Chronic Stress Induces the Expression of Inducible Nitric Oxide Synthase in Rat Brain Cortex

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 unknown. 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-independent NO synthase and induces the expression of iNOS in cortical neurons as seen by immunohistochemical and western blot analysis. Three weeks of repeated immobilization increases immunoreactivity for nitrotyrosine, a nitration product of peroxynitrite. Repeated stress causes accumulation of the NO metabolites NO 2 Ϫ ϩ NO 3 Ϫ (NO x Ϫ ) 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 inhibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) prevents NO x Ϫ accumulation in cortex, LDH release, and impairment of glutamate uptake in synaptosomes. 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 support a possible neuroprotective role for specific iNOS inhibitors in this situation.

Glutathione Depletion, Lipid Peroxidation and Mitochondrial Dysfunction Are Induced by Chronic Stress in Rat Brain

“…Therefore, the oxidative damage induced by stress may be either the cause or the consequence of the mitochondrial dysfunction. The reported increase in brain NO production by stress-induced iNOS expression (Olivenza et al 2000) is likely to inhibit reversibly mitochondrial respiration as it has been reported to occur in brain mitochondria (Lizasoain et al 1996). Interestingly, superoxide (O Ϫ 2 )-a by-product of mitochondrial respiratory chain-increases in the presence of inhibitors as NO (Poderoso et al 1996).…”

“…Tissues were homogenized as described (Olivenza et al 2000), and after centrifugation in a microcentrifuge for 15 min, the proteins present in the supernatant were loaded (10 g) and size-separated in 10% SDS-polyacrilamide gel electrophoresis (90 mA). The gels were processed against the Ags and after blotting onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) were incubated with a specific polyclonal iNOS antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:1000 dilution).…”

“…No immunostaining was found in sections taken from brains of control animals (data not shown). A more detailed explanation of the morphological changes based in immunohistochemical studies has been recently published (Olivenza et al 2000).…”

“…Garthwaite et al (1988) demonstrated that NMDA receptor activation generates nitric oxide (NO), and after this initial observation it was postulated that an overproduction of this molecule is the link between the actions of EAA and the subsequent cell damage (Dawson et al 1991;Nowicki et al 1991). In this context, not only constitutive formation of NO, but also inducible expression of iNOS has been found to occur in the brain during chronic stress Olivenza et al 2000).…”

Induction of Cyclooxygenase-2 Accounts for Restraint Stress-Induced Oxidative Status in Rat Brain

“…The high-output isoform of nitric oxide (NO) synthase (NOS-2) has been implicated in cellular toxicity in many cell systems, including the brain (for a review, see Gross and Wolin, 1995). In this context, we have demonstrated that chronic restraint stress induces the expression of NOS-2 in rat brain cortex and hippocampus and that its inhibition protects against stress-induced cell damage in this model (Olivenza et al, 2000). Once NOS-2 is expressed, the formation of large amounts of oxygen and nitrogen reactive species may account for the oxidation of cellular components found after chronic restraint stress in the rat brain (Liu et al, 1996;Madrigal et al, 2001b).…”