Rapid priming of calcium mobilization and superoxide anion production in human neutrophils by substimulatory concentrations of phorbol esters: A novel role for protein kinase C and

Cellular Signalliag Vol. 4, No. 5. tap. 511-523, 1992. 0698-6568/92 $5.00 + 0.00 Printed in Great Britain. ~ 1992 Pergamon Press Ltd

R A P I D P R I M I N G O F C A L C I U M M O B I L I Z A T I O N A N D S U P E R O X I D E

A N I O N P R O D U C T I O N I N H U M A N N E U T R O P H I L S BY

S U B S T I M U L A T O R Y C O N C E N T R A T I O N S O F P H O R B O L E S T E R S : A

N O V E L R O L E F O R P R O T E I N K I N A S E C A N D T Y R O S I N E

P H O S P H O R Y L A T I O N I N T H E U P - M O D U L A T I O N O F S I G N A L

T R A N S D U C T I O N

CAROLINE GILBERT, MURIELLE GAUDRY and PAUL H. NACCACHE*

Centre de Recherche en Inflammation, Immunologic et Rhumatoiogie, Centre de Recherche du CHUL, Department of Medicine, Universit6 Laval, 2705 Boulevard Laurier, Ste-Foy, Qutbec, Canada G1V 4G2

(Received 17 March 1992; and accepted 24 April 1992)

Abstract--The modulatory influences of phorbol esters on the functional responsiveness of human peripheral blood neutrophils have been investigated. These studies focused on measurements of the levels of cytoplasmic free calcium and of tyrosine phosphorylation as well as on their ability to mount an oxidative response. Short incubation times (< 1 min) with low concentrations of phorbol esters (5-50 nM) were shown to enhance the above indices of neutrophil responsiveness to chemotactic factors such as fMet-Leu-Phe and leukotriene B,. On the other hand, a time- and concentration-dependent inhibition of calcium mobilization and superoxide production was also observed. The effects of the phorbol esters were stereo-specific and were antagonized by a novel protein kinase C inhibitor (RO 318220) but were not affected by the oxidative burst inhibitor diphenyleneiodonium. Pre-incubation of the cells with phorbol 12,13-dibutyrate (PDBu) altered in a concentration-dependent manner the tyrosine phosphorylation pattern stimulated by fMet-Leu-Phe. In addition, the tyrosine kinase inhibitor erbstatin inhibited the priming of the mobilization of calcium induced by PDBu. These data demonstrate the rapidity of the effects of the activation of protein kinase C, their potential to modulate positively the early events of the excitation-response coupling sequence and the complexity of the functional interrelationships among the various cellular activation pathways available to human neutrophils and other non-muscle cells.

Key words: fMet-Leu-Phe, leukotriene B4, PDBu, RO 318220, erbstatin.

INTRODUCTION

CELL ACTIVATION results from the coordinated interplay among various biochemical transduction pathways, several details of which have been uncovered in the past few years. These include the phosphatidylinositol bifurcating pathway [1], the phospholipase D pathway [2], the cyclic nucleotide-dependent as well as tyrosine kinases [3], and the glycosyl-phosphatidylinositol-specific phospholipases [4]. Although the implication of several of these pathways in the activation of human polymorphonuclear neutrophils (neutrophils) has been demon-

*Author to whom correspondence should be addressed at: CHUL, Room 9800, 2705 Boulevard Laurier, Ste-Foy, Qurbee, Canada G I V 4G2.

strated [2, 5-9], most of the available informa- tion concerns the phospholipase C (phosphoinositidase C or PIC)-catalysed hydro- lysis of phosphatidylinositol 4,5-bisphosphate. The activation of the latter results in the generation of the intracellular mediators inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diglyceride (DG), increases of which lead to the mobilization of intracellular calcium and to the activation of protein kinase C, respectively.

Interrelationships among the two arms of the PIC-mediated transduction mechanisms have been demonstrated. For example, synergistic activation of neutrophil degranulation and of the activation of the oxidative burst can be observed upon the co-addition of sub-maximal concentrations of calcium ionophores and of

511

512 c. GILBERT et al.

activators of protein kinase C such as phorbol esters [e.g. 10] or of phorbol esters and chemotactic factors [11, 12], respectively. The mechanisms involved in the enhancement of neutrophil function by phorbol esters are presently incom- pletely understood but may involve the modulation of the binding affinities of chemotactic factors with their receptors [11] and/or augmented activation of protein kinase C [e.g. 10]. The complexity of the effects of phorbol esters is underscored by another set of results demonstrating that activation of protein kinase C by phorbol esters leads to a time- and concentration-dependent inhibition of chemotactic factor-induced calcium mobilization and degranulation in neutrophils [e.g. 12-14], possibly mediated by an inhibition of the activation of PIC [15]. Furthermore, phorbot esters have also been found to stimulate tyrosine phosphorylation in neutrophils [16] as well as in various other cells [e.g. 17, 18].

The purpose of the present investigations was to examine in detail the effects of the co-stimulation of neutrophils with chemotactic factors and phorbol esters on the generation of one of these cells' major second messengers, namely calcium. In contrast to the inhibitory effects described in previous investigations [12-15], we have observed an enhanced mobilization of calcium and production of superoxide anions in cells stimulated simultaneously with phorbol esters and chemotactic factors. The characteristics of the effects of the phorbol esters strongly suggest that their effects are mediated directly by protein kinase C and indirectly by tyrosine phosphorylation. These results identify a novel mechanism of modulation of the functional responsiveness of human neutrophils by phorbol esters and support the hypothesis of the existence of a subset of protein kinase C that is exquisitely sensitive to stimulation and involved in the amplification of the transduction machinery.

Phe, cytochrome C, superoxide dismutase, and phorbol 12,13-dibutyrate (PDBu) were obtained from Sigma Chemical Co. (St Louis, MO, U.S.A.). The inactive phorbol ester 4-ccphorbol 12,13-dibutyrate (~PDBu) was purchased from LC Services Corp. Woburn, MA, U.S.A. Fura-2/AM was obtained from Molecular Probes (Junction City, OR, U.S.A.). Leukotriene B4, diphenyleneiodonium (DPI), RO 318220 and erbstatin were generously provided by Drs R. Young (Merck-Frosst, Canada), O. T. C. Jones (Birmingham, U.K.), D. P. Clough (Roche Products Limited, Hertfordshire, U.K.) and K. Umezawa (Keio University, Yokohama, Japan), respectively. The concentration of leukotriene B4 was verified by UV spectrometry. The monoclonai antiphosphotyrosine antibody 05-321 was purchased from UBI (Lake Placid, NY, U.S.A.). All products used for SDS--PAGE and protein blotting were reagent grade and purchased from Pharmacia (Montr6al, Qu6bec, Canada). All the other reagents were of analytical grade.

Isolation o f neutrophils

Blood was obtained from the peripheral vein of healthy adults in preservative-free lithium heparin (10 IU/ml of blood). Neutrophils were isolated by means of 2% dextran sedimentation followed by standard techniques of FicoiI-Paque gradients and hypotonic lysis of erythrocytes. The final cell pre- parations contained at least 980 neutrophils. The cells were washed and resuspended in Hank's balanced salt solution (HBSS) containing 1.6mM calcium and supplemented with 10ram HEPES (pH 7.4).

Calcium mobilization

Intracellular free calcium was monitored using the fluorescent probe fura-2/AM as described in Grynkievicz et al. [19] and Faucher and Naccache [20]. Briefly, neutrophil suspensions (1 × 107 ceUs/ml) were incubated with 1/~M fura-2/AM for 30 rain at 37°C. The cells were then washed free of the extracel- lular probe, resuspended at 5x l0 s celis/ml and allowed to re-equilibrate for 10min at 37°C. They were then transferred to the thermostatted (37°C) cuvette compartment of the fluorimeter and the fluorescence monitored (excitation and emission wavelengths, 340 and 510rim, respectively). The internal calcium concentrations were calculated as described in Tsien et aL [21].

MATERIALS AND METHODS

Materials

Ficoll-Paque and Dextran T500 were from Pharmacia (Dorval, Quebec, Canada). fMet-Leu-

Superoxide formation

Superoxide production was monitored as described in Naccache et al. [7] as the (superoxide dismutase-sensitive) reduction of cytochrome C by a

Phorbol ester-induced priming of neutrophils 513

suspension of I x 106 neutrophiis using a slight modification of the method described in Metcalf et al. [22]. The absorption of cytochrome C was monitored at 550 and 540nm and the amount of superoxide anions produced was calculated from the difference between the optical densities at the two wavelengths using an extinction coefficient of 21.1.

Tyrosine phosphorylation

Neutrophii suspensions were incubated at 3 x 10 7 celis/ml for 15 s or 5 min with increasing concentrations of PDBu (0-500/~M) or with the appro- priate amounts of dimethylsulphoxide (DMSO). The cells were then stimulated with fMet-Leu-Phe (10-TM) for 15s and the reactions terminated by addition of 40/d of cell suspensions to an equivalent volume sample buffer [final concentrations: 5% fl- mercaptoethanol, 9% glycerol, 4% SDS in Tris-HCl, pH6.8, 25mM p-nitrophenoi phosphate, 25mM Na-orthovanadate, 12.5 mM phenylmethyl sulphonyl fluoride (PMSF), 125/tg/ml leupeptin and 125pg/ml aprotinin] at 95°C. The samples were denatured by boiling for 5 min, centrifuged for 3 min (12,000 g) and loaded on to a 7.5-15% SDS poly- acrylamide gel. Following electrophoresis of the samples, transfer to Immobilon PDVF membranes (Millipore Corp. Bedford, MA, U.S.A.) was carried out in electrophoretic transfer cells (Hoeffer Scientific Instruments, Canberra Packard, Mississauga, Ontario, Canada). Non-specific sites were blocked by a 1-h incubation at 37°C with 2% gelatin. The membranes were then incubated for l h at 37°C with the monocional antiphosphotyrosine antibody 05-321 (UBI, Lake Placid, NY, U.S.A.) at a final dilution of 1:5000. The membranes were washed five times at room temperature in Tris-buffered saline (TBS)-tween 20 solution (25 mM Tris-HCl, pH 7.6, 190mM NaCI, 1.5% v/v Twcen 20) for 1, 1, 15, 5 and 5 min, respectively. The washed membranes were then incubated with a sheep anti- mouse antibody covalently coupled to horseradish peroxidase (Amersham, Montrral, Qurbec, Canada) at a final dilution of 1:20,000 for 45 min at 37°C and washed again five times as described above. The phosphotyrosine-containing bands were then visual- ized using the Amersham ECL detection kit and Kodak X-omat films (i min pre-incubation, 30-60 s exposure).

RESULTS

C a l c i u m m o b i l i z a t i o n '

The effects of the addition of a premixed solution of fMet-Leu-Phe and PDBu on the levels of cytoplasmic free calcium were first

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examined. The increase in cytoplasmic free calcium induced by fMet-Leu-Phe was significantly and concentration-dependently increased in the presence of PDBu (Fig. 1). In accordance with previous findings with phorbol 12-myristate,13-acetate (PMA) [23], the addition of PDBu by itself did not cause any changes in the levels of cytoplasmic free calcium at any of the concentrations tested (results not shown). The effects of PDBu on the time courses of calcium mobilization are further illustrated in the inset of Fig. 1 in which the magnitude of the increases in cytoplasmic free calcium were normalized in order for the kinetics to be directly comparable. The phorbol ester did not alter appreciably the delay or the rate of mobilization of calcium. On the other hand, the rate of return of the levels of cytoplasmic free calcium to baseline was increased in a concentration-dependent manner by PDBu. The inactive stereo-isomer of PDBu, gPDBu, was without effect on the magnitude of the mobilization of calcium induced by fMet-Leu-Phe or on its kinetics.

The increases in the levels of cytoplasmic free calcium induced by leukotriene B4 (Fig. 2) were

514 C. GILBERT e t al.

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also increased in the presence of PDBu, but not by its inactive analogue gPDBu. The magnitude of PDBu (100nM)-induced enhancement of the amounts of calcium mobilization stimulated by leukotriene 04 ( 2 7 _ 9 % , m e a n _ S.E.M., n = 3, P = 0.05; one-tailed) was, however, smaller than that observed with fMet-Leu-Phe

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FIG. 3. Concentration-dependence of the effects of PDBu on the mobilization of calcium induced by fMet-Leu-Phe. In these experiments, the cells were stimulated simultaneously with the indicated concentrations of PDBu and 10-TM fMet-Leu-Phe. The data represent the percentage increases in the peak levels of calcium mobilization induced by 10 -7 fMet- Leu-Phe. Mean+S.E.M. of at least three separate determinations. The points at 20 (n = 8) and 100 (n = 3) nM PDBu had P values of less than 0.05 and that at 500 nM PBDu (n = 3) had a P value equal to 0.05 (Student's two-tailed t-test).

(62+19%, mean_S .E .M. , n = 3, P = 0.05; two-tailed). PDBu did not alter the rate of calcium mobilization induced by the lipid mediator but increased the rate of recovery of the levels of cytoplasmic free calcium.

The concentration-dependence of the effects of PDBu was further characterized. The results of these studies are summarized in Fig. 3. In these experiments, the cells were stimulated directly with a pre-mixed solution of fMet-Leu- Phe and the required concentrations of PDBu. A monotonic increase in the mobilization of calcium was observed in the presence of incre- mental concentrations of PDBu. This effect exhibited a PDBu threshold of about 5 nM and reached a plateau around 100 nM. The pattern of responsiveness was qualitatively different following a 5 min pre-incubation with PDBu (Fig. 4). Under these conditions, the threshold concentration was reduced to about 2-5 nM, a peak response attained at 10nM following which a decline and eventually an inhibition of the mobilization of calcium induced by fMet- Leu-Phe was observed.

The time-dependence of the effects of phorbol esters was investigated and the results are summarized in Fig. 5. In these experiments,

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the cells were either co-stimulated with fMet- Leu-Phe and the indicated concentrations of PDBu (0 min incubation) or pre-incubated with the phorbol ester for 1, 5, 10 or 20 min and then challenged with fMet-Leu-Phe. As illustrated in the preceding figures, when added simultaneously with fMet-Leu-Phe, PDBu (20, 100 or 500nM) increased the magnitude of the mobilization of calcium induced by the chemotactic factor (40-60% increase). The magnitude of the priming of calcium mobilization decreased with increasing time of pre-incubation, with the rate of decline being proportional to the concentration of phorbol ester used. While an inhibition of the mobilization of calcium induced by fMet-Leu-Phe became apparent following a 5-min incubation with 100 nM PDBu, a pre-incubation of 20 min with 20 nM PDBu was required for the same effect to manifest itself. Intermediate concentrations of the phorbol esters behaved accordingly. A similar inhibition of calcium mobilization by 500nM PDBu was observed in the presence of the oxidative burst inhibitor DPI [24-26] under conditions where the production of superoxide anions by the phorbol ester was completely inhibited (results not shown).

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Superoxide production

The addition of PDBu and chemotactic factors (as premixed solutions) resulted in a synergistic production of superoxide anions. The results of a representative experiment in which various concentrations of PDBu and fMet-Leu-Phe were added simultaneously to a suspension of human neutrophils and the resul- tant production of superoxide anions was monitored are shown in Fig. 6. It should be noted that the synergistic effects of the phorbol esters and of fMet-Leu-Phe could be studied as the oxidative response to the former exhibited its typical delay of about 200 s during which time the response to the chemotactic factor was essentially complete. The phorbol ester increased in a concentration-dependent manner the amount of the superoxide anions produced in response to fMet-Leu-Phe. Increases in the rate of superoxide production and decreases in the lag time were also apparent. As further demonstrated in Fig. 6, only the active isomer of PDBu is active in this respect; indeed, ~PDBu at concentrations as high as 100 nM did not alter the rate or the amount of superoxide anions produced in response to fMet-Leu-Phe.

An even more dramatic priming effect was observed when the effects of the phorbol ester

516 C. GILBERT et al.

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on the oxidative responses to leukotriene B 4

were investigated. As previously noted [e.g. 27], the lipid mediator did not significantly stimulate the oxidative burst of the cells by itself. However, when presented to the cells simultaneously with PDBu (as a premixed solution), a marked and sustained production of superoxide anions was observed that could clearly be distinguished from that stimulated by the phorbol ester, the response to which was significantly delayed in time compared to that of leukotriene B 4 (Fig. 7). The addition of ~PDBu (100 nM) did not augment the activation of the N A D P H oxidase by leukotriene B4 (results not shown).

Effects of the protein kinase C inhibitor RO 318220

We tested next the effects of RO 318220, a novel and potent protein kinase C inhibitor [28, 29] on the priming of calcium mobilization and of superoxide production induced by PDBu. The inhibitor did not significantly affect the mobilization of calcium induced by fMet-Leu- Phe (Fig. 8). On the other hand, RO 318220 inhibited the priming of the mobilization of calcium observed upon the simultaneous addition of the phorbol ester and of the chemotactic

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FIG. 8. Effect of RO 318220 on the PDBu-induced priming of calcium mobilization stimulated by fMet- Leu-Phe. The data represent the peak increases in cytoplasmic free calcium induced by l0 -7 M fMet- Leu-Phe. Where indicated, the cells were pre-incubated for 5rain with 3.3 x 10-7M RO 318220. The asterisk indicates that the (RO 318220 + PDBu) data were significantly different from the PDBu data (Student's t-test, P<0.05) . Mean+S.E.M. of at least three separate determinations.

factor. Furthermore, the increase in the rate of recovery of the calcium levels observed in the presence of PDBu (see Fig. 1) was also inhibited by RO 318220 (results not shown). A similar pattern was observed in the superoxide assay (Figs 9, 10). The addition of RO 318220 at 3 .3x 10-7M did not affect significantly the oxidative burst elicited by fMet-Leu-Phe (Fig.

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Phorbol ester-induced priming of neutrophils ~ 517

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seen to induce a concentration-dependent increase in the density of the same bands. Longer incubation times with PDBu led to stronger phosphorylation patterns (unpublished observations). Pre-incubation of the cells with PDBu for 5 min has two major effects on the fMet-Leu-Phe response. Firstly, a concentration-dependent synergistic stimulation of the tyrosine phosphorylation of the 115,000 mol. wt band and the appearance of a previously minor band of about 70,000 mol. wt was noted. Secondly, a concentration-dependent dephosphorylation of several bands in the 65,000- 55,000 mol. wt region was also observed. Essentially similar results, albeit of a slightly diminished magnitude, were obtained following a 15-s pre-incubation with PDBu (results not shown).

9). On the other hand, it abolished the production of superoxide anions induced by PDBu (Figs 9, 10), and significantly, but not completely, decreased the priming of the oxidative responses to fMet-Leu-Phe (Fig. 9) and leukotriene B 4 (Fig. I0).

Effect of PDBu on the tyrosine phosphorylation response of human neutrophils to fMet-Leu-Phe

Chemotactic factors (such as fMet-Leu-Phe [7, 30-32]) as well as phorbol esters [16] have previously been shown to stimulate tyrosine phosphorylation in human neutrophils. This, relatively new signalling pathway is thought to play an integral part in the activation sequence of human neutrophils. The effects of phorbol esters on the pattern of tyrosine phosphorylation induced by fMet-Leu-Phe were tested next (Fig. 11). In these experiments the cells were stimulated either with fMet-Leu-Phe (10 -7 M)

alone for 15 s or following a 5-min pre-incubation with increasing concentrations of PDBu (20-500 nM). A comparison of lanes 1 and 6 demonstrates that the chemotactic factor rapidly stimulates the tyrosine phosphorylation of several bands, most notably one of apparent molecular weight of about 115,000. A 5-rain incubation with the phorbol ester can also be

Effect of the tyrosine kinase inhibitor erbstatin

Erbstatin is a relatively specific tyrosine kinase inhibitor [33, 34]. It has been shown to inhibit the stimulation of tyrosine phosphorylation by chemotactic factors [7], granulocyte- macrophage colony-stimulating factor (GM- CSF) [35] as well as by phorbol esters (unpublished observations) in human neutrophils. On the other hand, erbstatin, under the conditions used in this study (5 #g/ml, 1-h pre-incubation), did not affect the ability of PDBu or PMA to induce a translocation of protein kinase C or to stimulate its activity (Gaudry and Naccache, unpublished observations) and only marginally inhibited the PMA-induced production of superoxide anions [7]. In view of the ability of PDBu to alter the pattern of tyrosine phosphorylation induced by fMet-Leu-Phe (Fig. 11), the effects of erbstatin on the priming of calcium mobilization induced by PDBu were therefore tested. The results of these studies are summarized in Fig. 12. As previously reported [7], erbstatin affected little, if at all, the calcium mobilizing ability of fMet-Leu-Phe (10 -7 M) in human neutrophils. PDBu induced its charac- teristic priming effects. Pre-incubation of the cells with erbstatin reduced to a significant extent (P < 0.05, unpaired Student's t-test) the

518 c. GILBERT et al.

increased mobilization of calcium observed in the simultaneous presence of the phorbol ester and of the chemotactic factor.

DISCUSSION

The activation of human neutrophils, as that of most other cell types, results from the interplay between the positive and negative influences of several enzymatic pathways. The elucidation of the respective importance of the various pathways implicated in cell activation (e.g. phosphatidylinositol-specific phospholipase C, phospholipase D, protein kinase C, tyrosine kinase) represents one of the foremost challenges in cell physiology. The results of the present study demonstrate that the activation of protein kinase C by phorbol esters in human neutrophils can, depending on the experimental conditions, lead to an augmentation or an inhibition of one of the primary events of the excitation-response coupling sequence, the mobilization of calcium. In addition, evidence implicating mediatory roles of tyrosine phosphorylation in the regulation of the priming of calcium mobilization by phorbol esters was also obtained. Thus, the same enzymatic pathway (that mediated by protein kinase C) can modulate both positively and negatively the functional responsiveness of human neutrophils, and possibly also that of various other cell types.

The negative modulation of stimulated calcium mobilization by protein kinase C activators has previously been well documented. Pre-incubation of cells with phorbol esters has been shown to inhibit, in a time- and concentration-dependent manner, the mobilization of calcium in several cell types including neutrophils [13, 14, 36-38], platelets [39-41] and other cell types [e.g. 42, 43]. This inhibitory effect of phorbol esters was probably mediated by protein kinase C as it was antagonized by inhibi- tots of this enzyme [14]. The inhibitory effects of phorbol esters were partly due to interference with the activation of the phosphatidylinositol 4,5-bisphosphate-specific phospholipase C [44] and partly to the compensating influence of the

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FIG. 12. Effect of erbstatin on the priming of calcium mobilization induced by fMet-Leu-Phe. The data represent the peak increases in cytoplasmic free calcium induced by 10 -~ M fMet-Leu-Phe. Where indicated, the cells were pre-incubated for 5 rain with 20 nM PDBu and/or 1 h with 5/~g/ml erbstatin. The asterisk indicates that the (erbstatin+PDBu) data were significantly different from the PDBu data (Student's t-test, P < 0.05). Mean_+S.E.M. of at least three separate determinations.

activation of buffering Ca2+-ATPases [45]. Inhibition of functional responsiveness (degranulation) as well as of second messenger generation [13, 14, 36-38] has been described.

The results of the present study demonstrate that the interactions of protein kinase C with PIC are more complex and include positive feedback loops that can amplify the early events of the excitation-response coupling sequence. Indeed, the addition of PDBu (but not aPDBu) to human neutrophils led to a rapid increase in the ability of chemotactic factors (fMet-Leu- Phe as well as leukotriene B4) to induce an increase in cytoplasmic free calcium and to stimulate the NADPH oxidase system. A similar stimulation of calcium mobilization was also observed with phorbol 12-myristate 13- acetate (results not shown). Furthermore, synergistic interactions between fMet-Leu-Phe and PDBu were also demonstrated at the level of tyrosine phosphorylation. The rapidity of the stimulatory effects of PDBu as well as the low concentrations required are particularly noteworthy and distinguish this response from those previously observed; indeed, the priming effects of PDBu on the mobilization of calcium were maximal within a few seconds of its addition

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[PDBu] 0. 20 50 100 500 0 20 50 100 500 (nM) i , t l

fMet-Leu-Phe + fMet-Leu-Phe (10"7M)

FIG. 11. Effect of PDBu on the pattern of tyrosine phosphorylation induced by fMet-Leu-Phe. Tyrosine phosphorylation was monitored as described in Materials and Methods. The cells were pre-incubated for 5 min with the indicated concentrations of PDBu or the equivalent amounts of DMSO and stimulated for 15 s with 10-TM fMet-Leu-Phe. The data are from a single experiment representative of three separate determinations.

519


and were evident at concentrations of the phorbol ester lower than those necessary for the detection of activation of protein kinase C. Longer pre-incubation times eventually allowed the previously observed inhibitory effects of phorbol ester on the mobilization of calcium to manifest themselves, with the length of the incubation times required being inversely proportional to the concentration of PDBu. It is of interest to note that no biochemical indices of protein kinase C activation (phosphorylation or translocation of enzymatic activity) can be measured under the conditions utilized herein to prime calcium mobilization (10-20nM PDBu, l min or less pre-incubation). These results imply that a subset of protein kinase C in close (physical and/or functional) proximity to physiologically relevant substrates can be rapidly activated by phorbol esters, leading to a profound modulation of the functional responsiveness of the cells. It is likely that the enhanced mobilization of calcium observed in PDBu-treated cells will exert a significant influence on various functional responses of these cells including the mounting of an oxidative burst, degranulation and possibly also adherence and phagocytosis.

Two lines of evidence indicate that despite the rapidity of onset of the positive modulation of the functional responsiveness of the cells by PDBu, this effect was nevertheless mediated by protein kinase C. Firstly, this effect was stereo- specific as ~PDBu (a stereo-isomer of PDBu of similar hydrophobicity and physico-chemical characteristics but which does not activate protein kinase C) did not reproduce the effects of PDBu even at the highest concentration tested (100nM). Secondly, the priming effects of PDBu on calcium mobilization and on the production of superoxide anions were antagonized by the protein kinase C inhibitor RO 318220 [28]. The structure of the latter compound was derived from that of the micro- bial products staurosporine and K252a and previously shown to exhibit an IC50 towards rat brain protein kinase C of about 100nM and towards bovine heart protein kinase A of 5.1 #M [28]. RO 318220 has the advantage over

the H-7 series of inhibitors and over staurosporine of being significantly more potent than the former (at least two orders of magnitude) and more selective towards protein kinase C than the latter. In addition, RO 318220, in contrast to staurosporine [46, 47] did not inhibit fMet-Leu-Phe-induced tyrosine phosphorylation in human neutrophils (Caon and Naccache, unpublished observations). It should be noted that RO 318220 only partially inhibited the priming of superoxide production induced by PDBu while it abolished the direct response to the phorbol ester. This result raises the possibility that some of the effects of phorbol esters may, as previously suggested [1 l], not be totally protein kinase C-mediated. The lack of effect of RO 318220 on the calcium and oxidative responses to fMet-Leu-Phe was consistent with previous findings with the protein kinase C inhibitors H-7 [14] and C-1 [48] and further indicated that the initiation (as opposed to the modulation) of these functions is not dependent on the activation of protein kinase C.

The priming of calcium mobilization by PDBu is likely to involve the participation of tyrosine phosphorylation. First, as demonstrated in Fig. l l, pre-incubation with the phorbol esters modifies the tyrosine phosphorylation pattern induced by fMet-Leu-Phe in human neutrophils. It is noteworthy that stimulation as well as inhibition of the tyrosine phosphorylation substrates have been observed. Although the nature of the tyrosine phosphory- lated substrates in human neutrophils are presently unknown, a correlation between the priming and inhibition of calcium mobilization and the changes in phosphorylation does appear to exist. Indeed, the synergistic phosphorylation of the ll5,000 mol. wt (and to a lesser extent of the 70,000 tool. wt) band was evident under conditions associated with priming. On the other hand, dephosphorylation of the bands in the 55,000-65,000 mol. wt region was closely correlated to the conditions under which calcium mobilization is inhibited by PDBu. However, we must also consider the possibility that the apparent changes in the

522 c. GILeFRT et al.

phosphorylation of the bands in the region of 55,000-65,000 and 70,000 mol. wt may be related and caused by the altered mobility of the same substrate due to its increased phosphorylation. The recent report of modification of the mobility of c-raf as a result of its phosphorylation (on serine and threonine residues, however) [49] provides additional support for the latter interpretation. Second, the tyrosine kinase inhibitor erbstatin diminished to a very large extent the priming effects of PDBu without affecting the basal response to fMet- Leu-Phe. This last set of data adds potential functional significance to the tyrosine phosphorylation data by tying them to the priming phenomenon.

Of potential relevance to the current findings are recent data demonstrating that the pre- incubation of human neutrophils with granulocyte-macrophage colony-stimulating factor also led to an increased mobilization of calcium [50] in the absence of a detectable activation of protein kinase C [51-55]. Evidence was also recently obtained suggesting that the effect of the growth factor was mediated by an up- regulation of phospholipase D [56]. Phorbol esters have been shown to stimulate phospholipase D in neutrophils [57] as well as in various other cell types [58]. Furthermore, the stimulation of the N A D P H oxidase has been related to the activation of phospholipase D [59]. These results raise the possibility that the priming of calcium mobilization and of the production of superoxide anions by phorbol esters may simi- larly be linked to a modulation of the activation of phospholipase D mediated by protein kinase C. The presently available results indicate that the further investigation of this possibility is warranted.

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CELLS 4:5-E

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Rapid priming of calcium mobilization and superoxide anion production in human neutrophils by substimulatory concentrations of phorbol esters: A novel role for protein kinase C and