphatidylethanolamine. The modified patterns of [ 3H]glycerol incorporation into islet phospholipids persisted in the absence of added Ca 2+, but were abolished by excess EDTA. Nutrient, but not neurotransmitter, secretagogues also stimulated the incorporation of [ 3H]glycerol into triacylglycerols. The results suggest that the stimulation of islets with the above classes of secretagogues is accompanied by enhanced de novo synthesis of acidic phospholipids.

Calcium-dependent hydrolyses of polyphosphoinositides in human erythrocyte membranes.

Incubation of erythrocytes with [32P]phosphate resulted in a linear incorporation of the label into PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), PtdIns4P (phosphatidylinositol 4-monophosphate), and PA (phosphatidic acid) over a period of 2 h at 37 degrees C. Exposure of 32P-labelled erythrocyte ghosts to calcium caused a loss of label from PtdIns(4,5)P2 and PtdIns4P, but not PA. The concentration of calcium required for half-maximal hydrolyses of both polyphosphoinositides was about 1 microM. Strontium, at higher concentrations, stimulated the hydrolyses of both polyphosphoinositides but barium, up to 1 mM, had little effect. Intact erythrocytes incubated in the presence of Ca-EGTA buffers and the ionophore A23187 did not show marked losses of [32P]PtdIns(4,5)P2 or [32P]PtdIns4P, but rather exhibited a dramatic increase in the level of [32P]PA. In contrast, cells which had been depleted of their ATP lost significant amounts of [32P]PtdIns(4,5)P2 and [32P]PtdIns4P and had less change in their levels of [32P]PA relative to intact cells. The calcium activation curve and the time course for [32P]PA synthesis in intact cells were similar to the calcium activation curve and the time course for the hydrolyses of [32P]PtdIns(4,5)P2 and [32P]PtdIns4P in ATP-depleted erythrocytes. These results strongly support a link between Ca2+-dependent polyphosphoinositide breakdown and PA synthesis in human erythrocytes.

Effects of insulin and protein synthesis inhibitors on phospholipid metabolism, diacylglycerol levels, and pyruvate dehydrogenase activity in BC3H-1 cultured myocytes.

BC3H-1 myocytes were cultured with 32PO4 for 3 days to label phospholipids to constant specific activity. Subsequent treatment with physiological concentrations of insulin provoked 40-70% increases in 32PO4 levels (reflecting increases in mass) in phosphatidic acid, phosphatidylinositol, and polyphosphoinositides, and, lesser, 20-25% increases in phosphatidylserine and the combined chromatographic area containing phosphatidylethanolamine plus phosphatidylcholine plus phosphatidylcholine. Insulin-induced increases in phospholipids were significant within 5 min and near-maximal at 15-30 min. Comparable rapid insulin-induced increases in [3H]phosphatidylinositol were observed in myocytes prelabeled with [3H]inositol. These insulin effects (as per prolonged pulse-chase experiments) were due to increase phospholipid synthesis rather than decreased phospholipid degradation. Cycloheximide (and puromycin) pretreatment prevented insulin-induced increases in phospholipids and rapidly reversed ongoing insulin effects on phospholipids and pyruvate dehydrogenase activity. Insulin also rapidly increased diacylglycerol levels. These findings suggest that: (a) insulin provokes rapid increases in de novo synthesis of phosphatidic acid and its derivatives, e.g. phosphoinositides and diacylglycerol; (b) protein synthesis inhibitors diminish phospholipid levels in insulin-treated (but not control) tissues by increasing phospholipid degradation (?phospholipase(s) activation); and (c) changes in phospholipids and diacylglycerol may be important for changes in pyruvate dehydrogenase and other enzymatic activities during treatment with insulin and/or protein synthesis inhibitors.

Changes in the platelet phosphoinositides during the first minute after stimulation of washed rabbit platelets with thrombin.

Experiments with washed platelets from rabbits demonstrate that stimulation with a low concentration of thrombin (0.1 unit/ml) that causes maximal aggregation and partial release of granule contents does not significantly decrease the amount of phosphatidylinositol 4,5-bisphosphate [ PtdIns (4,5)P2] at 10s; this contrasts with ADP stimulation. The amount of PtdIns (4,5)P2 was significantly decreased by a higher concentration of thrombin (0.3 unit/ml). Increased turnover of the PtdIns (4,5)P2 at 60s was indicated by changes in labelling with [3H]glycerol in platelets stimulated with both concentrations of thrombin. An unexpected observation with the lower thrombin concentration was a significant increase in the amount of phosphatidylinositol ( PtdIns ) at 10s. This contrasts with data from other laboratories, which indicate that thrombin causes a significant decrease in PtdIns . At 60s, with the lower concentration of thrombin, PtdIns was significantly decreased. With the higher concentration of thrombin there was a significant decrease in the amount of PtdIns at 10s, in keeping with the data from other laboratories. The initial increase in PtdIns may not have been observed by other investigators because higher concentrations of thrombin were used. The reaction involved in this initial increase in the amount of PtdIns does not appear to be increased degradation of PtdIns4P or PtdIns (4,5)P2, since their total amount was unchanged at 10s. The magnitude of the increase in PtdIns is such that more than the existing pool of phosphatidic acid would have to be converted into PtdIns to account for the increase. It is suggested that synthesis of phosphatidic acid de novo from dihydroxyacetone phosphate and glycerol 3-phosphate might be the source of phosphatidic acid, which leads to increased PtdIns at 10s with the lower concentration of thrombin. Thus it appears that the initial response of platelets to thrombin does not require an early change in PtdIns (4,5)P2 and may involve stimulation of synthesis de novo of PtdIns via phosphatidic acid.

Prostacyclin inhibition of phosphatidic acid synthesis in human platelets is not mediated by protein kinase C

The activation of protein kinase C in human platelets by phorbol-12,13-dibutyrate (PDBu) ∗ results in the phosphorylation of a 40,000 dalton protein. This phosphorylation is time- and concentration-dependent. Maximal phosphorylation is rapid and is not affected by indomethacin or prostacyclin. PDBu does not promote activation of the phosphodiesteratic cleavage (phospholipase C) of the inositol phospholipids and the subsequent formation of 1,2-diacylglycerol or its phosphorylated product, phosphatidic acid. If platelets exposed to PDBu are subsequently stimulated with thrombin, this stimulus does not initiate further 40,000 dalton protein phosphorylation but will promote the formation of phosphatidic acid and also the phosphorylation of a 20,000 dalton protein (myosin light chain). However, prostacyclin will prevent the subsequent stimulation of phosphatidic acid synthesis by thrombin in a concentration-dependent manner. The fact that prostacyclin can affect the response to thrombin, even in the presence of phorbol ester, supports the idea that the enzymes related to the formation of phosphatidic acid or inhibition of its synthesis are not related to the phosphorylated 40K protein.

Metabolic effects of β, α1, and α2 adrenoceptor activation on brown adipocytes isolated from the perirenal adipose tissue of fetal lambs

Brown adipocytes can be readily isolated by collagenase digestion of perirenal adipose tissue from fetal lambs. In isolated cells the addition of phenylephrine in the presence of alprenolol (to specifically stimulate α adrenoceptors) resulted in an increase in de novo synthesis of phosphatidylinositol and phosphatidic acid. The stimulatory effects were preferentially inhibited by prazosin while yohimbine had little effect, indicating that the adrenoceptors were α 1 in character. Isoproterenol stimulated cyclic adenosine monophosphate (AMP) accumulation and lipolysis as well as respiration. Forskolin also mimicked the effects of β adrenergic stimulation. Clonidine, a specific α 2 adrenergic agonist, inhibited lipolysis and cyclic AMP accumulation. Insulin inhibited cyclic AMP accumulation and stimulated glucose metabolism in the adipocytes. The present studies indicate that β, α 1, and α 2 adrenergic as well as insulin responses can be detected in ovine perirenal adipocytes.

The synthesis of linoleate and phosphatidic acid and its relationship to oil production in the microsomes of developing seeds of safflower ( Carthamus tinctorius, L. var. Gila)

Microsomes from the developing cotyledons of safflower seeds (var. Gila) were incubated, under desaturating conditions, with [1- 14C]oleoyl-CoA in the presence of glycerol 3-phosphate. The mass and specific radioactivity of oleate and linoleate in acyl-CoA, phosphatidylcholine and phosphatidic acid showed that (i) the oleate in oleoyl-CoA was selectively transferred to position 2 of phosphatidylcholine by acyl exchange where it underwent desaturation to linoleate; (ii) the newly formed linoleate was returned by further acyl exchange, to the acyl-CoA pool from which it was selectively utilised in the acylation of glycerol 3-phosphate to yield phosphatidic acid, a precursor of triacylglycerol. The results confirm that acyl exchange between acyl-CoA and phosphatidylcholine plays a major role in regulating the quality of C18 unsaturated fatty acids in the acyl-CoA pool for oil synthesis.

Facilitated utilization of endogenously synthesized lysophosphatidic acid by 1-acylglycerophosphate acyltransferase from Escherichia coli

Complete separation of glycerophosphate acyltransferase and 1-acylglycerophosphate acyltransferase from Escherichia coli was obtained by sequential extraction with Triton X-100. Solubilized glycerophosphate acyltransferase was reconstituted by the cholate dispersion and gel filtration method in small unilamellar vesicles. 1-Acylglycerophosphate acyltransferase could not be solubilized from the membranes and was used in endogenous membrane fragments after detergent removal. Mixing of the two preparations and subsequent incubation in the presence of glycerol 3-phosphate, palmitoyl-CoA and oleoyl-CoA resulted in the efficient synthesis of phosphatidic acid. Inclusion of exogenous lysophosphatitic acid in the assay medium resulted in a dilution of the newly synthesized lysophosphatidate. By contrast, the synthesis of phosphatidic acid from glycerol 3-phosphate by the acyltransferases present in native membrane vesicles was barely influenced by the presence of exogenous lysophosphatidic acid. When comparing the utilization of membrane-associated 14C-labeled and newly generated 3H-labeled lysophosphatidic acid, the latter appeared to be the preferred substrate. These results indicate that lysophosphatidic acid, synthesized by glycerophosphate acyltransferase, is utilized by 1-acylglycerophosphate acyltransferase without prior mixing with the total membrane-associated pool of lysophosphatidic acid, and suggest a close proximity of the two enzymes in native E. coli membranes. This property of the acyltransferases is lost upon separation and reconstitution of enzyme activities.

Inositol phospholipid metabolism and myoblast fusion.

The fusion of chick embryonic myoblasts has been studied in tissue culture. Myoblasts are maintained at 0.1 microM-Ca2+ for 50 h. During this time they achieve fusion competence. Fusion is initiated by raising the medium Ca2+ concentration to 1.4 mM. A rapid breakdown of the polyphosphoinositides was detected within 3 min of Ca2+ addition. Rapid synthesis of phosphatidic acid was also detected at this time. Breakdown of phosphatidylinositol and synthesis of 1,2-diacylglycerol were also detected. Other phospholipids were unaffected. Sr2+ could replace Ca2+ in this process but Mg2+ could not and also inhibited the Ca2+ effect. The Ca2+-ionophore A23187 stimulated further apparent polyphosphoinositide breakdown in the presence of Ca2+. 6. The results are discussed with respect to myoblast fusion.

Effect of retinoic acid on the acylation of phospholipids in granulocytes [formula omitted

The influence of retinoic acid on the incorporation of [1- 14C]palmitic acid and [1- 14C]arachidonic acid into phospholipids was examined in guinea pig peritoneal granulocytes. All- trans -retinoic acid inhibited the incorporation of both fatty acids into phsophatidic acid and phosphatidylinositol. However, it stimulated the incorporation of both fatty acids into phosphatidylcholine but not other phospholipids. All- trans -retinoic acid was more effective than 13- cis -retinoic acid. The influence of all- trans -retinoic acid on the acylation of phospholipids was concentration-dependent with significant effect occurring at 2.1 μM. The loss of labeled fatty acids from prelabeled phospholipids and the transport of labeled fatty acids into granulocytes were not responsive to the presence of retinoic acid in the incubation media. These results suggest that retinoic acid may affect the activities of acyltransferases involved in the synthesis of phosphatidic acid, phosphatidylinositol and phosphatidylcholine.

The role of the phosphatidate-inositide cycle in the action of steroidogenic agents

Most steroidogenic agents have been found to provoke rapid changes in the metabolism of phospholipids in the phosphatidate-inositide cycle. These changes include: (a) de novo synthesis of phosphatidic acid and its derivatives, phosphatidylglycerol, mono- and polyphosphoinositides and diglyceride; and (b) phosphatidylinositol hydrolysis and consequent generation of diglyceride and phosphatidic acid. The de novo phosphatidate synthesis effect occurs in the action of all tested steroidogenic agents (ACTH, LH. angiotensin, K +, serotonin) and may be induced by either of the “second messengers”, cAMP and Ca 2+ The phosphatidylinositol hydrolysis effect(s) occurs only in the action of steroidogenic agents that operate via Ca 2+ (angiotensin and K +) and may be important in Ca 2+ mobilization. The de novo phospholipid effect correlates well with changes in steroidogenesis and, like the latter, requires Ca 2+ and protein synthesis; from these and other results, it seems likely that this phospholipid effect plays an important role in the stimulation of steroidogenesis.

Biosynthesis of D-alanyl-lipoteichoic acid: role of diglyceride kinase in the synthesis of phosphatidylglycerol for chain elongation.

Lipophilic and hydrophilic D-alanyl-lipoteichoic acids are elongated in Lactobacillus casei by the transfer of sn-glycerol 1-phosphate units from phosphatidylglycerol to the poly(glycerophosphate) moiety of the polymer. These sn-glycerol 1-phosphate units are added to the end of the poly(glycerophosphate) which is distal to the glycolipid anchor; 1,2-diglyceride results from this addition. The presence of a diglyceride kinase was suggested by the ATP-dependent phosphorylation of 1,2-diglyceride to phosphatidic acid. Inorganic phosphate was used to initiate the synthesis of lipophilic lipoteichoic acid (LTA) and the elongation of both lipophilic and hydrophilic LTA. Three observations suggest that phosphate and other anions play a role in the in vitro synthesis of LTA and its precursors. First, the conversion of 1,2-diglyceride to phosphatidic acid by diglyceride kinase was stimulated. Second, the synthesis of phosphatidylglycerol was increased. Third, the elongation of lipophilic and hydrophilic LTA was enhanced. These observations indicated that one effect of phosphate might be to enhance the utilization of 1,2-diglyceride for the synthesis of phosphatidic acid. This phospholipid is a precursor of phosphatidylglycerol, the donor of sn-glycerol 1-phosphate for elongation of LTA.

Accumulation of phosphatidic acid in microsomes from propranolol-treated retinas during short-term incubations.

The pool size and synthesis of phosphatidic acid derived from [2-3H]glycerol were studied in bovine whole retinas and subcellular fractions. Microsomal preparations from retinas incubated with [2-3H]glycerol displayed the highest percentage labeling of phosphatidic acid at 5 min of incubation: labeling decreased rapidly thereafter. In drug-treated retinas, 0.5 mM propranolol increased the endogenous content of phosphatidic acid and stimulated [2-3H]glycerol labeling in whole retina and microsomal and postmicrosomal supernatant fractions. This effect was observed during short-term incubations and was reversible. In pulse-chase experiments, 60 min of reincubation greatly reduced the labeling effect, although propranolol still enhanced phosphatidic acid labeling. At the same time, endogenous phosphatidic acid accumulated, and reincubation without propranolol reversed the effect. During accumulation, the amount of palmitate increased and that of oleate decreased, whereas the relatively high level of docosahexaenoate in phosphatidic acid remained unchanged. It was concluded that this propranolol-induced effect is due to cationic amphiphilic drug activity in the endoplasmic reticulum that results in a partial inhibition of phosphatidic acid degradation and a stimulation of its de novo synthesis. Hence, net synthesis of phosphatidic acid can be assessed in the retina during short-term incubation with propranolol.

Kinetics of phosphatidylglycerol synthesis in isolated membrane vesicles from Escherichia coli containing different amounts of membrane-bound phosphatidic acid.

Cytoplasmic membrane vesicles of Escherichia coli containing various amounts of phosphatidic acid ranging from 0.2% to more than 50% (mol/mol) of the total lipid has been prepared by de novo synthesis of phosphatidic acid in the isolated membranes from acyl-CoA esters and sn-glycerol 3-phosphate. The kinetics of CTP-initiated phosphatidylglycerol synthesis in the phosphatidic-acid-enriched membranes has been studied. Phosphatidic acid pools with high and low reactivity as substrate for phosphatidylglycerol synthesis were present in the membranes. The two pools were found identical with respect to fatty acid composition and content of molecular species. The rate of phosphatidylglycerol synthesis from the highly reactive phosphatidic acid pool was independent of the phosphatidic acid concentration in membranes containing from 0.2-30% (mol/mol) phosphatidic acid. Ca2+-ions were found to inhibit the synthesis of phosphatidylglycerol. On the basis of the findings presented it is suggested that phosphatidic acid probably plays a minor role as a feed-back modulator of sn-glycerol 3-phosphate acylation in E. coli, and that phosphatidylglycerol synthesis can occur at near maximal rate in growing cells.

Inositol lipids and cell stimulation in mammalian salivary gland

The rat parotid salivary gland shows marked alterations in phospholipid metabolism when stimulated by certain agonists. These agonists are those which cause cellular Ca mobilization by activation of muscarinic, α-adrenergic or peptidergic (substance P) receptors. The phospholipid changes apparently reflect the activation of a phosphoinositide-phosphatidic acid cycle, the precise pathways of which are not known with certainty. The observed effects include (1) an increased labelling by 32PO4 of phosphatidylinositol and phosphatidic acid, (2) net synthesis of phosphatidic acid, (3) net breakdown of phosphatidylinositol and phosphatidylinositol-4,5-bisphosphate. These effects apparently do not require the presence of extracellular Ca or the release of internal Ca and cannot be produced by the artificial introduction of Ca into the cytosol with Ca ionophores. These findings are consistent with the view that a receptor-mediated alteration in phosphoinositide metabolism represents an early step in the stimulus-response pathway in the parotid acinar cell. It has been suggested that phosphatidic acid synthesis might be of central importance in mediating Ca influx and that PIP2 breakdown might play a role in activation of Ca release. Evidence for these latter ideas is for the present largely circumstantial.

Chain elongation and desaturation of eicosapentaenoate to docosahexaenoate and phospholipid labeling in the rat retina in vivo

The metabolism of [1- 14C]eicosapentaenoic acid in the retina after intravitreal injections in the adult rat eye was studied. The acylation of eicosapentaenoic acid and the appearance of labeled docosapentaenoate and docosahexaenoate in individual phospholipids was observed at 3, 5 and 30 min after injection. The elongation and desaturation products represented about 8 and 4%, respectively, of the total radioactivity of phospholipids 3 min after injection. The highest labeling was found in phosphatidylcholine, phosphatidylinositol and phosphatidic acid. The uneven labeling profiles and the specific activities in individual phospholipids suggested that, in addition to the deacylation-acylation route for the introduction of polyenoic acyl groups into phospholipids, acylation may also take place during the synthesis of phosphatidic acid, followed by channeling to phospholipids.

Effects of 12- o-tetradecanoylphorbol 13-acetate on glycerolipid metabolism in cultured myoblasts

We recently reported that treatment of differentiated chick embryo myoblasts in culture with the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a 2-fold increase in the level of 1,2-diacylglycerol in the plasma membrane fraction within 15–30 min (Grove, R.I. and Schimmel, S.D. (1981) Biochem. Biophys. Res. Commun. 102, 158–164). This system has been characterized further and the metabolic origin and fate of the stimulated diacylglycerol have been investigated. The stimulation of 1,2-diacylglycerol was insensitive to alterations of Ca 2+ concentration in the medium and to the presence of inhibitors of Ca 2+ flux, protein synthesis and prostaglandin synthesis. The fatty acid composition of the newly formed diacylglycerol was similar to that of phosphatidylcholine. In addition, the glycerol moiety of the diacylglycerol was shown to be derived from a lipid with metabolic turnover similar to that of phosphatidylcholine. The tumor promoter was also found to stimulate rapidly synthesis of phosphatidic acid, phosphatidylinositol and phosphatidylcholine. A possible model is proposed, therefore, in which the tumor promoter stimulates a membrane-associated phospholipase C which generates 1,2-diacylglycerol via the hydrolysis of phosphatidylcholine. The newly formed diacylglycerol is then metabolized back to phosphati-dylcholine or to phosphatidic acid and phosphatidylinositol.

Effect of undecanoic acid on phospholipid metabolisminTrichophyton rubrum

At its minimum inhibitory concentration, undecanoic acid (UDA) inhibits biosynthesis of phosphatidyl serine, phosphatidyl ethanolamine and polyphosphoinositol but does not inhibit the synthesis of phosphatidyl glycerol, phosphatidyl choline, phosphatidyl inositol and phosphatidic acid in Trichophyton rubrum. At higher concentration, however UDA inhibits biosynthesis of all phosphatides present in this dermatophyte. UDA also affects catabolism of these phosphatides. This inhibitory effect of UDA may be partially responsible for its toxic action on T. rubrum.

Adrenocorticotropin and adenosine 3',5'-monophosphate stimulate de novo synthesis of adrenal phosphatidic acid by a cycloheximide-sensitive, CA++-dependent mechanism.

We tested further our postulate that enhanced de novo synthesis of phosphatidic acid is responsible for ACTH- and cAMP-induced increases in adrenal phospholipids in the phosphatidate polyphosphoinositide pathway. During incubation of adrenal sections or cells in vitro, ACTH and cAMP increased the concentrations of and incorporation of [3H]glycerol and [14C]palmitate into phosphatidylcholine and phosphatidylethanolamine, two major phospholipids which are derived from phosphatidic acid, but are extrinsic to the inositide pathway. Thus, it is unlikely that ACTH and cAMP increase inositide phospholipids at the expense of other phospholipids. Similar to previously reported effects on phosphatidic acid and inositide phospholipids, cycloheximide blocked the effects of ACTH and cAMP on phosphatidylcholine and phosphatidylethanolamine. In addition, Ca++ was required for these effects, as well as for cAMP-induced increases in phosphatidic acid, inositide phospholipids, and steroidogenesis. Our findings strongly suggest that ACTH, via cAMP, stimulates de novo phosphatidate synthesis by a cycloheximide-sensitive, Ca++-dependent process, and this stimulation causes a rapid generalized increase in adrenal phospholipids. Moreover, the increased incorporation of labeled glycerol and palmitate into phospholipids suggests that ACTH and cAMP may stimulate the glycerol-3'-PO4 acyltransferase reaction. This stimulatory effect may play a central role in the steroidogenic and trophic actions of ACTH and cAMP.

Myotonic dystrophy: calcium-dependent phosphatidic acid synthesis in erythrocytes.

Recently it was reported that calcium-dependent phosphatidic acid synthesis in erythrocyte of patients with myotonic muscular dystrophy (MyD) is markedly impaired when compared to that in control subjects. Using 32P-loaded erythrocytes, we found no significant difference in the levels of 32P-phosphatidic acid synthesized after exposure to calcium and its ionophore A23187 between patients with MyD and controls. In a batch experiment typical of the experiments with 32P, and a twofold increase of phosphatidic acid in both groups was determined by inorganic phosphate measurements. Thus, the specific activity of the 32P-phosphatidic acid increased four- to five-fold in response to calcium Analyses of 32P-polyphosphoinositide breakdown in ghosts and in adenosine triphosphate-depleted erythrocytes also appeared normal for patients with myotonic muscular dystrophy. Possible discrepancies between the results presented here and those reported previously are discussed.