Chronic Stress Induces the Expression of Inducible Nitric Oxide Synthase in Rat Brain Cortex Raquel Olivenza, Marı ´a A. Moro, Ignacio Lizasoain, Pedro Lorenzo, *Ana P. Ferna ´ndez, *Jose ´ Rodrigo, ²Lisardo Bosca ´, and Juan C. Leza Departamento de Farmacologı ´a, Facultad de Medicina, Universidad Complutense, *Instituto Cajal, Consejo Superior de Investigaciones Cientı ´ficas, and ² Instituto de Bioquı ´mica, Facultad de Farmacia, Madrid, Spain Abstract: Long-term exposure to stress has detrimental effects on several brain functions in many species, including humans, and leads to neurodegenerative changes. However, the underlying neural mechanisms by which stress causes neurodegeneration are still un- known. We have investigated the role of endogenously released nitric oxide (NO) in this phenomenon and the possible induction of the inducible NO synthase (iNOS) isoform. In adult male rats, stress (immobilization for 6 h during 21 days) increases the activity of a calcium-inde- pendent NO synthase and induces the expression of iNOS in cortical neurons as seen by immunohistochem- ical and western blot analysis. Three weeks of repeated immobilization increases immunoreactivity for nitroty- rosine, a nitration product of peroxynitrite. Repeated stress causes accumulation of the NO metabolites NO 2 - + NO 3 - (NO x - ) accumulation in cortex, and these changes occur in parallel with lactate dehydrogenase (LDH) release and impairment of glutamate uptake in synaptosomes. Administration of the selective iNOS in- hibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) prevents NO x - accumulation in cortex, LDH release, and impairment of glutamate uptake in syn- aptosomes. Taken together, these findings indicate that a sustained overproduction of NO via iNOS expression may be responsible, at least in part, for some of the neurodegenerative changes caused by stress and sup- port a possible neuroprotective role for specific iNOS inhibitors in this situation. Key Words: Immobilization— Inducible nitric oxide synthase —Peroxynitrite—Stress- induced neurodegeneration. J. Neurochem. 74, 785–791 (2000). The response of the organism to stress includes both physical and behavioral adaptations, the syndrome de- scribed by Selye 60 years ago. Usually, cessation of stress terminates this response, and the organism returns to its original equilibrium. However, very intense or long-lasting stress results in a new biological equilibrium that can be either beneficial, e.g., exercise-induced con- ditioning of the cardiovascular system, or detrimental (hypertension or stroke) to the body (reviewed by Mc- Ewen, 1998). In the last few years, several reports indi- cated that long-lasting stress affects synaptic plasticity, dendritic morphology, and neurogenesis (reviewed by Kim and Yoon, 1998) in animals and induces both clin- ical and anatomical features of neurotoxic damage in humans, i.e., posttraumatic stress disorder (Sapolsky, 1996; Sheline et al., 1996). The precise mechanisms by which stress induces brain damage are still a matter of debate. The neurotoxic action of glutamate and other excitatory amino acids (EAAs) mainly through the NMDA receptor and the potentiation of their effects by glucocorticoids have been implicated in the pathogenesis of stress-induced brain injury (Sapol- sky et al., 1990; Moghaddam, 1993; Magarin ˜os and McEwen, 1995; Kim et al., 1996). After the initial observation that NMDA receptor ac- tivation generates nitric oxide (NO) (Garthwaite et al., 1988), it has been postulated that overproduction of this molecule is the link between the actions of EAAs and the subsequent cell damage (Dawson et al., 1991; Nowicki et al., 1991). NO is synthesized from L-arginine by the enzymes called NO synthases (NOSs): endothelial NOS, neuronal NOS, and an isoform expressed during inflammatory reactions, inducible NOS (iNOS). This third isoform is a high-output inducible isoform of NOS, which mediates cytotoxicity in many cell systems, including CNS (Moncada et al., 1991; Gross and Wolin, 1995). This NOS isoenzyme is expressed after exposure to cytokines and/or lipopolysaccharide (for review, see Knowles and Received June 15, 1999; revised manuscript received October 5, 1999; accepted October 5, 1999. Address correspondence and reprint requests to Dr. J. C. Leza at Departamento de Farmacologı ´a, Facultad de Medicina, Universidad Com- plutense, Madrid 28040, Spain. E-mail: jcleza@eucmax.sim.ucm.es A portion of this work was presented at the 6th International Meeting on Biology of Nitric Oxide, held September 5– 8, 1999, in Stockholm, Sweden. Abbreviations used: EAA, excitatory amino acid; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; NO, nitric oxide; NO 2 - , nitrite; NO 3 - , nitrate; NO x - , nitric oxide metabolites; NOS, nitric oxide synthase; ONOO - , peroxynitrite; PB, sodium phosphate buffer; PBS, phosphate-buffered saline. 785 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 2000 International Society for Neurochemistry