[en] Sphingolipids inhibit the activation of the neutrophil (PMN) NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR) and chemiluminescence (CL), we studied the effects of sphingosine (SPN) and ceramide analogues on phorbol 12-myristate 13-acetate (PMA, 5 x 10(-7)M) stimulated PMN (6 x 10(6) cells). By ESR with spin trapping (100 mM DMPO: 5,5-dimethyl-1-pyrroline-Noxide), we showed that SPN (5 to 8 x 10(-6)M), C(2)-ceramide (N-acetyl SPN) and C(6)-ceramide (N-hexanoyl SPN) at the final concentration of 2 x 10(-5) and 2 x 10(-4)M inhibit the production of free radicals by stimulated PMN. The ESR spectrum of stimulated PMN was that of DMPO-superoxide anion spin adduct. Inhibition by 5 x 10(-6)M SPN was equivalent to that of 30 U/ml SOD. SPN (5 to 8 x 10(-6)M) has no effect on in vitro systems generating superoxide anion (xanthine 50 mM/xanthine oxidase 110 mU/ml) or hydroxyl radical (Fenton reaction: 88 mM H(2)O(2), 0.01 mM Fe(2+) and 0.01 mM EDTA). SPN and N-acetyl SPN also inhibited the CL of PMA stimulated PMN in a dose dependent manner (from 2 x 10(-6) to 10(-5)M), but N-hexanoyl SPN was less active (from 2 x 10(-5) to 2 x 10(-4)M). These effects were compared with those of known PMN inhibitors, superoxide dismutase, catalase and azide. SPN was a better inhibitor compared with these agents. The complete inhibition by SPN of ESR signal and CL of stimulated PMN confirms that this compound or one of its metabolites act at the level of NADPH-oxidase, the key enzyme responsible for production of oxygen-derived free radicals.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Mouithys-Mickalad, Ange ; Université de Liège - ULiège > Centre de l'oxygène : Recherche et développement (C.O.R.D.)
Deby, Ginette ; Université de Liège - ULiège > Centre de l'oxygène : Recherche et développement (C.O.R.D.)
Hoebeke, Maryse ; Université de Liège - ULiège > Département de physique > Spectroscopie biomédicale
Mathy, Marianne ; Université de Liège - ULiège > Département des sciences de la motricité > Unité de recherche sur l'os et le cartillage (U.R.O.C.)
Lamy, Maurice ; Centre Hospitalier Universitaire de Liège - CHU > Anesthésie et réanimation
Deby, C.
Language :
English
Title :
Effects of sphingosine and sphingosine analogues on the free radical production by stimulated neutrophils: ESR and chemiluminescence studies.
Publication date :
1997
Journal title :
Mediators of Inflammation
ISSN :
0962-9351
eISSN :
1466-1861
Publisher :
Hindawi Publishing Corporation, Sylvania, United States - Ohio
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