PEROXYNITRITE FORMATION FROM ACTIVATED HUMAN-LEUKOCYTES

We showed direct evidence of peroxynitrite formation from polymorphonu clear cells (PMN) with the nitration of 4-hydroxpphenylacetic acid (HP A) to 4-hydroxy-3-nitrophenylacetic acid (NO(2)HPA). Human PMN from he althy volunteers was stimulated with phorbol-12-myristate-13-acetate ( PMA, 10 ng/ml) at 37 degrees C in 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid-buffered Hank' s balanced salt solution (pH 7.4) w ith HPA (1 mM). NO(2)HPA was detected under PMA stimulation only in th e presence of myeloperoxidase inhibitor. NO(2)HPA was eliminated by N- monomethyl-L-arginine (100 mu M). The inhibition of myeloperoxidase ap pears to be essential to demonstrate the production of NO(2)HPA since myeloperoxidase itself or its product, hypochlorite, reacted with pero xynitrite and hampered the formation of NO(2)HPA. (C) 1996 Academic Pr ess, Inc.