Vol. 189,‘No. 2, 1992

December 15, 1992

BIOCHEMICAL AND BIOPHYSICAL 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

9% Summary: Staurosporine potentiates y+ a sustained elevation of intracellular Ca

forma . n of platelet-activating factor (PAF) and causes ([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 1992 Academic 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-L- leucyl-l-phenylalanine; NDGA, nordihydroguaretic acid; PAF, platelet-activating factor (l-O- alkyl-2-acetyl-sn-glycero-3-phosphocholine); PBS, phospate-buffered saline.

0006-291X/92 $4.00

771 Copyright 0 1992 by Academic Press, Inc.

All rights of reproduction in any form reserved.

Vol. 189, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNKATIONS

MATERIALS AND METHODS

Bovine serum albumin (fatty acid free), FMLP and FURA-2/AM were supplied by Materi& 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

I! room temperature. The cells twice and resuspended in PBS to a density of 5 x 10 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 FMLP-

stimulated [3H]PAF-biosynthesis. This suggested that a regulatory process involving

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Vol. 189, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-0 600 t ,. + 100 nM FMLP I

x

$500

8 - 400 .;

2 2

300

,x .P 200 D I

2 100

F - 0 Conlrol witt lout 0.03 0.3 3 IO

:“hlb,tOi iVEB 2086 (jd) NOGA (PM)

Fig. 1. Effect of staurosporine, WEB 2086 and NDG& on FMLP-stimulated [3H]PAF- formation in human neutrophils. Neutrophils (5 x 10 cells/OS ml), pretreated with 1 FM staurosporine or vehicle in the presence of WEB 2086 or NDGA for 5 mig at 37 “C, ?ere stimulated for 10 min with 100 nM FMLP in the presence of 20 &i/ml [ HIacetate. [ H]PAF was extracted and quantitated as described in “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]PAF-

formation 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, + 100 nM FMLP

0 1 2 3 4 5 0 1 2 3 4 5 Time (min) Time (min)

Effect of WEB 2086 on [Ca2+]i in staurosporine-pretreated neutrophils after Fig. 2. stimulation with FMLP. FURA-2/AM loaded cells (5 x 10 cells/ml) were pretreated with 1 PM staurosporine and/or 3 PM WB 2086 for 5 min at 37 “C prior to stimulation with 100 nM FMLP. Measurement of fluorescence and calculation of [Ca2+Ji were done as described in “Materials and Methods”. The tracings represent a single experiment out of three. similar experiments performed using neutrophils obtained from different donors.

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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 [3H]PAF-formation 6 @pm)

[Ca2+]i after 5 min (nM)

1. PAF (100 nM) 92* 17 17+ 5 2. PAF (100 nM) +

staurosporine (1 PM) 165 f 33 64 + 18

Experiments for measurement of [3H]PAF-fqrmation and [Ca2+]i were dol)e+as described in the legends for Fig. 1 and 2. Values for 6 [Ca2 ]i represent differences in [Ca min after FMLP challenge. Values represent the mean f S.E. (n = 3).

]i before and at 5

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Vol.‘18’9, ho. 2, 1992 BIOCHEMICAL AND 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 - WEB 2086 + WEB 2086 (3 PM)

Cell pellet 1. Cells + vehicle not detected not detected 2. Cells + 100 nM FMLP 1.60 f 0.14 1.45 &- 0.14 3. Cells + 100 nM FMLP +

1 PM staurosporine 8.20 f 0.74 2.90 f 0.29 Sunematant

4. Cells + vehicle not detected not detected 5. Cells + 100 nM FMLP not detected not detected 6. Cells + 100 nM FMLP +

1 PM staurosporine not detected not detected

Neutrophils (5 x lo6 cells/O.5 ml), pretreated with 1 PM staurosporine or DMSO as vehicle in the presence or absence of 3 PM WEB 2086 for 5 min at 37 “C, were stimulated for 5 min with 100 nM FMLP. Incubations were stopped by 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.

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