Vol. 189,‘No. December
2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
15, 1992
RESEARCH COMMUNICATIONS Pages 771-776
ENHANCEMENT BY STAUROSFORINE OF PLATELET-ACTIVATING FACTOR FORMATION IN N-FORMYL PEPTIDECHALLENGED HUMAN NEuTRomIs MEDIATED BY INTRACELLULAR PLATELET-ACTIVATING FACTOR BINDING SITES Stefan Mtiller and Santosh Nigam * Eicosanoid Research, Department of Gynecology, Universibltsklinikum Steglitz, Free University Berlin, Hindenburgdamm 30, D-1000 Berlin 45, Germany Received
September
8,
1992
Summary: Staurosporine potentiates y+ forma 9% . n of platelet-activating factor (PAF) and causes a sustained elevation of intracellular Ca ([Ca Ii). WEB 2086., a specifi5 $AF-receptor antagonist, inhibits both potentiation of PAF formatton and elevation of [Ca ]i by 78% and 65 %, respectively. Moreover, the PAF produced by FMLP and/or Staurosporine was completely retained in the cell. This suggests that the effect of staurosporine in FMLP-stimulated neutrophils may 5yediated by the action of endogenously produced PAF, which in turn leads to an increase in [Ca ]i and PAF formation. We conclude that PAF is the major product of human neutrophils which reacts via specific intracellular PAF binding sites to stimulate the phospholipase A2, and its synthesis is under control of a staurosporine-sensitive protein kinase. 0 1992Academic Press.Inc.
Upon stimulation with the chemotactic peptide N-formyl-methionine-leucin-phenylalanine (FMLP) human neutrophils produce platelet-activating factor (PAF) [l-3]. PAF itself is a potent stimulus for several cell types including the neutrophil and may act therefore as an autacoid [4]. Several lines of evidence suggest also the involvement of intracellular functions for PAF in stimulated human neutrophils [5,6]. However both, the role of endogenously produced PAF and the regulation of its reactive potential in stimulated neutrophils are poorly understood. We reported recently that staurosporine, a potent and unspecific inhibitor of protein kinase C [7l, potentiated the formation of products of the phospholipase C, phospholipase D and phospholipase A2 pathways including PAF in FMLP-stimulated human neutrophils [3]. The inhibition of a negative feedback mechanism by a staurosporine-sensitive protein kinase was therein postulated, which led to a sustained elevation of [Ca2+]i [8] and an enhanced biosynthesis of PAF. In the present study, we report that staurosporine also enables endogenously formed PAF to act as a stimulus via specific intracellular PAF binding sites in FMLP-stimulated human neutrophils. * To whom correspondence should be addressed. Abbreviations [Ca2+]i, cytosolic free Ca2+ concentration; FMLP, N-formyl-L-methionyl-Lleucyl-l-phenylalanine; NDGA, nordihydroguaretic acid; PAF, platelet-activating factor (l-Oalkyl-2-acetyl-sn-glycero-3-phosphocholine); PBS, phospate-buffered saline.
771
0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
189,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
MATERIALS
AND METHODS
RESEARCH
COMMUNKATIONS
Materi& Bovine serum albumin (fatty acid free), FMLP and FURA-2/AM were supplied by Sigma, FRG and Percoll by Pharmacia, FRG. Staurosporine was purchased by Calbiyhem, FRG. PAF was purc$ased from Bachem, Switzerland. The radiolabeled compounds [ HIacetate (3.3 Ci/mmol) and [ H]PAF (44 Ci/mmol) were supplied by Amersham-Buchler, FRG. WEB 2086 was a generous gift from Boehringer Ingelheim, FRG. Stock solutions of FMLP and staurosporine were prepared in dimethyl sulfoxide. Appropriate concentrations were made by dilution with phosphate buffered saline (PBS). Final concentrations of dimethyl sulfoxide (< 0.2%) did not affect the neutrophil functions in our experiments. Prenaration of neutrophils Human neutrophils were isolated from venous blood of healthy volunteers by a discontinous percoll gradient as described [9]. After lysis of contaminating red cells, neutrophils were washed twice with PBS and resuspended in PBS supplemented with 0.25% bovine serum albumin and 0.17~1 ucose. Prior to incubation experiments, the medium was supplemented with 1.2 mM Ca . More than 97% cells were viable as tested by the exclusion of trypan blue. PAF-svnthesis Neutrophil suspensions (lo7 cells/ml) were pretreated for 5 min at 37+“C with 20 pCi/ml [3H]acetate. To a 0.5 ml aliquot of the suspension containing 1.2 mM Ca preincubated for 5 min at 37 “C with staurosporine and/or WEB 2086 was added 100 nM FMLP to start the reaction. Incubations were stopped by addition of methanol/chloroform/acetic acid (2: 1:0.03 v/v) and the samples were extracted according to Bligh. and Dyer [lo]. The purification of PAF was achieved by HPLC as described [9]3 Fractions contammg PAF were collected at a retention time of 9 + 1 min and quantified for [ HI-radioactivity. In selected experiments endogenous P@ was isolated by the procedure described above and the amount of PAF was determined by [ Clserotonin release from prelabeled rabbit platelets as described [9]. Measurement of rCa2+1; [Ca2+]i was determined as described [8]. Briefly, neutrophil suspensions were incubated with 5 PM FURA-2/AM for 45 min room temperature. The cells twice and resuspended in PBS to a density of 5 x 10I! cells/ml. After addition of 1.2 and pretreatment with staurosporine and/or WEB 2086 for 5 min at 37 “C, neutrophils were challenged with 100 nM FMLP. The fluorescence was measured using a fluorescence spectrophotometer (Hitachi F-4OOO&pan), exciting the suspension at 340 nm while the emission wavelegth was kept at 505 nm. [Ca ]i was quantified as described elsewhere [11,12]. RESULTS AND DISCUSSION In a previous study, we have shown that pretreatment of human neutrophils with 1 PM staurosporine caused a significant increase of the FMLP-induced r3H]PAF formation and [ 14C]AA release [3]. Since 5-lipoxygenase products and PAF are potent agonists of neutrophils [13,14], we investigated whether these mediators participated in the enhanced production of PAF by staurosporine. Whereas NDGA, a 5-lipoxygenase-inhibitor,
was almost ineffective, a dose-
dependent inhibition was observed by WEB 2086, a specific PAF-receptor antagonist (Fig. 1) [ 153. WEB 2086 at a concentration of 3 PM caused a maximal inhibition of 78% of staurosporine-potentiated [3H]PAF-formation
in human neutrophils challenged with 100 nM
FMLP. In the absence of staurosporine WEB 2086 was ineffective as inhibitor of FMLPstimulated [3H]PAF-biosynthesis.
This suggested that a regulatory process involving 772
Vol.
189, No. 2, 1992
BIOCHEMICAL
-0
600 x
AND BIOPHYSICAL
+ 100 nM FMLP
,.
t
RESEARCH COMMUNICATIONS
I
$500 8
.;
400
2 300 2 ,x .P 200 D I 2 100 F -
0
Conlrol
witt lout
0.03
:“hlb,tOi
0.3 iVEB 2086
3
IO NOGA (PM)
(jd)
Fig. 1. Effect of staurosporine, WEB 2086 and NDG& on FMLP-stimulated [3H]PAFformation in human neutrophils. Neutrophils (5 x 10 cells/OS ml), pretreated with 1 FM staurosporineor vehicle in the presenceof WEB 2086 or NDGA for 5 mig at 37 “C, ?ere stimulated for 10 min with 100 nM FMLP in the presenceof 20 &i/ml [ HIacetate. [ H]PAF was extracted and quantitated as describedin “Materials and Methods”. Values represent the mean f S.E. (n = 4).
intracellularly synthesized PAF was operative in presence of staurosporine, and which could be inhibited by WEB 2086. Since, the sustained elevation of [Ca2+]i,
indirectly mobilized by
staurosporine [16], has been suggested to be responsible for the potentiation of [3H]PAFformation in FMLP-challenged neutrophils [3,8], we examined the influence of WEB 2086 on the alteration of [Ca2+]i in presence and absence of staurosporine. Indeed, pretreatment of human B + 100 nM FMLP 4,
0
1
2 Time
3 (min)
+ 100 nM FMLP
4
5
0
1
2 Time
3
4
5
(min)
Fig. 2. Effect of WEB 2086 on [Ca2+]i in staurosporine-pretreated neutrophils after stimulation with FMLP. FURA-2/AM loaded cells (5 x 10 cells/ml) were pretreated with 1 PM staurosporineand/or 3 PM WB 2086 for 5 min at 37 “C prior to stimulation with 100 nM FMLP. Measurement of fluorescenceand calculation of [Ca2+Ji were done as described in “Materials and Methods”. The tracings representa single experiment out of three.similar experiments performed using neutrophils obtained from different donors. 773
Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
neutrophils with 3 PM WEB 2086 prevented the staurosporine-induced elevation of [Ca2+]i by 65% five min after stimulation with FMLP (Fig. 2B), whereas no effect on FMLP-stimulated [Ca2+]i was observed in the absence of staurosporine (Fig. 2A). Therefore, we conjectured that the effects of staurosporine were predominantly due to the action of intracellularly synthesized PAF in FMLP-stimulated cells, which in turn led to a sustained elevation of [Ca2+]i and potentiation of [3H]PAF-biosynthesis.
In a previous report we have demonstrated that WEB 2086
inhibited the enhancement of [ 14C]AA release by putative PKC inhibitors, e.g. H-7 and Polymyxin B [3]. Since both Lyso-PAF and AA are released from the same precursor [l, 171, it can be implicated that the intracellularly synthesized PAF in FMLP-stimulated human neutrophils is a key mediator for the regulation of phospholipase A2. Strikingly,
staurosporine also induced a sustained elevation of [Ca2+]i and potentiation of
[3H]PAF-biosynthesis
m human neutrophils stimulated by exogenous PAF (Table 1) revealing
that the basic receptor-coupled mechanisms, such as receptor-G protein-phospholipase C coupling and/or alteration of [Ca2i]i-homeostasis,
may also be affected by staurosporine, as discussed
previously [8]. This is in contradiction to a recent report, in which staurosporine has been shown to inhibit partially PAF-induced AA release and eicosanoid formation in rat Kupffer cells [18]. Since the total detectable PAF produced by FMLP and/or staurosporine was completely retained in the cell (Table 2), we believe that the action of intracellular PAF was mediated by intracellular PAF receptor binding sites, as suggested by other authors too [4], which could be blocked by WEB 2086 (Fig. 1). These receptor binding sites may not be available to exogenously added PAF and are regulated by staurosporine-sensitive kinases. However, it cannot be ruled out that other mediators besides PAF may also be involved in intracellular processes in human neutrophils [19].
Table 1. Effect of staurosporine on [Ca2+]i and[3H]PAF-formation in neutrophils after stimulation with PAF
Incubation
1. PAF (100 nM) 2. PAF (100 nM) + staurosporine(1 PM)
[3H]PAF-formation @pm)
6 [Ca2+]i after 5 min (nM)
92*
17
17+ 5
165 f
33
64 + 18
Experiments for measurementof [3H]PAF-fqrmation and [Ca2+]i were dol)e+asdescribed in the legends for Fig. 1 and 2. Values for 6 [Ca2 ]i representdifferences in [Ca ]i before and at 5 min after FMLP challenge. Values representthe mean f S.E. (n = 3). 774
Vol.‘18’9,
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BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Table 2. Effect of staurosporin on the release of intracellularly synthesized PAF in neutrophils challenged with FMLP PAF-forTtion (pmoll5.10 cells) Incubation Cell pellet 1. Cells + vehicle 2. Cells + 100 nM FMLP 3. Cells + 100 nM FMLP + 1 PM staurosporine Sunematant 4. Cells + vehicle 5. Cells + 100 nM FMLP 6. Cells + 100 nM FMLP + 1 PM staurosporine
- WEB 2086
+ WEB 2086 (3 PM)
not detected 1.60 f 0.14
not detected 1.45 &-0.14
8.20 f 0.74
2.90 f 0.29
not detected not detected
not detected not detected
not detected
not detected
Neutrophils (5 x lo6 cells/O.5 ml), pretreated with 1 PM staurosporineor DMSO as vehicle in the presenceor absenceof 3 PM WEB 2086 for 5 min at 37 “C, were stimulated for 5 min with 100 nM FMLP. Incubations were stoppedby centrifuging the cells at 4OC. For extraction and determination of PAF in pellet and supematant see “Materials and Methods”. Values represent the mean f S.E. (n = 3).
In conclusion, we demonstrated that the potentiation by staurosporine of PAF-formation in FMLP-challenged human neutrophils is predominantly contributed by the intracellularly synthesized PAF, which acts via specific intracellular PAF binding sites. In addition, we showed that the stimulation of human neutrophils by FMLP and PAF and the subsequent action of intracellular PAF in FMLP-stimulated neutrophils seemed to be regulated by the same staurosporine-sensitive kinase(s). Our observations thus may have far reaching implications for using staurosporine and other similar PKC inhibitors as pharmacological modulators of neutrophil functions. Acknowledgments: This study was generously supported by the Association for International Cancer Research, U.K. Authors wish to thank Drs. Stanislav Svetlov and Almut Roscher for the helpful discussion and Gabriele Beyer for the expert technical assistance.
i: :: 5. 4:
Chilton, F.H., and Connolly, T.R. (1988) J. Biol. Chem. 263, 5260-5265. Naccache, P.H., Molski, M.M., Volpi, M., Shefcyk, J., Molski, T.F.P., Loew, L., Becker, E.L., and Sha’afi, R.I. (1986) J. Leukoc. Biol. 40, 533-48. Miiller, S., and Nigam, S. (1992) Eur. J. Pharmacol. 218, 251-258. Stewart, A.G., Dubbin, P.N., Harris, T., and Dusting, G.J. (1990) Proc. Natl. Acad Sci. USA 87, 3215-3219. Tool, A.T., Verhoeven, A.J., Roos, D., and Koenderman, L. (1989) FEBS-Lett. 259, 209-212. Doebber, T.W., and Wu, M.S. (1987) Proc. Natl. Acad. Sci. 84, 7557-61. Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M., and Tomita, F. (1986) Biochem. Biophys. Res. Comm. 135, 397-402. 775
Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUhlldAilOk
Nigam, S., Miiller, S., and Walzog, B. (1992) B&him. Biophys. Acta. 1135, 301-308. i: Bligh, Miiller, S., and Nigam, S. (1990) J. Lipid Med. 3, 239-341. E.G., and Dyer, W.D. (1959) Canadian J. B&hem. Physiol. 8, 911-917. fY: Yano, Scanlon, M., William, D.A., and Fay, F.S. (1987) J. Biol. Chem. 262, 63086312. S., and Nozawa, Y. (1983) FEBS Lett. 161, 296-300. 2 Sttz2yt K.,A.G.,Nakashima, Harris, T., DeNichilo, M., and Lopez, A.F. (1991) Immunology 72, 14. 15. ;4* 1s: 19.
Nigam, S., Fiore, S., Luscinskas, F.W., and Serhan, C.N. (1990) J. Cell. Phys. 143, 512-523. Casals-Stenzel, J., Muacevic, G. and Weber, K.H. (1987) J. Pharmacol. Exp. Therap. 241, 974-981. Wong, K., Kwan-Yeung, L., and Turkson, J. (1992) B&hem. J. 283, 499-505. Snyder, F. (1990) Am J. Physiol. Cell Physiol. 259, C697-C708. Wei, C. ,Heling, L., Donald, J.H., and Merle, S.O.(1992) J. Biol. Chem. 267, 6725-6734. Agwu, D.E., McPhail, L.C., Sozzani, S., Bass, D.A.,andMcCall, C.E. (1991) J. Clin. Invest. 88, 531-539.
776
2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
15, 1992
RESEARCH COMMUNICATIONS Pages 771-776
ENHANCEMENT BY STAUROSFORINE OF PLATELET-ACTIVATING FACTOR FORMATION IN N-FORMYL PEPTIDECHALLENGED HUMAN NEuTRomIs MEDIATED BY INTRACELLULAR PLATELET-ACTIVATING FACTOR BINDING SITES Stefan Mtiller and Santosh Nigam * Eicosanoid Research, Department of Gynecology, Universibltsklinikum Steglitz, Free University Berlin, Hindenburgdamm 30, D-1000 Berlin 45, Germany Received
September
8,
1992
Summary: Staurosporine potentiates y+ forma 9% . n of platelet-activating factor (PAF) and causes a sustained elevation of intracellular Ca ([Ca Ii). WEB 2086., a specifi5 $AF-receptor antagonist, inhibits both potentiation of PAF formatton and elevation of [Ca ]i by 78% and 65 %, respectively. Moreover, the PAF produced by FMLP and/or Staurosporine was completely retained in the cell. This suggests that the effect of staurosporine in FMLP-stimulated neutrophils may 5yediated by the action of endogenously produced PAF, which in turn leads to an increase in [Ca ]i and PAF formation. We conclude that PAF is the major product of human neutrophils which reacts via specific intracellular PAF binding sites to stimulate the phospholipase A2, and its synthesis is under control of a staurosporine-sensitive protein kinase. 0 1992Academic Press.Inc.
Upon stimulation with the chemotactic peptide N-formyl-methionine-leucin-phenylalanine (FMLP) human neutrophils produce platelet-activating factor (PAF) [l-3]. PAF itself is a potent stimulus for several cell types including the neutrophil and may act therefore as an autacoid [4]. Several lines of evidence suggest also the involvement of intracellular functions for PAF in stimulated human neutrophils [5,6]. However both, the role of endogenously produced PAF and the regulation of its reactive potential in stimulated neutrophils are poorly understood. We reported recently that staurosporine, a potent and unspecific inhibitor of protein kinase C [7l, potentiated the formation of products of the phospholipase C, phospholipase D and phospholipase A2 pathways including PAF in FMLP-stimulated human neutrophils [3]. The inhibition of a negative feedback mechanism by a staurosporine-sensitive protein kinase was therein postulated, which led to a sustained elevation of [Ca2+]i [8] and an enhanced biosynthesis of PAF. In the present study, we report that staurosporine also enables endogenously formed PAF to act as a stimulus via specific intracellular PAF binding sites in FMLP-stimulated human neutrophils. * To whom correspondence should be addressed. Abbreviations [Ca2+]i, cytosolic free Ca2+ concentration; FMLP, N-formyl-L-methionyl-Lleucyl-l-phenylalanine; NDGA, nordihydroguaretic acid; PAF, platelet-activating factor (l-Oalkyl-2-acetyl-sn-glycero-3-phosphocholine); PBS, phospate-buffered saline.
771
0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
189,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
MATERIALS
AND METHODS
RESEARCH
COMMUNKATIONS
Materi& Bovine serum albumin (fatty acid free), FMLP and FURA-2/AM were supplied by Sigma, FRG and Percoll by Pharmacia, FRG. Staurosporine was purchased by Calbiyhem, FRG. PAF was purc$ased from Bachem, Switzerland. The radiolabeled compounds [ HIacetate (3.3 Ci/mmol) and [ H]PAF (44 Ci/mmol) were supplied by Amersham-Buchler, FRG. WEB 2086 was a generous gift from Boehringer Ingelheim, FRG. Stock solutions of FMLP and staurosporine were prepared in dimethyl sulfoxide. Appropriate concentrations were made by dilution with phosphate buffered saline (PBS). Final concentrations of dimethyl sulfoxide (< 0.2%) did not affect the neutrophil functions in our experiments. Prenaration of neutrophils Human neutrophils were isolated from venous blood of healthy volunteers by a discontinous percoll gradient as described [9]. After lysis of contaminating red cells, neutrophils were washed twice with PBS and resuspended in PBS supplemented with 0.25% bovine serum albumin and 0.17~1 ucose. Prior to incubation experiments, the medium was supplemented with 1.2 mM Ca . More than 97% cells were viable as tested by the exclusion of trypan blue. PAF-svnthesis Neutrophil suspensions (lo7 cells/ml) were pretreated for 5 min at 37+“C with 20 pCi/ml [3H]acetate. To a 0.5 ml aliquot of the suspension containing 1.2 mM Ca preincubated for 5 min at 37 “C with staurosporine and/or WEB 2086 was added 100 nM FMLP to start the reaction. Incubations were stopped by addition of methanol/chloroform/acetic acid (2: 1:0.03 v/v) and the samples were extracted according to Bligh. and Dyer [lo]. The purification of PAF was achieved by HPLC as described [9]3 Fractions contammg PAF were collected at a retention time of 9 + 1 min and quantified for [ HI-radioactivity. In selected experiments endogenous P@ was isolated by the procedure described above and the amount of PAF was determined by [ Clserotonin release from prelabeled rabbit platelets as described [9]. Measurement of rCa2+1; [Ca2+]i was determined as described [8]. Briefly, neutrophil suspensions were incubated with 5 PM FURA-2/AM for 45 min room temperature. The cells twice and resuspended in PBS to a density of 5 x 10I! cells/ml. After addition of 1.2 and pretreatment with staurosporine and/or WEB 2086 for 5 min at 37 “C, neutrophils were challenged with 100 nM FMLP. The fluorescence was measured using a fluorescence spectrophotometer (Hitachi F-4OOO&pan), exciting the suspension at 340 nm while the emission wavelegth was kept at 505 nm. [Ca ]i was quantified as described elsewhere [11,12]. RESULTS AND DISCUSSION In a previous study, we have shown that pretreatment of human neutrophils with 1 PM staurosporine caused a significant increase of the FMLP-induced r3H]PAF formation and [ 14C]AA release [3]. Since 5-lipoxygenase products and PAF are potent agonists of neutrophils [13,14], we investigated whether these mediators participated in the enhanced production of PAF by staurosporine. Whereas NDGA, a 5-lipoxygenase-inhibitor,
was almost ineffective, a dose-
dependent inhibition was observed by WEB 2086, a specific PAF-receptor antagonist (Fig. 1) [ 153. WEB 2086 at a concentration of 3 PM caused a maximal inhibition of 78% of staurosporine-potentiated [3H]PAF-formation
in human neutrophils challenged with 100 nM
FMLP. In the absence of staurosporine WEB 2086 was ineffective as inhibitor of FMLPstimulated [3H]PAF-biosynthesis.
This suggested that a regulatory process involving 772
Vol.
189, No. 2, 1992
BIOCHEMICAL
-0
600 x
AND BIOPHYSICAL
+ 100 nM FMLP
,.
t
RESEARCH COMMUNICATIONS
I
$500 8
.;
400
2 300 2 ,x .P 200 D I 2 100 F -
0
Conlrol
witt lout
0.03
:“hlb,tOi
0.3 iVEB 2086
3
IO NOGA (PM)
(jd)
Fig. 1. Effect of staurosporine, WEB 2086 and NDG& on FMLP-stimulated [3H]PAFformation in human neutrophils. Neutrophils (5 x 10 cells/OS ml), pretreated with 1 FM staurosporineor vehicle in the presenceof WEB 2086 or NDGA for 5 mig at 37 “C, ?ere stimulated for 10 min with 100 nM FMLP in the presenceof 20 &i/ml [ HIacetate. [ H]PAF was extracted and quantitated as describedin “Materials and Methods”. Values represent the mean f S.E. (n = 4).
intracellularly synthesized PAF was operative in presence of staurosporine, and which could be inhibited by WEB 2086. Since, the sustained elevation of [Ca2+]i,
indirectly mobilized by
staurosporine [16], has been suggested to be responsible for the potentiation of [3H]PAFformation in FMLP-challenged neutrophils [3,8], we examined the influence of WEB 2086 on the alteration of [Ca2+]i in presence and absence of staurosporine. Indeed, pretreatment of human B + 100 nM FMLP 4,
0
1
2 Time
3 (min)
+ 100 nM FMLP
4
5
0
1
2 Time
3
4
5
(min)
Fig. 2. Effect of WEB 2086 on [Ca2+]i in staurosporine-pretreated neutrophils after stimulation with FMLP. FURA-2/AM loaded cells (5 x 10 cells/ml) were pretreated with 1 PM staurosporineand/or 3 PM WB 2086 for 5 min at 37 “C prior to stimulation with 100 nM FMLP. Measurement of fluorescenceand calculation of [Ca2+Ji were done as described in “Materials and Methods”. The tracings representa single experiment out of three.similar experiments performed using neutrophils obtained from different donors. 773
Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
neutrophils with 3 PM WEB 2086 prevented the staurosporine-induced elevation of [Ca2+]i by 65% five min after stimulation with FMLP (Fig. 2B), whereas no effect on FMLP-stimulated [Ca2+]i was observed in the absence of staurosporine (Fig. 2A). Therefore, we conjectured that the effects of staurosporine were predominantly due to the action of intracellularly synthesized PAF in FMLP-stimulated cells, which in turn led to a sustained elevation of [Ca2+]i and potentiation of [3H]PAF-biosynthesis.
In a previous report we have demonstrated that WEB 2086
inhibited the enhancement of [ 14C]AA release by putative PKC inhibitors, e.g. H-7 and Polymyxin B [3]. Since both Lyso-PAF and AA are released from the same precursor [l, 171, it can be implicated that the intracellularly synthesized PAF in FMLP-stimulated human neutrophils is a key mediator for the regulation of phospholipase A2. Strikingly,
staurosporine also induced a sustained elevation of [Ca2+]i and potentiation of
[3H]PAF-biosynthesis
m human neutrophils stimulated by exogenous PAF (Table 1) revealing
that the basic receptor-coupled mechanisms, such as receptor-G protein-phospholipase C coupling and/or alteration of [Ca2i]i-homeostasis,
may also be affected by staurosporine, as discussed
previously [8]. This is in contradiction to a recent report, in which staurosporine has been shown to inhibit partially PAF-induced AA release and eicosanoid formation in rat Kupffer cells [18]. Since the total detectable PAF produced by FMLP and/or staurosporine was completely retained in the cell (Table 2), we believe that the action of intracellular PAF was mediated by intracellular PAF receptor binding sites, as suggested by other authors too [4], which could be blocked by WEB 2086 (Fig. 1). These receptor binding sites may not be available to exogenously added PAF and are regulated by staurosporine-sensitive kinases. However, it cannot be ruled out that other mediators besides PAF may also be involved in intracellular processes in human neutrophils [19].
Table 1. Effect of staurosporine on [Ca2+]i and[3H]PAF-formation in neutrophils after stimulation with PAF
Incubation
1. PAF (100 nM) 2. PAF (100 nM) + staurosporine(1 PM)
[3H]PAF-formation @pm)
6 [Ca2+]i after 5 min (nM)
92*
17
17+ 5
165 f
33
64 + 18
Experiments for measurementof [3H]PAF-fqrmation and [Ca2+]i were dol)e+asdescribed in the legends for Fig. 1 and 2. Values for 6 [Ca2 ]i representdifferences in [Ca ]i before and at 5 min after FMLP challenge. Values representthe mean f S.E. (n = 3). 774
Vol.‘18’9,
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1992
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BIOPHYSICAL
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COMMUNICATIONS
Table 2. Effect of staurosporin on the release of intracellularly synthesized PAF in neutrophils challenged with FMLP PAF-forTtion (pmoll5.10 cells) Incubation Cell pellet 1. Cells + vehicle 2. Cells + 100 nM FMLP 3. Cells + 100 nM FMLP + 1 PM staurosporine Sunematant 4. Cells + vehicle 5. Cells + 100 nM FMLP 6. Cells + 100 nM FMLP + 1 PM staurosporine
- WEB 2086
+ WEB 2086 (3 PM)
not detected 1.60 f 0.14
not detected 1.45 &-0.14
8.20 f 0.74
2.90 f 0.29
not detected not detected
not detected not detected
not detected
not detected
Neutrophils (5 x lo6 cells/O.5 ml), pretreated with 1 PM staurosporineor DMSO as vehicle in the presenceor absenceof 3 PM WEB 2086 for 5 min at 37 “C, were stimulated for 5 min with 100 nM FMLP. Incubations were stoppedby centrifuging the cells at 4OC. For extraction and determination of PAF in pellet and supematant see “Materials and Methods”. Values represent the mean f S.E. (n = 3).
In conclusion, we demonstrated that the potentiation by staurosporine of PAF-formation in FMLP-challenged human neutrophils is predominantly contributed by the intracellularly synthesized PAF, which acts via specific intracellular PAF binding sites. In addition, we showed that the stimulation of human neutrophils by FMLP and PAF and the subsequent action of intracellular PAF in FMLP-stimulated neutrophils seemed to be regulated by the same staurosporine-sensitive kinase(s). Our observations thus may have far reaching implications for using staurosporine and other similar PKC inhibitors as pharmacological modulators of neutrophil functions. Acknowledgments: This study was generously supported by the Association for International Cancer Research, U.K. Authors wish to thank Drs. Stanislav Svetlov and Almut Roscher for the helpful discussion and Gabriele Beyer for the expert technical assistance.
i: :: 5. 4:
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