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

Abstract

Inhibition of glutamine synthesis reduces astrocyte swelling and associated physiological abnormalities during acute ammonium acetate infusion in anesthetized rats. We tested the hypothesis that inhibition of glutamine accumulation during more prolonged ammonium acetate infusion in unanesthetized rats reduces cortical astrocyte swelling and immunohistochemical changes in astrocytic proteins. Rats received a continuous i.v. infusion of either sodium acetate or ammonium acetate for 24 h to increase plasma ammonia from about 30-400 μmol/l. Cohorts were pretreated with vehicle or L-methionine-S-sulfoximine (MSO; 0.83 mmol/kg). MSO reduced glutamine synthetase activity by 57% and glutamine synthetase immunopositive cell number by 69%, and attenuated cortical glutamine accumulation by 71%. Hyperammonemia increased the number of swollen astrocytes in cortex and MSO reduced this increase to control values. The number of glial fibrillary acidic protein immunopositive cells in cortex was greater in hyperammonemic rats and the increase in superficial cortical layers was attenuated by MSO. Immunoreactivity for the gap junction protein connexin-43 in the neuropil, assessed by optical density, was greater in the hyperammonemic group compared with controls, but this increase was not attenuated by MSO. No changes in the optical density of GLT1 glutamate transporter immunoreactivity in cortex were detected in any group. We conclude that glutamine synthetase inhibition reduces astrocyte swelling and ameliorates some of the reactive astroglial cytoskeletal alterations seen at 24 h of hyperammonemia, but that gap junction changes in astrocytes occur independently of glutamine accumulation and swelling. Keywords ammonia; astrocyte; connexin; cerebral edema; glial fibrillary acidic protein; glutamine 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 swelli...

Ammonia induces RNA oxidation in cultured astrocytes and brainin vivo

“…3,30 Thus, astrocytes may be another source of ROS/RNOS in the acutely hyperammonemic brain in vivo. In line with this, in vivo inhibition of glutamine synthetase prevents not only ammonia-induced astrocyte swelling 31 but also cerebral NO production 27 and protein tyrosine nitration. 32 Remarkably, ROS/RNOS produced by astrocytes can affect neuronal function.…”

Effect of Glutamine Synthetase Inhibition on Brain and Interorgan Ammonia Metabolism in Bile Duct Ligated Rats

“…These results are promising for manipulating brain metabolism during hyperammonia since the evidence of glutamine playing a significant role in the development of HE is substantial [7][8][9][10][11][12][13] and animal studies points to a beneficial effect of inhibition GS by MSO. [17][18][19][20][21] In support of this, two recent reviews considered the potential use of MSO in patients with acute liver failure and limited treatment offers. 36,37 It seems unlikely, though, that MSO will be considered as a possible treatment for hyperammonemia in patients with chronic liver disease as convulsions have been observed in animals administered MSO 38,39 but the possibility of designing analogs of MSO that inhibit GS without having the undesired side effects produced by MSO should be considered.…”

Astroglial Glutamate–Glutamine Shuttle Is Involved in Central Sensitization of Nociceptive Neurons in Rat Medullary Dorsal Horn

“…MSO has been widely used as a GS inhibitor in astroglia in studies of hyperammonemia, memory consolidation, and seizures, and its effects can be counteracted by glutamine application (Bacci et al, 2002;Suarez et al, 2002;Gibbs and Hertz, 2005;Tanigami et al, 2005;Liang et al, 2006). One recent study has used MSO to examine the effect of inflammatory stimuli on activation of GS in astroglia and found that GS activation is related to glutamatergic receptor activation (Muscoli et al, 2005).…”

“…This shuttle includes the uptake of excessive extrasynaptic glutamate and the production [via glutamine synthetase (GS)] and release from astroglia of glutamine, which is then taken up by neuronal elements to replenish the supply of glutamate (Zwingmann and Leibfritz, 2003;Hertz, 2004;Hertz and Zielke, 2004;Fonseca et al, 2005). A potent inhibitor of GS in astroglia is methionine sulfoximine (MSO), and its effects can be counteracted by glutamine application (Bacci et al, 2002;Blin et al, 2002;Suarez et al, 2002;Shin et al, 2003;Gibbs and Hertz, 2005;Tanigami et al, 2005;Liang et al, 2006). However, only one study has used MSO to investigate the involvement of astroglial GS in the effects of noxious stimuli on glutamatergic receptor-related events in astroglia (Muscoli et al, 2005), and no studies have tested its effects on central sensitization in functionally identified nociceptive neurons.…”