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

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 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.