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Proc. Nati. Acad. Sci. USA Vol. 84, pp. 7557-7561, November 1987 Cell Biology Platelet-activating factor (PAF) stimulates the PAF-synthesizing enzyme acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine 02-acetyltransferase and PAF synthesis in neutrophils (degranulation/platelet-activating factor antagonist/platelet-activating factor receptor/l-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase) THOMAS W. DOEBBER AND MARGARET S. WU Department of Biochemical Regulation, Merck Sharp & Dohme Research Laboratories, Rahway, NJ 07065-0900 Communicated by Edward M. Scolnick, August 12, 1987

ABSTRACT Platelet activating factor (1-alkyl-2-acetyl-sn- associated with inflammatory responses: opsonized zymo- glycero-3-phosphocholine; PAF) induced in isolated rat peri- san- and complement component C5a-stimulated neutrophils toneal and human peripheral neutrophils a rapid and potent (9, 10), complement component C5a-stimulated eosinophils activation of the PAF biosynthetic enzyme acetyl-CoA:1-alkyl- (11), immune complex-activated and opsonized zymosan- sn-glycero-3-phosphocholine 02-acetyltransferase (EC 2.3.1. activated macrophages (12), and antigen-stimulated mast 67). The PAF-induced activation of the neutrophil acetyltrans- cells (13). PAF is not stored as the bioactive phospholipid, ferase (8-10 times basal neutrophil activity) was maximal but it is very rapidly synthesized upon cell stimulation. The within 30 sec after PAF addition, as was the PAF-stimulated published experimental evidence supports a two-enzyme degranulation. After 1 min of PAF stimulation, the elevated pathway involving first a phospholipase A2, which cleaves acetyltransferase activity steadily decreased. Within 2 min of the fatty acid from 1-alkyl-2-acyl-glycero-3-phosphocholine stimulation ofneutrophils with 10-' M PAF, the 7-fold increase to form 1-alkyl-glycero-3-phosphocholine, which is then in acetyltransferase activity was coincident with substantial acetylated to form PAF by acetyl-CoA:1-alkyl-sn-glycero-3- PAF synthesis (as measured by [3H]acetate incorporation into phosphocholine 02-acetyltransferase (EC 2.3.1.67) (14). This PAF), which was 14% ofthe PAF synthesis induced by the Ca21 acetyltransferase was first demonstrated and partially char- ionophore A23187 at 10-5 M. PAF activation of the acetyl- acterized in rat spleens by Wykle et al. (15). Alonso et al. (16) transferase and PAF synthesis required intact neutrophils as demonstrated a severalfold activation of the acetyltrans- they did not occur in cells broken by sonication. The neutrophil ferase in human neutrophils upon stimulation with opsonized acetyltransferase was 10-30 times more sensitive to activation zymosan. The time course and zymosan dose-dependency of by PAF than was degranulation as the acetyltransferase acti- this acetyltransferase induction was very similar to the stimu- vation was evident with 10-9 M PAF and was about maximal lated PAF production in the same cells. Subsequently, Lee et with 3 x 10-j M PAF. The unstimulated and PAF-induced al. (11) demonstrated the activation of acetyltransferase in acetyltransferase exhibited the same Km for acetyl-CoA (67 human eosinophils by the eosinophil chemotactic factor of ,M), but the Vm.x for the PAF-induced enzyme (1667 anaphylaxis, complement component C5a, fMet-Leu-Phe, or pmol/min per 107 cells) was 10 times that of the unstimulated the Ca+2 ionophore A23187. Lenihan and Lee (17) and Gomez- enzyme (175 pmol/min per 107 cells). The PAF induction of the Cambronero et al. (18) presented evidence that the acetyltrans- acetyltransferase was less sensitive to inhibition by the specific ferase in rat spleen microsomes can be activated by kinase- PAF receptor antagonist L-652,731 than was PAF-induced catalyzed phosphorylation and deactivated by phosphatase- degranulation. This, along with the differing sensitivities to catalyzed dephosphorylation. In this paper, we demonstrate PAF, suggests that acetyltransferase activation and degranula- that PAF itself stimulates acetyltransferase and PAF tion induced by PAF either involve two different PAF receptors potently or involve one receptor type with different receptor occupancy synthesis in intact neutrophils. The nature of the acetyltrans- requirements. Escherichia coli alkaline phosphatase, which ferase activation and its relation to PAF-induced degranulation greatly decreased the activity of the acetyltransferase in spleen in the same cells are presented. microsomes, had little or no effect on the basal or PAF-induced neutrophil acetyltransferase. Thus, by stimulating the activity MATERIALS AND METHODS of acetyltransferase, PAF induces in neutrophils the synthesis of more PAF, thereby probably augmenting the neutrophil Rat peritoneal polymorphonuclear neutrophils were elicited response to the initial PAF. and isolated by the method of Cunningham et al. (19) utilizing 8 ml of 12% sodium caseinate per rat. Female Wistar rats Platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero- (200-250 g) were from Charles River Breeding Laboratories. 3-phosphocholine) has been implicated as an important Human peripheral neutrophils were isolated from blood from mediator of pathophysiological reactions in animal disease normal volunteers by the method of Boyum (20) utilizing models and in human disease for at least two general reasons. dextran sedimentation. For both neutrophil isolations, eryth- First, PAF is a very potent stimulator of neutrophil degran- rocytes were lysed by a 6-min treatment with 0.75% NH4Cl, ulation (1), platelet aggregation (2), and cardiac dysfunction and the neutrophils were finally suspended in Hanks' bal- (3) in vitro and systemic hypotension (4), increased vascular anced salt solution/0.25% bovine serum albumin. Rat spleen permeability (5), neutropenia (6), bronchoconstriction (7), microsomes were prepared by the method of Wykle et al. and elevated plasma lysosomal hydrolase levels (8) in vivo. (15). For experiments in which exogenous phosphatase Second, PAF is produced by several different cell types (Sigma) was used (Fig. 5), Hanks' balanced salt solution was replaced with 15 mM Hepes, pH 7.4/0.14 M NaCI/5 mM glucose. The publication costs of this article were defrayed in part by page charge KCI/0.1% payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: PAF, platelet-activating factor. 7557 Downloaded by guest on September 30, 2021 7558 Cell Biology: Doebber and Wu Proc. Natl. Acad. Sci. USA 84 (1987) For the PAF-induction of the neutrophil acetyltransferase, form/glacial acetic acid (2:1:0.04, vol/vol). The [3H]PAF was 5 x 101 neutrophils in 0.4 ml of buffer containing 2.5 ,ug of separated from [3H]acetate by two sequential extractions cytochalasin B in 1.5-ml polypropylene tubes were preincu- utilizing the procedure of Bligh and Dyer (24). The [3H]PAF bated at 37°C for 15 min. One hundred microliters of a was identified by thin-layer chromatography using the sol- solution containing PAF (Calbiochem) in Hanks' solution/ vent system of Wykle et al. (15). A [3H]PAF standard that 0.25% bovine serum albumin/3 mM CaCl2/5 mM MgCl2 was was added to 3.8 ml of methanol/chloroform/glacial acetic added for a further 30-sec incubation (except in Fig. 1), and acid containing 0.8 ml of the neutrophil suspension was 0.2 ml was immediately removed for pelleting cells in a processed in the same manner. This was used for the microcentrifuge. Then 0.3 ml of0.1 M Tris-HCI (pH 7.2) was identification of PAF and the determination of the percent added to the remaining cell suspension, and the suspension recovery from the two extractions and thin-layer chromatog- was sonicated for 15 sec (Branson Sonifier Cell Disruptor, raphy. One-centimeter sections of each lane (from sample W-350). The supernatant from the pelleted cells was assayed origin to solvent front) of the chromatography plate were for lactate dehydrogenase (21) as a measure of cell viability scraped and counted for 3H activity. as well as B-glucuronidase (22) and myeloperoxidase (23) as The PAF receptor antagonist L-652,731 [trans-2,5- a measure ofdegranulation. The lactate dehydrogenase in the bis(3,4,5-trimethoxyphenyl)tetrahydrofuran] was prepared supernatant was always low (-6% of total) and did not and kindly supplied by Tesfaye Biftu (25). increase with PAF or any other experimental conditions. The The data presented in the figures are representative of resulting sonicated neutrophils (200 ,ul in a final assay volume experiments that were performed two to four times. of 1.0 ml) were assayed for acetyltransferase activity by the procedure of Wykle et al. (15) utilizing 5 ,uM 1-alkyl-glycero- RESULTS 3-phosphocholine (Avanti Polar Lipids) and 100 ,uM [3H]- acetyl-CoA (0.3 ,Ci; 1 Ci = 37 GBq; New England Nuclear) The acetyltransferase in rat neutrophils stimulated with 10-6 and a 10-min incubation at 37°C. Reactions were stopped by M PAF underwent a very rapid 8.5-fold activation that was the lipid extraction procedure of Bligh and Dyer (24). In a maximal within 30 sec (Fig. 1). The PAF-induced f3- control study, 91% ofthe total radioactive lipid formed by the glucuronidase secretion was equally rapid. After 1 min, the PAF-induced neutrophil acetyltransferase comigrated with elevated acetyltransferase activity decreased steadily so that [1-alkyl-1',2'-3H]PAF (New England Nuclear) upon thin- by 5 min it was 40% of the maximal level but was still 3.5 layer chromatography using the solvent system of Wykle et times the basal level (before PAF stimulation). 1- al. (15). The same peak recovery was obtained for the Alkylglycerophosphocholine at 10-6 M was completely in- [3H]PAF standard upon thin-layer chromatography. Conse- active in stimulating the acetyltransferase in intact neutro- quently, the CHC13 layer from the lipid extraction was phils. evaporated and then counted directly as a measure of In order to determine if PAF induction of the neutrophil [3H]PAF synthesized by the acetyltransferase for all exper- acetyltransferase results in synthesis of new PAF, [3H]- iments except for the one in Fig. 5. For the latter, [14C]acetyl- acetate was added to neutrophil suspensions before the CoA (New England Nuclear) was the acetate-donor sub- addition of PAF, and the production of [3H]PAF was exam- strate, and all samples were analyzed by thin-layer chroma- ined. Table 1 demonstrates that neutrophils incubated for 2 tography to identify the [14C]PAF. min with 10-6 M PAF synthesized substantial amounts of For the study of PAF synthesis by intact neutrophils, 3.8 [3H]PAF. In identical tubes that lacked the [3H]acetate, PAF x 107 rat peritoneal neutrophils in 0.8 ml of Hanks' solu- addition increased the acetyltransferase activity 7-fold. Neu- tion/0.25% bovine serum albumin were preincubated at 37°C trophils that were lysed by sonication before PAF addition for 10 min. Fifty microcuries of [3H]acetate (New England did not synthesize (3H]PAF or incur increased acetyltrans- Nuclear; 7.7 Ci/mmol) was added to each tube. After an ferase activity. The effects of the Ca2l ionophore A23187 on additional 5-min preincubation, 200 ,ul of the Ca2l ionophore the same neutrophil responses were also determined in the A23187 or PAF in Hanks' solution/0.25% bovine serum same experiments since A23187 is so far the inducer of the albumin/3 mM CaC12/5 mM MgCl2 was added for a 2-min greatest amount of PAF production in neutrophils. Exposure incubation. The neutrophil suspensions were then trans- of neutrophils to 10-5 M A23187 for 2 min resulted in an ferred to glass tubes containing 3.8 ml of methanol/chloro- 8.5-fold increase in acetyltransferase and the expected large amount of [3H]PAF synthesis (Table 1). The PAF dose-response stimulation of the acetyltransferase and 83-glucuronidase secretion for both rat elicited peritoneal neutrophils and human peripheral neutrophils is 20 demonstrated in Fig. 2. Since it requires only nanomolar 0 X-c levels of PAF, the acetyltransferase activation in both rat 16 ° -na) E (Fig. 2A) and human (Fig. 2B) neutrophils was substantially c 0 -0 more sensitive to PAF than degranulation, which requires a 12 w T 10-30 times higher PAF concentration. The dip in the .E 0) acetyltransferase activation curve for human neutrophils at I- 0) E 8 cn 0n -o Table 1. Activation of acetyltransferase and PAF synthesis in 0c L07 2 neutrophils by PAF and the Ca2+ ionophore A23187 4 ,, O3 Acetyltransferase activity, pmol of [3H]PAF synthesized, Agonist PAF/min per 10' cells dpm Time after PAF, min Buffer 108 + 12 0 PAF, 10-6 M 736 ± 100 14,700 ± 1400 FIG. 1. Time course of the 10-6 M PAF-induced activation of A23187, 10-5 M 918 ± 110 105,000 ± 5600 acetyltransferase and degranulation in rat neutrophils. The 0 min points were the values for neutrophils that did not receive PAF. The Values are the means ± SEM from duplicates of two separate ,B-glucuronidase activity secreted after PAF addition (0.5-5 min) was experiments (four experimental values). The [3H]PAF synthesized is 11% of the total cellular content. that from 3.8 x 107 neutrophils. Downloaded by guest on September 30, 2021 Cell Biology: Doebber and Wu Proc. Natl. Acad. Sci. USA 84 (1987) 7559

0 A PAF levels is not currently known, but cell toxicity does not seem likely as the lactate dehydrogenase levels in the GJ I,- were low for the whole of PAF 0 en supernatants uniformly range am ~ concentrations. 0 V The activity of the basal and PAF-induced acetyltransfer- 0 E ases with regard to the effect ofthe acetyl-CoA concentration -E 0~ is illustrated in Fig. 3. At all levels of acetyl-CoA, the PAF-induced enzyme synthesized six times more PAF than the unstimulated enzyme (Fig. 3A). From the inverse plot in Fig. 3B, it was determined that the Km remained the same (67 100 AM) for the basal and stimulated enzyme, whereas the Vmax > increased almost 10-fold from 175 pmol of PAF/min per 107 0 80 ° cells for the basal enzyme to 1667 pmol/min per 107 cells for -s c the PAF-induced enzyme. 0 60 The PAF-induced activation of the neutrophil acetyltrans- N ferase was probably mediated by a cell surface receptor for .E (,, PAF since the activation occurred at such low PAF levels; a o E 40 cell surface PAF receptor on neutrophils has been demon- 0~n.L C strated (26), which has been shown to be responsible for all 20 ' PAF-induced cellular and tissue responses so far examined. O Thus, the potent and specific PAF-receptor antagonist 0 Q L-652,731 (27) should inhibit the neutrophil acetyltransferase 0-8 3x1( activation induced by PAF. Fig. 4A demonstrates that 0.4 and PAF, M 10 AM L-652,731 substantially inhibited the acetyltransferase activation by PAF concentrations of 10-8 and 3 x 10-8 M. At FIG. 2. PAF dose-response activation of acetyltransfern se PAF concentrations of lo- and 3 x i0- M, L-652,731 was degranulation in rat (A) and human (B) neutrophils. At 10-6 A anFd the 3-glucuronidase activity secreted was 20% and 29% of ti hie total much less inhibitory. However, L-652,731 markedly inhibit- cellular content for the rat and the human neutrophils, respe(ctively. ed neutrophil degranulation induced by all PAF concentrations as illustrated in Fig. 4B. Specifically, 10 ,uM L-652,731 inhibited acetyltransferase activation by only 37% and 17% lo-7 and 3 x i0- M PAF was reproducible and repr*esents 1500j a difference from the rat neutrophil acetyltransferase re- A 0 sponse. The reason for this human cell response at thes e high _en 1200j 1200r A 0 - 900[ 0 - - 10'00 __ -- ~.0/10-. ~ ~ o -5 -0 8( 600; 0j(;~~~~ N0c 0C 0~ 6C 00 300[ / I LC 4cDO00- *-- ~~~~~~~~~I--, 01 / 0 ' 3X109 10-8 3x108 10-7 3X10-7

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FIG. 4. Effect of PAF-receptor antagonist L-652,731 on the PAF dose-response activation of rat neutrophil acetyltransferase (A) and degranulation (B). During neutrophil preincubation at 370C, 1/ocetyl -CoA,mM-l L-652,731 in a dimethyl sulfoxide/water (1:1) solution was added to cell suspensions to result in a final concentration of 0.4 ,4M (o) or 10 FIG. 3. Effect of acetyl-CoA concentration on the activity of FLM (A). Control cells (o) received vehicle (dimethyl sulfoxide/H2O, acetyltransferase from unstimulated (o) and lo-7 M PAF-stimulated 1:1). PAF was then added for the subsequent 30-sec incubation. In (e) rat neutrophils. (A) Direct plot ofdata. (B) Double-reciprocal plot the absence of L-652,731, 3 x 10-' M PAF induced the secretion of of data for determination of Km and Vmax. 27% of the total cellular )3-glucuronidase. Downloaded by guest on September 30, 2021 7560 Cell Biology: Doebber and Wu Proc. Natl. Acad. Sci. USA 84 (1987) for 10-7 and 3 x i0-' M PAF (Fig. 4A), respectively, but explain the results of Bourgain et al. (28) in their in vivo study inhibited f3-glucuronidase secretion by 100% and 87% for the of PAF-induced thrombosis. In vivo, the site ofPAF-induced same respective PAF concentrations (Fig. 4B). PAF synthesis and the site ofaction ofthe newly formed PAF Recently published studies of Lenihan and Lee (17) and could be the same cells or different cells and tissues located also Gomez-Cambronero et al. (18) indicated that the ace- quite distant from each other. For example, the PAF that has tyltransferase in isolated spleen microsomes can be activated been demonstrated to mediate some of the rat anaphylactic by a protein kinase-catalyzed phosphorylation and deacti- responses to soluble immune complex infusion (29) may vated by a phosphatase-catalyzed dephosphorylation. IfPAF result initially from Kupffer cells stimulated by the immune activation of neutrophil acetyltransferase involves phospho- complexes and secondly from other cells (e.g., neutrophils) rylation ofthe enzyme, then treatment ofthe PAF-stimulated stimulated by PAF that originated from the Kupffer cells. acetyltransferase with alkaline phosphatase should substan- This cycle could continue until a PAF antagonist blocks PAF tially decrease the activity. A 5-min preincubation of the receptor stimulation or cells become desensitized to further neutrophil lysate at 370C did decrease the activity ofthe basal PAF stimulation. and PAF-stimulated acetyltransferase, but inclusion of 10 Other investigators have not observed PAF induction of units of Escherichia coli alkaline phosphatase in the prein- PAF synthesis. One reason is that not all cells may exhibit cubation did little to decrease further the activity, especially this PAF response. We observed PAF induction of acetyl- for the PAF-induced enzyme (Fig. 5). However, in the same transferase in peripheral rabbit neutrophils but not in rabbit experiment, alkaline phosphatase markedly decreased the platelets (unpublished observations). Second, the time of activity ofthe spleen microsomal acetyltransferase as report- neutrophil incubation with PAF is of critical importance. ed by Lenihan and Lee (17) and Gomez-Cambronero et al. Sisson et al. (30) reported that a 30-min incubation of human (18). As a control for any phosphatase effect, 25 mM neutrophils with 10-6 M PAF did not result in any measurable inorganic phosphate was included in separate preincuba- PAF synthesis. The work presented in this paper demon- tions, which completely inhibited the alkaline phosphatase strating the very transient activation of acetyltransferase when assayed with the synthetic substrate p-nitrophenyl indicates that by 30 min after PAF addition the neutrophil phosphate. The inorganic phosphate completely blocked the acetyltransferase has probably returned to basal activity and phosphatase effect on the spleen microsomal acetyltrans- thus does not catalyze the synthesis of PAF. Also, any PAF ferase and, curiously, substantially blocked the loss in synthesized at earlier times (e.g., 2 min) may very likely be activity incurred by the PAF-induced acetyltransferase dur- degraded by the PAF acetylhydrolase. That PAF has now ing the preincubations with and without phosphatase. been demonstrated to induce the synthesis of additional PAF certainly adds to the proinflammatory nature of this phospholipid. DISCUSSION There was some variation in the absolute acetyltransferase The experimental results in this paper demonstrate that PAF activity of unstimulated and PAF-induced neutrophils, as is a potent and very rapid stimulator of its own biosynthetic seen in the different figures. However, the magnitude of the enzyme, acetyltransferase, in isolated neutrophils from rats relative increase in the PAF-induced acetyltransferase rela- and humans. Coincident with the PAF induction of acetyl- tive to the basal enzyme level was quite consistent. transferase in neutrophils was the stimulation of PAF syn- The acetyltransferase stimulation by PAF was indicated to thesis. The PAF induction ofPAF synthesis was suggested to be PAF receptor-mediated. The stimulation occurred with

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Basal PMNs PAF-Induced PMNs Spleen Microsornes FIG. 5. Effect of preincubation with E. coli alkaline phosphatase on acetyltransferase from uninduced and i0-0 M PAF-induced rat neutrophils and spleen microsomes. Sonicated neutrophil and spleen microsomes were assayed for acetyltransferase activity either directly (Control), after a 5-min preincubation at 37°C in assay buffer (Preinc.), after a 5-min preincubation at 37°C with 10 units of phosphatase (Preinc. + Phos.), or after a 5-min preincubation at 37°C with 10 units of phosphatase and 25 mM phosphate (Preinc. + Phos. + Pi). The preincubation pH was 7.2. One unit of phosphatase hydrolyzes 1 ,umol of p-nitrophenyl phosphate per min at 37°C (pH 10.4). PMN, polymorphonuclear leukocyte. O Downloaded by guest on September 30, 2021 Cell Biology: Doebber and Wu Proc. Natl. Acad. Sci. USA 84 (1987) 7561 very low PAF levels (starting at 10-9 M). The PAF stimula- siastic support of this work, and Mrs. Joan Kiliyanski for excellent tion was inhibited by a specific PAF receptor antagonist. secretarial assistance. Finally, this PAF stimulation required the neutrophil to be intact, which makes this response different from the phos- 1. O'Flaherty, J. T., Wykle, R. L., Miller, C. H., Lewis, J. C., Waite, M., Bass, D. A., McCall, C. E. & DeChatelet, L. R. pholipase A2 activation by fMet-Leu-Phe, complement com- (1981) Am. J. Pathol. 103, 70-79. ponent C5a, or leukotriene B4 that occurs in isolated 2. Benneniste, J., LeCouedic, J. P. & Kamoun, P. (1975) Lancet neutrophil membranes (31). i, 344. The findings that the acetyltransferase induction relative to 3. Levi, R., Burke, J. A., Guo, Z.-G., Hattori, Y., Hoppens, degranulation required much lower PAF levels (Fig. 2) and C. M., McManus, L. M., Hanahan, D. J. & Pinckard, R. N. was inhibited to a lesser extent by the PAF antagonist (1984) Circ. Res. 54, 117-124. L-652,731 (Fig. 4) suggest two explanations. First, two 4. Blank, M. L., Snyder, F., Beyers, W., Brooks, B. & different neutrophil receptors for PAF trigger these two Muirhead, E. E. (1979) Biochem. Biophys. Res. Commun. 90, responses, whereby the receptor leading to acetyltransferase 1194-1200. 5. Sanchez-Crespo, M., Alonso, F., Inarrea, P., Alvarez, V. & activation requires much lower PAF concentrations for signal Egido, J. (1982) Immunopharmacology 4, 173-185. transduction and has a substantially lower affinity for 6. McManus, L. M., Hanahan, D. J., Demopoulos, C. A. & L-652,731 compared to the receptor associated with Pinckard, R. N. (1980) J. Immunol. 124, 2919-2924. degranulation. The second and more likely possibility is that 7. Vargaftig, B. B., Lefort, J., Chignard, M. & Benveniste, J. one PAF receptor type exists on neutrophils, but different (1980) Eur. J. Pharmacol. 65, 185-192. receptor occupancy requirements are involved. Consequent- 8. Doebber, T. W., Wu, M. S. & Shen, T. Y. (1984) Biochem. ly, acetyltransferase activation might require a substantially Biophys. Res. Commun. 125, 980-987. smaller fraction of total PAF receptors to be occupied for 9. Lynch, J. M. & Henson, P. M. (1986) J. Immunol. 137, signal transduction to occur compared to degranulation. 2653-2661. a PAF a greater 10. Camussi, G., Aglietta, M., Coda, R., Bussolino, W., Then, antagonist would have to block Piacibello, W. & Tetta, C. (1981) Immunology 42, 191-199. fraction of PAF receptors in order to block acetyltransferase 11. Lee, T. C., Lenihan, D. J., Malone, B., Roddy, L. L. & activation, which would result in less potent inhibition of Wasserman, S. 1. (1984) J. Biol. Chem. 259, 5526-5530. acetyltransferase activation relative to degranulation. In 12. Mencia-Huerta, J.-M. & Benveniste, J. (1981) Cell. Immunol. support of this hypothesis, Sklar et al. (32) reported that 57, 281-292. different neutrophil responses (e.g., membrane depolariza- 13. Mencia-Huerta, J.-M., Lewis, R. A., Razin, E. & Austen, tion, degranulation, and aggregation) to fMet-Leu-Phe occur K. F. (1983) J. Immunol. 131, 2958-2964. at distinct and very different levels of receptor occupancy. 14. Chilton, F. H., Ellis, J. M., Olson, S. C. & Wykle, R. L. The rapid activation and subsequent deactivation of the (1984) J. Jiol. Chem. 259, 12014-12019. 15. Wykle, R. L., Malone, B. & Snyder, F. (1980) J. Biol. Chem. acetyltransferase (Fig. 1) suggest that covalent modifications 255, 10256-10260. of this enzyme are responsible. Neutrophil stimulation by 3 16. Alonso, F., Gil, M. G., Sanchez-Crespo, M. & Mato, J. M. x 10-9 M PAF resulted in almost the same time course of (1982) J. Biol. Chem. 257, 3376-3378. activation/deactivation ofacetyltransferase as did the 10-6 M 17. Lenihan, D. J. & Lee, T.-C. (1984) Biochem. Biophys. Res. PAF utilized in Fig. 1. As mentioned previously, exogenous Commun. 120, 834-839. enzymes that could phosphorylate and dephosphorylate the 18. Gomez-Cambronero, J., Velasco, S., Mato, J. M. & Sanchez- spleen microsomal acetyltransferase were indicated to acti- Crespo, M. (1985) Biochim. Biophys. Acta 845, 516-519. vate and deactivate, respectively, the latter enzyme (17, 18). 19. Cunningham, F. M., Smith, M. J. H., Ford-Hutchinson, Thus, it was anticipated that PAF-induced activation of the A. W. & Walker, J. R. (1979) J. Pathol. 128, 15-20. 20. Boyum, A. (1967) Scand. J. Clin. Lab. Invest. Suppl. 97, neutrophil acetyltransferase might also involve phosphoryl- 77-89. ation. If so, addition of exogenous phosphatase to lysates of 21. Morganstein, S., Flor, R., Kessler, G. & Klein, B. (1965) Anal. PAF-activated neutrophils should decrease the activity. Biochem. 13, 149-161. However, E. coli alkaline phosphatase added to the prein- 22. Stahl, P., Rodman, J. J. & Schlesinger, P. (1976) Arch. Bio- cubation of lysates from PAF-induced neutrophils did not chem. Biophys. 177, 594-605. cause any decrease in acetyltransferase activity compared to 23. Worthington Biochemical Corp. (1972) Worthington Enzyme the control preincubation (Fig. 5). The same phosphatase in Manual (Worthington, Freehold, NJ), p. 43. 24. Bligh, E. G. & Dyer, W. J. (1959) Can. J. Biochem. Physiol. the same experiment did substantially reduce the activity of 37, 911-917. the spleen microsomal acetyltransferase. The phosphatase 25. Biftu, T., Gamble, N. F., Doebber, T., Hwang, S.-B., Shen, inhibitor inorganic phosphate completely blocked the phos- T.-Y., Snyder, J., Springer, J. P. & Stevenson, R. (1986) J. phatase action on the spleen microsomal enzyme and sub- Med. Chem. 29,1917-1921. stantially reduced the inactivation of the PAF-induced 26. Valone, F. H. & Goetzl, E. J. (1983) Immunology 48, 141-149. neutrophil enzyme that occurred during the control prein- 27. Hwang, S.-B., Lam, M.-H., Biftu, T., Beattie, T. R. & Shen, T. Y. (1985) J. Biol. Chem. 260, 15639-15645. cubation. Possibly, there is an endogenous neutrophil phos- 28. Bourgain, R. H., Maes, L., Braquet, P., Andries, R., Touqui, phatase that dephosphorylated the acetyltransferase from L. & Braquet, M. (1985) Prostaglandins 30, 185-197. PAF-induced neutrophils to result in the reduced activity 29. Doebber, T. W., Wu, M. S. & Biftu, T. (1986) J. Immunol. seen in the control preincubation. Upon addition ofinorganic 136, 4659-4668. this phosphatase would be inhibited 30. Sisson, J. H., Prescott, S. M., McIntyre, T. M. & Zimmer- phosphate, proposed man, G. A. (1987) J. Immunol. 138, 3918-3926. resulting in decreased deactivation of the PAF-induced 31. Bormann, B. J., Huang, C.-K., Mackin, W. M. & Becker, neutrophil acetyltransferase during the preincubation. E. L. (1984) Proc. Natl. Acad. Sci. USA 81, 767-770. 32. Sklar, L. A., Hyslop, P. A., Oades, Z. G., Omann, G. M., The authors express their appreciation to Dr. Stephen Prescott for Jesaitis, A. J., Painter, R. G. & Cochrane, C. G. (1985) J. Biol. his helpful recommendations, Mr. Alfred W. Alberts for his enthu- Chem. 260, 11461-11467. Downloaded by guest on September 30, 2021