Combined Effects of Ammonia and Manganese on Astrocytes in Culture

Abstract

Ammonia has been strongly implicated in the pathogenesis of hepatic encephalopathy (HE), and astrocytes appear to be the primary target of ammonia neurotoxicity. Recent work has shown that manganese also plays a role in the pathogenesis of HE and causes astrocyte morphologic and functional changes similar to ammonia. We therefore investigated whether a combination of these compounds could produce additive/synergistic effects. Cultured astrocytes treated with 5 mM ammonia (NH4Cl) along with 100 microM manganese acetate (MnAc) for 3 h showed a 55-65% increase in free radical production over ammonia or manganese alone (P < 0.05). There was also a 50% decrease in the mitochondrial membrane potential (beta psi(m)) at 24 h following treatment with NH4Cl (5 mM) plus MnAc (50 microM) (P < 0.05), as compared to ammonia or manganese alone. Astrocytes treated with ammonia or manganese alone for 24 h showed no cell death, as determined by LDH release and light microscopic examination. However, cultures treated with ammonia plus manganese showed 80-90% necrotic cell death as estimated by light microscopy and 59% cell death as determined by LDH release. LDH release by ammonia plus manganese was blocked by the antioxidant superoxide dismutase (25 units/ml) as well as by the nitric oxide synthase inhibitor N(omega)-nitro-L-argininemethyl ester (500 microM). In conclusion, ammonia plus manganese exert additive/synergetic effects on the induction free radicals, mitochondrial inner membrane depolarization and cellular integrity, which may contribute to the tissue injury associated with chronic forms of HE.