Bioassay of paf-acether by rabbit platelet aggregation.
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Animals↗
Biomedical subjects
E Ninio
Publications and source records attributed to E Ninio.
Quantitation of paf-acether by release of endogenous platelet serotonin assessed by liquid chromatography with electrochemical detection.
A new method to quantitate paf-acether (paf) was developed. It is based on the measurement of serotonin released from washed rabbit platelets challenged with paf. Platelets (1 X 10(8)/ml) were exposed with or without stirring to various concentrations of paf (26-130 pM) at 37 degrees C or at room temperature. Supernatants were submitted to a 4-min liquid chromatography run and serotonin was measured by electrochemical detection. We quantitated paf from three different biological sources, human neutrophils, mouse peritoneal macrophages, and cultured mast cells, comparing a classical method, i.e., platelet aggregation with the electrochemical detection of endogenous serotonin. We found similar results since, when compared with the aggregation method, the results differed by 12 to 47%. The sensitivity of both methods was 26 pM. The between-day variation coefficient was 23 and 14% (n = 12) for the aggregation method and the serotonin release, respectively, whereas the within-day variation coefficient for serotonin quantitation was less than 5% (n = 12). The superiority of the new method lies in its simplicity, the economy of platelets, and its possibility of automation. It can be applied to any agonist or any mechanism capable of releasing serotonin from platelets and more generally when a simple and fast method for measuring serotonin is desirable.
Animals↗
Biosynthesis of paf-acether. XIV. Paf-acether output in murine peritoneal macrophages is regulated by the level of acetylhydrolase.
Paf-acether (paf) synthesis was previously shown to be impaired in 24 hr-adherent and Bacillus Calmette-Guérin-activated murine peritoneal macrophages as compared to resident macrophages. We report here that the induction of acetylhydrolase was responsible for the decreased paf output in 24 hr-adherent macrophages. The kinetic analysis of the enzymes derived from 2 hr-, 24 hr- and BCG-activated adherent macrophages and from plasma revealed that the Km for paf was similar whatever the source of the acetylhydrolase whereas the Vmax was five-fold increased in 24 hr-cultured macrophages. The acetylhydrolase activity was Ca2+-independent and was not inhibited by addition of alkyl-acyl (long chain)-glycero-phosphocholine suggesting that the enzyme was not a phospholipase A2.
1-Alkyl-2-acetylglycerophosphocholine Esterase↗
Biosynthesis of paf-acether. X. Phorbol myristate acetate-induced paf-acether biosynthesis and acetyltransferase activation in human neutrophils.
We tested the hypothesis that protein kinase C might play a role in the biosynthesis of platelet-activating factor (paf-acether) in human neutrophils. PMA but not its inactive analog 4-alpha-phorbol-12,13-didecanoate induced lyso paf-acether production, followed by acetyltransferase activation, leading to paf-acether synthesis and release. Moreover, PMA was twice as powerful compared to opsonized zymosan (OPZ). 1-Oleoyl-2-acetyl-glycerol also induced acetyltransferase activation and paf and lyso paf production. The paf-acether formed by PMA or OPZ stimulation was composed of alkyl chains C16:0 (84.3 +/- 5% and 80.7 +/- 3.5%, respectively, and C18:0 (15.7 +/- 5% and 19.3 +/- 3.5%, respectively, means +/- SEM) as assessed by gas chromatography-electron capture detection. The inhibitor of protein kinase C, D-sphingosine, markedly decreased paf and lyso paf production and acetyltransferase activation in PMA- as well as OPZ-stimulated neutrophils. These results strongly suggest the involvement of protein kinase C in signal transduction during cell stimulation, leading to the paf biosynthesis.
Acetyltransferases↗
Capillary gas chromatography and tandem mass spectrometry of paf-acether and analogs: absence of 1-O-alkyl-2-propionyl-sn-glycero-3-phosphocholine in human polymorphonuclear neutrophils.
Fast atom bombardment-tandem mass spectrometry was used to identify molecular species of paf-acether (paf) produced by human polymorphonuclear neutrophils. Using this biological material, normal phase high performance liquid chromatography was necessary prior to the fast atom bombardment-tandem mass spectrometry step. Gas liquid chromatography/electron capture detection after hydrolysis with phospholipase C and conversion to heptafluorobutyrate derivatives was used to confirm the results. The results indicated the presence of mainly 1-O-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphocholine, acyl analogs of paf and only trace amounts of other alkyl analogs of paf. We did not detect the 2-propionyl analog of paf. Moreover, supplementation of human polymorphonuclear neutrophils with sodium propionate did not result in formation of the 2-propionyl analog of paf.
Electrons↗
Biosynthesis of paf-acether. XI. Regulation of acetyltransferase by enzyme-substrate imbalance.
Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase is the key enzyme in paf-acether (paf) biosynthesis, since it yields the active mediator from its nonacetylated precursor, lyso-paf. In microsomal fractions obtained from the ionophore A23187-stimulated human polymorphonuclear neutrophils, the optimal conditions allowing the full acetylation of lyso-paf were: 2-2.5 mg.ml-1 bovine serum albumin, 40 microM lyso-paf, 200 microM acetyl-CoA and acetyltransferase of high specific activity, at least 18 nmol.min-1.mg protein- -1. The reaction frequently stopped before the substrate was consumed due to spontaneous decay of the enzyme activity at 37 degrees C and inhibition of the enzyme by the paf formed in the reaction. However, low concentrations of acetyltransferase substrates (lyso-paf or lysophosphatidylcholine) and the antioxidant dithiothreitol, but not the inhibitors of proteinases or phosphatases, protected the enzyme against decay. In contrast, high concentrations of those lyso substrates inhibited the enzyme activity in the assay. This inhibition as well as that due to paf was overcome by raising the concentration of the enzyme contained in the microsomal fraction or the bovine serum albumin in the assay. These results suggest that the biosynthesis of paf in cell-free assay and most probably in intact cells might be controlled to a larger extent by the acetyltransferase concentration rather than by that of its substrates.
Acetyltransferases↗
Ketotifen inhibits paf-acether biosynthesis and beta-hexosaminidase release in mouse mast cells stimulated with antigen.
Acetyl-CoA acetyltransferase (1-O-alkyl-sn-glycero-3-phosphocholine) is a key enzyme in paf-acether biosynthesis. Its immunological activation as related to paf-acether formation was investigated in mast cells derived from mouse bone marrow. The action of ketotifen, a prophylactic anti-asthma drug, on the antigen-induced activation of acetyltransferase and on the release of paf-acether and beta-hexosaminidase was studied in mast cells. Mast cells were sensitized with dinitrophenyl-specific monoclonal IgE and preincubated for 15 min at 37 degrees C with various concentrations of ketotifen or vehicle prior to challenge with dinitrophenyl coupled to bovine serum albumin (40 ng/ml). Acetyltransferase activity and mediator formation and release were measured. Ketotifen inhibited dose dependently the antigen-induced paf-acether formation and release, beta-hexosaminidase release and acetyltransferase stimulation. The IC50 values were 20.0 +/- 4.4, 11.8 +/- 6.2, 8.8 +/- 3.8 and 20.5 +/- 3.4 microM (mean +/- S.E.M., n = 3) respectively. Mast cells were preincubated with 50 microM ketotifen for 15 min at 37 degrees C then washed prior to antigen challenge. The release of paf-acether and beta-hexosaminidase and the stimulation of acetyltransferase were inhibited by 90.0 +/- 15.0, 91.0 +/- 15.0 and 88.0 +/- 11.0% (n = 3) respectively. In addition, Ca2+ entry was inhibited by 100% as assessed from Quin-2 fluorescence. Thus, the release of a preformed granular enzyme beta-hexosaminidase is inhibited by ketotifen together with the enzymatic formation of a newly formed mediator.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetyltransferases↗
Biosynthesis of paf-acether. IX. Role for a phosphorylation-dependent activation of acetyltransferase in antigen-stimulated mouse mast cells.
Mouse bone marrow-derived mast cells passively sensitized with monoclonal IgE released paf-acether (platelet-activating factor) and beta-hexosaminidase when challenged with the specific antigen. The formation and the release of paf-acether followed an early increase in the activity of the acetyltransferase, the main enzyme in paf-acether biosynthesis. The antigen-induced activation of the acetyltransferase was dependent on physiologic temperature and on the presence of Ca2+. By using microsomal fractions from unchallenged and challenged mast cells, the Vmax values were 3.5 and 12.0 nmol/min/mg of protein, respectively, whereas in both cases a Km value for acetyl-coenzyme A of 172 microM was measured. The stimulation of acetyltransferase could be mimicked in vitro under experimental conditions which favor phosphorylation, i.e. adding ATP and Mg2+ to lysates from unchallenged mast cells. In contrast, ATP and Mg2+ were uneffective on lysates from challenged cells that exhibited high level of acetyltransferase activity, suggesting that phosphorylation of the enzyme already took place at the time of cell stimulation. Moreover, addition of alkaline phosphatase to microsomal fraction obtained from either antigen-challenged mouse bone marrow-derived mast cells or unchallenged cells, resulted in 52% and 43% loss of acetyltransferase activity, respectively. Phorbol myristate acetate treatment of cells doubled the enzyme activity supporting the phosphorylation hypothesis. Thus, we report on the immunologic activation of a key enzyme for paf-acether synthesis and on the mechanism of this activation in a pure mast cell population. A link between bridging of IgE receptors and the activation of an enzyme critical to the formation of a lipid mediator is thereby evidenced.
Acetyltransferases↗
Biosynthesis of paf-acether: VIII: Impairment of paf-acether production in activated macrophages does not depend upon acetyltransferase activity.
Activated peritoneal macrophages (M phi) from mice injected with Bacilli Calmette-Guérin, trehalose dimycolate, a defined immunostimulant derived from Mycobacterium tuberculosis, or streptococci C74 (St), synthesized two to three times less paf-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in response to a zymosan challenge than did (0.7 +/- 0.2 nmol/mg protein) resident-M phi (R-M phi). To assess at which step the paf-acether biosynthetic pathway was impaired, the content in phospholipid paf-acether precursors was evaluated. The alkyl-acyl-glycerophosphocholine content was comparable in R-M phi (50.4 +/- 18.6 nmol/mg protein) and activated-M phi (40.0 +/- 6.5 to 63.9 +/- 7.7 nmol/mg protein), as well as the lyso paf-acether content (0.85 +/- 0.18 nmol/mg protein for R-M phi vs 0.63 +/- 0.16 to 1.23 +/- 0.21 nmol/mg protein for activated macrophages). The nonlimiting rate of the phospholipid substrate was strengthened by experiments showing that incubation of the various populations with lyso paf-acether did not yield increased amounts of paf-acether. Similarly, incubation of R-M phi or St-M phi with sodium acetate increased paf-acether production to the same extent in both populations, ruling out the acetate substrate deficiency as the cause of the impaired production. The level of acetyltransferase activity, the enzyme that transfers an acetate moiety of acetyl coenzyme A (acetyl-CoA) onto lyso paf-acether, was very low in thioglycolate (TG)-elicited M phi but high in R-M phi and activated ones. In all cases, it was increased by two to three times upon zymosan challenge. This suggested that increased paf-acether catabolism and not impaired anabolism could be responsible for the marked reduced formation noted in activated macrophages. The addition of acetyl-CoA (200 microM) to the various macrophage monolayers restored paf-acether formation by activated cells to R-M phi values but with delayed kinetics as compared with paf-acether formation induced by zymosan. The enhancing effect of acetyl-CoA on paf-acether production was inhibited upon oleyl-CoA addition, suggesting that acetyl-CoA may increase paf-acether production by preventing the reacylation of lyso paf-acether resulting from paf-acether degradation. In conclusion, the paf-acether output in some inflammatory macrophages may be regulated by the level of acetyltransferase activity, because it is observed in TG-M phi. However, we present the first evidence for another mechanism of regulation, most probably related to the deacylation/reacylation of paf-acether precursors and metabolites.
Acetates↗
Decrease in IgE Fc receptor expression on mouse bone marrow-derived mast cells and inhibition of paf-acether formation and of beta-hexosaminidase release by dexamethasone.
The effect of dexamethasone (DM) on the immunologic and nonimmunologic release of paf-acether and of the granule marker beta-hexosaminidase (BHEX) from mouse bone marrow-derived mast cells (BMMC) was studied. BMMC (1 X 10(6] in a modified Tyrode's solution containing 0.25% bovine serum albumin (BSA) were sensitized with an optimal dose of dinitrophenyl (DNP)-specific monoclonal IgE, and were washed before challenge with 40 ng/ml of DNP coupled to BSA. Preincubation of BMMC for 24 hr with 1 nM to 1 microM DM inhibited in a dose-dependent fashion the immunologic release of paf-acether and of BHEX as compared with control cells, with a half-maximal effect at 20 nM and 4 nM respectively. By contrast, the ionophore A23187 (1 microM)-induced release of paf-acether and of BHEX was unaffected by DM pretreatment. Finally, the antigen-induced increase in acetyltransferase activity, used as an index of cellular activation, was inhibited by 37 +/- 16% in 1 microM DM-treated BMMC as compared with untreated cells. Preincubation of BMMC with DM for 24 hr caused a dose-dependent inhibition of 125I-IgE binding to the cells, with a half-maximal effect at 14 nM. As determined by Scatchard analysis, the number of IgE Fc receptors was decreased by 55% in 1 microM DM-treated BMMC as compared with untreated cells, although the dissociation constants were comparable (control: 12.6 +/- 4.1 nM; DM-treated cells: 14.1 +/- 6.7 nM; mean +/- 1 SD; n = 3). Cytofluorometer analysis of BMMC sensitized with a saturating amount of purified monoclonal IgE, followed by addition of a fluoresceinated anti-mouse IgG (heavy and light chains), revealed a single cellular population for both DM-treated and untreated BMMC. This demonstrates that the DM-induced decrease in IgE Fc receptor expression was exhibited by every BMMC. The possible link between the decreased sensitization of the cells consequent to the reduction in IgE Fc receptor expression and the alteration of the secretory response and acetyltransferase activity was investigated. BMMC were incubated with IgE under experimental conditions giving half-sensitization of the cells. Upon antigen challenge, a 10.5 +/- 3.7% decrease in acetyltransferase activity and a 29.2 +/- 3.5% decrease in paf-acether release were observed with half-sensitized cells as compared with cells sensitized with a saturating amount of IgE.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Transient activation of the acetyltransferase necessary for paf-acether biosynthesis in thrombin-activated platelets.
Thrombin-activated platelets formed paf-acether (1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine), a molecule susceptible to play a role in haemostasis and thrombosis, and its deacetylated analogue, lyso paf-acether, a biologically inactive molecule. We presently show the presence in human and rabbit platelet lysates of an acetyltransferase which transfers the acetyl moiety of acetyl-coenzyme A (acetyl-CoA) onto synthetic lyso paf-acether, yielding the fully active paf-acether molecule. Under our optimal standard conditions, 0.36 +/- 0.23 nmol paf-acether/10 min/mg proteins was formed by the acetyltransferase from resting human platelets. Upon thrombin stimulation, the acetyltransferase activity doubled within 30 s, reaching a maximum at 2 min (1.17 +/- 0.31 nmol paf-acether/10 min/mg proteins) and decreased progressively. Similar results were obtained using rabbit platelets. In addition we demonstrated that the activation and deactivation of the acetyltransferase correlated with the kinetics of paf-acether formation by thrombin-activated rabbit platelets. It is hypothesized that this enzyme may represent one of the regulating mechanism of paf-acether formation by platelets.
Acetyltransferases↗
Evidence that biosynthesis of platelet-activating factor (paf-acether) by human neutrophils occurs in an intracellular membrane.
Human polymorphonuclear leukocytes (PMN) were incubated in the absence or presence of the calcium ionophore A23187 (6 microM) for 10 min at 37 degrees C. They were then lysed by nitrogen cavitation and fractionated using Percoll gradients. Three major fractions of increasing density corresponding to plasma membrane, intracellular membranes and secretory granules were detected using [3H]concanavalin A, NADH-dehydrogenase and beta-D-glucuronidase as respective markers. In both cases, the acetyltransferase activity responsible for biosynthesis of paf-acether (platelet-activating factor of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was detected in the intermediary fraction, the enzyme activity being increased 3-4-fold in stimulated cells. From the comparison with the distribution of various markers, it is concluded that in human PMN the final step of paf-acether assembly occurs in an intracellular membrane, possibly the endoplasmic reticulum.
Acetyltransferases↗
Biosynthesis of platelet-activating factor. VI. Precursor of platelet-activating factor and acetyltransferase activity in isolated rat kidney cells.
Isolated glomeruli and tubular and medullary cells obtained from perfused kidneys from Wistar rats were stimulated with ionophore A 23187 (0.5 to 6 microM) or kept overnight at pH 9.5. The amount of platelet-activating factor (Paf-acether) formed was measured in the ethanolic cell extracts using aggregation of washed rabbit platelets. 2-Lyso Paf-acether present in cells was transformed into Paf-acether by chemical acetylation and measured in the same manner as Paf-acether. Microsomes from glomeruli and medullary and tubular cells were prepared, and the acetyltransferase activity was measured. Paf-acether was formed in a dose-dependent fashion by glomeruli and medullary cells, and maximal formation with 3 microM ionophore A 23187 was 1.9 +/- 0.2 and 1.1 +/- 0.2 pmoles/mg of protein, respectively. Paf-acether was not recovered from tubular cells. Three cell types produced large amounts of 2-lyso Paf-acether when incubated at alkaline pH. Only glomeruli generated appreciable quantity of 2-lyso Paf-acether upon ionophore A 23187 stimulation. The acetyltransferase specific activities in ionophore A 23187-stimulated glomeruli and medullary and tubular cells were 3.8 +/- 0.8, 0.3 +/- 0.1, and 0.2 +/- 0.1 nmoles of Paf-acether/10 min/mg of protein, respectively. This study demonstrates the formation of Paf-acether by two distinct populations of kidney cells, pointing out the glomerular cells, besides the already known medullary cells, as capable of forming Paf-acether.
Acetyltransferases↗
Biosynthesis of Paf-acether (platelet-activating factor). VII. Precursors of Paf-acether and acetyl-transferase activity in human leukocytes.
Human polymorphonuclear neutrophils, monocytes, and lymphocytes were studied for their ability to synthesize Paf-acether when stimulated with the ionophore A 23187 (Io) or with specific secretagogues. When stimulated with Io, neutrophils produced 100 +/- 8.5 pmol Paf-acether 1 X 10(6) cells (mean +/- 1 SD, n = 5); monocytes were less efficient (44 +/- 3.3 pmol Paf-acether/1 X 10(6) cells), whereas lymphocytes were practically unable to form this mediator (1.0 +/- 0.4 pmol Paf-acether/1 X 10(6) cells). Neutrophils and monocytes released in the extracellular medium 49 and 37% of Paf-acether that they formed, respectively. We attempted to correlate the amount of Paf-acether produced by the various cell types with that of its precursors, 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine and 1-O-alkyl-sn-glycero-3-phosphocholine (2-lyso Paf-acether). In the three cell types, the amount of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine was sufficient to ensure the formation of 2-lyso Paf-acether and consequently that of Paf-acether. The quantity of 2-lyso Paf-acether formed appeared to be the limiting factor only in the case of the neutrophils. These cells increased their synthesis of Paf-acether in the presence of exogenous 2-lyso Paf-acether. To investigate the failure of lymphocytes to produce the mediator, the acetylating step of Paf-acether formation was studied, and we found a very weak activity (0.5 +/- 0.1 nmol Paf-acether/10 min/mg protein) in this cell type as opposed to monocytes (4.0 +/- 2.3 nmol Paf-acether/10 min/mg protein) and neutrophils (17.8 +/- 5.3 nmol Paf-acether/10 min/mg protein). These activities were doubled in Io-stimulated cells. Thus, the modulation of acetyl-transferase activity appears to be a key step in the regulation of Paf-acether biosynthesis. Also, the availability of 2-lyso Paf-acether could regulate Paf-acether synthesis in human neutrophils.
Acetyltransferases↗
Biosynthesis of platelet-activating factor (PAF-acether). V. Enhancement of acetyltransferase activity in murine peritoneal cells by calcium ionophore A23187.
The activity of the acetyltransferase capable of transferring the acetyl moiety of acetyl-CoA onto 2-lyso PAF-acether (1-alkyl-sn-glycero-3-phosphocholine) to form PAF-acether was compared in ionophore A23187-stimulated and in non-stimulated rat peritoneal cells. Stimulation resulted in a doubling of the acetyltransferase activity within 30 s. This effect was abolished in the presence of EDTA (1 mM) or EGTA (1 mM) and restored by addition of Ca2+ (10 mM). The specificity of acetyltransferase measured in ionophore-stimulated as well as in untreated cells is the same. In both situations we observed the same Km values for acetyl-CoA, whereas the Vmax values were different. The wide similarities of the two enzyme preparations lead us to conclude that stimulation by the ionophore involves an increase in the number of enzyme molecules rather than a change in the kinetic parameters of the acetyltransferase.
Acetyltransferases↗
Biosynthesis of platelet-activating factor. I. Evidence for an acetyl-transferase activity in murine macrophages.
Platelet-activating factor (PAF-acether; 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) is released from murine peritoneal adherent cells by inflammatory and non-inflammatory stimuli. We have found, in extracts from these cells, an enzyme activity that synthesizes. PAF-acether from synthetic lyso-PAF-acether by transferring the acetyl moiety of acetyl-coenzyme A onto the lyso-PAF-acether molecule. The enzyme is stabilized by 1 mM dithiothreitol, is calcium-dependent, has an apparent Km of 172 microM for acetyl-CoA and is active in a 6-8 pH range. When the acetyl-CoA substrate is replaced by propionyl-CoA, an ether lipid is produced which turns out to be as potent an aggregating agent as PAF-acether. In all cases, the products of the reaction were characterized by their behaviour in platelet-aggregation tests and their high-pressure liquid chromatography (HPLC) elution profiles. The precise definition of this acetyl-transferase is of primary importance for the development of new pharmacological agents capable of moduling a potent platelet aggregating factor.
Acetyltransferases↗
Is platelet-activating factor (PAF-acether) synthesis by murine peritoneal cells (PC) a two-step process?
PAF-acether (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) is released from several cell sources simultaneously with an inactive non-acylated compound (1-O-alkyl-glyceryl-3-phosphorylcholine) (lyso-PAF-acether). Formation of the latter probably results from the activation of a phospholipase A2 (PLA2). Indeed, the PLA2 inhibitors, bromophenacyl bromide (BPB), mepacrine, 874CB (100 microM), and EDTA (5 mM), blocked the zymosan-induced release of PAF-acether from PC. EDTA and BPB also markedly reduced the release of lyso-PAF-acether. To verify this hypothesis, acetyl coenzyme A (acetyl-CoA) was added to stimulated PC. This enhanced the release of PAF-acether in a dose-dependent fashion from 1 microM acetyl-CoA to reach a maximal increase - 200% - at 100 microM. Furthermore, using 3H acetyl-CoA, incorporation of labelled acetate into PAF-acether was suggested by (1) identical chromatographic patterns of biological activity and radioactivity; (2) disappearance of these activities after treatment with PLA2, but not after exposure to lipase from Rhizopus arrhizus. PAF-acether was also obtained when both acetyl-CoA (100 microM) and synthetic lyso-PAF-acether (0.2 microM) were added to unstimulated PC previously treated with BPB (100 microM for 10 min). These results suggest that the release of PAF-acether is the consequence of at least two different steps: (1) hydrolysis of 1-O-alkyl-2-acyl-glyceryl phosphorylcholine by PLA2; (2) enzymatic acetylation of the hydrolysis product.
Acetophenones↗