Effect of glutamine synthetase inhibition on astrocyte swelling and altered astroglial protein expression during hyperammonemia in rats.

Astrocyte swelling is a distinctive feature of the histological finding in humans who die from severe liver disease or urea cycle disorders. Experimental models of liver dysfunction and hyperammonemia are also marked by watery swelling of astrocytes and cellular hypertrophy of astrocyte cell bodies without necessarily presenting with overt neuronal pathology. Thus, hyperammonemia is considered to be a major factor contributing to the cellular changes seen in severe hepatic encephalopathy (Norenberg, 1998). Tracer studies reveal that blood-borne ammonia is rapidly incorporated into brain glutamine, and that this process is markedly slowed in vivo by inhibiting glutamine synthetase (GS) with methionine sulfoximine (MSO), thereby disrupting compartmentation of nitrogen metabolism (Cooper et al., 1979). Based on the observations 1) that increased brain glutamine concentration is one of the most consistent biochemical change seen both clinically and experimentally with liver disease, urea cycle disorders or pure hyperammonemia (Cooper, 2001), and 2) that GS is enriched in astrocytes (Norenberg and Martinez-Hernandez, 1979), we postulated that glutamine accumulated in astrocytes may act as an osmolyte that contributes to astrocyte swelling (Brusilow and Traystman, 1986). In support of this hypothesis, we found that inhibiting glutamine synthesis by pretreating rats with MSO prevented increases in cortical water content (Takahashi et al., 1991) and intracranial pressure (Takahashi et al., 1992) during 6 h of hyperammonemia. Electron microscopy revealed diminished watery swelling of astrocyte processes and less enlargement of the nucleus with MSO treatment, although other aspects of cell hypertrophy were still present (Willard-Mack et al., 1996). Functionally, astrocytic control of extracellular K+ activity (Sugimoto et al., 1997) and cerebrovascular reactivity to CO2 (Hirata et al., 1996, 1999) were improved by MSO pretreatment. Others have shown that MSO attenuates cerebral edema and intracranial hypertension induced by a 3-h infusion of ammonium acetate (NH4Ac) in portacaval shunted rats (Blei et al., 1994), and that MSO increases cerebral glucose consumption in portacaval-shunted rats (Hawkins et al., 1993). Thus, some aspects of astrocyte morphological and functional changes during acute hyperammonemia are related to glutamine accumulation rather than direct effects of ammonia. Existing studies of MSO treatments in hyperammonemic rats have caveats. One limitation of these studies with MSO treatment in hyperammonemic rats is that a relatively short period of NH4Ac infusion was used. Effects of MSO may not be sustained with longer periods. Another limitation in some of these studies was the use of pentobarbital anesthesia, which could have influenced the results through its depressant effects on energy metabolism or its alteration of GABAergic neurotransmission. To overcome these limitations in the present study, we used a more prolonged, 24 h infusion of NH4Ac in the absence of anesthesia. We tested the hypothesis that pretreatment with MSO will attenuate ammonia-induced increases in glutamine concentration, astrocyte swelling and tissue water content over a 24 h period of hyperammonemia. In addition, we determined whether changes in astrocyte markers were evident by 24 h of hyperammonemia and if these changes could be ameliorated by MSO pretreatment. Specifically, we evaluated immunoreactivity for GS, the intermediate filament glial fibrillary acidic protein (GFAP), connexin-43 present in astrocyte gap junctions (Rash et al., 2001), and the astroglial glutamate transporter GLT1. Immunohistochemistry was used to localize changes in these proteins on a cortical laminar basis.