James D. Winkler

The signal transduction system of the leukotriene D4 receptor

During the past several years, substantial progress in understanding the receptors and signal transduction processes for peptidyl leukotrienes has been reported. Receptors have been identified and characterized, the major steps in the signal transduction pathway have been described, and the genetic and epigenetic regulatory processes have been characterized. Very recent studies have defined the mechanisms by which LTE4 acts as a partial agonist at the LTD4 receptor. The cloning of the genes for the proteins involved in the major steps of the signalling process has also been initiated. Stanley Crooke and co-authors summarize this recent progress and present their current notions about the LTD4 receptor signalling process.

Characterization of CoA-independent transacylase activity in U937 cells

Coenzyme A-independent transacylase (CoA-IT) mediates the transfer of polyunsaturated fatty acids from the sn-2 position of a donor phospholipid to the sn-2 position of an acceptor lyso-phospholipid. We have characterized this activity in U937 cells, a human monocytic cell line. The microsomes of these cells contained CoA-IT activity which demonstrated a fatty acid preference for transferring arachidonic acid into exogenously added 1-alkyl-2-lyso-GPC. This enzymatic activity was optimum between pH 6.5 and 9, was heat labile and displayed an apparent Km for 1-alkyl-2-lyso-GPC of 0.4 microM. This activity was not dependent on Ca2+, Mg2+, CoA or ATP, was not inhibited by 2-mercaptoethanol nor by addition of product, 1-alkyl-2-acyl-GPC. The activity of this enzyme was not altered by differentiation of U937 cells towards the macrophage with Me2SO. Treatment of U937 cells with dexamethasone had no effect on transacylase activity. The activity of this enzyme was decreased by the serine esterase inhibitors phenylmethyl-sulfonyl fluoride and N-tosyl-L-phenylalanine chloromethyl ketone and by the histidine modifier diethyl pyrocarbonate, suggesting that CoA-IT may belong to a family of acyltransferase enzymes typified by LCAT. CoA-IT activity was not affected by compounds that affect PLA2 activity, such as quinacrine, aristolochic acid and arachidonic acid, suggesting a mechanism of action for CoA-IT different from classical, low molecular weight PLA2 enzymes. In conclusion, U937 cells contain CoA-IT activity and this study extends our previous knowledge of this enzyme by demonstrating the differences between CoA-IT and PLA2 enzymes and suggesting similarities between CoA-IT and LCAT.

Association between leukotriene B4-induced phospholipase D activation and degranulation of human neutrophils

We have explored the role of phospholipase D (PLD) activation in leukotriene B4 (LTB4)-induced Ca2+ mobilization and degranulation of human neutrophils. Stimulation of [3H]alkyl-acyl-phosphatidylcholine-labeled neutrophils with LTB4 resulted in a rapid accumulation of [3H]alkyl-phosphatidic acid (PA) as well as a somewhat slower accumulation of [3H]alkyl-diglyceride (DG). In the presence of ethanol, PLD catalyzed a transphosphatidylation reaction in which LTB4 increased [3H]alkyl-phosphatidylethanol formation and simultaneously decreased LTB4-induced PA and DG accumulation. This pattern of lipid metabolism is consistent with the conclusion that LTB4 stimulates PLD activity in human neutrophils. Additional studies in which the extracellular and intracellular concentrations of Ca2+ were varied indicated that maximal LTB4-induced PLD activation was dependent upon Ca2+ and potentiated by inhibitors of protein kinase C. The time-course and concentration-response curves for LTB4-induced PLD activation were different from those for LTB4-induced Ca2+ mobilization, as measured by fura-2 fluorescence. On the other hand, the concentration-response curve for LTB4-induced PLD activation was similar to that for LTB4-induced degranulation. Preincubation of the cells with ethanol inhibited LTB4-induced PA and DG accumulation, as well as degranulation, suggesting that one or both of these metabolites were important for this response. In contrast, ethanol had no effect on LTB4-induced Ca2+ mobilization. Propranolol, an inhibitor of phosphatidate phosphohydrolase, abolished DG accumulation in response to LTB4 but had no effect on degranulation, suggesting that PA is more important than DG as a mediator of degranulation. Taken collectively, these data indicate that LTB4-induced activation of PLD in human neutrophils is mediated by a Ca(2+)-dependent mechanism, but not by protein kinase C. In addition, PLD activation in these cells may induce degranulation, but not Ca2+ mobilization.

CoA-independent transacylase activity is increased in human neutrophils after treatment with tumor necrosis factor α

CoA-independent transacylase (CoA-IT) appears to play a critical role in lipid mediator generation by rapidly moving arachidonate (AA) between phospholipid pools during cell activation. Tumor necrosis factor-alpha (TNF) pretreatment of human neutrophils increases agonist-induced production of inflammatory mediators. The current study tested if the TNF-induced increase in lipid mediator production may be, in part, due to altered CoA-IT activity. Neutrophils were treated with TNF (250 U/ml, 30 min), homogenates prepared, and CoA-IT activity measured by the ability of these homogenates to acylate 1-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine (GPC). There was an increased CoA-IT activity, from 9.1 +/- 1.1 to 13.7 +/- 1.4 pmol/mg per min in control vs. TNF-treated samples, respectively. Varying the concentration of 1-alkyl-2-lyso-GPC revealed an increased CoA-IT activity in microsomes that was due to an increased Vmax, from 26 to 54 pmol/mg per min. The ability of TNF to increase CoA-IT activity was concentration-dependent, with maximal response observed at 25 U/ml. This effect on CoA-IT appears to be specific, in that TNF treatment of neutrophils had no effect on CoA-dependent acylation of 1-acyl-2-lyso-sn-glycero-3-phosphocholine, using either AA-CoA or linolenoyl-CoA as substrates. In the intact cell, the movement of [3H]AA from other phospholipids into PE in fMLP-stimulated neutrophils was greatly enhanced after TNF treatment, demonstrating a functional consequence of increased CoA-IT activity. In addition, TNF treatment doubled platelet-activating factor production in response to the chemotactic peptide fMLP, as measured by [3H]acetate incorporation, while the response to A23187 remained unchanged. Taken together, these results provide the first evidence of modulation of CoA-IT activity by a proinflammatory cytokine and suggest that one mechanism for augmented lipid mediator formation is through increases in CoA-IT activity.

Arachidonate—Phospholipid Remodeling and Cell Proliferation

It has been recognized since the mid-1980’s that arachidonic acid is taken up and remodeled between as many as 20 different glycerolipid molecular species in most cell types [1]. However, it has been difficult to understand why it is necessary for mammalian cells to maintain so many different arachidonate-containing glycerolipids and the ramifications of remodeling arachidonate between different glycerolipid molecular species. It is known that arachidonate-phospholipid remodeling occurs at a relative“slow” rate in most resting cells but this rate can be accelerated many fold during priming or stimulation of cells [2, 3]. Recently, we have begun to utilize newly-discovered inhibitors of the enzyme, CoA-independent transacylase (CoA-IT), which is thought to remodel arachidonate into the phospholipid subclass 1-alkyl-2-arachidonoyl-GPC and ethanolamine-containing phospholipids. Using these inhibitors of remodeling, it has been discovered that rapid remodeling of arachidonate is probably necessary to support continued arachidonic acid release and PAF generation which is initiated by phospholipase A2.

Evidence for different mechanisms involved in the formation of lyso platelet-activating factor and the calcium-dependent release of arachidonic acid from human neutrophils

Recent studies suggest that the first step in platelet-activating factor (PAF) biosynthesis, 1-alkyl-2-lyso-GPC (lyso PAF) formation, may be initiated by the selective transfer of arachidonate from 1-alkyl-2-arachidonoyl-GPC to an acceptor lyso phospholipid by a CoA-independent transacylase activity (CoA-IT). The present study was designed to determine whether the formation of 1-alkyl-2-lyso-GPC and the release of arachidonic acid can occur by different mechanisms. These experiments examined both the formation of 1-[3H]alkyl-2-lyso-GPC from 1-[3H]alkyl-2-acyl-GPC and the release of arachidonic acid from membrane phospholipids as determined by GC/MS in neutrophil homogenates under various conditions. The addition of unlabelled lyso phospholipids to neutrophil homogenates stimulated the time-dependent formation of 1-[3H]alkyl-2-lyso-GPC from 1-[3H]alkyl-2-acyl-GPC. Without exogenous lyso phospholipids, little 1-[3H]alkyl-2-lyso-GPC was formed in this reaction. The activity which catalyzed the formation of 1-[3H]alkyl-2-lyso-GPC had characteristics identical to CoA-IT as indicated by the fact that both reactions were: independent of Ca2+, Mg2+, CoA and CoA fatty acids, located in microsomal fractions, and stable in 10 mM dithiothreitol. In sharp contrast to the aforementioned reaction, addition of lyso phospholipids did not affect the quantity of arachidonic acid released from membrane phospholipids. Furthermore, there was a Ca(2+)-independent release of arachidonic acid from membrane phospholipid that was increased 4 to 5-fold after the addition of 5 mM Ca2+. Finally, Ca(2+)-dependent arachidonic acid release was inhibited by putative phospholipase A2 inhibitors, aristolochic acid and scalaradial, at concentrations where neither the production of 1-[3H]alkyl-2-lyso-GPC nor Ca(2+)-independent arachidonic acid release was altered. Together these data imply that there may be different mechanisms involved in the formation of 1-alkyl-2-lyso-GPC and arachidonic acid from membrane phospholipids.

Mechanisms of Regulation of Receptors and Signal Transduction Pathways for the Peptidyl Leukotrienes

Studies in our laboratory and others have defined the receptors and signal transduction pathways for the peptidyl leukotrienes (for review, see reference 1 ) . We have proposed a model that describes a number of steps in the signal transduction process for LTD, and have systematically restricted the process-adding steps and clarifying mechanisms as required to explain our observations. FIGURE 1 shows the most recent iteration of our model. LTD, receptors are cell-surface localized macromolecules.2~3 LTD, receptors are highly specific and they interact selectively with LTD, and minimally with LTC,.4-6 LTE, also interacts with LTD, receptors and is a partial a g o n i ~ t . ~ The LTD, receptors observed in cultured cells of various types have properties comparable to those of LTD, receptors in guinea pig and human l ~ n g , ~ ~ but they differ from LTC, binding sites and LTD, receptors in guinea pig When LTD, interacts with the LTD, receptor, it causes a transient increase in intracellular calcium (Ca++ transient), along with activation of a phosphoinositidespecific phospholipase C (PI-PLC).3.5-7 In some cells and various tissues, these effects have been shown to depend on a guanine nucleotide binding protein (G-protein) susceptible to pertussis toxin (Ci).I8 In other cells and tissues, a G-protein that is insensitive to pertussis toxin has been shown to be involved.I6 Subsequent to the calcium transient, diacylglycerol, and inositol phosphate generation induced by LTD,, protein kinase C is Protein kinase C activation may play a role in regulation of LTD, receptor activity and may be involved in propagation of the LTD,-induced signal. For example, topoisomerase I , but not 11, is activated after LTD4 interacts with its receptors and appears to be involved in enhancing transcription of the gene for phospholipase A, activating protein (PLAP).z1-23 Phospholipase A, activating protein induces an increase in the activity of a phosphatidylcholine-selective phospholipase A, (PC-PLA2).24 The increased activity may be due to interactions of PLAP with either the enzyme or substrate (or both) and this results in enhanced interaction of arachidonic acid, which is metabolized via various pathways with the predominant metabolites varying as a function of the phenotype of the cell..2

Phorbol 12-myristate 13-acetate inhibition of leukotriene D4-induced signal transduction was rapidly reversed by staurosporine

Activation of leukotriene D4 receptors results in phospholipase C-mediated breakdown of phosphatidylinositol and increases in intracellular Ca2+ in U-937 cells. Treatment (10 min) with phorbol 12-myristate 13-acetate blocked leukotriene D4-induced phosphatidylinositol metabolism and Ca2+ mobilization (IC50 = 0.2 nM). Treatment with 10 nM phorbol 12-myristate 13-acetate produced blockade which was complete within 1 min and no recovery was observed over 7 days. Addition of the protein kinase C inhibitor staurosporine (100 nM) to U-937 cells pretreated with phorbol 12-myristate 13-acetate for 5 min or 24 hr resulted in a rapid reappearance of leukotriene D4-induced Ca2+ mobilization. Half of the response recovered within 2 min, with complete recovery in 20 min. Staurosporine produced a concentration-related recovery of signal transduction, with an EC50 of 30 nM. These data describe cells which have a novel response to phorbol 12-myristate 13-acetate in that the inhibition of leukotriene D4 signal transduction is persistent and yet rapidly reversed by staurosporine.

Inhibitors of protein kinase C selectively enhanced leukotriene D4-induced calcium mobilization in differentiated U-937 cells

U-937 cells differentiated with dimethylsulphoxide for 3-4 days express receptors for leukotriene D4 (LTD4), which are coupled to Ca2+ mobilization and phosphatidylinositol (PI) metabolism. Treatment of U-937 cells with an inhibitor of protein kinase C (PKC) [staurosporine (100 nM)] augmented the Ca2+ mobilized by LTD4. The peak concentration of the LTD4-induced increase in [Ca2+]i was 1500 nM in untreated cells and 3000 nM in cells treated with staurosporine for 30 s. Maximal mobilization responses were observed at 1-10 microM LTD4 in both control and staurosporine-treated cells. The increased Ca2+ response to LTD4 after staurosporine treatment occurred within 30 s and was attributable to both intracellular and extracellular stores. Additionally, a second phase of Ca2+ mobilization occurred after stimulation with LTD4, which was elevated by pretreatment with staurosporine--this effect was maximal after 5-10 min of treatment. Staurosporine either had no effect or decreased the Ca2+ mobilization response of differentiated U-937 cells to other agonists, such as LTB4, platelet activating factor, ATP or the chemotactic peptide f-Met-Leu-Phe. Although staurosporine alone had no effect on basal PI metabolism it increased LTD4-induced PI metabolism. Staurosporine did not prevent the tachyphylaxis observed upon second challenge with LTD4, nor did it prevent LTD4-induced homologous densensitization. Other compounds which inhibit PKC (sphingosine and 1-O-hexadecyl-2-O-methylglycerol), also enhanced the Ca2+ response of U-937 cells to LTD4, but not to other agonists. These data show that inhibition of PKC enhanced responses of LTD4, suggesting that PKC plays a role in determining the responsiveness of LTD4 receptors.

Inhibitors of Arachidonate Metabolism and Effects on PAF Production

Our understanding of the enzyme Coenzyme A-independent transacylase (CoA-IT) has increased dramatically over the last 10 years. The enzyme catalyses the removal of the fatty acyl group from the sn-2 position of glycerophospholipids (GPL) and transfers it into 1-radyl-2-lyso GPL.1 The enzyme shows striking selectivity for transfer of arachidonate and other long-chain, unsaturated fatty acyl groups. It also shows strong preference for phosphocholine-and phosphoethanolamine-containing GPL, along with a preference for using 1-ether GPL as acceptors for the transferred arachidonate.2 The mechanism of action of CoA-IT has yet to be defined at the molecular level, but CoA-IT is hypothesized to be a member of the family of tranferases typified by lecithin-cholesterol acyl transferase.3 Based on these characteristics, CoA-IT has been presumed to play a role in the movement of arachidonate between GPL that occurs in inflammatory cells.4–7