Effect of staurosporine on fMet-Leu-Phe-stimulated human neutrophils: dissociated release of inositol 1,4,5-triphosphate, diacylglycerol and intracellular calcium

BiochimicaetBiol~stcaA¢za, !135(1992)301-308 © i992 ElsevierScience PublishersB.V. All rightsreserved0167-4889/92/$05.00

301

BBAMCR 13164

Effect of staurosporine oa ~qVIet-Leu-Phe-stimulated human neutrophils: dissociated release of inositol 1,4,5-trisphosphate, diacylglycerol and intracellular calcium S a n t o s h N i g a m , S t e f a n Miiiler a n d B a r b a r a W a l z o g Eicosanoid Research, Depar~nu'ntof Gynecology. Unil'ersit~tsklinikumSteglitz. Free Unit'ersi~"Bedin. Bedin (Germany)

(Received 28 January 1992) Keywords: Neutroghil;(Human);,Slaurosporine;lnositol IA,5-trisphosphate;Diacylglycerot;lntracellularcalcium Staurosporine, a microbial alkaloid, enhances inositol 1,4,5-trisphnsphate (IP3) and 1,2-diacylglycerol (DG) production rapidly and dose-dependently in [Met-l..eu-Ph¢ (F'MLP)-stimulated human neutrophils showing maximal effects at ! pM concentr~tion. The IP3 increase was specific for staurnsporine as three other putative protein kinase C (PKC) inhibitors, !-17,sphingos;zieand palmitoylcarnitine were unable to enhance the IP3 generation in FMLP-stimulated human neutrophi!s. Stanrosporine, at concentrations 0.3-1.0 pM, did not affect the initial mobilization of FMLP-induced intracellular Ca2+ (Ca.+), although a sustained elevation of cytosolic Caz+ level was observed within 5 min. This effect could not be suppressed, even by l /.tM phorbolmyrislate 12,13-acetate (PMA). Whereas lower concentrations of staurosporine (_< 100 nM) were unable to affect FMLP-induced IP~ production, DG accumulation and Ca~÷, the PMA-inhibited initial Ca2+ signal ~nd IP3 formation triggered by FMLP were almost completely restored. At higher concentrations (>_ 31~ nM) staurosporine reversed the inhibitory effect of other protein kinascs, distinct from the PMA-inducible one, which may be responsible for the phosphatidyl inositol 4,5-bisphosphate (P!~) breakdown, thus causing accumulation of IP3 and DG and an elevation of Cai2+ level. Whereas IP3 declined to basal leval .vithia 5 rain. the DG level remained elevated during the same period. This phenomenon is attributed to phnspholipa.~_~. D (PLD) stimulation by stanrnsporine, which augments the IX} synthesis, in part through PA degradation via phosphatidic acid (PA) phosphoh-jdrolase.

Introduction The receptor-mediated activation of many cell types by iigands, such as the chemotactic tripeptide FMLP, immune complexes or complement CSa, is associated with the activation of PLC and PLA 2 [1,2]. The stimulation of PLC occurs through a specific receptor coupled via a G-protein to PLC, which hydrolyzes PIP2 thus generating the two messengers DG and IP3 [3]. Whereas DG activates cytosolie PKC ~n the presence of phosphatidyiserine and C.a2+ [4], IP3 diffuses into

Abbreviations: PKC protein kinase C; FMLP. fMet-Lcu-Phe; PLC phospholipase C; PLA2, phospholipas¢ A2; PLD, phospholipas~ D;

PMA, phorbolmyristateL?..13~_cetate;Ca2., intracellularCa2+; DG, 1.2-diacylg]yccrol; IP3, inosito) 1.4.5-trisphospbate; PIP2, phosphatidylinositol 4..¢bisphosphale; Pi, phosphatidylinositol; PA. phosphatidic acid; PAl:. platelet-aCl~-ating factor. SOD. supero]dd¢ dismutase. Correspondence: $. Nigam. Eicosanoid Re.arch. DepL of Gynecology. Unh,'ersilS.tsldinikumSteglitz. Free University Berlin, D-1000

Bedin 45, Germany.

the ~-wtoplasm and mobilizes Ca. + from the dense tubula, ~stem [5]. DG can be substituted by the turnout promoter PMA, an activator of PKC, which causes binding and activation of PKC [6,7] as well as enhancement of O~ production in human neutrophils [6]. Several results characterizing the functions of PKC in signal transduction have been reported by using PMA. Accordingly, PKC has been suggested to be responsible for controlling agonist-stimulated signal transduction via PLC pathways by regulating the PLC activity [8] and Ca,2-+ homeostasis [10] in human neu. trophils. Staurosporine is a very potent and nonspecifie inhibitor of PKC [11,12], which antagonizes PMA.induced effects on neutrophils at low concentrations [13]. Furthermore, staurosporine was found to induce the association of purified PKC with human erythrocyte membranes and inhibit the membrane-associated enzyme [14]. Also, it inhibits the protcolytically generated catalytic domain of PKC from rat brain without affecting the phorboiestcr binding [15]. We ttscd staurosporine to study the role of PMA-inducible and PMA-noninducible protein kinases on the synthesis

302 and release of IP3, DG and Ca~ + in FMLP-stimulated human neutrophils. Materials and Mctlmds

Materials FMLP, PMA, HT, palmitoyi carnitine, sphingosine and 8-bromo-cycfic AMP were supplied by Sigma, FRG. F U R A - 2 / A M was obtained from Serva, FRG and pertussis toxin from Calbiochem, FRG. The radiolabeled compounds were supplied by Amersham, FRG. The PKC-inlu'bitor, staurnsporine, was a generous gift from Prof. E. Ferber, Max Planck Institute for Immunology, Fre~urg, FRG. Staurosporine and FMLP were dissolved in w a t e r / dimetbyl sulfo~de (9: l). Final concentrations of dimethylsulfox/de ( < 0.2%) d~d not affect the formation of IP 3 and DG, or C.v~" release. Isolation o f human neutrophils Blood (40 ml) was collected from healthy donors and neutrophils were isolated as described [16]. The viability of cells, examined by Trypan blue exclusion test, was >_96%. Measurement o f i~.ositol L 4,5-trisphosphate Aliquots (0.1 ml) of a neutrophil suspension (2.5-106 cells) we~e preincubated with indicated concentrations of staurosporine a n d / o r PMA for 5 min at 37~C. Next, incubations were performed with 100 nM FMLP. The reaction was stopped by addition of 150 /.d ice-cold 10% (v/v) perehlorie acid and the suspension extracted with tri-n-octylamine/Freon (1:1, v / v ) mixture as described [17]. IP 3 was determined in a 100/zl aliquot of the upper phase with a commercial 3H assay system (Amersham, FRG). Cross reactivities for inositol 1,3,4-trisphosphate and inositol 1,3,4,5-tetrakisphosphate (IP4) were 0.22% and 6.4%, respectively. Assay o f diacylglycerol Cells (5-106/ml) were preincuhated with staurnsporine (1/zM) for 5 min at 3"PC before starting the reaction by addition of FMLP (100 nM). The reaction was stopped at the indicated time and the mixture was extracted according to Bligh and Dyer [18]. Dia~iglycerol was assayed as described [19,20]. Measurement o f PLD-mediated DG and PA Cells ( 2 - 1 0 7 / m l PBS) were labeled with [3H]lysoPAF in a shaking water bath for 75 min at 37:C. Ceils were washed twice with PBS and resuspeaded in PBS to a density of 5-106 ceils/ml. 1 ml of neutrophii suspension was then treated with 2 0 0 / t M propranolol a n d / o r 1/zM staurosporine for 5 min at 37°(= prior to stimulation with 100 nIVi FMLP. The reaction was stopped after 5 rain by addition of 3.75 ml methanol/ chloroform (2:1, v / v ) and extracted accordiat~ m Bligh

and Dyer [18]. The sepa:atioo of DG and PA was achieved with a two-step thin-layer chromatography on silica-gel 60 plates (Merck, FRG). First, t~e ~amples were separated with a mobile phase ethyl acetate/isooctane/acetic acid/water (110:50:20:100, v/v) and the plates were dried for 15 min at room temperature. Second, the plates were separated by aJJoi.hvl mobile phase chloroform/methanol/acetic acid/water (80: 13:8:0.3). DG and PA were visualized with the help of standards in an iodine chamber and scraped. The radioactivity was counted in a scintillation counter (Beckman, FRG).

Me ~ay.uronento f intracellular Ca 2 + Neutrophil suspensions were incubated with 5 / z M F U R A - 2 / A M for 45 rain at room temperature. The cells were washed twice and resuspended in PBS to a density of 5 - 106 cells/ml. After equilibration for another 10 rain, the neutrophils were challenged with 100 nM FMLP at 3"PC. The fluorescence was measured using a fluorescence spectrophotometer (Hitachi F4000, Japan), exciting the snspen~ion at 340 nm while the emission wavelength was kept at 505 rim. Ca 2+ concentrations were quantified, as described elsewhere [21,22]. When indicated, neutrophils were incubated ~,i;:; stanrosporine a n d / o r PMA or 8-bromo-cyciic AMP for 5 win before stimulation with FMLP. Measurement o f 45Ca2 ~" inflttx and efflwc Ca e+ influx and efflux in neutrophils were detarmined as described [23]. Briefly, fGr the measurement of 45CaZ÷ influx, neutrophils (1 - 107 ceils) were stimulated with 100 nM FMLP in the presence of 2 ttCi 45Ca2+ with and without the pretreatment of staurosporine for 5 rain. The reaction was stopped after 5 rain by the addition of ice-cold 20 mM EDTA (1 m!) and centrifuged at 200 × g for 2 rain at 4°C. Next, the pellet was washed twice with PBS and the radioactb.~ty was counted. The Ca ~÷ effiux was measured on the neutrophils prelabeled with 4 0 / t C i 4sCa2+ for 60 rain at 37°C. Cells were washed twice before the addition of 100 nM FMLP. After 5 rain the reaction was stopped with 2 ml ice-cold 150 mM NaCI containing 1.5% albumin, 2.5 mM LaCI 3 and 5% sucrose, and centrifuged at 1 6 0 0 × g for 2 rain. The supernatant was measured for the radioactivity. Superoxide anion production Supero~de an:on production was measured by SOD-inhibitable reduction of cytochrome c as determined spectrophotometrically at 550 nm with a microtitre plate reader (Molecular Devices, USA) [24]. Kinetic measurements, with 2.5-106 cells/200 ILl assay volume, were performed between 0.5 min and 2.5 min after addition of 100 nM PMA at 37°C. Assays were performed in duplicate.

303

zooo -~

t

~o , ' ~

5,0 -

o Veh~:le Stc~o- FMLP FMLP. FMLR. soc~e Stouro- Stou~o(111M) ~oonme ~on~e (lOr~M) (300~M)

!

m

FMLP* S;c~o_~oonne t I~M)

Fig. 1. Dose-dependent enhancement by staurosporine of IP~ production in FMLP-stimulated human neutrophils. Cells (2.5- I0 6) were

preincubated with various concentrations of staumsporinc for 5 min at 3"PC. Cells were stimulated for 30 s with 100 nM FMLP. For details see Materials and Methods. Values represent the mean_+S.Do of three sepa~te experiments.

Statistical ecaluation Results w e r e expressed as m e a n values ± S.E. o r S.D. o f : h r e e s e p a r a t e experiments. Statistical signific a n c e w a s o b t a i n e d b y p e r f o r m i n g S t u d e n t ' s t-test with P values P < 0.05. Results

Effect o f staurosporine on IP~ and DG formation W e assessed t h e P I P z breakdo~.~ in h u m a n n e u trophils b y m e a s u r i n g t h e f o r m a t i o n o f IF 3 a n d D G a f t e r stimulation with 100 n M F M L P . F i g u r e 1 shows the d o s e - d e p e n d e n t synthesis o f IP 3 by F M L P - s t i m u lated n e u t r o p h i l s in r e s p o n s e t o various c o n c e n t r a t i o n s o f s t a u r o s p o ~ n e . AILhough ~ a u r o s p o ~ n e a l o n e did not activate the f o r m a l l o n o f I ~ , it c a u s e d in F M L P stimulated neutrophiLs m o r e t h a n a 0.5-fold increase o f IP 3 within 30 s a t a c o n c e n t r a t i o n o f 300 nM. M a x i m u m level o f IP 3 ( > 2.5-fold) w a s o b t a i n e d a t 1 / z M c o n c e n tration o f s t a u r o s p o r i n e . T i m e - c o u r s e stddics l c v e a l e d IP 3 f o r m a t i o n a n d D G release as r a p i d events (Figs. 2, 3). S t a u r u s p o r i n e at 1 p M c o n c e n t r a t i o n c a u s e d m o r e t h a n a 2-fold increase o f IP 3 f o r m a t i o n o v e r th~ F M L P value in neutrophi!s. Moreover, the rise in IP 3 r e a c h e d with s t a u r o s p o r i n e m a x i m u m a t 30 s, as c o m p a r e d to the p e a k level observed a t 10 s, w h e n F M L P w a s a p p l i e d alone. T h e basal levels o f IP 3 with o r w i t h o u t s t a u r o s p o r i n e w e r e r e a c h e d within 5 min. Fig. 3 shows the time c o u r s e o f D G p r o d u e q o n in response to F M L P in s t a u r o s p o r i n e p r e t r e a t e d cells. In c o n t r a s t t o IP3 g e n e r a t i o n , n o significant m c r e a s ¢ in

010 30

50

120 180 Time (~eco~ds)

2~0

~0

Fig. 2. Effect of staurosporine on the time course of IP3 formation in FMLP-stimulated human neutrophils. Cells (5-t0~/m[ PBS) were stimulated by FMLP (100 nM) in the pr=sence of staurosporine (I gM) for different lengths of time. Fer details see Materials and Methods. Each point represents the mean value~S.E, of three separate experiments.

D G p r o d u c t i o n by s t a u r o s p o r i n e w a s o b s e r v e d within 30 s. Moreover, a 2-fold ittcrease over the F M L P value w a s o b s e r v e d at 1 min, r e a c h i n g a m a x i m u m level (2.4-fold) at 2 min, w h i c h r e m a i n e d c o n s t a n t d u r i n g t h e follow-up period. T h e s e different time courses a r e indicative o f a different m e c h a n i s m for the g e n e r a t i o n o f D G , w h i c h m a y be s e c o n d a r y to the effect o f s t a u r o s p o r i n e o n IP 3 formation. P r e t r e a t m e n t o f cells with R 59022, a D G kinase inhibitor, c a u s e d only a small increase in a c c u m u l a t i o n o f D G with a c o n c o m i t a n t l y small d e c r e a s e o f P A ( d a t a not shown).

300-

~200-

loo-

0-1

t

i

1

10

30

60

j

120 Time heconds)

i:

i

300

Fig. 3. Effect of staumsporine on the time course of DG formation in FMLP-stimulatvd human neutrophils. Cells (5" 10~/ml)were preincubated for 5 rain at 37~C either wi!hout (open circles) or with (circles with dot) staurosporine (1 izM) prior to stimulation by i00 nM FMLP for different lengths of time. Open squares denote the effect u; i/zM staurosporine alone and squares with dot denote the effect of vehicle. For details see Materials and Methods. The F'MLP values represent the mean_S.E, of four separate experiments and other values mean + S.E. of three separate experiments.

304

Fig. 4. Effect of s t a u ~ n e ON the release of Ca." in ~LP-slimnlated hUmaN neutroohils. F U R A - 2 / A M loaded celts (5- l('6/mlJ were preincu&ated for 5 min with 30 n M (A). 100 n M {B), 300 n M (C) and lfi~0n M (D) s~irosporine prior to stimulation with 100 n M FMLP. For

details see Materials and MetJ~ods.The tracings represent a single experiment out of three separate experiments with idcntic.Jl results.

Effect o f stauro~porine o~! the actiration o f P L D pathway Fig. 5 shows t h a t n e u t r o p h i l s p r e t r e a t e g w'.th 1 /zM s t a n r o s p o r i n e d o u b l e d the p h o s p h a t i d i c acid p r o d u c tion a n d a h n o s l tripled ihe diradylglyceroi synthesis. However, p r e t r e a t m e n t o f n e u t r o p h i l s with 200 # M p r o p r a n o l o l r e s u h e d in c o m p l e t e i n h ~ i t i o n o f diradylgbjceroi ~nd c o n ¢ o m i t m t d y e n h a n c e m e n t o f p h o p h a tidie :~c'icLwhich pinpoints the activation o f P L D p a t h way.

Effect o f odzer PKC inhibit'ors on IP3 production Next, we d e t e r m i n e d t h e effect o f o t h e r P K C intfibi*,ors o n t h e I ~ p r o d u c t i o n in F M L F - s t i m u l a t e d h u m a n neutrophils. T h e results a r e s u m m a r i z e d in

[M! ~

acid (own x *,~}

A

il

0~ ~

T a b l e !. In c o n t r a s t to s t a u r o s p o r i n e t h e c o m p o u n d s H 7 ( 5 0 / z M ) , p a l m i t o y l c a m i t i n e ( 2 0 / t M ) a n d sphingosine (2.5 F-M) c a u s e d a n inhibition o f 4 6 % , 11% a n d 15% in F M L P - i n d u c e d l P 3 f o r m a t i o n , respectively. N o n e o f these c o m p o u n d s a l o n e c a u s e d a n a l t e r a t i o n in basal IP 3 lc,~els.

Effect o f staurospcrine on Ca 2+ concentration and 4SCa" ÷ fluxes In o r d e r t o investigate if s t a u r o s p o r i n e , w h i c h a u g m e n t e d the release o f 1P3 a n d D G , also increased Ca~ + as a c o n s e q u e n c e o f IP3 g e n e r a t i o n , n e u t r o p h i l s w e r e exposed to various c o n c e n t r a t i o n s o f staur o s p o r i n e f o r 5 rain b e f o r e c h a l l e n g i n g with F M L P . F M L P (100 n M ) i n d u c e d a r a p i d a n d t r a n s i e n t increase ip C a 2+ level o f a p p r o x i m a t e l y 200 n M within 10 s, w h i c h s u b s e q u e n t l y d e c r e a s e d to the b a s a l level within 2 - 3 m i n (Fig. 4). In cells p r e t r e a t e d with s t a u r o s p o r i n e , the r a p i d rise in C a 2+ is followed by a g r a d u a l decline, w h i c h lasted f o r a b o u t 5 min, t h u s resulting in a s u s t a i n e d elevation o f a p p r o x . 70 n M C a 2 + a b o v e t h e basal level (Fig. 4, T a b l e II). A l t h o u g h n o significant effect o f s t a u r o s p o r i n e at c o n c e n t r a t i o n s a p p l i e d w a s

TABLE 1 Comparison of different P K C inhibitors on, d:e FMLP-stimulated IPj generation • h~an neutrophOs

o-~~~ Co~rd~

l(~aM. FMLP

Cells {2.5- !0 6 /ml) were preincubated with the inhibitor for 5 rain at 3 7 ~ prior to stimulation with 100 nM FMLP for 30 s. For experimental details see Materials and Methods. Values represent the differences between basal lP3 conten~ and that of stimulated cells ~ind repr~w.e_nithe mean_+S.E, of four separate experiments. B-sal IP3 level was 0.4+_0.t "mini/t06 celts. Significance was assasseci by comparison with FMLP-stimulated ~,,dues. lfl(~M I:MI.P * 1 ~M Stourospcdm

Fig. 5. Effect of stanrc~porine on the the PA (A) and diradylglycerol (B) formation with (dark bars) and without (white bars) 200 /.tM propranoIol in FMLP-stimulated human neutrophits. Cells (5106/rid) were labeled with [3H]lyso-PAF and incubated wiG:. 200 /xM propranolol and/er I ~M staurosporine for 5 min at 3~C prior to stimulation ~'ith 1OO ,,M FMLP. For details see Materials and Methods. The values represent mean+S.E, of three separate expe.riments.

PKC inhibitor

6 IP3 (pmol/lO6 celts)

Difference (%)

Vehicle Stanrosporine (1/zM) H7 (50 ttM) Sphingosiae (2.5/zM) Palmitoylcamitine (20/.tM)

1.14_+_0.17 2.85_+0.61a 0.61 _+0.17 a 0.96+0.11 1.04+_0.20

:t:0 + 150 -46 -- 15 - Ii

P <_O.05.

~5 TABLE I1 Effect of staurosporine and~or PMA on CaZ~+ in htanan neutrophils at tO s and 300 s following ~imulation ttqth FMLP

"rh,~LE ili

Cells (5- lO°/ml) were equilibrated for tO min before stat!ing the reaction by addition of 100 nM FMLP. Preineubations with stau:'oslmrine and/or PMA were performed for 5 rain before stim,Jlat~qn. Values reprc-oent differences in Ca~÷ concentrations before and after FMLP challenge as means_+S.D,of three separate experi~.,:nts. Significance was assessed by comparison with FMLPostimulated values.

For experimental details see Materials and Methods. Values represent the mean+S.E, of three separate cxperiments.

Incubation

6 Ca~+ (nM)

FMLP (100 nM) FMLP ( 100 riM)+ staurosporine (300 nM) FMLP (100 nM)+ PMA(i p.M) FMLP riO0 nM)+ PMA(I p-M)+ staurosporine (300nM)

224±28

10± 12

208 ± 40

68 4 6 ~

124_+30~

0_+ 0

187±46

Effect of staurosporbze on ~ e transrnembranous Ca z + fluxes F~LP-.'timulated human neutrophils

Incubation

45C.a2+ iniiux (cpm/107 cells)

4SCa2+efflux (cpm/107 cells)

Vehicle Staurosporine (1/zM) FMLP (100 nM) FMLP (100 nM)+ smurosporine (I p.M)

146+ 32 192± 48 1812± 166

846+ 113 682± 93 6269± 41!

1922±238

6112±386

rosporine, at a ¢oticentration of 100 nM, restored these functions to 97% and 85% o f the F M L P value, respectively (Table IV). T h e antagonizing properties o f stau-

70_+16 ~

a P .<. 0.05.

TABLE IV

observed on the initial mobilization of Ca,2-+ by FMLP,

Concentration of Ca~ + and IP3 in human neutrophils 10 s after stimulation with FMLP in presence and absence of staumsporine and/or PMA

the concentrations o f 100-3.'}0 n M staurosporine reversed the P M A - i n d u c e d diminution o f C a 2+ by 75% (Fig. 6, T a b l e II). P M A (1 p.M), however, did not alter the sustained elevation o f C a 2+ (Table ll) and the e n h a n c e d IP 3 formation ( d a t a not shown). Since the rapid initial mobilization o f C a 2+ by staurosporine r e m a i n e d upaffected, it was o f interest to investigate the effect o f staurosporine on t r a n s m e m b l a n o u s Ca z+ fluxes in FMLP-stimulated h u m a n neutrophils. Experiments using 4SCa2+ for the d e t r m i n a tion o f Ca 2+ fluxes showed that staurosporine ( 1 / z M ) did not alter t r a n s m e m b r a n o u s fluxes o f 4SCa2+ within 5 rain (Table III). E f f e c t o f stanrosporine o n P M A - i n d u c e d m o d u l a t i o n o f neutrophil fu,'u:tions PMA, at a concentration o f 100 nM, caused an inhibition o f the FMLP-induced Ca2+i signal a n d IP3 accumulation by 38% a n d 72%, respectively. Stau-

-Ik

J

-

A

'

q

/

~

I-

Cells (5-10~/ml) were preincubated with staurosporine and/or PMA for 5 rain prior to stimulation with 100 nM FMLP. ~ Ca2÷ represents the value between basal arid stimulated C.a~+ concentrations; fi IP3 represenLsthe value between stimulated and control ceils. Basal levels were determined as i65+16 nM for Ca2* and 0.57±0.14 pmot/106 cells for IP3. Values are mean±S.D, of three separate experiments. Significance was assessed by comparison with FMLP value. Incubation

8 CalZ+ (nM) 8 IP3 (pmol/10* cells) at 10 s at 10 s

FMLP (100 nM) FMLP (100 nM)+ staurosporine (100 nM) FMLP ( I•0 nM) + PMA (100 nM) FMLP ( 100 nM) + PMA (100 nM)+ staurosporine (100 nM)

187+42

2.58±0.16

117+11 a

0.73_+0.38a

181 ± 35

2.23±0.41

~P~O~.

"

V-~r

2.64±0.7.9

190±46

I-

I s~l,

o

'

.

,

I

,

.

!

Fig. 5. Reversalof PMA-induced inhibition of Ca2+ by staurosporine in FMLP-stimulated human neutrophils. FURA-2 loaded cells (5-106/ml) were preincubated for 5 rain with 100 riM staurosporlne prior to stimulation with 100 nM FMLP. For details see Materials and Methods. The tracings representa single experiment out of three separateexperimentswith identical results.

306

41

+

PMA (100 nM)

g, 0-

÷

S~urosporine (1O0riM) Fig. 7. Inhibition of PMA-induced 0 2 p~aductic~l by staurosporine in FMLP-stimulated human neutrophils. Cells (2.';-l~/mlt "~e~e preincubated for 5 rain with vehicle or 100 nM staurosl~rine in microtitre prate wells. Kinetic measuremenls ::'ere performed at 550 nm between 0.5 min and 2.5 mio, folio~ug addition of 100 nM PMA. Values represent the mean+S.D, of four separate experiments. rosporine to P M A have also gained support from the i n h ~ i t i o n of PMA-induced O ~ production by 100 n M staurosporine (Fig. 7). However, 100 nM staurosporine alone were ,.'ncapable of affecting the Ca 2+ signal and IP3 formation in FMLP-challenged h u m a n neutrophils, suggesting that PMA-induced mechanisms do not play a predominant role in the regulation of the PLC ttansduction pathway initiated by FMLP. Next, we investigated if st:,urosporine is acting via inhibition of protein kinase A. Preincubation of neutrophils with 8-bromo-cAMP, however, caused no reversal of staurosporine-induced Ca 2+ signal (Table V k Discussion Staurosporine has been demcmstrated to inhibit PKC from several cellular sources [11,14,15]. It inhibits protein phosphorylation as well as the respiratory burst triggered by P M A and F M L P [!3,25]. But, staurosporine is not a selective inhibitor for PKC and suppresses a variety of other protein kinases in vitro [H,26]. In this study, we present strong evidence that the regulation of the PLC pathway occurs by one or more protein kinases, which are inhibited only at higher concentrations of stanrosporine. These protein kinases are distinct from those which are inducible by PMA. Further, our data demonstrated that stanrosporine increased IP 3 formation dose-dependently in FMLPchallenged neutrophils (Fig. 1). This has been previously shown in PAF- and thrombin-stimulated platelets [27,28]. However, the temporal formation of IP 3 by stanrosporine showed a shift of peak IP 3 from 10 s to 30 s (Fig. 2). W h e r e a s FMLP-stimulated neutrophils

alone showed significant degradation of 11"3 at 30 s, their pretreatment with 1 I.tM staurosporinc enhanced the IP3 concentration almost 2-fold during the same period. The accumulation of IP 3 may be associated with the reversal of suppressive effects of one or more protein kinases by staurosporine, e.g., uncouplir~s of FMLP recepmr-G protcin-PLC, phosphorylation of 5phosphomonoesterase with subsequent acceleration of I P 3 degradation and i n h ~ i t i o n of IP3 kinase in platelets [29-31]. Since the D G formation was also enhanced by staurosporine (Hg. 3), it could be conjectured that the PLC activity was increased by staurosporine. Strikingly, the temporal effect of staurosporine showed a dissociation between IP 3 gene:ation and the concurrent D G formatio:t (Figs. 2, 3). In contrast to IP3, which declined to basal level between 30 s and 300 following stimulation, the D G production increased after 30 s and remained elevated during the follow-up period. This led us to the follb-vdng assumptions: (1) there exists a n additional source, besides the PI pool, for D G production through PLC [32,33]; (2) a different mechanism for D G formation is operative, such as P L D / p h o s p h o h y d r o l a s e pathway [34]. The measurement of P A and diradylglyce~ol from neutrophils, pre!aheled with [3H]lyso-PAF after stimulating with FMLP, .~howed tbe e n h a n c e m e n t of both by staurosporine (Fig. 5), suggesting a PLD-mediated pbosphatidylcholine metabolism. The evidence for PLD pathway was further supported by p r e t r e a t m e n t of neutrophils with 200 p.M propranolol, which blocked

TABLE V Effe~l of staurosporine on Ca"" in presence of &bromo-cAMP in FMLP-stlmulatcd human neutrophils

Ceils (5-IO6/ml) were preincubated with slaurosporine and/or 8bromo-cAMP (100/tM or i mM) for 5 min prior to stimulation with 1O0 nM FMLP. ~ Ca~÷ represents the value between basal and stimulated CaT- concentration.~. Basal level for Ca~÷ was aeiurmined as 141+_13 nM. Values represent mean-+S.D, of three separate experiments. Significance was assessed by comparison with FMLP value. Incubation FMLP (100 nM) FMLP (100 nM)+ Staurosporioe (300 nM) FMLP (10OnM) + 8-bromo-cAMP ( IU0/t M) FMLP (100 nM) + 8-bromo-cAMP (100 #M)+ stanrosporine (300 nM) FMLP (100 nM)+ 8-bromo-cAMP (i mM)+ stanrosporine (300 nM) aP
8 Ca~ ÷ (nM) at 10s at 300 s 244_+56 25+_ 9 264+_21

68 -+10 a

299+77

34+12

281 + 45

56_+10 a

259_+52

49_+ 8 a

307 P A transformation to D G via PA-phosphohydrolase and resulted in P A accumulation and D G reduction (Fig. 5). Pretreatments of cells with R 59022, a D G kinase inhibitor, affected D G and P A formation only slightly (data not shown). This excludes the possibility of D G accumulation in o u r system due to D G kinase inhibition. Also, it was of interest to know whether the staurosporine effects are common to other PKC inhibitors. Use of H7, palmitoylcarnitine and sphingosine, which inhibit PKC via different mechanisms, showed, in contrast to staurosporine, an inhibition of IP3 by H7, but almost no effect by the other two inhiI~itors. Evaluation of Cai2+ concentrations revealed no additionai rapid mobilization of Ca~+ by staurosporine (Fig. 4). This p h e n o m e n o n does not coincide with the o b s e ~ a t i o n s m a d e by others in thrombin-stimulate¢ platelets, where cytoplasmic accumulation of Ca~+ was reported [28]. However, the effect on platelets was achieved with a 10-fold higher concentration of staurosporine than in our studies, i n our system, the gradual decline of Ca 2+ caused by staurosporine showed no link to transmembranons Ca2+ fluxes (Table II). W e believe that the staurosporine-induced time lag for generation of I P 3 might have caused the gradual decline of Ca~+ levels, i n addition, it calmot be ruled out that stanrosporine inhibited the Ca2+ reentry in intracellular pools, which is apparently regulated by a PKC-iinked Ca2+ A T P a s e [10]. Despite o u r observations of only minimal effects by 100 nM staurosporine on the neutrophil functions, we could restore the PMA-inhibited initial Ca 2+ signal ~ d I P 3 response to nearly 90% of the F M L P value (Fig. 6, Table IV). Moreover, we could block the PMA-induced respiratory burst with the same concentration (Fig. 7). But, the clear differentiation o f effects observed at high concentrations o f staurosporine suggests that the PMA-induced mechanisms play only a minor role in the regulation of FMLP-activated PLC and PLD pathways. W e conclude that higher concentrations of staurosporine ( > 300 nM) enhance IP 3 and D G formation in FMLP-stimulated h u m a n neutrophils. Dissociation of temporal synthesis o f I P 3 and D G synthesis by staurosporlne led to a n assumption of the presence of more than one source for D G production. In this p r o c e ~ a major part of D G is presumably contr:buted by the P L D pathway. IP3 accumulation causes the sustained elevation o f Ca2+. Lower concentrations of staurosporine (_< 100 nM) reversed the PMA-induced effects on human neutrophils without affecting the FMLP-activated PLC pathway. W e believe that the PMA-inducible protein kinase C, and also protein kinase A may not be responsible for the regulation of early responses of the PLC pathway in FMLP-challenged neutrophils. The role of the protein kinase,

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