Evidence for the activation of phospholipases during acrosome reaction of human sperm elicited by calcium ionophore A23187

Incubation of washed human sperm with [3H]- or [14C]arachidonic acid allowed a major incorporation of the label into phospholipids, provided that the final concentration of the fatty acid did not exceed 20 microM. A further challenge with calcium ionophore A23187 of spermatozoa suspended in a calcium-containing medium led to phospholipid hydrolysis, which could account for 10-12% of total cell radioactivity. Degradation products were identified as free, unconverted arachidonic acid, occurring with some diacylglycerol. Phospholipid hydrolysis was significant after 15 min of incubation and became maximal after 120 min. It was found to be calcium dependent, diacylglycerol and free arachidonate production occurring maximally at 2 mM and 5 mM CaCl2, respectively. Phosphatidylcholine and phosphatidylinositol were the most significantly degraded phospholipids after 60 min of incubation. Similar incubations conducted with 32P-labeled sperm confirmed the selective hydrolysis of phosphatidylcholine and revealed an increase production of phosphatidic acid probably due to a phosphorylation of diacylglycerol. Under the same conditions, one third of the cells remained motile and electron microscopy revealed that acrosome reaction was completed in 40% of the cells and displayed an intermediary state in 40-50% of the spermatozoa. Furthermore, a good parallelism was observed between the extent of the acrosome reaction and the extent of phospholipid hydrolysis promoted by increasing concentrations of A23187. It is concluded that calcium entry into the cells activates both a phospholipase A2 and a phospholipase C, leading to the production of substances, like lysophospholipid, diacylglycerol or phosphatidic acid, which may or may not be involved in acrosome reaction.     

Apparent specificity of the thrombin-stimulated deacylation of endothelial glycerolipids for polyunsaturated fatty acids with a delta-5 desaturation

Human umbilical vein endothelial cells readily incorporate exogenous polyunsaturated fatty acids. Subsequent stimulation with thrombin results in the release of both arachidonate and eicosapentaenoate from cellular phospholipids. The present study has investigated the utilization of 8,11,14-[¹⁴C]eicosatrienoate, the precursor of prostaglandin E₁. Analysis of released ¹⁴C-fatty acids by radio-gas chromatography indicated that thrombin stimulated the release of 6–10% of the [¹⁴C]arachidonate synthesized by desaturation of the [¹⁴C]eicosatrienoate, but did not stimulate release of [¹⁴C]eicosatrienoate per se (less than 1%). As determined by digestion of cellular lipid extracts with pancreatic phospholipase A₂, both 8,11,14-[¹⁴C]eicosatrienoate and [¹⁴C]arachidonate were esterified primarily in the 2-position. Similarly, separation of phospholipid classes by two-dimensional thin-layer chromatography did not indicate any major differences in the distribution of the incorporated ¹⁴C-fatty acids. Experiments with additional ¹⁴C-fatty acids indicated that 5,8,11-eicosatrienoate is released in response to thrombin but that 8,11,14,17-eicosatetraenoate is not. These results suggest that the delta-5 double bond is required for the thrombin-stimulated release of free fatty acids from endothelial phospholipids and their subsequent availability as substrates for eicosanoid synthesis.     

Manoalide,a Phospholipase A2 Inhibitor,Inhibits Arachidonate Incorporation and Turnover in Brain Phospholipids of the Awake Rat

The Fatty Acid method was used to determine whether incorporation of plasma radiolabeled arachidonic acid into brain phospholipids is controlled by phospholipase A₂. Awake rats received an i.v. injection of a phospholipase A₂ inhibitor, manoalide (10 mg/kg), and then were infused i.v. with [1-¹⁴C]arachidonate or [³H]arachidonate. Animals were killed after infusion by microwave irradiation, and tracer distribution was analyzed in brain phospholipid, neutral lipid and acyl-CoA pools. Calcium-independent phospholipase A₂ activity in brain homogenate was reduced by manoalide, whereas phospholipase C activity was unaffected. At 60 min but not at 20 or 40 min after its injection, manoalide had significantly decreased by 50% incorporation of unesterified arachidonate into and turnover within brain phospholipids, taking into account dilution of the brain arachidonoyl-CoA pool by recycled arachidonate. Manoalide also increased by 100% the net rate of unesterified arachidonate incorporation into brain triacylglycerol. This study indicates that manoalide can be used to inhibit brain phospholipase A₂ in vivo, and that phospholipase A₂ plays a critical role in arachidonate turnover in brain phospholipids and neutral lipids.     

Release of arachidonic acid from vascular endothelial cells: fatty acyl specificity is observed with receptor-mediated agonists and with the calcium ionophore A23187 but not with melittin

Vascular endothelial cells respond to a variety of physiological and pharmacological stimuli by releasing free arachidonic acid from membrane phospholipids, thus initiating synthesis of prostacyclin. Previous work in our laboratory has demonstrated that the thrombin-stimulated deacylation is specific for arachidonate and structurally similar polyunsaturated fatty acids that contain a delta-5 double bond. We now report that histamine, bradykinin, and the calcium ionophore A23187 exhibit the same fatty acid specificity as does thrombin. Experiments with both human umbilical vein and calf pulmonary artery endothelial cells indicate that these agonists stimulate the release of previously incorporated [14C]arachidonate but not 8,11,14-[14C]eicosatrienoate or [14C]docosatetraenoate. By contrast, melittin stimulates the release of 8,11,14-eicosatrienoate, docosatetraenoate, and oleate as well as arachidonate. These results suggest that histamine, bradykinin, and A23187 activate a common calcium-dependent phospholipase A2. Melittin appears either to alter the substrate specificity of the receptor-linked phospholipase A2 activity or to activate additional enzymes as well.     

Activation of (arachidonyl) phosphatidylinositol turnover in rabbit neutrophils by the calcium ionophore A23187.

The addition of the Ca2+ ionophore A23187 to rabbit neutrophils stimulated [14C]arachidonic acid incorporation into phosphatidylinositol and lysosomal enzyme secretion. A significant increase in phosphatidylinositol labelling was observed after a 2 min exposure to 0.1 microM-ionophore A23187. Maximum increases in rate of labelling were obtained with 1 microM-ionophore A23187 within 1 min, declining to basal rates after 15 min. Similarly, maximum rate of enzyme release occurred during the first 2 min of exposure to ionophore and release was essentially complete by 15 min. Threshold and peak ionophore A23187 concentrations for stimulating both processes were identical. In contrast with the specificity of phosphatidylinositol labelling induced by 1 microM-ionophore A23187 in the absence of cytochalasin B, ionophore also significantly stimulated labelling of phosphatidylserine and phosphatidylethanolamine in the presence of cytochalasin B. With a threshold ionophore concentration (0.1 microM), the enhanced incorporation of arachidonate was relatively specific for phosphatidylinositol in cytochalasin-treated cells. Ionophore A23187 did not accelerate labelling of phosphatidylinositol by [14C]acetate or [14C]glycerol, indicating that ionophore A23187 does not stimulate phosphatidylinositol synthesis de novo, although it did promote [14C]palmitate and [32P]Pi incorporation into neutrophil phosphatidylinositol. However, the increase in phosphatidylinositol labelling with these latter precursors was generally slower in onset and much more modest in magnitude than that observed with arachidonic acid. These results support the hypothesis that a Ca2+-dependent phospholipase, which acts on the arachidonate moiety of phosphatidylinositol, is responsible for initiating at least certain of the membrane events coupled to the release of secretory product from the neutrophil.     

Selective changes in fatty acid composition of phosphatidylserine in rat erythrocyte membrane induced by nitrate

The relationship between nitrate which is formed from inhaled nitrogen dioxide, a common air pollutant, and changes in fatty acid metabolism of phosphatidylserine in rat erythrocytes has been examined. When erythrocytes were incubated at 37°C for 60 min with fatty acid, the incorporation rate of [1-¹⁴C]arachidonic acid and [9,10-³H]palmitic acid into phosphatidylserine was 15% (80 pmol/h per μmol lipid phosphorus) and 20% (12 pmol/h per μmol lipid phosphorus) of those into phosphatidylethanolamine, respectively. By the addition of 1.0 mM sodium nitrate or 0.5 μM ionophore A23187 to the incubation mixture, the rate of incorporation of both arachidonic acid and palmitic acid into phosphatidylethanolamine was stimulated 1.45-fold. On the other hand, the incorporation of palmitic acid into phosphatidylserine was little affected, while that of arachidonic acid was stimulated 1.35-fold. An increase in arachidonic acid of phosphatidylserine was also found by the addition of nitrate or ionophore A23187. This increase was dependent on the concentration of extracellular calcium and observed by the addition of other chaotropic anions in the order SCN >CIO₄^? > NO₃^?. It seems likely, therefore, that nitrate causes changes in erythrocyte membranes to facilitate calcium uptake. Increasing the concentration of intracellular calcium may cause stimulation of acyl-CoA:lysophospholipid acyltransferase and/or endogenous phospholipase A₂.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

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.     

Asymmetry of arachidonic acid metabolism in the phospholipids of the human platelet membrane as studied with purified phospholipases

(1) Human platelets were incubated with high density lipoproteins (HDL) doubly labelled with either free [¹⁴C]arachidonate/[³H]arachidonoylphosphatidylcholine or free [¹⁴C]oleate/[³H]oleoylphosphatidylcholine. Whereas [¹⁴C]arachidonate was incorporated at a 10–15 times higher rate than [¹⁴C]oleic acid, the exchange of both species of phosphatidylcholine occurred to the same extent. In both cases, free ³H-labelled fatty acids were generated during the labelling procedure, indicating phospholipase A₂ hydrolysis. A redistribution of radioactivity to other phospholipids was noted after exchange of [³H]arachidonoylphosphatidylcholine only. (2) The exchange of phosphatidylcholine to platelets was confirmed using [¹⁴C]choline-labelled dipalmitoyl- and 1-palmitoyl-2-arachidonoylphosphatidylcholines. (3) Non-lytic degradation of platelet phospholipids by phospholipases revealed that free fatty acids were incorporated at the inside of the cells, whereas exchange was taking place on the platelet outer surface. However, 2-arachidonoylphosphatidylcholine displayed a more rapid movement towards the cell inside. The above findings suggest a topological asymmetry for the two pathways (acylation and exchange) of fatty acid renewal in platelets. The possible mechanisms and physiological relevance of the translocation of the external arachidonic acid pool across the membrane are discussed.     

Hexachlorocyclohexanes affect the arachidonic acid release from phosphatidylinositol but not from other phospholipid classes in tubular cell cultures

Gamma- and delta-isomers of hexachlorocyclohexane caused marked decreases in the levels of radioactive phospholipids, and increases in the levels of [³H]arachidonate incorporated into free fatty acids in rat renal tubular cells. The increased radioactivity of free fatty acids arises from the decrease of [³H]arachidonate incorporated into phosphatidylinositol, but not into phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. This fact suggests that phosphatidylinositol can be broken down to the fatty acid from the sn-2 position and lysophospholipid by a phospholipase activity increased by hexachlorocyclohexanes. The observed specific toxicant action could be achieved in two ways: (a) operating upon a specific phospholipase A₂ that acts on phosphatidylinositol, but not on other phospholipids as substrates and/or (b) involving substrate-phospholipase A₂ interactions. Interestingly, the observed effect of the -isomer was more pronounced than that of the -one.     

Pathways of arachidonic acid liberation in thrombin and calcium ionophore A23187-stimulated human endothelial cells: respective roles of phospholipids and triacylglycerol and evidence for diacylglycerol generation from phosphatidylcholine

Cultured endothelial cells from human umbilical vein were incubated for 20 h at 37 degrees C in the presence of [U-14C]arachidonic acid. Around 60-70% of the radioactive fatty acid was incorporated into cell lipids and was predominantly found in phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and triacylglycerol (39%, 33%, 13% and 6.5% of total incorporated radioactivity, respectively). Stimulation of the cells with human thrombin (2 U/ml) or calcium ionophore A23187 (5 microM) promoted the release into supernatants of arachidonic acid, 6-ketoprostaglandin F1 alpha, prostaglandins E2 and F2 alpha, in decreasing order of importance. The amount of secreted material was 4-fold higher with A23187, compared to thrombin. Parallel to the liberation process, phosphatidylcholine underwent a rapid decrease of radioactivity with both agonists, suggesting the involvement of a Ca2+-dependent phospholipase A2. Phosphatidylethanolamine displayed a minor decrease with A23187, whereas some reacylation was observed at 10 min with thrombin. Phosphatidylinositol was non-significantly affected in thrombin-stimulated cells, whereas A23187 promoted an early but minor decrease, followed by resynthesis. In contrast to A23187, thrombin was also able to promote a significant hydrolysis of triacylglycerol, which might thus be implicated in the process of arachidonate liberation. Finally, radioactive phosphatidic acid and diacylglycerol appeared in endothelial cells, in response to the two agonists. However, diacylglycerol formation did not parallel that of phosphatidic acid, especially with A23187. Determination of the 14C/3H ratio of the different lipids upon cell labelling with both [14C]arachidonic acid and [3H]palmitic acid revealed that diacylglycerol and phosphatidic acid are hardly derived from inositol-phospholipid breakdown by phospholipase C. Other possible pathways involving for instance phospholipase C splitting of phosphatidylcholine are discussed.     

Arachidonate mobilization in diacyl, alkylacyl and alkenylacyl phospholipids on stimulation of rat platelets by thrombin and the Ca2+ ionophore A23187.

Platelet stimulation by thrombin or Ca2+ ionophore induces mobilization of arachidonate from lipid stores. We have previously shown that, in [14C]arachidonic acid-prelabelled resting platelets, [14C]arachidonate was transferred from diacyl-sn-glycerophosphocholine to ethanolamine and choline-containing ether phospholipids. This transfer reached an equilibrium after 5 h incubation [Colard, Breton & Bereziat (1984a) Biochem. J. 222, 657-662]. [14C]Arachidonate-prelabelled platelets having reached this transfer equilibrium were used to study the mobilization of arachidonate in etheracyl and diacyl phospholipids. Upon thrombin stimulation, arachidonate decreased in diacyl-sn-glycero-3-phosphoinositol, in alkylacyl- and diacyl-sn-glycero-3-phosphocholine and increased in alkenylacyl- and diacyl-sn-glycero-3-phosphoethanolamine. Upon challenge with Ca2+ ionophore A23187, arachidonate decreased in diacyl-sn-glycero-3-phosphoethanolamine, in diacyl- and alkylacyl-sn-glycero-3-phosphocholine and increased in alkenylacyl-sn-glycero-3-phosphoethanolamine. We also compared arachidonate mobilization in platelets stimulated immediately after [14C]arachidonic acid chase with platelets stimulated after 5 h reincubation. We observed that the arachidonate newly incorporated into diacyl-sn-glycero-3-phosphocholine and triacylglycerols was rapidly released upon stimulation. This suggests the presence in these two lipids of a rapidly-turning-over arachidonate pool.     

Epidermal growth factor stimulates phospholipase A2 in vasopressin-treated rat glomerular mesangial cells.

Epidermal growth factor (EGF) enhances vasopressin- and ionophore-A23187-induced prostaglandin production and arachidonate release by rat glomerular mesangial cells in culture. The purpose of the present study was to delineate the phospholipid pathways involved in this effect. In cells labelled with [14C]arachidonate, EGF significantly enhanced the free arachidonate released in response to A23187 or vasopressin without enhancing the production of [14C]arachidonate-labelled diacylglycerol. EGF increased the [14C]arachidonate-labelled phosphatidic acid formed in response to vasopressin, but to a much smaller extent than it increased free arachidonate release. These results indicate that activation of phospholipase C is not sufficient to explain the increase in free arachidonate release observed on addition of EGF. To examine if EGF enhanced phospholipase A2 activity, mesangial cells were labelled with [2-2H]glycerol and [14C]-arachidonate, and the formation of arachidonate-poor lysophospholipids was studied. When combined with vasopressin, EGF significantly enhanced the formation of arachidonate-poor lysophospholipids as compared with vasopressin alone. The fate of exogenously added lysophosphatidylcholine was not altered after stimulation with vasopressin plus EGF, indicating that decreased deacylation or reacylation of the lysophospholipids was not responsible for their accumulation. Taken together, these results indicate that EGF enhances free arachidonate release by activation of phospholipase A2. The signalling mechanism responsible for the change in phospholipase A2 activity is not known, but could conceivably involve phosphorylation of modulating proteins such as lipocortin or G-proteins.     

Studies on the enzymatic pathways of calcium ionophore-induced phospholipid degradation and arachidonic acid mobilization in peritoneal macrophages

Exposure of mouse peritoneal macrophages to ionophore A23187 caused a rapid and extensive Ca2+-dependent phospholipid degradation and mobilization of arachidonic acid. Phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine all contributed to the arachidonic acid release, although the ethanolamine phospholipids incorporated [3H]arachidonic acid more slowly during the prelabeling period, particularly the plasmalogen form. Several enzymatic pathways could be positively identified as contributing to the ionophore-induced phospholipid degradation by the use of several different radiolabeled phospholipid precursors: (i) a phospholipase A-mediated deacylation, (ii) a phosphodiesterase (phospholipase C) reaction, rapidly generating diacylglycerol units from inositol phospholipids, and (iii) enzymatic processes generating diacylglycerol and CDP- and phosphocholine/ethanolamine from phosphatidylcholine/ethanolamine. The diacylglycerol formed was in part phosphorylated and in part hydrolyzed to monoacylglycerol, with retention of its arachidonic acid. These, and other, results indicate that the Ca2+-ionophore activates several apparently distinct phospholipid-degrading processes, in contrast to stimuli acting via cellular receptors.     

Fatty acid and glycerol labeling of glycerolipids of leukocytes in response to ionophore A23187.

The effect of divalent cation ionophore, A23187, on the incorporation of [1-14C]palmitic acid, [1-14C]linoleic acid and [U-14C]glycerol into glycerolipids of polymorphonulcear leukocytes was examined. Ionophore A23187 stimulated the labeling of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, and diacylglycerol by both labeled fatty acids and glycerol. [1-14C]Palmitic acid and [1-14C]linoleic acid incorporation into phosphatidylcholine and triacylglycerol was reduced by the presence of the ionophore in the incubation medium, while [U-14C]glycerol labeling of these lipids was not significantly changed under identical conditions. These data reflect that the acylation of sn-glycerol 3-phosphate is activated, and the acylations of lysophosphatidyl-choline and endogenous diacylglycerol are inhibited in cells incubated with ionophore A23187. External calcium was not required for the ionophore effect on the incorporation of labeled fatty acids and glycerol. It is suggested that the ionophore alters the metabolism of the fatty acid and glycerol moieties of glycerolipids by changing the distribution of intracellular calcium of leukocytes.     

Alterations of fatty acyl turnover in macrophage glycerolipids induced by stimulation. Evidence for enhanced recycling of arachidonic acid

Glycerophospholipid biosynthesis by the de novo pathway was assessed in mouse peritoneal macrophages by pulse-labeling with [U-¹⁴C]glycerol. Phosphatidylcholine (PC), which amounts to about 35% of total cellular phospholipids, exhibited the highest rate of glycerol uptake, followed by phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Remodeling of PC molecular species by deacylation/reacylation was established by determining the redistribution of glycerol label over 2 h after a 1 h pulse of [U-¹⁴C]glycerol and by determining incorporation of ¹⁸O from H₂ ¹⁸O-containing media. These data suggest that stearic and arachidonic acid enter PC primarily by the remodeling pathway but that small amounts of highly unsaturated molecular species, including 1,2-diarachidonoyl PC, are rapidly synthesized de novo, and subsequently remodeled or degraded. Treatment of the cells with the ionophore A23187 resulted in the selective enhancement of arachidonate turnover in PC, PI and neutral lipid, as well as enhanced de novo PI synthesis. [U-¹⁴C]Glycerol labeling experiments suggest that arachidonic acid liberated by Ca²⁺-dependent phospholipase A₂ activity is also reacylated in part through de novo glycerolipid biosynthesis, leading to the formation and remodeling of 1,2-diarachidonoyl PC and other highly polyunsaturated molecular species.     

Phospholipase C activity in rat kidney. Effect of deoxycholate on phosphatidylinositol turnover

Rat renal cortical and medullary slices incorporate [14C]arachidonate into phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and triacylglycerols. The percent distribution of [14C]arachidonate among the various phospholipids is similar in renal cortex and medulla, although the total amount of radioactively labeled phospholipids is higher in the renal medulla. Subsequent incubation of prelabeled slices in the presence of deoxycholate induces a loss of radioactivity from [14C]phosphatidylinositol, with a concomitant increase in 1,2-[14C]diacylglycerol. Neutral lipids are not affected. The degradation of phosphatidylinositol to [14C]diacylglycerol indicates the presence of phospholipase C activity. Renal medulla seems to be more sensitive to deoxycholate than the renal cortex. Deoxycholate also induces slightly the disappearance of some 14C radioactivity from phosphatidylethanolamine and phosphatidylcholine, which might reflect activation of phospholipase A2. The activity of the phospholipase C could constitute the first step in the sequence of reactions that leads to the release of arachidonic acid.     

Metabolism of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human promyelocytic leukemic HL60 cells. Stimulated expression of phospholipase A2 and acetyltransferase requires differentiation

Human promyelocytic leukemia (HL60) cells can be induced to differentiate into mature granulocytes by exposure to dimethyl sulfoxide. The addition of N-formylMet-Leu-Phe or the Ca2+ ionophore A23187 to these differentiated cells generated 15-30 pmol of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC)/10(6) cells as quantified by platelet aggregation assays. Under identical conditions, uninduced cells produced little alkylacetyl-GPC. Upon the addition of ionophore A23187, differentiated cells, and not uninduced ones, released [14C]arachidonate from prelabeled phospholipids including ether-linked phosphatidylcholines, formed both 3H-labeled 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC) and [3H]alkylacetyl-GPC from endogenous 3H-labeled 1-O-alkyl-2-(long chain) acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC), and incorporated exogenously added [3H]acetate or [3H]alkyllyso-GPC into alkylacetyl-GPC. These results are suggestive that both phospholipase A2 and acetyltransferase activities are involved in alkylacetyl-GPC biosynthesis by HL60 cells and that these activities appear during differentiation. However, when measured in cell extracts, the activities of phospholipase A2 and acetyltransferase of uninduced cells were virtually indistinguishable from those of differentiated cells. Uninduced cells exhibited enhanced incorporation of [3H]alkyllyso-GPC or [3H]alkylacetyl-GPC into alkylacyl-GPC and of [14C]arachidonate and [14C]oleate into various phospholipids including phosphatidylcholine. However, such enhanced expression of acylation reactions could not account for the lack of accumulation of arachidonate or of alkylacetyl-GPC by uninduced cells. Furthermore, analyses of phospholipid classes by phosphorus determination showed no significant alterations in phospholipid composition of HL60 cells during differentiation. Together these data are suggestive that mechanisms regulating the activation of phospholipase A2 and acetyltransferase activities are defective in uninduced cells and that an increased concentration of cytosolic free Ca2+ alone is not a sufficient requirement for these mechanisms.     

Phospholipid metabolism of stimulated lymphocytes: Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [³H]arachidonate and [¹⁴C]oleate or [³H]arachidonate and [¹⁴C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3–7-fold) and phosphatidylethanolamine (2–3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

The alpha 1-adrenergic transduction system in hamster brown adipocytes. Release of arachidonic acid accompanies activation of phospholipase C.

Previous studies of brown adipocytes identified an increased breakdown of phosphoinositides after selective alpha 1-adrenergic-receptor activation. The present paper reports that this response, elicited with phenylephrine in the presence of propranolol and measured as the accumulation of [3H]inositol phosphates, is accompanied by increased release of [3H]arachidonic acid from cells prelabelled with [3H]arachidonic acid. Differences between stimulated arachidonic acid release and formation of inositol phosphates included a requirement for extracellular Ca2+ for stimulated release of arachidonic acid but not for the formation of inositol phosphates and the preferential inhibition of inositol phosphate formation by phorbol 12-myristate 13-acetate. The release of arachidonic acid in response to phenylephrine was associated with an accumulation of [3H]arachidonic acid-labelled diacylglycerol, and this response was not dependent on extracellular Ca2+ but was partially prevented by treatment with the phorbol ester. The release of arachidonic acid was also stimulated by melittin, which increases the activity of phospholipase A2, by ionophore A23187, by lipolytic stimulation with forskolin and by exogenous phospholipase C. The arachidonic acid response to phospholipase C was completely blocked by RHC 80267, an inhibitor of diacylglycerol lipase, but this inhibitor had no effect on release stimulated with melittin or A23187 and inhibited phenylephrine-stimulated release by only 40%. The arachidonate response to forskolin was additive with the responses to either phenylephrine or exogenous phospholipase C. These data indicate that brown adipocytes are capable of releasing arachidonic acid from neutral lipids via triacylglycerol lipolysis, and from phospholipids via phospholipase A2 or by the sequential activities of phospholipase C and diacylglycerol lipase. Our findings also suggest that the action of phenylephrine to promote the liberation of arachidonic acid utilizes both of these reactions.