Effect of protein kinase C inhibitors on calcium ionophore-induced arachidonic acid mobilization in human leukocytes

Effect of protein kinase C inhibitors on calcium ionophore-induced arachidonic acid mobilization in human leukocytes

Immunopharmacology, 16 (1988) 63 69 Elsevier 63 IMO00415 Effect of protein kinase C inhibitors on calcium ionophore-induced arachidonic acid mobili...

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Immunopharmacology, 16 (1988) 63 69 Elsevier

63

IMO00415

Effect of protein kinase C inhibitors on calcium ionophore-induced arachidonic acid mobilization in human leukocytes Gillian M.P. Galbraith Department of Microbiology and Immunology, 171 Ashley A venue, Medical University of South Carolina, Charleston, SC 29425, U.S.A. (Received 6 October 1987; accepted 26 May 1988)

Abstract: Arachidonic acid mobilization in human polymorphonuclear leukocytes stimulated with calcium ionophore A23187 was amplified by synthetic diacylglycerol and, to a much lesser extent, by phorbol ester. The effect was synergistic and dependent upon influx of calcium ions. Thin layer chromatographic analysis of phospholipids of stimulated cells revealed a loss of arachidonic acid associated with phosphatidylinositol and phosphatidylcholine. The synergistic response was unaffected by treatment of cells with two inhibitors of protein kinase C, namely, polymyxin B and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. Moreover, both agents consistently augmented the cellular response to A23187 alone. These findings suggest that A23187-induced arachidonic acid mobilization in leukocytes is independent of protein kinase C activity. Key words:

Leukocytes; Calcium ionophore; Diacylglycerol; Protein kinase C inhibitors; Arachidonic acid mobilization

Introduction

The release and metabolism of arachidonic acid (AA; 20:4, n-6) by cells exposed to appropriate external signals has been widely investigated. Calcium ionophores such as A23187 and ionomycin induce AA release in several cell types including polymorphonuclear leukocytes (PMN) (Borgeat and Samuelsson, 1979; Walsh et al., 1981; Godfrey et al., 1987), platelets (Rittenhouse and Horne, 1984), Correspondence: Gillian M.P. Galbraith, M.D., Department of Microbiology and Immunology, 171 Ashley Avenue, Medical University of South Carolina, Charleston, SC 29425, U.S.A. Abbreviations." AA, arachidonic acid; PMN, polymorphonuclear leukocytes; PLA2, phospholipase A2; PLC, phospholipase C; PKC, protein kinase C; H-7, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine; PXB, polymyxin B sulfate; PMA, phorbol 12-myristate 13-acetate; OAG, 1-oleoyl-2-acetyl-rac-glycerol; PBS, phosphate buffered saline; TLC, thin layer chromatography; PC, phosphatidylcholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; MMPE, monomethylphosphatidylethanolamine; LPC, lysophosphatidylcholine; fMLP, formylmethionylleucylphenylalanine.

monocytes and lymphocytes (Hoffman et al., 1987). This phenomenon has been ascribed to the activation of calcium-dependent phospholipase A 2 (PLA2). In addition, calcium ionophore treatment of PMN results in activation of phospholipase C (PLC) (Meade et al., 1986); the diacylglycerol produced by PLC-mediated degradation of phosphoinositides does not appear to be a major source of liberated AA in these cells (Meade et al., 1986). However, diacylglycerol is the physiological agonist of phospholipid/Ca2+-dependent protein kinase C (PKC) (Nishizuka, 1986) and activation of PKC does also occur in PMN and platelets exposed to calcium ionophore (Kang et al., 1985; Liles et al., 1987; Sano et al., 1985). The possibility that PKC activity may play a role in AA release is suggested by reports that A23187induced AA mobilization is enhanced in PMN by tumor-promoting phorbol esters (Volpi et al., 1985; McColl et al., 1986) and in platelets by synthetic diacylglycerols (Halenda et al., 1985), which directly activate PKC (Castagna et al., 1982; Nishizuka,

0162-3109/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

64 1984). Furthermore, in PMN treated with A23187, AA metabolism by the 5-1ipoxygenase pathway is also amplified by phorbol esters and diacylglycerol (Liles et al., 1987). The mechanisms by which PKC could modulate phospholipase activities, and thus AA release, remain undefined, but theoretically may reside in the PKC-mediated phosphorylation of associated proteins such as lipocortin, a putative endogenous PLA 2 regulatory protein (Hirata et al., 1985). Inhibition of PKC activity might therefore be anticipated to exert a secondary inhibitory effect on ionophoreinduced AA release. In the present study, this hypothesis was tested in normal human PMN by use of two agents known to inhibit PKC activity, namely, polymyxin B (Wrenn and Wooten, 1984) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (Hidaka et al., 1984). In contrast to expectation, both compounds consistently augmented AA mobilization in ionophore-treated PMN. Furthermore, neither agent inhibited the enhancement of AA release by diacylglycerol in such cells. These results suggest that ionophore-induced AA mobilization in human PMN is independent of PKC activity.

Materials and Methods

Reagents Calcium ionophore A23187 (free acid), phorbol 12myristate, 13-acetate (PMA), 1-oleoyl-2-acetyl-racglycerol (OAG), polymyxin B sulfate (PXB), 1-(5isoquinolinylsulfonyl)-2-methylpiperazine (H-7), and N-formylmethionylleucylphenylalanine (fMLP) were obtained from Sigma Chemicals, St. Louis, MO. [5,6,8,9,11,12,14,15-3H]AA, specific activity 163-196 Ci/mmol, was purchased from Amersham Corporation, Arlington Heights, IL and Econofluor scintillation fluid from New England Nuclear, Boston, MA.

Cell challenge procedures Peripheral heparinized venous blood was obtained with informed consent from normal healthy adult

volunteers. PMN suspensions were prepared by sequential dextran sedimentation, ficoll-hypaque density centrifugation, and hypotonic lysis of residual erythrocytes. The resulting 16reparation contained greater than 95% viable PMN. Aliquots of 1-2 x 1 0 7 PMN were incubated for 30 min at 37°C with 0.5 /~Ci [3H]AA sonicated in PBS, pH 7.2, for 10 min at 4°C, then washed three times in PBS. These cells were exposed to different concentrations and combinations of A23187, fMLP, PMA, OAG, PXB and H-7 or to buffer alone in a final volume of 1 ml, for different times, at 37°C in a humidified atmosphere containing 5% CO 2. The above agents were initially dissolved in DMSO and brought to final concentration in buffer consisting of 10 mM glucose, 150 mM NaC1, 3 mM KC1, 2 mM CaC12 and 5 mM Hepes, pH 7.4. In certain experiments, the buffer included 2 mM or 10 mM EDTA and no calcium. Upon completion of incubation procedures, cell suspensions were centrifuged at 800 x g for 10 min at 22°C. The cell supernatants were vortexed for 1 rain with 3 ml chloroform:methanol (2:1) and centrifuged at 800 x g for 10 min at 22°C. The chloroform phase of each mixture was retrieved, evaporated under a stream of nitrogen, redissolved in scintillation fluid and subjected to beta counting. The residual cell pellets were suspended in 0.5 ml methanol, dried on glass filter paper for 20 h at 22°C and subjected to beta counting. Alternatively, the pellets were subjected to lipid extraction according to Folch (1957) and examined by two-dimensional TLC as described below. All assays included control cells exposed to buffer alone but subjected to manipulations identical to those of experimental cells, in order to obtain baseline levels of release of radiolabelled products. Resuits were expressed as percent release relative to the total incorporated [3H]AA, or as cpm released.

Two-dimensional thin layer chromatograph), ( TLC) Chloroform extracts of cells were resolved by TLC on preformed silica gels (Analtech, Newark, DE) using chloroform:methanol:ammonium hydroxide (65:35:5.5) solvent for separation in the first dimen-

65 sion, and butanol:acetone:acetic acid:water (5:5:1:1) in the second d i m e n s i o n . The c h r o m a t o g r a m s were d e v e l o p e d with iodine v a p o r , a n d app r o p r i a t e areas o f silica were r e m o v e d a n d e x a m ined by liquid scintillation. L i p i d s t a n d a r d s included p h o s p h a t i d y l c h o l i n e , p h o s p h a t i d y l i n o s i tol, p h o s p h a t i d y l e t h a n o l a m i n e , m o n o m e t h y l p h o s phatidylethanolamine and lysophosphatidylcholine. Results were expressed as c p m a s s o c i a t e d with each p h o s p h o l i p i d examined. T o correct for variation between experiments, these d a t a were n o r m a l ized to the m e a n values o b t a i n e d with cells i n c u b a t ed with buffer alone.

Analysis of data D a t a o b t a i n e d in release e x p e r i m e n t s were a n a l y s e d for c o n f o r m a t i o n to a n o r m a l d i s t r i b u t i o n a n d c o m p a r e d by S t u d e n t ' s t test for p a i r e d samples.

Results

Release of radiolabelled products by PMN Cells prelabelled with [3H]AA a n d e x p o s e d to buffer, O A G , P M A , PXB, H-7 o r low m o l a r i t y (0.5/~M) A23187 a l o n e generally released 0 . 5 - 2 % o f incorp o r a t e d label into the s u s p e n d i n g m e d i u m . In contrast, c o i n c u b a t i o n o f P M N with 0.5 p M A23187

a n d O A G resulted in a release o f r a d i o a c t i v i t y which was O A G c o n c e n t r a t i o n - d e p e n d e n t , with significant increase in release being o b t a i n e d at 12.5 50 p M O A G (p < 0.01 c o m p a r e d to control, Stud e n t ' s t test; Fig. 1). T h e a p p a r e n t decrease in response at the higher O A G c o n c e n t r a t i o n s used was n o t statistically significant. The synergistic response to these agents was s u b s t a n t i a l l y reduced in the presence o f 2 m M E D T A (Fig. 1). In o t h e r experiments, P M N were e x p o s e d to different c o n c e n t r a tions o f A23187 in the presence o r absence o f 12.5 p M O A G . The i o n o p h o r e d o s e - d e p e n d e n t mobiliz a t i o n o f [3H]AA was a u g m e n t e d by 12.5 # M O A G at all A23187 molarities (Fig. 2). Similar studies in which P M A was used gave less consistent results. A t P M A c o n c e n t r a t i o n s between 25 a n d 200 n M , m i n o r e n h a n c e m e n t o f the A23187 response was o b s e r v e d with high m o l a r i t y (100-200 n M ) P M A ; higher c o n c e n t r a t i o n s o f P M A r e d u c e d cell viability. Thus, release o f ~H with 0.5 p M A23187 was increased in the presence o f 100 n M P M A from 2.4 + 0.5 to 3.5 • 0.7% (mean i S E M , n = 8). A n a l y s i s by S t u d e n t ' s t test revealed that this small difference was statistically significant (p < 0.05). Expression o f the same d a t a in terms o f net c p m released (for c o m p a r i s o n with results o f a previously r e p o r t e d study; M c C o l l et al., 1986) showed an increase from 252 + 113 to 995 + 251 c p m (mean + S E M , n = 8). In c o n t r a s t , P M N cos t i m u l a t e d with 0.5 p M A23187 a n d 12.5/tM O A G

30-

2O

2-

lO

0 10

20

30

4'0

50

OAG (t~M)

/ 1

Fig. [. Release of [3HIAA by PMN: dose-dependent enhancement by OAG of A23187 effect. Cells were treated for 10 min with 0.5 #M A23187 + 0-50/tM OAG ( x , mean + SEM, n = 4); 5 50 #M OAG alone (ll, mean, n = 2); or 0.5 #M A23187 + 5-50 pM OAG + 2 mM EDTA (O, mean, n = 2). Treatment with 0.5 pM A23187 alone resulted in 1.4 :k 0.3% release.

2

3

4

5

A23187 (~LM)

Fig. 2. Release of [3H]AA by PMN: effect of OAG on dose response to A23187. Cells were treated for 10 min with 0.5 5/~M A23187 alone (O, mean ± SEM, n = 4); or 12.5 pM OAG + 0.5-5 pM A23187 ( I , mean + SEM, n = 4). Treatmenl with 12.5 pM OAG alone resulted in 1.5 ± 0.[% release.

66 released 6062 + 1091 c p m (n = 6). In o r d e r to investigate the effect o f P K C inhibitors on the synergistic effect o f A23187 a n d O A G , P M N were e x p o s e d to these agents either in the presence o f P X B (12.5-100 /~M) o r following inc u b a t i o n o f cells with H-7 (15 120/~M) for 10 min at 37°C. N o difference in release o f r a d i o l a b e l l e d p r o d u c t s was found. H o w e v e r , b o t h P X B a n d H-7 a p p e a r e d to a u g m e n t 3H release by P M N stimulated with A23187 alone (Fig. 3). T h e e n h a n c e m e n t by these agents was d o s e - d e p e n d e n t , with m a x i m a l effect being o b t a i n e d at 100 tzM P X B a n d 60/~M H-7 (not shown). In c o n t r a s t , b o t h agents at these conc e n t r a t i o n s significantly inhibited f M L P - i n d u c e d [3H]AA m o b i l i z a t i o n . Thus, P M N e x p o s e d to l0 n M f M L P released 7.2 + 0.5% i n c o r p o r a t e d 3H; this was r e d u c e d to 2.0 4- 0 . 7 % in the presence o f 100/~M PXB, a n d to 2.7 4- 0.5% in cells preincub a t e d with 60/~M H-7 ( m e a n 4- S E M , n = 6, p < 0.001, S t u d e n t ' s t test).

Distribution of FH]AA in PMN phospholipids T w o - d i m e n s i o n a l T L C o f F o i c h - e x t r a c t e d P M N revealed t h a t [3H]AA was i n c o r p o r a t e d p r i m a r i l y into p h o s p h a t i d y l c h o l i n e a n d p h o s p h a t i d y l i n o s i t o l (Table 1). E x p o s u r e o f cells to 12.5 # M O A G o r 0.5/~M A23187 a l o n e resulted in no a p p r e c i a b l e loss o f radiolabei f r o m the P M N p h o s p h o l i p i d s . In c o n t r a s t , 14" 1210-

6" ~;

4" 2" 00 1

2A23187 ( ~

4

5

Fig. 3. Release of [3H]AA by PMN stimulated with A23187: effect of PXB and H-7. Cells were treated for 10 min with 0.5-5 ktM A23187 alone ([], mean + SEM, n = 4), with ionophore in the presence of 100/~M PXB, (11, mean + SEM, n = 4) or with ionophore following 10 min preincubation with 60/~M H-7 ( x, mean J: SEM, n = 4). Treatment with 100/~M PXB alone or 60 pM H-7 alone, resulted in 1.2 + 0.2 and 0.8 ± 0.5% release, respectively.

e x a m i n a t i o n o f cells c o i n c u b a t e d with 12.5 /~M O A G a n d 0.5 ~ M A23187 revealed a r e d u c t i o n o f r a d i o l a b e l associated with p h o s p h a t i d y l i n o s i t o l a n d p h o s p h a t i d y l c h o l i n e ; similar results were f o u n d in cells s t i m u l a t e d with an o p t i m a l c o n c e n t r a t i o n (5 /zM) o f i o n o p h o r e a l o n e (Table I).

Discussion The synergistic b e h a v i o r o f calcium i o n o p h o r e s a n d p h o r b o l esters or diacylglycerols has been a m p l y d e m o n s t r a t e d in studies o f different cell functions in several cell types, a n d is generally c o n s i d e r e d to reflect a response r e q u i r e m e n t for two signals - calcium m o b i l i z a t i o n a n d P K C a c t i v a t i o n which are p h y s i o l o g i c a l l y p r o v i d e d by inositol t r i p h o s p h a t e a n d diacylglycerol, the p r o d u c t s o f m e m b r a n e rec e p t o r - m e d i a t e d P L C a c t i v a t i o n a n d hydrolysis o f p h o s p h o i n o s i t i d e s ( C a s t a g n a , 1987). In the present study, O A G , a synthetic diacylglycerol, a u g m e n t e d A A m o b i l i z a t i o n in h u m a n P M N s t i m u l a t e d with the calcium i o n o p h o r e A23187. This e n h a n c e m e n t a p p e a r e d to be d e p e n d e n t on influx o f calcium, since it was a b r o g a t e d in the presence o f 2 m M E D TABLE I Distribution of [3H]AA in PMN phospholipids Stimulus

PC

PI

PE

Buffer (n - 5) 0.5 #M A23187 (n = 4) 12.5 #M OAG (n = 4) 0.5/~M A23187 + 12.5/~M OAG (n = 3) 5 pM A23187 (n = 3)

5106 + 1071 7017 :t: 1029 2283 :t: 362 4911 5_ 120

7798 ± 795

3100 4- 522

3930 ± 765

7830 ± 913

1933 + 276

2794 + 96

3066 ± 203

2648 ± 71

2468 4- 239

3567 :t: 365

1646 :t: 79

Two-dimensional TLC was performed on extracts of stimulated PMN as described in the text. Results are expressed as cpm, mean ± SEM for indicated number of experiments. Lysophosphatidylcholine and monomethylphosphatidylethanolamine together incorporated < 2% of label; for simplicity, these data are not shown.

67 TA. The effect was synergistic, in that substantial release of radiolabelled products occurred in cells treated with concentrations of A23187 and OAG insufficient to effect release by each agent alone. Thin-layer chromatography revealed that the release of radioactivity into the medium was mirrored by a loss of radiolabel from PMN phosphatidylinositol and phosphatidylcholine. Although OAG markedly augmented ionophoremediated [3H]AA mobilization, the enhancement obtained with PMA was minor and less consistent. These findings appear to conflict with those of previous investigators who reported synergism between A23187 and PMA in similar studies (Volpi, et al., 1985; McColl et al., 1986). However, comparison of present data with those of McColl et al., who also studied human PMN, revealed marked similarity. Thus, in the present study, mean net 3H release was increased from 252 to 995 cpm upon costimulation of PMN with 0.5/~M A23187 and I00 nM PMA; McColl et al. reported an increase from 570 to 1530 cpm in similar assays. The reasons for the greater efficacy of OAG in this system are not clear. Both OAG and phorbol esters activate PKC directly and increase the affinity of the enzyme for calcium (Castagna, 1987). However, OAG is more rapidly metabolized and does not possess tumor-promoting properties, and thus its action may be considered to mimic more closely naturally produced diacylglycerol. Although the synergism between ionophore and diacylglycerol or phorbol esters is not fully understood, current evidence suggests that each may potentiate the action of the other. For example, the action of the ionophore may satisfy the calcium requirement of PKC, and A23187 has been shown to increase the affinity of interaction between PKC and its agonist in a calcium-dependent manner (French et al., 1987). Conversely, PKC has been implicated (Hirata et al., 1984) in the phosphorylation and subsequent inactivation of lipocortin which in certain cell types appears to inhibit the activity of PLA 2 (Hirata et al., 1985). This reasoning necessarily assumes that PKC plays a primary role in cellular responses elicited by diacylglycerol and calcium ionophores.

In order to test the hypothesis that PKC activity may be involved in ionophore-induced AA release by PMN, the effects of two PKC inhibitors were investigated. The results indicated that both PXB and H-7 augmented the response to A23187 in a dose-dependent manner, and neither agent modulated the synergistic effect of A23187 and OAG. In contrast, these agents significantly inhibited AA mobilization in PMN stimulated with the chemotactic peptide, fMLP. These differential effects of PKC inhibitors may reflect the fact that although phospholipase C activation occurs in cells stimulated with either fMLP or A23187, the response to fMLP is mediated by a receptor-ligand interaction. The mechanisms responsible for the potentiating effects of PXB and H-7 in ionophore-induced AA mobilization in PMN are unknown. PXB is an amphipathic peptolipid which associates with phospholipids and therefore may alter the phospholipase substrate (Theretz et al., 1984). In addition, PXB has been shown to enhance calcium influx in cultured cells (Burt et al., 1983) which could account for augmentation of an ionophore effect. However, H-7 is a potent and relatively selective inhibitor of PKC, which not only suppressed fMLP-induced AA mobilization in this study, but also has been shown to inhibit both PKC activity and A23187-induced superoxide formation in human neutrophils under experimental conditions similar to those described here (Fujita et al., 1986), Thus, the results of the present study can be interpreted in several ways. First, if PKC is involved in AA mobilization by A23187, then the enzyme involved is unresponsive to PXB or H-7. Several molecular species of PKC exist (Coussens et al., 1986); this may account for differences in protein phosphorylation patterns observed with diacylglycerol and phorbol esters (Castagna, 1987). Second, other species of PKC which are inhibited by these agents may be responsible for the phosphorylationactivation of (unidentified) regulatory proteins, which could account for the enhancement of AA mobilization by PXB and I-t-7. A comparable scenario has been described in platelets, in which phorbol ester-induced serotonin release is enhanced by H-7 by means of selective inhibition of phosphory-

68 lation of myosin light chain (Inagaki et'al., 1984). A final alternative is that in human PMN, AA mobilization in response to calcium ionophore and its augmentation by diacylglycerol are not dependent upon PKC activation.

Acknowledgements I thank Kathy Haines for expert technical assistance. This work was supported in part by South Carolina Appropriations for Biomedical Research.

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