PAF-induced chemiluminescence in guinea-pig macrophages is mediated by oxygen radicals and inhibited by RP 59227, a PAF antagonist

692

F&h, ~~~-P~~~

A., Cormao, C. and Cavero, I.

San@ CRVA, 13, quui ~~~-~~,

BP t4,94403

Vitqw&-Seince

&e&x, France

~a~~p~ag~ possess platelet activating factor (PAF)receptors, the stimulation of which leads to the generation of

1985). These energetic species produce a chemil~escence phenomenon (CL) by ~=cterize the PAF-induced CL response in sea-pig of this pre~n~tion is to n,h,__. macrophages and to determine its mechanism. Male mea-pigs were given mineral oil i-p. (10 ml) and 2 to 8 days later the macrophages which had migrated into the peritoneal cavity were harvested. Optimal conditions for CL ~n~n~a~on-r~~nse curves to PAF (1.6-1600 nM) were found to be 5 x 106 macrophages/ml harvested on the 4th day and suspended in Hanks balanced salt solution, to which 100 FM 1~01 and 0.25 BSA were added. The role of oxygen radicals in CL responses to PAF (16 nM: concentration producing approximately 80% of the maximal effect) was evaluated by studying the effects of superoxide dismutasc (SOD) (C$ scavenger), catalase (IQ& scavenger) and defe~x~~e (iron ion chelating agent). Each reported response was obtained in at least’5 independent preparations. Under the present experimental conditions, the EC, for PAF was found to be 4.3 10.5 nM. SOD, catalase and deferoxamine concentration dependently inhibited the effects of PA with IC,, values of 0.87 f 0.09 U/ml, 5434 f 1436 U/ml and 29 j, 4 PM, respectively. RP 59227, an antagonist of PA-induced human platelet (PRP) aggregation @A, = 7.02 f 0.04) was a potent inhibitor of PAF-induced chemiluminescencc (I&, = 6.5 f 1.3 nM). Furthermore, PP 59227 (Marquis et al., 1989) as well as another PA antagonist, 2086 (Casals-Stenzel et al., 1987) depressed the maximum of the ~n~ntration-r~~nse curve to PA. Their calculated pD,p values were 7.7 and 8.1, resp~tively. Zpowever,the noncompetitive antagonism of PP 59227 was reversible since the inhibitory effects vis-i-vis PA were reduced by 75% after washing macrophages exposed to PP 59227 twice with fresh Hanks balanced salt solution. In conclusion, this study indicates that chemihuniuescence is a suitable parameter for measuring PA-evoked activation of macrophages. This phenomenon is due to formation of oxygen radicals which are very reactive species that can mediate cellular damage, particularlyin ischemic tissues. Consequently, blockade of PAF receptors with PP 59227 can afford a new therapeutic approach to prevent deleterious effects due to oxygen radicals generated by PA which is released during rny~~~~ or cerebral i~he~a and perfusion.

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Hayashi,H. et al., 1985,J. Biochem.97,1737. Marquis,0. et al., 1989, J. Pbarrnacol.m_ T&r. 250,293. CasabStml,

J. et al., 1987, J. Phamacd. Exp. Ther. 241,374.

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Santus *, R., Perdrix * *, L., Labtid * *, C., Morli&e * * *, P. and Mazitk *, J.C. * MuGum National d%listoire NaturelIe* Loborotoire de Physicochimie de I’Adaptation Biologique (INSERM U312). Paris, * * IRIS 24 Rue du Pant N~~I~/Sei~e

and * * * C.H. U. Henri ~ondoF, ~~ratoire

de ~~rrnat~og~e (INSERM

U312) Cr&eil; France

The protectiveeffect of S 5682 (flavonoid fraction, ~cro~s~, consisting of 90% diosmin and 10% h~pe~din~ against lipid peroxidation has been compared to that of vitamin E (Vit E). At 10 pM, S 5682 inhibits 40% of the