Floyd H. Chilton

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.

Biological effects of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil☆

The synthesis of large quantities of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) relative to 1-alkyl-2-acetyl-GPC (PAF; platelet-activating factor) has been demonstrated in several inflammatory cells. The present study has examined agonist and antagonist activities of 1-acyl-2-acetyl-GPC in the human neutrophil. 1-Acyl-2-acetyl-GPC induced a rapid increase in cytosolic calcium in the neutrophil; this effect was detected at 2 x 10(-9) M and was maximal at 10(-6) M. The peak response induced by 1-acyl-2-acetyl-GPC was similar to that induced by PAF although the potency of 1-acyl-2-acetyl-GPC was 300-fold lower than that of PAF. The dose response curves for both 1-acyl-2-acetyl-GPC and PAF were shifted in a parallel fashion by L-652,731 (10(-6) M), a PAF receptor antagonist, suggesting that both 1-acyl-2-acetyl-GPC and PAF act on the same receptor. High concentrations of 1-acyl-2-acetyl-GPC (10(-5) M) induced the release of beta-glucuronidase and lysozyme from the human neutrophil. The percent release of lysozyme induced by 1-acyl-2-acetyl-GPC was consistently higher than that of beta-glucuronidase. Prior stimulation of neutrophils with 1-acyl-2-acetyl-GPC dose-dependently inhibited the increase in cytosolic calcium induced by a subsequent challenge with an optimal concentration of PAF. Similarly, preincubation of neutrophils with 1-acyl-2-acetyl-GPC dose-dependently inhibited beta-glucuronidase and lysozyme release induced by a subsequent stimulation with PAF. The inhibitory effect on degranulation could not be surmounted even by concentrations of PAF 10-fold higher than that of 1-acyl-2-acetyl-GPC. The inhibition appeared to be selective for PAF since 1-acyl-2-acetyl-GPC did not affect f-met peptide-induced degranulation. This study suggests that 1-acyl-2-acetyl-GPC may act as a naturally-occurring specific inhibitor of PAF-induced activation of the human neutrophil.

Relative amounts of 1-O-alkyl- and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in stimulated endothelial cells

The specific precursor for platelet-activating factor, 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, constitutes 10 per cent of the 1-radyl-2-acyl-sn-glycero-3-phosphocholines in endothelial cells. Stimulation of endothelial cells results in accumulation of PAF and its sn-1-acyl- analog (acylPAF), with acylPAF the predominant product. Mass spectrometry confirmed these relative amounts and confirmed that stimulated endothelial cells accumulate 1-3 ng PAF per million cells. These data suggest that stimulated endothelial cells accumulate both PAF and acylPAF and that the PAF synthetic pathway in endothelial cells is not highly selective for the specific PAF precursor (1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine).

Recent advances in our understanding of the biochemical interactions between platelet-activating factor and arachidonic acid.

In the last few years, it has become increasingly apparent that the biochemistry of PAF (platelet-activating factor) and that of arachidonic acid are interrelated in a number of inflammatory cells. Experiments presented here further point out that arachidonic acid plays a crucial role in the catabolism and biosynthesis of PAF. In addition, they suggest that the same phospholipid molecular species may serve as a source for both arachidonic acid and 1-alkyl-2-lyso-sn-glycero-3-phosphocholine during cell activation. Finally, they reveal that there may be common regulatory mechanisms for the biosynthesis of PAF and arachidonic acid metabolites. Taken together, studies examining the relationship between PAF and arachidonic acid suggest it may be difficult to consider the biochemistry of PAF without considering arachidonic acid metabolism and vice versa.

Arachidonic acid metabolism during antigen and ionophore activation of the mouse bone marrow derived mast cell

This study has examined the metabolism of arachidonic acid in the mouse bone marrow-derived mast cell (BMMC) during immunologic and nonimmunologic activation. The predominant pools of endogenous arachidonate in the mast cells were found in ethanolamine (46%), choline (39%) and inositol (14%) containing glycerolipids. Initial studies established conditions where equilibrium labelling of these major phospholipids in the BMMC could be reached. Upon challenge, arachidonate was lost from all major phospholipid classes (phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylinositol). There was a small but significant increase in the amount of label associated with phosphatidic acid during cell activation. Arachidonate was distributed among 1-acyl, 1-alkyl and 1-alk-1-enyl-linked subclasses of PC and PE. The rank order of loss of labelled arachidonate from the major PE and PC subclasses during antigen and ionophore activation was 1-alk-enyl-2-arachidonoyl-GPE greater than 1-acyl-2-arachidonoyl-GPC greater than 1-acyl-2-arachidonoyl-GPE greater than 1-alkyl-2-arachidonoyl-GPC. Labelled products released into the supernatant fluids and free arachidonic acid within the cell accounted for the bulk of arachidonate lost from phospholipids. Labelled products in the supernatant fluids were composed of LTB4, LTC4, PGD2 and free arachidonic acid. BMMC phospholipids were also labelled for 24 hr with [3H]choline, [3H]myoinositol or [14H]ethanolamine and labelled 2-lyso phospholipids were measured after cell activation. Radioactivity in lysophospholipids from PC, PE and PI increased significantly between 30 s and 2 min after antigen activation and then declined. Taken together, these studies suggest that arachidonate is mobilized predominantly from PE and in particular 1-alk-1-enyl-2-arachidonoyl-GPE by the direct removal of arachidonate from the sn-2 position of the molecule. Most of this arachidonate is then released from cells as eicosanoids or free fatty acid.

A comparative study of the effects of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine and platelet activating factor on histamine and leukotriene C4 release from human leukocytes

BACKGROUND IgE-mediated stimulation of human basophils and lung mast cells causes the synthesis of larger amounts of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) than 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor [PAF]). METHODS To study the biologic activity of 1-acyl-2-acetyl-GPC, we compared its effects and those of PAF on histamine and leukotriene C4 (LTC4) release from human mixed leukocytes that contained basophils. RESULTS 1-Acyl-2-acetyl-GPC (0.1 to 10 mumol/L) failed to release significant amounts of histamine (> or = 10%) in most donors tested (20 of 24), whereas PAF (0.01 to 1 mumol/L) was active in 58%. 1-Acyl-2-acetyl-GPC (0.1 to 10 mumol/L) was a stimulus for LTC4 release (132 +/- 30 ng/micrograms of histamine) with a potency of about 1000 times less than PAF. The kinetics of 1-acyl-2-acetyl-GPC-activated LTC4 release were similar to those of PAF (half-life approximately equal to 2 minutes). The specific PAF receptor antagonist, WEB 2086 (10 nmol/L to 10 mumol/L), inhibited both 1-acyl-2-acetyl-GPC- and PAF-mediated LTC4 release with the same potency (inhibitory concentration of 50% approximately equal to 1.5 mumol/L). Brief (2-minute) cell preincubation with 1-acyl-2-acetyl-GPC in the absence of extracellular Ca2+ induced a decrease in the subsequent Ca2+ dependent activation of PAF. Similarly, 1-acyl-2-acetyl-GPC (0.1 to 10 mumol/L) caused a concentration-dependent inhibition of PAF-activated histamine secretion (inhibitory concentration of 50% approximately equal to 0.2 mumol/L). CONCLUSIONS Our data suggest that 1-acyl-2-acetyl-GPC may represent, under certain circumstances, a modulator of human basophil mediator release via mechanisms shared with PAF.

Mechanisms of prostanoid synthesis in human synovial cells: Cytokine-peptide synergism

Bradykinin (BK)2 and interleukin-1 (IL-1) interact synergistically to stimulate prostaglandin synthesis in human synovial fibroblast-like cells. The effect of BK is rapid and correlates with its capacity to elevate cytosolic levels of calcium ([Ca2+]i), while IL-1s effect is slow and is dependent upon de novo protein synthesis. The mechanism of this synergistic interaction was investigated. In the basal state, high levels of arachidonic acid (AA) were spontaneously released from synovial cells but near absent levels of cyclooxygenase activity prevented metabolism of AA to prostanoid. BK was a potent stimulus for elevating AA, but not prostaglandins, above basal levels. IL-1, in contrast, increased prostaglandins, but not AA, above basal levels. IL-1 treatment was not associated with a loss or redistribution of AA among phospholipid classes.These results are consistent with high basal phospholipase activity in synovial cells and demonstrate the ability of BK, presumably via its ability to raise [Ca2+]i, to further elevate this activity(ies). Metabolism of AA to prostanoid is minimal in resting and BK-stimulated synovial cells, however, without the concomitant induction of cyclooxygenase activity by IL-1. These studies clarify the different, but synergistic, mechanisms of action of a peptide and cytokine in stimulating prostanoid synthesis in synovial cells. In addition, these data extend the results of previous investigations in demonstrating that basal phospholipase activity provides sufficient AA substrate for IL-1 induced prostanoid synthesis without invoking the concomitant induction of phospholipase activity by IL-1.

Influence of immunologic activation and cellular fatty acid levels on the catabolism of platelet-activating factor within the murine mast cell (PT-18)

The present study has examined the catabolism of 1-O-[3H]hexadecyl-2-acetyl-GPC (C16-PAF) and of 1-O-octadecyl-2-acetyl-GPC (C18-PAF) in spleen-derived PT-18 murine mast cells (mast cells). Mast cells catabolized exogenous PAF into two inactive metabolites, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lysoPAF) and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC). The rate of conversion of C16-PAF to metabolites was more rapid than that of C18-PAF. Analysis of the acyl composition of 1-O-alkyl-2-acyl-GPC formed during the metabolism of PAF revealed that arachidonic acid (20:4) was the major fatty acyl chain incorporated at the sn-2 position. However, 25% of newly formed 1-O-alkyl-2-acyl-GPC was reacylated with docosahexaenoic acid (22:6). The influence of cellular fatty acid content on PAF catabolism was further explored in mast cells in which the ratio of fatty acids within cellular phosphoglycerides had been altered by supplementing the cells with various fatty acids in culture. Mast cells supplemented with 20:4 or 22:6 converted PAF to 1-O-alkyl-2-acyl-GPC at a significantly higher rate than non-supplemented cells. In contrast, cells supplemented with linoleic acid (18:2) metabolized PAF at rates similar to non-supplemented cells. Analysis of the acyl composition of 1-O-alkyl-2-acyl-GPC derived from the metabolism of PAF in 20:4-supplemented cells indicated that 20:4 was incorporated exclusively into the sn-2 position. Conversely, 22:6-supplemented cells incorporated predominantly 22:6 at the sn-2 position of 1-alkyl-2-lyso-GPC. Supplementation with 18:2 had no effect on the acylation pattern seen in newly formed 1-O-alkyl-2-acyl-GPC. Activation of passively sensitized mast cells with antigen or with ionophore A23187 significantly enhanced the rate of catabolism of exogenously-provided PAF but had no effect on the acylation pattern of 1-O-alkyl-2-acyl-GPC. Experiments performed with the soluble fraction of the cells showed that acetyl hydrolase activity was increased in mast cells stimulated with antigen. In addition, supernatant fluids from antigen or ionophore-treated mast cells converted PAF to lysoPAF, suggesting that acetyl hydrolase activity was released during cell activation. These data indicate that the ability of mast cells to catabolize PAF to inactive metabolites is influenced by cell activation and by the cellular levels of certain fatty acids.

Metabolism of platelet-activating factor in the guinea-pig heart

Platelet-activating factor (PAF; 1-alkyl-2-acetyl-glycero-phosphocholine) is a biologically active phospholipid which is synthesized by a variety of blood cells and organ systems. PAF exerts many effects on the cardiovascular system including hypotension, depression of myocardial contractility and coronary constriction. The present study has examined the capacity of the guinea-pig heart to regulate the levels of exogenous PAF in two different models: isolated perfused heart and isolated ventricular myocytes. In the first model, isolated hearts were perfused with labeled PAF (10(-10) M) in a recirculating manner at flow rates of 15 ml/min (normal flow perfusion; NFP) and 2 ml/min (low flow perfusion, LFP). Exogenously provided PAF appeared in the tissue in a time-dependent manner. The rate of extraction of PAF was higher during LFP than during NFP. PAF was metabolized by the heart to two major products, lyso-PAF and 1-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-alkyl-2-acyl-GPC). Lyso-PAF was found primarily in the perfusion buffer while both lyso-PAF and 1-alkyl-2-acyl-GPC were detected in the tissue. No qualitative difference in the metabolic products derived from PAF catabolism was observed between hearts undergoing NFP and LFP. Acetyl hydrolase activity was detected in the perfusion fluid at both flow rates, probably accounting for the formation of lyso-PAF in the perfusate. However, perfusion fluid from LFP contained a higher acetyl hydrolase activity, per micrograms of protein as compared to fluid from NFP. Isolated ventricular myocytes incubated with labeled PAF (3 x 10(-9) M) also converted it to 1-alkyl-2-acyl-GPC. Kinetic experiments suggested that PAF was initially deacetylated to form lyso-PAF and that this intermediate was then rapidly reacylated with a fatty acyl moiety at the sn-2 position. HPLC analysis of the fatty acids inserted at the sn-2 position of 1-alkyl-2-acyl-GPC revealed that the myocytes reacylated lyso-PAF predominantly with arachidonic acid. These data indicate that the guinea-pig heart may regulate PAF levels by at least two mechanisms: (1) it may release acetyl hydrolase into the vascular compartment, particularly under low flow conditions; and (2) the ventricular myocyte has the capacity to take up PAF and catabolize it to inactive products.