Synergism between Diacylglycerol in the Anion Production in Polymorphonuclear
Platelet-Activating Factor and Induction of Superoxide Guinea Pig Leukocytes’
of Physiological Chemistry, Hiroshima
Medicine, Kasumi, Minami-ku,
14, 1989, and in revised form .January :3, ISSO
The superoxide anion (0,) production of guinea pig polymorphonuclear leukocytes (PMNL) by platelet-activating factor (PAF) was greatly enhanced by the addition of 1-oleoyl-2-acetylglycerol (OAG), even at low concentrations of OAG at which O2 production was little induced. The enhanced production was biphasic, depending on concentrations of PAF. One was saturable at a lower concentration range of PAF and probably mediated through a putative PAF receptor-islet-activating protein-sensitive GTP-binding protein (G protein) pathway. Another was increased in a concentration-dependent manner at a higher concentration range of PAF and apparently mediated through both receptor-G protein-dependent and -independent pathways. These results suggest that at least two types of action of PAF are involved in the synergistic stimulation of 0, I(# 1990 Academic production by PAF and OAG in PMNL. Press,
Polymorphonuclear leukocytes (PMNL)3 respond to a number of stimulants and produce active oxygen species, such as 0,) H,02, etc., which play an important role in
’ This work was supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and a Grant from Terumo Life Science Foundation. ” To whom correspondence should be addressed. .’ Abbreviations used: PMNL, polymorphonuclear leukocytes; Oz, superoxide anion; FMLP, N-formyl-methionyl-leucyl-phenylalanine; PMA, phorbol 1%myristate 1%acetate; OAG, 1-oleoyl-2-acetylglycerol; PAF, platelet-activating factor (l-O-alkyl-2-O-acetyl-sn-glycero3-phosphocholine); ONO-6240, l-O-hexadecyl-(2fZ,S)-0.ethyl-3-0-7. (thiazolinoheptyl)glyceromethanesulfonate; H-7, 1-(5.isoquinolinesulfonyl)methylpiperazine dihydrochloride; IAP, islet-activating protein; G protein, GTP-binding protein; Pipes, 1,4-piperazinediethanesulfonic acid. 000:3-9861/90 $3.00 Copyright (L 1990 hy Academic Press, All rights ~~f’reproduction in any form
the microbicidal activity (1). The production is due to the activation of NADPH oxidase embedded in the plasma membranes (2). Recently, it was suggested that protein kinase C, which may be involved in many responses in various cell types (3), regulates activation of this enzyme (4,5). However, a precise regulatory mechanism for the activation has not been fully elucidated yet. We have previously shown that a protein kinase C activator, l-oleoyl-2-acetylglycerol (OAG), and arachidonate-releasing agents, A23187 and FMLP, synergistically stimulate 0, production in accordance with an increase in the arachidonate release and protein phosphorylation by protein kinase C (6). We have also reported that 0, production in PMNL is greatly enhanced by the combination of OAG and arachidonate at low concentrations at which each of them alone is little effective (7). These results suggest that intrinsically released arachidonate and protein phosphorylation by protein kinase C act synergistically in the stimulatory signal transduction process to induce 0, production in PMNL. On the other hand, it has generally been accepted that the endogenous stores of arachidonate are at the 2-position of phosphoglycerides (8) and that arachidonate or its metabolites are released by phospholipases (i.e., phospholipase A, and/or C) (9, 10). Since a recent report (11) shows that PMNL contain a large amount of arachidonate as a component of ether-linked lipid and have significant capacity to mobilize arachidonate of this form, it is plausible that not only arachidonate itself but also platelet-activating factor (PAF) and/or leukotrienes, which are produced in accordance with an activation of arachidonate release, is involved in the synergistic stimulation of 0, production by protein kinase C. Thus, in the present study, we have investigated the effect of PAF on 0, production in PMNL and found that PAF induces a significant stimulation of the production in the presence of OAG in PMNL. 21
22 5 8
“0 30 K .r E $ 20 E 5 5 ‘G 10 s -0 e a
FIG. 1. Stimulatory effects of PAF or PAF analogs on 0.j production in PMNL in combination with OAG. After preincubation at 37°C for 5 min, PMNL (2 X lo6 cells/ml) were stimulated by the indicated concentrations of PAF or PAF analogs in the presence (0) or absence (0) of 10 PM OAG. Bars indicate SD of the means (n = 3). No bar means that the SD is within the range of the symbol.
Materials. L-o-Lysophosphatidylcholine y-O-alkyl (lyso-PAF), Lo-phosphatidylcholine fi-acetyl-y-oleoyl (l-acyl-PAF) and H-7 were purchased from Sigma Chemical Co.; PAF from Avanti Polar-Lipids, Inc. ONO-6240 was a kind gift from Ono Pharmaceutical Co., Ltd. (Osaka, Japan). All other chemicals were of reagent grade from standard commercial sources. OAG, PAF, and PAF analogs were dissolved in dimethylsulfoxide. H-7 and ONO-6240 were used as aqueous solutions. Preparation of guinea pig PMNL and islet-actiuating protein (IAP) treatment. PMNL were obtained from peritoneal cavities of female guinea pigs of the Hartley strain as reported previously (12). PMNL (10’ cells/ml) were suspended in Hanks’-Pipes buffer (pH 7.3) containing 8 mM Pipes, 137 mM NaCl, 5.4 mM KCl, 0.81 mM MgS04, 1.28 mM CaCl,, 0.43 mM Na,HPO,, 0.44 mM KH,PO,, and 5.5 mM glucose, then incubated at 37°C for 3 h with or without 500 rig/ml IAP. After the 3-h incubation, PMNL were washed twice with ordinary Hanks’Pipes buffer and then resuspended to the indicated concentration in the same medium. Measurements of 0, production by PMNL. 0, production by PMNL was assayed on the basis of superoxide dismutase-inhibitable reduction of cytochrome c (final concentration, 0.1 mM) by the anion produced (13), as reported previously (6, 7).
tration range for PAF and regains further at a higher PAF concentration. Though the same synergistic stimulation of 0; production was also observed in the case of treatment with OAG and lyso-PAF, the production was strongly elicited only at a higher concentration range for lyso-PAF (Fig. 1B). Lyso-PAF did not antagonize the synergistic stimulatory effect of PAF and OAG on 0; production (data not shown). l-Acyl-PAF did not induce the production so far even in the presence of OAG (Fig. 1C). These results indicate that PAF and lyso-PAF synergistically stimulate 0, production with OAG in a structure-related manner. Figure 2 shows, however, that OAG itself has little stimulatory effect on 0, production in PMNL at concentrations up to 10 PM. But OAG markedly potentiated the stimulatory effect of PAF even at a concentration as low as 1 PM. Effect of IAP Pretreatment on Stimulation of 0; Production by a Combination of PAF and OAG Since it is suggested that an IAP-sensitive GTP-binding protein (G protein) is involved in the action of PAF (14, E), we examined the effect of IAP pretreatment on 0, production stimulated by PAF. As shown in Fig. 3, PAF-stimulated 0, production was completely inhibited by the pretreatment at all concentrations of PAF examined. The increase in PAF-mediated 0, production by OAG was also inhibited by the IAP treatment, especially at lower concentrations of PAF. The synergistic stimulation of the production by a combination of PAF and OAG, however, was not completely abolished at higher concentrations of PAF, and the extent of activity suppressed was almost the same irrespective of the con-
Stimulatory Effect of PAF in Combination on 0; Production in PMNL
Treatment of guinea pig PMNL with PAF induced substantial stimulation of 0, production in a concentration-dependent manner (Fig. IA). However, neither lyso-PAF nor l-acyl-PAF had such an effect in concentrations up to 5 pg/ml (Figs. 1B and 1C). The addition of OAG to a concentration of 10 PM markedly enhanced the stimulatory effect of PAF on 0, production (Fig. 1A). This synergistic stimulation shows a biphasic pattern; the activity increase is saturated at a lower concen-
OAG(NO FIG. 2. Dependency of the synergistic stimulatory effect of PAF on 0; production in PMNL on OAG concentration. After preincubation at 37°C for 5 min, PMNL (2 X lo6 cells/ml) were stimulated by the indicated concentrations of OAG in the presence of 0 rig/ml (O), 50 rig/ml (A) and 5 @g/ml (Cl) of PAF. Bars indicate SD of the means (n = 3). No bar means that the SD is within the range of the symbol.
/“/ i/’ / 0
PAF PAF (rig/ml)
FIG. 3. Effect of IAP pretreatment on PAF-stimulated 0, production in PMNL. PMNL were pretreated with (A, A) or without (0, 0) 500 rig/ml IAP as described under Materials and Methods, and then 0; production was measured in the presence (0, A) or absence (0, a) of 10 FM OAG as described in Fig. 1. Bars indicate SD of the means (n = 3). No bar means that the SD is within the range of the symbol.
centration of PAF. On the other hand, stimulation of 0, production by FMLP in both the presence and the absence of OAG was completely inhibited by IAP pretreatment, while the stimulation by PMA was not inhibited by the pretreatment (Table I). These results suggest that the action of PAF involved in the stimulation of 0, production is mediated through IAP-sensitive and -insensitive pathways, both independent of the activation of protein kinase C. Effect of ONO-6240 and H-7 on Stimulation of 0, Production by a Combination of PAF and OAG To investigate the involvement of the putative PAF receptor in the synergistic stimulation of 0, production with OAG, the effect of ONO-6240, a PAF receptor an-
FIG. 4. Effect of ONO-6240 and H-7 on 0; production stimulated by a combination of PAF and OAG. PMNL (2 X 10” cells/ml) were preincubated with (0) or without (0) 1 pM ONO-6240 (A) or 100 PM H-7 (B) at 37°C for 5 min, and then 0, production was measured in the presence of both indicated concentrations of PAF and 10 pM OAG. Bars indicate SD of the means (n = 3). No bar means that the SD is within the range of the symbol.
tagonist (16), was examined. Figure 4A shows that 1 PM ONO-6240 inhibits the production induced by a combination of 5 rig/ml PAF and 10 FM OAG. The synergistic stimulation, however, was not completely abolished by ONO-6240 in the presence of 500 rig/ml PAF, and the extent of activity suppressed was almost the same irrespective of the concentration of PAF. Such a mode of the inhibition by the PAF receptor antagonist is similar to that observed in the case of IAP pretreatment. ONO6240 had little effect on PMA-induced 0, production at the concentration used (data not shown). In marked contrast, the increased PAF-induced 0, production by OAG was inhibited by H-7, an inhibitor of protein kinase C (17), irrespective of the concentration of PAF (Fig. 4B), indicating the significance of protein kinase C in the OAG stimulation. DISCUSSION
Effect of IAP Pretreatment on FMLP- and PMA-Stimulated 0, Production in PMNL 0; production (nmol/5 min/lO’cells)
Stimulants None OAG 10 pi FMLP 5 X lo-? M OAG + FMLP PMA 312 nM
Control 1.0 f 0.1 2.4 6.7 22.0 18.8
f +t +
0.3 0.4 1.6 0.7
500 rig/ml IAP-treated 1.1 + 0.4 2.8 + 0.2
1.3 + 0.6 3.8 f 0.4 19.9 + 0.1
Note. Experimental conditions were as described in the legend to Fig. 3. The results are expressed as the means i- SD (n = 3).
We have shown in this report the synergism in stimulating 0, production between PAF and low concentrations of OAG, the latter of which enhance protein phosphorylation but, by themselves, have little effect on O2 production (6, 7). The results obtained also suggest that there are at least two pathways in the action of PAF which lead to synergistic stimulation of 0, production with OAG in PMNL. At lower concentrations of PAF, the action is mediated by the receptor and IAP-sensitive G protein. This type of action of PAF is very similar to that of FMLP. And such a receptor-mediated effect of PAF on synergistic 0, production with FMLP was also reported (18). At higher concentrations of PAF, however, the action is apparently mediated through both receptor-G protein-independent and -dependent pathways. The result suggests the possibility that PAF by
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