Inhibitory effects of N-acetylcysteine on superoxide anion generation in human polymorphonuclear leukocytes.
It has been suggested that reactive oxygen species released by activated polymorphonuclear leukocytes (PMN) in man is one mechanism of tissue injury. Therapeutic action aimed at increasing antioxidant defence mechanisms is still a clinical challenge. This study examines the activity of N-acetylcysteine, a known antioxidant, in the protection of PMN exposed in-vitro to the chemoattractant peptide fMet-Leu-Phe (FMLP), the protein kinase C activator phorbol myristate acetate or the lipid peroxidation promoter t-butyl hydroperoxide. FMLP (3-300 nM) and phorbol myristate acetate (160 pm-160 nM) induced concentration-related superoxide anion generation. Pre-treatment with N-acetylcysteine (33-333 microM) resulted in concentration-related inhibition of superoxide production induced by FMLP (30 nM) or phorbol myristate acetate (16 nM);-log IC50 values were 3.97 +/- 0.07 and 3.91 +/- 0.10, respectively. Changes in intracellular calcium ion concentration ([Ca2+]i) induced by FMLP (30 nM) were studied in fura-2-loaded human PMN. FMLP produced a transient calcium response, i.e. a peak followed by decay to a residual value above baseline. N-Acetylcysteine (333 microM) did not affect either basal [Ca2+]i values or changes in [Ca2+]i values after treatment with FMLP. Activation by phorbol myristate acetate caused a reduction in glutathione levels from 5.94 +/- 0.86 (control) to 1.84 +/- 0.51 nmol/3 x 10(6) cells (P < 0.05 compared with control). Pre-treatment with N-acetylcysteine (333 microM) fully reversed the reduction in glutathione levels induced by phorbol myristate acetate (4.83 +/- 0.68 nmol/3 x 10(6) cells; P > 0.05 compared with control). Exposure to t-butyl hydroperoxide (0.5 mM, 30 min) markedly increased malondialdehyde levels (from 0.03 +/- 0.02 to 0.73 +/- 0.07 nmol/10(6) cells), and index of lipid peroxidation. Malondialdehyde levels were significantly reduced in PMN treated with N-acetylcysteine (333 microM; 0.55 +/- 0.04 nmol/10(6) cells; P < 0.05 compared with untreated cells exposed to t-butyl hydroperoxide). In conclusion, N-acetylcysteine reduces superoxide generation in response to FMLP and phorbol myristate acetate and partially protects against lipid peroxidation in PMN from man. The protection afforded by N-acetylcysteine is not related to alteration of the intracellular calcium signal but might be effected by replenishment of the intracellular glutathione levels.
Villagrasa
V
V
Departamento de Farmacología, Facultad de Medicina y Odontología, Universitat de València, Spain.
Cortijo
J
J
Martí-Cabrera
M
M
Ortiz
J L
JL
Berto
L
L
Esteras
A
A
Bruseghini
L
L
Morcillo
E J
EJ
D000111:Acetylcysteine / Q000494:pharmacology*; D000838:Anions; D002118:Calcium / Q000097:blood; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000097:blood; D006801:Humans; D015227:Lipid Peroxidation / Q000187:drug effects; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism*; D010545:Peroxides / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D013481:Superoxides / Q000097:blood*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020122:tert-Butylhydroperoxide
D000838:Anions; D010545:Peroxides; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D020122:tert-Butylhydroperoxide; D011493:Protein Kinase C; D005978:Glutathione; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D000111:Acetylcysteine