Ammonia increases nitric oxide, free Zn(2+) and metallothionein mRNA expression in cultured rat astrocytes.
ABSTRACT Abstract Ammonia is a major player in the pathogenesis of hepatic encephalopathy (HE) and affects astrocyte function by triggering a self-amplifying cycle between osmotic and oxidative stress. We recently demonstrated that hypoosmotic astrocyte swelling rapidly stimulates nitric oxide (NO) production and increases intracellular free Zn(2+) concentration ([Zn(2+)](i)). Here we report effects of ammonia on [Zn(2+)](i) homeostasis and NO synthesis. In cultured rat astrocytes NH(4)Cl (5 mmol/l) increased within 6h both, cytosolic and mitochondrial [Zn(2+)]. The [Zn(2+)](i) increase was transient and was mimicked by the non-metabolizable CH(3)NH(3)Cl, and was dependent on NO-formation, as evidenced by the sensitivity towards the NOS inhibitor L-NMMA. The NH(4)Cl-induced NO-formation was sensitive to the Ca(2+)-chelator BAPTA-AM and both, the increase of NO and [Zn(2+)](i) were blocked by the NMDA receptor antagonist MK-801. The NH(4)Cl-triggered increase of [Zn(2+)](i) was followed by a Zn(2+)-dependent nuclear appearance of the transcription factor MTF-1 and metallothionein mRNA induction. Metallothionein mRNA was also increased in vivo in rat cerebral cortex 6h after an NH(4)Ac challenge. NH(4)Cl increased peripheral-type benzodiazepine receptor (PBR) protein expression, whereas PBR mRNA levels were decreased in a Zn(2+)-independent manner. The Zn(2+)-dependent upregulation of metallothionein following ammonia intoxication may reflect a cytoprotective response, whereas the increase in PBR expression may augment HE development.