Mechanism of arachidonic acid release in human polymorphonuclear leukocytes

Previous studies have demonstrated that [³H]arachidonic acid is released from prelabeled human neutrophil phospholipids when the cells are stimulated by calcium ionophore A23187 or by opsonized zymosan. Neither lysophospholipid generated by phospholipase A₂ activity, diacylglycerol nor monoacylglycerol produced via phospholipase C/diacylglycerol lipase action have been identified following neutrophil challenge. The inability to detect any intermediates during the release of arachidonate is due to either rapid reacylation of lysophospholipid or conversion of diacylglycerol (monoacylglycerol) to cellular acylglycerols. The addition of exogenous [¹⁴C]fatty acid at the time of challenge was employed to determine the involvement of either phospholipase A₂ or phospholipase C activities. Neutrophil stimulation with calcium ionophore A23187 resulted in an incorporation of exogenous [¹⁴C]arachidonate into phosphatidylinositol and phosphatidylcholine, those phospholipids which specifically release arachidonate. When the saturated fatty acid, [¹⁴C]stearate, replaced [¹⁴C]arachidonate, very little [¹⁴C]fatty acid was incorporated into any of the phospholipid species. Lipid phosphorus measurements revealed no significant mass change in any phospholipid class following ionophore challenge. Production of [¹⁴C]phosphatidic acid was not detected, as would be expected if diacylglycerol kinase and de novo phospholipid metabolism were significantly involved.     

Cyclic adenosine 3',5'-monophosphate and prostacyclin inhibit membrane phospholipase activity in platelets.

[¹⁴C]-Arachidonic acid is incorporated mainly into phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of horse platelet membranes. Treatment of washed platelets with thrombin leads to a rapid loss of radioactivity from these phospholipids. The liberated [¹⁴C]-arachidonate is immediately transformed into hydroxyacids and thromboxanes. Treatment with dibutyryl cyclic AMP, cyclic AMP phosphodiesterase inhibitors or prostacyclin, a newly discovered prostaglandin that stimulates platelet adenylate cyclase, prevents the action of thrombin on phospholipid break-down as well as on platelet aggregation. Dibutyryl cyclic AMP does not affect the metabolism of exogenous [¹⁴C]-arachidonic acid. Cyclic AMP may thus play a crucial role in the regulation of platelet phospholipase acitivity, and this could explain at least in part the inhibition of aggregation caused by substances which, like prostacyclin, raise the levels of cyclic AMP.     

The turnover of molecular species of phosphatidylinositol in ehrlich ascites tumor cells

It has been shown previously that ³²P_i is incorporated into phosphatidylinositol 30 times faster than into the other phospholipid classes in Ehrlich ascites tumor cells, whereas [1-¹⁴C]glycerol is incorporated at almost the same rate (Waku, K., Nakazawa, Y. and Mori W. (1976) J. Biochem. 79, 407–411). It was therefore suggested that there is a recirculating system (phosphatidylinositol → diacylglycerol → phosphatidic acid → CDPdiacylglycerol → phosphatidylinositol) of phosphatidylinositol in Ehrlich ascites tumor cells. In this work, ³²P_i or [1-³H]glycerol was injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor cells from which the lipids were extracted after selected periods. Phosphatidylinositol was prepared and fractionated in the form of dimethylphosphatidic acid into six molecular species by AgNO₃-impregnated TLC. The specific radioactivities of the fractionated species were determined. ³²P_i was incorporated into diene molecular species and [1-³H]glycerol into monoene species with a higher rate than the other species and both precursors were incorporated into tetraene species rather slowly. ³²P/³H values appeared to be at almost the same for each molecular species, although monoene species showed slightly lower values. These results suggest that there could be a recirculating of the phosphorylinositol moiety in each of the molecular species of phosphatidylinositol.     

Bradykinin stimulates phospholipase C in rat renal medullary slices

Rat renal medullary slices prelabeled with [14C]arachidonic acid generate [14C]diacylglycerol within 1 min of exposure to bradykinin action. Production of [14C]diacylglycerol is transient. 2 min after the addition of bradykinin, the levels of metabolite reach the maximum, but decrease thereafter. Simultaneously, bradykinin induces a parallel decrease of the radioactivity in phosphatidylinositol. No degradation of other phospholipids is observed, and triacylglycerol is not affected. The degradation of [14C]phosphatidylinositol to [14C]diacylglycerol indicated the presence of phospholipase C activity. Preincubation of prelabeled slices with 2 mM dibutyryl cyclic AMP prevents both the generation of diacylglycerol and the degradation of phosphatidylinositol. Neither mepacrine nor indomethacin block diacylglycerol production and phosphatidylinositol breakdown. We conclude that, when rat renal medullary slices are stimulated with bradykinin, phosphatidylinositol-specific phospholipase C is activated.     

1,25-Dihydroxyvitamin D3 or dexamethasone modulate arachidonic acid uptake and distribution into glycerophospholipids by normal adult human osteoblast-like cells

The effects of treatment with the osteotropic steroids 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 17β-estradiol, or dexamethasone on [1-¹⁴C]arachidonic acid (AA) uptake and distribution into glycerophospholipid classes by normal adult human osteoblast-like (hOB) cells were investigated. Total uptake of [1-¹⁴C]AA was decreased in cells treated with dexamethasone when assayed after a 24-, 48-, or 96-h exposure to the hormone. Specific radiolabel incorporation into phosphatidylcholine was reduced by a 48-h treatment with dexamethasone with a concurrent increase in the radiolabeling of phosphatidylethanolamine. However, these changes were transient, and by 96 h of dexamethasone treatment the distribution of the radiolabeled fatty acid had reequilibrated to resemble the pattern found for vehicle treated samples. Total uptake of [1-¹⁴C]AA was diminished by 96-h treatment with 1,25(OH)₂D₃ (79 ± 3% of control, P < 0.01); at that time point, a significant decrease in the proportional radiolabeling of the phosphatidylinositol pool was identified (92 ± 2% of control, P < 0.05). The 1,25(OH)₂D₃-dependent decrease in total uptake and in phosphatidylinositol incorporation of [1-¹⁴C]AA were found to be hormone dose dependent. Treatment with 24,25(OH)₂D₃ was without effect on either total [1-¹⁴C]AA uptake or the specific [1-¹⁴C]AA radiolabeling of the phosphatidylinositol pool. 1,25(OH)₂D₃ treatment decreased hOB cell uptake of [1-¹⁴C]oleic acid and decreased its proportional incorporation into the phosphatidylinositol pool. Gas chromatographic analyses revealed no 1,25(OH)₂D₃-dependent effects on total phosphatidylinositol lipid mass or on the mole percent of arachidonic acid within the phosphatidylinositol pool, leaving the mechanism of the effects of the secosteroid on hOB cell AA metabolism unexplained. 17β-Estradiol had no effects on the parameters of AA metabolism measured. As a consequence of their modulation of arachidonic acid uptake and its distribution into hOB cellular phospholipids, steroids might alter the biological effects of other hormones whose actions include the stimulated production of bioactive AA metabolites, such as prostaglandins or the various lipoxygenase products.     

Reduced labeling of brain phosphatidylinositol,triacylglycerols, and diacylglycerols by [1-14C]arachidonic acid after electroconvulsive shock: Potentiation of the effect by adrenergic drugs and comparison with palmitic acid labeling

The effect of electroconvulsive shock on the labeling of phospholipids and neutral lipids in mice brains was examined after intracerebral injection of [1-¹⁴C] arachidonic acid or [1-¹⁴C]palmitic acid. Electroconvulsive shock reduced greatly the removal of radiolabeled arachidonic acid from the free fatty acid pool. At the same time, the incorporation of arachidonic acid was partially inhibited in triacylglycerol, diacylglycerol, and phosphatidylinositol, whereas the incorporation of [1-¹⁴C]palmitic acid was not affected. Pretreatment with desipramine and pargyline potentiated the lipid effect of electroconvulsive shock in neutral glycerides. These electroconvulsive shock-induced changes reflect alterations in the metabolism of intracerebrally injected arachidonic acid, but not of similarly injected palmitic acid. From the available data whether decreased ATP, enzyme inhibition or other factors are involved cannot be ascertained. Moreover, the electroconvulsive shock-enhanced endogenous free arachidonic acid may possibly dilute the injected radiolabeled fatty acid, thus decreasing its availability for arachidonoyl-coenzyme A synthesis. Hence, a partial inhibition of the activation-acylation of these fatty acids, primarily arachidonic acid, also may be involved in the seizure-induced accumulation of free fatty acids in the brain.     

Effect of calmodulin antagonists on lysosomal enzyme secretion and phospholipid metabolism in guinea-pig macrophages

The effects of calmodulin antagonists on the secretion of lysosomal enzyme and lipid metabolism in guinea-pig peritoneal macrophages were studied. Calmodulin antagonists, such as trifluoperazine, dibucaine and quinacrine, inhibited the secretion of N-acetyl-β-d-glucosaminidase from cytochalasin B-treated macrophages when the macrophages were stimulated by the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (f Met-Leu-Phe) or the Ca²⁺ ionophore A23187. The effect of calmodulin antagonists on the incorporation of [³²P]P_i or [³H]glycerol into glycerolipids as well as on the redistribution of [¹⁴C]glycerol or [³H]arachidonic acid in [¹⁴C]glycerol- or [³H]arachidonic acid-prelabelled lipids were examined. Trifluoperazine, dibucaine or quinacrine stimulated [³²P]P_i incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) without significant effect on the labelling of phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), lysophosphatidylcholine (lyso-PtdCho) and lysophosphatidylethanolamine (lyso-PtdEtn). The incorporation of [³²P]P_i into phosphatidylcholine (PtdCho) was, on the contrary, inhibited. When calmodulin antagonists were added to macrophages stimulated by fMet-Leu-Phe, [³²P]P_i incorporation into PtdIns and PtdA was synergistically increased compared with that induced only by calmodulin antagonists. Trifluoperazine inhibited the incorporation of [³H]glycerol into PtdCho, triacylglycerol and PtdEtn. Also in this case, the incorporation of [³H]glycerol into PtdA and PtdIns was greatly enhanced. But [³H]glycerol incorporation into PtdSer, lyso-PtdEtn and lyso-PtdCho was not affected by the drug. On the other hand, diacylglycerol labelling with [³H]glycerol was maximally activated by 10μm-trifluoperazine and levelled off with the increasing concentration. When the effect of calmodulin antagonists on the redistribution of [¹⁴C]glycerol among lipids was examined in pulse-chase experiments, no significant effect on [¹⁴C]glycerol redistribution in PtdEtn, PtdCho, PtdIns, PtdSer, PtdA and tri- and di-acylglycerol could be detected. When macrophages prelabelled with [³H]arachidonic acid were treated with trifluoperazine, dibucaine or quinacrine, the [³H]arachidonic acid moiety in PtdEtn and PtdCho was decreased and that in PtdA was increased. The formation of [arachidonate-³H]diacylglycerol and non-esterified [³H]-arachidonic acid was also enhanced, but the increase in [³H]arachidonic acid was only observed at concentrations between 1 and 50μm. [Arachidonate-³H]PtdIns was not significantly affected. The activated formation of [arachidonate-³H]PtdA, diacylglycerol and non-esterified arachidonic acid by these drugs was synergistically enhanced in the presence of fMet-Leu-Phe.     

Turnover of protein and diacylglycerol components of lipophorin in insect haemolymph

Lipophorin, radiolabelled in the protein or diacylglycerol moiety, was purified from adult locusts injected previously with [¹⁴C]protein hydrolysate or sodium[1-¹⁴C]palmitate. The radiolabelled lipophorin was injected into adult male locusts and haemolymph samples taken periodically to determine the rate of disappearance of radioactivity from the haemolymph. Lipophorin was also purified from locusts that had been injected four days previously with ([¹⁴C]protein)-lipophorin to demonstrate that the radioactivity observed in the haemolymph at this time is due to radiolabelled lipophorin. The results indicate that the half-life of the protein component of lipophorin in resting insects is about 5–6 days whereas that of the diacylglycerol component is only about 2–3 hr.The results are consistent with the hypothesis that lipophorin functions as a “reusable shuttle” to transport a variety of lipid classes between sites of absorption, storage and utilisation.     

In Vivo Pathway of Oleate and Linoleate Desaturation in Developing Cotyledons of Cucumis sativus L. Seedlings

Exogenous [1-¹⁴C]oleic acid and [1-¹⁴C]linoleic acid were taken up and esterified to complex lipids by greening cucumber (Cucumis sativus L.) cotyledons. Both ¹⁴C-labeled fatty acids were initially esterified to phosphatidylcholine prior to eventual accumulation in triacylglycerols and galactolipids. Kinetic data suggest that esterification occurs prior to desaturation and that phosphatidylcholine is the initial site of both [¹⁴C]-oleate and [1-¹⁴C]linoleate esterification and of [1-¹⁴C]oleate desaturation to [1-¹⁴C]linoleate. [1-¹⁴C]Linoleic acid was esterified more rapidly than [¹⁴C]oleic acid and its desaturation product, [1-¹⁴C]α-linolenate, occurred mainly on monogalactosyl diacylglycerol, although some was also observed on the other major acyl lipids, including phosphatidylcholine.     

Induction of cell DNA replication in G1-specific ts mutants by microinjection of SV40 DNA

Treatment of Xenopus laevis follicles with 50–100 units/ml of human chorionic gonadotropin causes rapid stimulation of [¹⁴C]glucose uptake. Studies with these follicles showed that the stimulation of uptake occurred with a wide range of concentrations of [¹⁴C]glucose or its nonmetabolizable analog [¹⁴C]3-O-methylglucose. Approx. 70% of the glucose taken up in both hormone-treated and control cells becomes incorporated into glycogen within 1 h. The uptake of sugar by these follicles was also stimulated by bovine-luteinizing hormone—but not by folliclestimulating hormone, progesterone or insulin. Human chorionic gonadotropin stimulated sugar uptake by follicles containing medium-sized oocytes (stages 3,4 and 5 according to Dumont) which cannot be induced to undergo meiotic maturation by this hormone. After 4–6 h treatment of fully grown X. laevis follicles with either progesterone or human chorionic gonadotropin, glucose uptake suffers a drastic decrease to below basal levels. This inhibition of uptake is coincident with the breakdown of the germinal vesicle of the oocyte and is clearly related to meiotic maturation, since it is not observed with medium-sized follicles which cannot mature.     

Diacylglycerol kinase and lipase activities in rat brain subcellular fractions

Phosphatidic acid synthesis via diacylglycerol kinase and free fatty acid release via diacylglycerol lipase were investigated in rat brain subcellular fractions using membrane-bound [I-¹⁴C]arachidonoyl-diacylglycerol as substrate. Labeled diacylglycerol was generated by incubating brain membranes containing [I-¹⁴C]arachidonoyl-phosphatidylinositols in the presence of deoxycholate and Ca²⁺. Incubation of the prelabeled synaptosomes enriched in [1-¹⁴C]arachidonoyl-diacylglycerols or incubation of brain subcellular fractions with heat-treated prelabeled membranes resulted in the release of free fatty acids from the diacylglycerols. When incubations were carried out in the presence of ATP, MgCl₂ and NaF, both free fatty acid release and conversion of diacylglycerols to phosphatidic acids were observed. The conversion of diacylglycerols to phosphatidate or their hydrolysis to free fatty acids were linear with time for at least 15 min. In three brain subcellular fractions examined, diacylglycerol kinase activity indicated a pH maximum of 7.4. The free fatty acid release was enhanced slightly by Ca²⁺ (1 mM), but Ca²⁺ (0.5–4 mM) in the presence of Mg²⁺ (10 mM) was inhibitory to the diacylglycerol kinase reaction. Phosphatidate formation was also inhibited by an excessive amount of deoxycholate added to the incubation mixture. Among the brain subcellular fractions, diacylglycerol kinase was more active in synaptic vesicles and cytosol than in the microsomal fraction, whereas diacylglycerol lipase activity was higher in the cytosol fraction than in the membrane fractions. Upon washing the membranes by centrifugation, a substantial portion of the diacylglycerol kinase activity was removed after the first washing, whereas the diacylglycerol lipase activity remained essentially unchanged. The metabolic role of arachidonoyl-diacylglycerols in brain membranes in relation to the biosynthesis of phosphatidate and the release of arachidomic acid is discussed.     

Tumor necrosis factor-alpha stimulates phosphatidylinositol breakdown by phospholipase C to coordinately increase the levels of diacylglycerol, free arachidonic acid and prostaglandins in an osteoblast (MC3T3-E1) cell line

The effects of (human recombinant) tumor necrosis factor-alpha on phosphatidylinositol breakdown, release of 1,2-diacylglycerols, mobilization of arachidonate from diacylglycerol and prostaglandin synthesis were examined in a model osteoblast cell line (MC3T3-E1). Tumor necrosis factor-alpha (10 nM) caused a specific (30%) decrease in the mass of phosphatidylinositol (and no other phospholipids) within 30 min of exposure. Tumor necrosis factor-alpha doubled the rate of incorporation of [32P]orthophosphoric acid into phosphatidylinositol, indicating that the turnover of inositol phosphate was enhanced, and increased the content of diacylglycerol in parallel with phosphatidylinositol breakdown. The cytokine (10-50 nM; 4 h) also promoted a specific release of 24-34% of the [3H]arachidonate from prelabeled phosphatidylinositol, a release of 80% of the 3H-fatty acid from the diacylglycerol pool, and a 30-fold increase in the synthesis of prostaglandin E2. The tumor necrosis factor-alpha induced liberation of [3H]arachidonate from diacylglycerol, cellular arachidonate release and the synthesis of prostaglandin E2 were each blocked by an inhibitor of diacylglycerol lipase, the compound RHC 80267 (30 microM). Therefore, we conclude that, in the MC3T3-E1 cell line, tumor necrosis factor-alpha activates a phosphatidylinositol-specific phospholipase C (phosphatidylinositol inositolphosphohydrolase; EC 3.1.4.3) to release diacylglycerol, and increases the metabolism of diacylglycerol to liberate arachidonate for prostaglandin synthesis.     

CMP-Dependent incorporation of [14C]glycerol 3-phosphate into phosphatidylglycerol and phosphatidylglycerol phosphate by rabbit lung microsomes

Rabbit lung microsomes were found to catalyze CMP-dependent incorporation of [¹⁴C]glycerol 3-phosphate into a total lipid extract. The radioactively labeled products in the lipid extract were identified as phosphatidylglycerol and phosphatidylglycerol phosphate. CMP-dependent incorporation of [¹⁴C]glycerol 3-phosphate by lung microsomes proceeded optimally at pH 7.4 and required Mn²⁺ The apparent K_m value for CMP in this reaction was calculated to be 0.19 mM. No other cytidine nucleotide could substitute completely for CMP in supporting [¹⁴C]glycerol 3-phosphate incorporation into lipid. Cytosine-β-d-arabinofuranoside-5'-monophosphate-dependent incorporation of [¹⁴C]glycerol 3-phosphate was observed at pH 8.5 but not at pH 6.8. CMP-dependent incorporation of [¹⁴C]glycerol 3-phosphate by microsomes was inhibited by inositol. The optimal in vitro rates of CMP-dependent and CDPdiacylglycerol-dependent incorporation of [¹⁴Ciglycerol 3-phosphate into lipid were similar (approximately 1 nmol·mg^{−1 protein·h−1}) and were not additive. Both CMP-dependent and CDPdiacylglycerol-dependent incorporation of [¹⁴C]glycerol 3-phosphate by lung microsomes appeared to involve CDPdiacylglycerol:glycerol-3-phosphate phosphatidyltransferase. However, the specific activity of this enzyme in a particular subcellular fraction did not relate directly to the extent of CMP-dependent [¹⁴C]glycerol 3-phosphate incorporation in that fraction. Pre-incubation of lung microsomes with 5 mM CMP plus 3 mM phosphatidylinositol increased CMP-dependent incorporation of [¹⁴C]glycerol 3-phosphate. When lung microsomes were depleted specifically of phosphatidylinositol by incubating with a phosphatidylinositol-specific phospholipase C, CMP-dependent incorporation was diminished. The Mn²⁺ requirement for CMP-dependent incorporation of [¹⁴C]glycerol 3-phosphate, its phosphatidylinositol requirement and its inhibition by Triton X-100 (0.2%) were not features shared by CDPdiacylglycerol-dependent incorporation of [¹⁴Ciglycerol 3-phosphate but were characteristics of the reverse reaction catalyzed by CDPdiacylglycerol; inositol phosphatidyltransferase. Together with the previous finding of a developmental increase in the CMP content of fetal rabbit lung, these observations are consistent with a role for CMP in the regulation of the phosphatidylinositol and phosphatidylglycerol content of lung surfactant during lung maturation.     

Human platelets have cholesteryl ester hydrolytic activity resulting in esterification of [1-14C]oleate to individual phospholipids of platelets

Human platelet cholesteryl ester hydrolytic (CEH) activity was determined toward cholesteryl [1-¹⁴C]oleate resulting in esterification of [1-¹⁴C]oleate to individual platelet phospholipids: choline-containing phospholipids (PC); ethanolamine-containing phospholipids (PE); phosphatidylserine (PS); phosphatidylinositol (PI); and sphingomyelin (SPH). Liberation of [1-¹⁴C]oleate and esterification of [1-¹⁴C]oleate to platelet phospholipids was enhanced by 100 nM iloprost (a stable analogue of prostacyclin that increases platelet cyclic adenosine monophosphate (c-AMP)), inhibited by 30 μM H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide)) (a specific c-AMP dependent protein kinase (CADPK) inhibitor) and 500 μM 2′,5′ dideoxyadenosine (DDA) (an inhibitor of iloprost-induced rise in platelet c-AMP), but unaffected by 150 mM chloroquine diphosphate. These observations suggest that the CEH activity is mediated by a CADPK phosphorylation of an enzyme with the phosphorylated state representing the active form of the enzyme and that the CEH activity is extralysosomal.     

SELECTIVE SYNTHESIS OF MOLECULAR CLASSES OF PHOSPHATIDIC ACID, DIACYLGLYCEROL AND PHOSPHATIDYLINOSITOL IN RAT BRAIN

—Phosphatidic acids of rat brain were shown to be predominantly of the monoenoic class while diacylglycerols and phosphatidylinositols were constituted mainly by tetraenes. Metabolic inter-relationships were examined after intraventricular injection of [¹⁴C]glycerol, [³H]arachidonate and [9,10-³H₂]stearate. In each case, diacylglycerols were most highly labelled, although a small pool of arachidonate was located in brain triacylglycerols, mainly esterified to a primary carbinol, with extremely high turnover rate. Fractionation of the lipids showed a preferential synthesis of disaturated, monoenoic and polyenoic classes (>4 double bonds) of phosphatidic acid, diacylglycerol and phosphatidylinositol. The high flux of [³H]stearate through disaturated species of phosphatidic acid and diacylglycerol could be partially suppressed by simultaneous injections of unsaturated fatty acids, both probably consequences of perturbing the very small brain pool of free fatty acids. Kinetics of labelling of phosphatidylinositols were consistent with formation of arachidonoyl-containing species by acyl transfer mechanisms with disaturated and oligoenoic classes serving as precursors. Although the profile of molecular classes of diacylglycerol and phosphatidylinositol strongly suggests a metabolic relation, there was no obvious evidence for this in the kinetic studies of the whole brain lipids. Such relation, however, may have been masked by the rapid flow of radioactivity from phosphatidic acids to diacylglycerols.     

Changes in [3H]inositol-labelled phosphoinositides of pig platelets in response to thrombin

Pig platelet phosphoinositides have been labelled with [³H]inositol and then treated with thrombin in the absence of Ca²⁺. There was a loss of labelled phosphatidylinositol 4,5-bisphosphate between 30 and 60 s after the addition of thrombin but the general picture was of increased labelling over a 4-min period. Labelling of phosphatidylinositol 4-phosphate showed no period of loss but there was an early loss of phosphatidylinositol and no increased labelling during the 4-min incubation. The small amount of lysophosphatidyl[³H]inositol in the platelets was not affected by thrombin treatment. Thrombin caused loss of [¹⁴C]arachidonate-labelled phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol.     

Synthesis of mono- and digalactosyldiacylglycerol in isolated spinach chloroplasts

Purified, intact chloroplasts of Spinacia oleracea L. synthesize galactose-labeled mono- and digalactosyldiacylglycerol (MGDG and DGDG) from UDP-[U-¹⁴C]galactose. In the presence of high concentrations of unchelated divalent cations they also synthesize tri- and tetra-galactosyldiacylglycerol. The acyl chains of galactose-labeled MGDG are strongly desaturated and such MGDG is a good precursor for DGDG and higher oligogalactolipids. The synthesis of MGDG is catalyzed by UDP-Gal:sn-1,2-diacylglycerol galactosyltransferase, and synthesis of DGDG and the oligogalactolipids is exclusively catalyzed by galactolipid:galactolipid galactosyltransferase. The content of diacylglycerol in chloroplasts remains low during UDP-Gal incorporation. This indicates that formation of diacylglycerol by galactolipid:galactolipid galactosyltransferase is balanced with diacylglycerol consumption by UDP-Gal:diacylglycerol galactosyltransferase for MGDG synthesis. Incubation of intact spinach chloroplasts with [2-¹⁴C]acetate or sn-[U-¹⁴C]glycerol-3-P in the presence of Mg²⁺ and unlabeled UDP-Gal resulted in high ¹⁴C incorporation into MGDG, while DGDG labeling was low. This de novo made MGDG is mainly oligoene. Its conversion into DGDG is also catalyzed, at least in part, by galactolipid:galactolipid galactosyltransferase.     

The utilisation of fatty-acid substrates in triacylglycerol biosynthesis by tissue-slices of developing safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) cotyledons

Developing cotyledons of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) readily utilised exogenously supplied ¹⁴C-labelled fatty-acid substrates for the synthesis of triacylglycerols. The other major radioactive lipids were phosphatidylcholine and diacylglycerol. In safflower cotyledons, [¹⁴C]oleate was rapidly transferred to position 2 of sn-phosphatidylcholine and concomitant with this was the appearance of radioactive linoleate. The linoleate was further utilised in the synthesis of diacyl- and triacyl-glycerol via the reactions of the so-called Kennedy pathway. Supplying [¹⁴C]linoleate, however, resulted in a more rapid labelling of the diacylglycerols than from [¹⁴C]oleate. In contrast, sunflower cotyledons readily utilised both labelled acyl substrates for rapid diacylglycerol formation as well as incorporation into position 2 of sn-phosphatidylcholine. In both species, however, [¹⁴C]palmitate largely entered sn-phosphatidylcholine at position 1 during triacylglycerol synthesis. The results support our previous in-vitro observations with isolated microsomal membrane preparations that (i) the entry of oleate into position 2 of sn-phosphatidylcholine, via acyl exchange, for desaturation to linoleate is of major importance in regulating the level of polyunsaturated fatty acids available for triacylglycerol formation and (ii) Palmitate is largely excluded from position 2 of sn-phosphatidylcholine and enters this phospholipid at position 1 probably via the equilibration with diacylglycerol. Specie differences appear to exist between safflower and sunflower in relation to the relative importance of acyl exchange and the interconversion of diacylglycerol with phosphatidylcholine as mechanisms for the entry of oleate into the phospholipid for desaturation.Abbreviations FW fresh weight - TLC thin-layer chromatography     

The polyphosphoinositides,phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate,are present in nuclei isolated from carrot protoplast

Summary Nuclei were isolated from carrot protoplasts and the distribution of [³H]inositol-labeled phospholipids was analyzed by thinlayer chromatography. Phosphatidylinositol (PI), lysophos-phatidylinositol (LPI), phosphatidylinositol monophosphate (PIP), lysophosphatidylinositol monophosphate (LPIP), and phosphatidylinositol bisphosphate (PIP₂) were 55.7%, 12.3%, 5.0%, 11.5%, and 3.6% of the respective [³H]inositol-labeled lipids recovered from the nuclear fraction. While both the plasma membrane and nuclear fraction contained polyphosphoinositides, the distribution of the phosphoinositides and the amount of inositol-labeled lipid were distinct. For example, the nuclear fraction had a higher percentage of LPI and PIP₂ and less PI and LPIP than the plasma membrane fraction. The amount of [³H]inositol-labeled lipid recovered from the nuclear fraction per mg protein was an order of magnitude lower than that recovered from either the plasma membrane of lower phase fraction isolated by aqueous two-phase partitioning, or from whole cells and protoplasts. In addition, when the ratio of the [³H]inositol-labeled lipid was compared to total [¹⁴C]myristate-labeled lipid recovered there was three to ten fold less [³H] relative to [¹⁴C] in the nuclear fraction.These data indicate that while the polyphosphoinositides are a relatively high percentage of the inositol lipid in the nuclear fraction, the inositol lipid was only a small portion of the total lipid in the nuclei. Despite this low concentration of inositol lipid, when [ ³²P]-ATP was added to the isolated nuclei,³²P-labeled PIP and PIP₂ were synthesized. Thus, the carrot nuclei contained PI and PIP kinase as well as the polyphosphoinositides.Abbreviations PI phosphatidylinositol - LPI lysophosphatidylinositol - PIP phosphatidylinositol monophosphate - LPIP lysophosphatidylinositol monophosphate - PIP₂ phosphatidylinositol bisphosphate - DAG diacylglycerol - IP₃ inositol 1,4,5-trisphosphate     

Phospholipid turnover in isolated rat pancreatic acini. Consideration of the relative roles of phospholipase A2 and phospholipase C

The purpose of the present study was to explore the interaction of phosphatidylinositol breakdown and the turnover of arachidonic acid in isolated rat pancreatic acini by using receptor agonists and the calcium ionophore ionomycin. Acini prelabelled with myo-[³H]inositol in vivo responded to carbachol with a rapid breakdown of phosphatidylinositol. In the presence of [³²P]P_i, carbachol increased labelling of phosphatidic acid and phosphatidylinositol within 1 and 5 min respectively. Carbachol also rapidly stimulated the incorporation of [¹⁴C]arachidonic acid into phosphatidylinositol within 2 min, and the peptidergic secretagogue caerulein caused the loss of radioactivity from phospholipids prelabelled with arachidonic acid. Ca²⁺ deprivation partially impaired the stimulatory action of carbachol on arachidonic acid turnover. In contrast with its stimulatory effects on [³²P]P_i and [¹⁴C]arachidonate incorporation, carbachol inhibited the incorporation of the saturated fatty acid stearic acid into phosphatidylinositol. Whereas ionomycin stimulation of phosphatidylinositol breakdown and [³²P]P_i labelling of phospholipids was slower in onset and less effective than carbachol stimulation, the ionophore effectively promoted (arachidonyl) phosphatidylinositol turnover within 2 min. These results implicate two separate pathways for stimulated phosphatidylinositol degradation in the exocrine pancreas, involving phospholipases A₂ and C. Whereas mobilization of cellular Ca²⁺ appears sufficient to cause activation of phospholipase A₂ and amylase secretion, additional events triggered by receptor activation may be required to act in concert with Ca²⁺ to optimally stimulate phospholipase C. The nature of the interaction between phospholipases A₂ and C and their specific physiological roles in pancreatic secretion remain to be elucidated.