Ammonia increases nitric oxide, free Zn2+, and metallothionein mRNA expression in cultured rat astrocytes

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 Zn2+ concentration ([Zn2+]i). Here we report effects of ammonia on [Zn2+]i homeostasis and NO synthesis. In cultured rat astrocytes, NH4Cl (5 mm) increased within 6 h both cytosolic and mitochondrial [Zn2+]. The [Zn2+]i increase was transient and was mimicked by the nonmetabolizable CH3NH3Cl, and it was dependent on NO formation, as evidenced by the sensitivity toward the nitric oxide synthase inhibitor N G-monomethyl-l-arginine. The NH4Cl-induced NO formation was sensitive to the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester and increases in both NO and [Zn2+]i were blocked by the N-methyl-d-aspartate receptor antagonist MK-801. The NH4Cl-triggered increase in [Zn2+]i was followed by a Zn2+-dependent nuclear appearance of the metal response element-binding transcription factor and metallothionein messenger RNA (mRNA) induction. Metallothionein mRNA was also increased in vivo in rat cerebral cortex 6 h after an NH4Ac challenge. NH4Cl increased peripheral-type benzodiazepine receptor (PBR) protein expression, whereas PBR mRNA levels were decreased in a Zn2+-independent manner. The Zn2+-dependent upregulation of metallothionein following ammonia intoxication may reflect a cytoprotective response, whereas the increase in PBR expression may augment HE development.