80 is a potent inhibitor of phospholipase C and a dual modulator of phospholipase A2 from human platelet

Biochimica et Biophysics Acta 920 (1987) 301-305

301

Elsevier

BBA Report

BBA 50208

Compound 48/80 is a potent inhibitor of phospholipase C and a dual modulator of phospholipase A 2 from human platelet Christian Bronner a, Charles Wiggins b, Didier Mont6 ‘, Fritz Mtirki d, AndrC Capron ‘, Yves Landry a and Richard C. Franson b ’ Laboratoire d’Allergopharmacoiogie, Universitk Louis Pasteur, Strasbourg (France) b Department of Biochemistry Medical College of Virginia, Richmond, VA (U.S.A.), ’ Centre d’lmmunologie et de Biologie Parasitaire, CJnitPMixte INSERM U 167~CNRS 624, Lille (France) d Pharmaceutical Research Department, Ciba-Geigy, Base1 (Switzerland) (Received

Key words:

Compound

48/80;

Phospholipase

17 March

1987)

C; Phospholipase

A 2; Platelet aggregation;

(Human)

Compound 48/80 inhibited phosphatidylinositol-specific phospholipase C activity from human platelets. Whereas 1 gg/ml of compound 4t3/80 slightly stimulated Ca2+-dependent phospholipase A,, higher concentrations led to dose-dependent inhibition of this platelet enzyme. This biphasic effect was confirmed with phospholipases A, purified from rat liver and human synovial fluid. The aggregation of human platelets induced by ADP and PAF-acether was inhibited by compound 48/80, whereas the aggregation induced by ionophore A23187 was not modified by this compound. These results demonstrate that the inhibition of platelet aggregation by compound 48/80 is not due solely to effects on calmodulin as previously reported, but that inhibition of phospholipases and probably arachidonate mobilization may also be involved.

Compound 48/80 is a mixture of condensation products of N-methyl-p-methoxyphenethylamine with formaldehyde [l]. Described as a potent histamine liberator [2], compound 48/80 is now considered to be a selective inducer of histamine release from serosal as opposed to mucosal mast cells [3]. The biochemical mechanisms of this effect remains unknown. Compound 48/80 was more recently shown to inhibit calmodulinactivated enzymes with a low efficiency on the basal enzyme activities [4,5] and was thus proposed as a specific tool for studying the involve-

Abbreviations: PAF-acether, adenosine diphosphate.

platelet

activating

factor;

ADP,

Correspondence: Y. Landry, Laboratoire d’Allergopharmacologie, UniversitC Louis Pasteur, B.P. lo,67048 Strasbourg Cedex, France.

0005-2760/87/$03.50

0 1987 Elsevier Science Publishers

ment of calmodulin in biological functions [4,5], as the use of other calmodulin inhibitors has been questioned [6,7]. The ability of compound 48/80 to inhibit platelet aggregation has also been ascribed to the inhibition of calmodulin-dependent processes [8]. The aggregating agents used in this latter study were ADP, thrombin, collagen and PAF-acether, i.e., ligands of plasma membrane receptors. We have also studied the effects of 48/80 on platelets and obtained an inhibition of the second phase of aggregation induced by ADP and PAF-acether (Fig. 1). Previous results [8] showed the full inhibition by compound 48/80 of the effect of ADP. Compound 48/80 did not affect platelets in the absence of inducers of aggregation. Moreover, Fig. 1 shows that the aggregation induced with A23187, a divalent cation ionophore [9], was not affected by compound 48/80. The second wave of ADPinduced aggregation has been shown to be related

B.V. (Biomedical

Division)

302

A23187

1

25

10 Wml

[Compound U/801

(pg/ml)

PAF-ACETHER

0

25

50 I-

75I-L

Control 1

0

1

I

12

Time

I

I

3

4

after

0

12

induction

3

4

(min)

Fig. 1. Effect of compound 48/80 on the aggregation of human platelets induced by ADP. PAF-acether and ionophore A23187. Platelet-rich plasma (PRP) was prepared by centrifugation (100x g, 15 min) of titrated blood obtained from healthy volunteers. Each test tube was filled with 400 ~1 platelet-rich plasma and placed at 37 o C in a dual-channel aggregometer (Chrono-Log-Corporation) for 5 min. Compound 48/80 (10 ~1) was preincubated 5 min before adding the aggregating agents. No aggregation was observed during this preincubation. Representative tracings of the aggregation of human platelets in the absence (control) or presence of 10 or 50 pg/ml compound 48/80 are shown in the upper left and lower panels. Compound 48/80 slightly modified the kinetics of aggregation induced by ionophore A23187 but did not modify its intensity. The dotted line corresponds to the control. Dose-effect curves for compound 48/80 on platelet aggregation induced by ionophore 5 PM A23187 (m); 10 FM PAF-acether (0) and 2.5 PM ADP (A), are shown in the upper right panel. Values are means of three experiments performed in duplicate and expressed as percentage of control in the absence of compound 48/80.

to the secondary release of arachidonic acid metabolites due to the activation of phospholipase A, (Ref. 10 for review). The ionophore A23187 is usually considered to bypass receptor-dependent coupling phenomena. In platelets, A23187 induces the phosphorylation of 20 and 40 kDa proteins

[ll]. These proteins have been identified as myosin light chain [12] and lipocortin, an anti-phospholipase A, protein [13], which are the substrates of a specific calmodulin-dependent protein kinase and of protein kinase C, respectively. Moreover, a recent study [14] showed that A23187 induces

phosphorylation of the 40 kDa protein in the absence of phospholipase C activation. This provides evidence that an increase of intracellular calcium can activate protein kinase C without the formation of 1,2-diacylglycerol by phospholipase c [14]. Altogether, these results suggested that phospholipase A, and phospholipase C might be involved, besides calmodulin, in the effects of compound 48/80 on platelet aggregation. Phospholipase C is predominantly localized in the supernatant fraction of sonicated human platelets, but has also been found in the particulate fraction [15,16]. The importance of enzyme localization in platelet aggregation remains unclear. Fig. 2 shows that compound 48/80 inhibits both cytosolic and particulate phosphatidylinositol-specific phospholipase C activities with a similar efficiency; IC,, values were 2.1 pg/rnl (supernatant) and 5.0 pg/ml (particulate fraction). The concentrations of compound 48/80 required to observe platelet aggregation were slightly higher. This might be due to the polymeric structure of compound 48/80, hindering its incorporation into platelet membranes. Interestingly, incubations with phospholipase A, from human platelet purified 3500fold demonstrate that the neutral-active and Ca2+-dependent phospholipase A, was inhibited to the same extent by compound 48/80 (IC,, = 2.2 pg/ml). A significant stimulatory effect was observed with 1 pg/ml of compound 48/80. We examined the effect of compound 48/80 on phospholipases A 2, isolated from other sources (Fig. 3). Ca*+-dependent neutral-active phospholipase A 2 purified 12 OOO-foldfrom human synovial fluid was stimulated at concentration no greater than 6 pg/ml, while higher concentrations were inhibitory. By contrast, acid-active and ion-independent phospholipase A, from rat liver lysosomes was slightly stimulated by low concentrations of compound 48/80, while concentrations greater than 0.2 pg/ml produced dose-dependent inhibition (IC,, = 0.9 pg/ml). In contrast to the phosphoinositol-specific phospholipase C, an acid-active and nonspecific phospholipase C (sphingomyelinase) was insensitive to compound 48/80 (Fig. 3). The variable effects of compound 48/80 on in vitro phospholipase A, and phosphatidylinositol-specific phospholipase C activities are not

2

e t;

lou

8

“0

Fig. 2. Effect of compound 48/80 on phosphatidylinositolspecific phospholipase C and phospholipase A, activities from human platelets. Phospholipase C activities were measured in the soluble (0) and the particulate (0) fractions. Platelets were suspended in 10 mM Tris-maleate buffer (pH 6.0) and sonicated for twice 20 s with a Branson sonifier. The homogenate was centrifuged at 105 000 X g for 1 h at 4’ C. The supematant was stored at -20 o C until measurement. The particulate fraction was resuspended in the buffer, homogenized with a Potter-Elvehjem homogenizer, and centrifuged as above. This procedure was repeated twice. The last pellet was resuspended in buffer and stored at - 20 ’ C in order to test the particulate activity. Phospholipase C activity was assayed in 1 ml of Tris-maleate 25 mM (pH 6.0) with 0.2 mM CaCl, according to Mauco et al. [U]. The substrate was a mixture of 2.5 nCi/ml of phosphatidyl[‘4C]inositol (Amersham, 270 mCi/mmol) and 20 nmol/ml of unlabelled phosphatidylinositol (Sigma) which were evaporated to dryness under nitrogen and sonicated in water just prior to use. Reactions were started by addition of 0.1 ml of the platelet fraction and were stopped 10 min later by adding 0.05 ml of 0.5 M HCl and 5 ml of chloroform/methanol (2 : 1, v/v). The tubes were mixed vigorously and centrifuged to separate the aqueous phase. r4C was counted in this phase in order to measure soluble inositol phosphate released during the reaction. Specific activities in the absence of inhibitor were 6.9 (supematant) and 4.4 (particulate fraction) mnol/min per mg protein. Human platelet phospholipase A, was purified 3500-fold as described in Ref. 17 and its activity (A) was measured in the presence of compound 48/80 by using l-[l14C]oleate-labelled autoclaved Escherichia coli [18]. Values are means of three experiments performed in duplicate and expressed as percent of control in the absence of compound 48/80. The asterisks indicate significant potentiation of phospholipase A, activity by compound 48/80 (P < 0.05).

readily explained by the pH or ion requirements nor the extent of purification of the enzymes. These data point to a complex interaction of drug

\

n

b l

but more recent studies show that the calcium regulation of rat and human phospholipase A, is independent of calmodulin [28]. The stimulatory effect at low concentrations of compound 48/80 may be of interest considering the role of phospholipase A, in the drug mediated stimulation of secretion in mast cells. The authors wish to thank Dr M. Joseph from the Centre d’Immunologie et de Biologie Parasitaire Lille, and Dr M. Chignard from the Institut Pasteur, Paris, for helpful discussions. This work was supported in part by grants from the CNRS (ATP 3385) and from the INSERM (CRE 86-5009), the Fondation pour la Recherche MCditale and Ciba-Geigy Limited, Basel, Switzerland. References

[Compound

48/801

(pg/ml)

Fig. 3. Effect of compound 48/80 on human synovial fluid A, and phosphohpase A 2, rat liver lysosol phospholipase sphingomyelinase (PLC) activities. Human synovial fluid phospholipase was 12000-fold purified as described in Ref. 18. Rat-liver lysosol phospholipase A, activities were determined by using I-[1-r4C]oleate-labelled autoclaved Escherichia coli as previously described [19] in the presence of different doses of compound 48/80, for synovial fluid phospholipase A, (0) rat liver lysosol phospholipase A, (A). Sphingomyelinase activity (m) was measured by an established method [20], using [Nmerhy/-‘4 Clsphingomyelin. Values are means of two experiments performed in duplicate and expressed as percent of control in the absence of compound 48/80. The asterisks indicate significant potentiation of phospholipase A z activity by campound 48/80 ( * P < 0.05, * * P < 0.01).

with enzyme or lipid substrate which may not only influence in vitro phospholipase activity but also platelet aggregation. Thus, compound 48/80 can no longer be considered a specific tool for studying calmodulin-dependent processes. Indeed, similar concentrations of this compound inhibit phosand phosphadiylinositol-specific pholipase A, phospholipase C as well as calmodulin-dependent enzymes. Similar results were obtained for the calmodulin antagonist trifluoperazine, which also nonspecifically inhibits phospholipase activities [21-231 and platelet aggregation [21,24-261. PreviA, were p hospholipases ously, Ca *+-dependent considered calmodulin-dependent enzymes [27],

1 Baltzly, R., Buck, J.S., DeBeer, E.J. and Webb, F.J. (1949) J. Am. Chem. Sot. 71, 1301-1305 2 Paton. W.D.M. (1951) Br. J. Pharmacol. 6, 499-508 P. and 3 Befus, A.D., Pearce, F.L., Gauldie, J., Horsewood, Bienenstock, J. (1982) J. Immunol. 128, 2471-2480 4 Gietzen, K. (1983) Biochem. J. 216, 611-616 5 Gietzen, K., Adamczyk-Engelmann, P.. Wiithrich, A.. Konstantinova, A. and Bader, H. (1983) B&him. Biophys. Acta 736, 109-118 6 Roufogalis, B.D. (1981) Biochem. Biophys. Res. Commun. 98, 607-613 7 Landry, Y., Amellal, M. and Ruckstuhl, M. (1981) Biothem. Pharmacol. 30, 2031-3032 R.E. (1984) Thromb. Res. 8 Heimich, J. and Zimmermann, 36, 475-480 9 Reed, P.W. and Lardy, H.A. (1972) J. Biol. Chem. 247, 6970-6977 B.B., Chignard, M. and Benveniste. J. (1981) 10 Vargaftig, Biochem. Pharmacol. 30, 263-271 K., Takai, Y., Sawamura, M., Moshijima, M., 11 Kaibuchi, Fujikura, T. and Nishizuka, Y. (1983) J. Biol. Chem. 258, 6701-6704 12 Daniel, J.L., Halmsen, H. and Adelstein, R.S. (1977) Thromb. Haemost. 38, 984-989 13 Touqui, L., Rothhut, B., Shaw, A.M., Fradin, A., Vargaftig, B.B. and Russo-Marie, F. (1986) Nature 321, 177-180 14 Lapetina, E.G., Reep, B. and Watson, S.P. (1986) Life Sci. 39, 751-759 L. (1979) FEBS 15 Mauco, G., Chap, M. and Douste-Blazy, Lett. 100, 367-370 E.G. (1983) Biochim. Biophys. 16 Siess, W. and Lapetina, Acta 752, 329-338 R.C. (1979) Biochim. Biophys. 17 Jesse, R.L. and Franson, Acta 575, 467-470 18 Fawzy, A.A. and Franson, R.C. (1986) Biophys. J. 49, 533a R., Patriarca, P. and Elsbach, P. (1974) J. Lipid 19 Franson, Res. 15, 380-388

305 20 Hysmith, R.M. and Franson, R.C. (1982) B&him. Biophys. Acta 711, 26-32 21 Walenga, R.W., Opas, E.E. and Feinstein, M.B. (1981) J. Biol. Chem. 256, 12523-12528 22 Thakkar, J.K., East, J. and Franson, R.C. (1984) Biol. Reprod. 30, 679-686 23 Bartolf, M. and Franson, R.C. (1984) Biochim. Biophys. Acta 793, 379-386 24 Kindness, G., Williamson, F.B. and Long, W.F. (1980) Thromb. Res. 17, 549-554

25 Rao, G.H.R., Reddy, K.R. and White, J.G. (1980) Prostaglandins Med. 5, 221-234 26 White, G.C. and Raynor, S.T. (1980) Thromb. Res. 18, 279-284 27 Wong, P.Y.-K. and Cheung, W.Y. (1979) B&hem. Biophys. Res. Commun. 90, 473-480 28 Withnall, M.T., Brown, T.J. and Diocee, B.K. (1984) Biothem. Biophys. Res. Commun. 121, 507-513