Hypoosmotic swelling increases protein tyrosine nitration in cultured rat astrocytes

Abstract

Astrocyte swelling is observed in different types of brain injury. We studied a potential contribution of swelling to protein tyrosine nitration (PTN) by using cultured rat astrocytes exposed to hypoosmotic (205 mosmol/L) medium. Hypoosmolarity (2 h) increases total PTN by about 2-fold in 2 h. The hypoosmotic PTN is significantly inhibited by the NMDA receptor antagonist MK-801, the nitric oxide synthase (NOS) inhibitor L-NMMA, the extracellular Ca2+ chelator EGTA and the calmodulin antagonist W13, suggesting the involvement of NMDA receptor activation, influx of extracellular Ca2+ and Ca2+/calmodulin-dependent NO synthesis. Further, superoxide dismutase plus catalase and uric acid strongly inhibit hypoosmotic PTN, suggesting the involvement of the toxic metabolite peroxynitrite (ONOO-) as a nitrating agent. Hypoosmotic astrocyte swelling rapidly stimulates generation of reactive oxygen intermediates; this process is prevented by MK-801 and EGTA. In addition, MK-801 inhibits the hypoosmotic elevation of [Ca2+]i. The findings support the view that astrocyte swelling as induced, for example, by toxins relevant for hepatic encephalopathy is sufficient to produce oxidative stress and PTN and thus contributes to altered astroglial and neuronal function.

Ammonia induces RNA oxidation in cultured astrocytes and brain in vivo†

“…Immunoreactivity of the neuron-specific nuclear NeuN was not detectable in the astrocytes culture. After starvation for 1 hour in serum-free medium NH 4 Cl, diazepam or TNF-␣ were added to the culture medium. Alternatively, hypoosmolarity (205 mOsm/L) was achieved by dilution of the medium with the appropriate volume of NaCl-free medium.…”

“…Slices were placed immediately into carbogen (5% CO 2 , 95% O 2 )-pregassed Dulbecco's minimum essential medium containing 1000 mg/L Dglucose without phenol red (Gibco BRL, Life Sciences, Gaithersburg, MD). Three to four hours after slice preparation, NH 4 Cl and TNF-␣ were added to the culture medium. During the entire experimental procedure, incubation media were continuously oxygenated with carbogen.…”

“…(A) North-western blot analysis of concentration dependence (n ϭ 3). Astrocytes were exposed to the indicated concentrations of NH 4 Cl for 6 hours. (B) Reversibility of RNA-oxidation.…”

“…In another set of experiments, astrocytes were exposed for 1 hour to 1 mmol/L NH 4 Cl or were untreated (control2). Thereafter the medium was exchanged and the culture was continued in both cases for another 71 hours in the absence of ammonia ("control2" and "NH 4 sodium citrate, pH 7.0). At the end of the transfer, the membrane was heated for 1 hour at 70°C.…”

“…4,5 Because astrocyte swelling produces oxidative stress 4,5 and oxidative stress enhances astrocyte swelling, 6 a self-amplifying cycle between astrocyte swelling and oxidative stress has been proposed. 3 Consistently, hypo-osmolarity and HE-precipitating factors that stimulate astrocyte swelling increase protein tyrosine nitration in cultured rat astrocytes and rat brain in vivo, respectively.…”