Characterization of calcineurin in human neutrophils. Inhibitory effect of hydrogen peroxide on its enzyme activity and on NF-kappaB DNA binding.
We describe here a specific calcineurin activity in neutrophil lysates, which is dependent on Ca2+, inhibited by trifluoroperazine, and insensitive to okadaic acid. Immunoblotting experiments using a specific antiserum recognized both the A and B chains of calcineurin. Neutrophils treated with cyclosporin A or FK 506 showed a dose-dependent inhibition of calcineurin activity. The effect of oxidant compounds on calcineurin activity was also investigated. Neutrophils treated with hydrogen peroxide (H2O2), where catalase was inhibited with aminotriazole, exhibited a specific inhibition of calcineurin activity. However, the addition of reducing agents to neutrophil extracts partially reversed the inhibition caused by H2O2. A similar inhibitory effect of H2O2 on calcineurin activity was observed to occur in isolated lymphocytes. This is the first demonstration that redox agents modulate calcineurin activity in a cellular system. In addition, electrophoretic mobility shift assays revealed that lipopolysaccharide-induced activation of NF-kappaB in human neutrophils is inhibited by cell pretreatment with H2O2 in a dose-dependent manner. These data indicate that calcineurin activity regulates the functional activity of lipopolysaccharide-induced NF-kappaB/Rel proteins in human neutrophils. These data indicate a role of peroxides in the modulation of calcineurin activity and that the H2O2-dependent NF-kappaB inactivation in neutrophils occurs in concert with inhibition of calcineurin.
Carballo
M
M
Departamento de Bioquímica Médica y Biología Molecular, Facultad de Medicina, Universidad de Sevilla, 41009 Seville, Spain.
Márquez
G
G
Conde
M
M
Martín-Nieto
J
J
Monteseirín
J
J
Conde
J
J
Pintado
E
E
Sobrino
F
F
D019703:Calcineurin / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007166:Immunosuppressive Agents / Q000494:pharmacology; D066298:In Vitro Techniques; D016328:NF-kappa B / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects* / Q000201:enzymology; D019319:Okadaic Acid / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D011485:Protein Binding; D014268:Trifluoperazine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007166:Immunosuppressive Agents; D016328:NF-kappa B; D016877:Oxidants; D019319:Okadaic Acid; D014268:Trifluoperazine; D004247:DNA; D006861:Hydrogen Peroxide; D019703:Calcineurin