Journal of Neurochemistry, 2001, 76, 532±538 Inducible nitric oxide synthase expression in brain cortex after acute restraint stress is regulated by nuclear factor kB-mediated mechanisms Jose  L. M. Madrigal,* Marõ Âa A. Moro,* Ignacio Lizasoain,* Pedro Lorenzo,* Antonio Castrillo,² Lisardo Bosca ² and Juan C. Leza* *Departmento de Farmacologõ Âa, Facultad de Medicina, Universidad Complutense (UCM), ²Instituto de Bioquõ Âmica, UCM ± Consejo Superior de Investigaciones Cientõ ®cas, Madrid, Spain Abstract The underlying mechanisms by which physical or psycho- logical stress causes neurodegeneration are still unknown. We have demonstrated that the high-output and long-lasting synthesizing source of nitric oxide (NO), inducible NO synthase (iNOS), is expressed in brain cortex during stress and that its overexpression accounts for the neurodegenera- tive changes seen after 3 weeks of repeated stress. Now we have found that acute stress (restraint for 6 h) increases the activity of a calcium-independent NOS and induces the expression of iNOS in brain cortex in adult male rats. In order to elucidate the possible mechanisms involved in this induction, we studied the role of transcription nuclear factor kB (NF-kB), which is required for iNOS synthesis. We have observed that an acute restraint stress session stimulates the translocation of the NF-kB to the nucleus after 4 h and that the administration of the NF-kB inhibitor pyrrolidine dithiocarba- mate [PDTC, 75 and 150 mg/kg intraperitoneally (i.p.)] at the onset of stress inhibits the stress-induced increase in iNOS expression. Since glutamate release and subsequent NMDA (N-methyl-D-aspartate) receptor activation has been recog- nized as an early change after exposure to stressful stimuli, and glutamate has been shown to induce iNOS in brain via a NF-kB-dependent mechanism, we studied the possible role of excitatory amino acids in the induction of iNOS in our model. Pretreatment with the NMDA receptor antagonist dizocilpine (MK-801, 0.1 and 0.3 mg/kg i.p.) inhibits the stress-induced NF-kB activation as well as the stress-induced increase in iNOS expression. Taken together, these ®ndings indicate that excitatory amino acids and subsequent activation of NF-kB account for stress-induced iNOS expression in cerebral cortex, and support a possible neuroprotective role for speci®c inhibitors in this situation. Keywords: dizocilpine (MK-801), inducible nitric oxide synthase, nuclear factor-kB, restraint stress. J. Neurochem. (2001) 76, 532±538. Inducible nitric oxide synthase (iNOS) is a high-output isoform of NOS, calcium- and calmodulin-independent, which is expressed after exposure of cells to several noxious agents such as cytokines or lipopolysaccharide (reviewed in Nathan and Xie 1994). This NOS isoenzyme mediates cytotoxicity in many cell systems (Moncada et al. 1991; Gross and Wolin 1995). In this context, we have recently demonstrated that restraint stress induces a generalized increase in NO production (Leza et al. 1998) and that iNOS is expressed in cerebral cortex of rats exposed to stress (Olivenza et al. 2000). In support of the idea that iNOS is one of the mechanisms responsible for the functional damage, we have also demonstrated that aminoguanidine, a selective inhibitor of iNOS, protects against cell damage 532 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 532±538 Received July 25, 2000; accepted September 8, 2000. Address correspondence and reprint requests to J. C. Leza, Departamento de Farmacologõ Âa, Universidad Complutense, Madrid 28040, Spain. E-mail: jcleza@med.ucm.es Part of this manuscript was presented as a poster at the IV European Meeting on Glial Cell Function in Health and Disease, 24±27 May, 2000, Barcelona, Spain. Abbreviations used: BH 4 , 5,6,7,8-tetrahydrobiopterin; EAA, excita- tory amino acids; EMSA, electrophoretic mobility shift assay; iNOS, inducible NOS; MK-801, dizocilpine; NF-kB, nuclear factor kB; L-NAME, N v -nitro-l-arginine methyl ester; NOS, nitric oxide synthase; PDTC, pyrrolidine dithiocarbamate; PMSF, phenylmethylsul- phonyl ¯uoride; SDS, sodium dodecyl sulphate; TLCK, Na-p-tosyl-l- lysine-chloromethyl ketone.