AIM： To assess whether portacaval anastomosis （PCA） in rats affects the protein expression and/or activity of glutaminase in kidneys, intestines and in three brain areas of cortex, basal ganglia and cerebellum and ...AIM： To assess whether portacaval anastomosis （PCA） in rats affects the protein expression and/or activity of glutaminase in kidneys, intestines and in three brain areas of cortex, basal ganglia and cerebellum and to explain the neurological alterations found in hepatic encephalopathy （HE）. METHODS： Sixteen male Wistar rats weighing 250-350 g were grouped into sham-operation control （n=8） or portacaval shunt （n = 8）. Twenty-eight days after the procedure, the animals were sacrificed. The duodenum, kidney and brain were removed, homogenised and mitochondria were isolated. Ammonia was measured in brain and blood. Phosphate-activated glutaminase （PAG） activity was determined by measuring ammonia production following incubation for one hour at 37 ℃ with O-phthalaldehyde （OPA） and specific activity expressed in units per gram of protein （pkat/g of protein）. Protein expression was measured by immunoblotting. RESULTS： Duodenal and kidney PAG activities together with protein content were significantly higher in PCA group than in control or sham-operated rats （duodenum PAG activity was 976.95±268.87μkat/g of protein in PCA rats vs 429.19±126.92.μkat/g of protein in shamoperated rats; kidneys PAG activity was 1259.18±228.79 μkat/g protein in PCA rats vs 669.67±400.8 μkat/g of protein in controls, P〈0.05; duodenal protein content： 173% in PCA vs sham-operated rats; in kidneys the content of protein was 152% in PCA vs sham-operated rats）. PAG activity and protein expression in PCA rats were higher in cortex and basal ganglia than those in shamoperated rats （cortex： 6646.6 ±1870.4 μkat/g of protein vs 3573.8± 2037.4 μkat/g of protein in control rats, P〈 0.01; basal ganglia, PAG activity was 3657.3± 1469.6 μkat/g of protein in PCA rats vs 2271.2±384 μkat/g of protein in sham operated rats, P〈0.05; In the cerebellum, the PAG activity was 2471.6±701.4 μkat/g of protein vs 1452.9 ±567.8 μkat/g of protein in the PCA and sham rats, respectively, P〈0.05; content of protein： cerebral cortex： 162% ±40% vs 100% ± 26%, P〈 0.009; and basal ganglia： 140% ±39% vs 100% ±14%, P〈0.05; but not in cerebellum： 100% ±25% vs 100% ± 16%, P= ns）. CONCLUSION： Increased PAG activity in kidney and duodenum could contribute significantly to the hyperammonaemia in PCA rats, animal model of encephalopathy. PAG is increased in non-synaptic mitochondria from the cortex and basal ganglia and could be implicated in the pathogenesis of hepatic encephalopathy. Therefore, PAG could be a possible target for the treatment of HE or liver dysfunction.展开更多
基金Supported by funding from the Spanish Ministry of Health (grants # PI040384 and # 03/155-2002) awarded to the Spanish Network of Hepatic Encephalopathy Research and a grant from PAI (CTS-532)
文摘AIM： To assess whether portacaval anastomosis （PCA） in rats affects the protein expression and/or activity of glutaminase in kidneys, intestines and in three brain areas of cortex, basal ganglia and cerebellum and to explain the neurological alterations found in hepatic encephalopathy （HE）. METHODS： Sixteen male Wistar rats weighing 250-350 g were grouped into sham-operation control （n=8） or portacaval shunt （n = 8）. Twenty-eight days after the procedure, the animals were sacrificed. The duodenum, kidney and brain were removed, homogenised and mitochondria were isolated. Ammonia was measured in brain and blood. Phosphate-activated glutaminase （PAG） activity was determined by measuring ammonia production following incubation for one hour at 37 ℃ with O-phthalaldehyde （OPA） and specific activity expressed in units per gram of protein （pkat/g of protein）. Protein expression was measured by immunoblotting. RESULTS： Duodenal and kidney PAG activities together with protein content were significantly higher in PCA group than in control or sham-operated rats （duodenum PAG activity was 976.95±268.87μkat/g of protein in PCA rats vs 429.19±126.92.μkat/g of protein in shamoperated rats; kidneys PAG activity was 1259.18±228.79 μkat/g protein in PCA rats vs 669.67±400.8 μkat/g of protein in controls, P〈0.05; duodenal protein content： 173% in PCA vs sham-operated rats; in kidneys the content of protein was 152% in PCA vs sham-operated rats）. PAG activity and protein expression in PCA rats were higher in cortex and basal ganglia than those in shamoperated rats （cortex： 6646.6 ±1870.4 μkat/g of protein vs 3573.8± 2037.4 μkat/g of protein in control rats, P〈 0.01; basal ganglia, PAG activity was 3657.3± 1469.6 μkat/g of protein in PCA rats vs 2271.2±384 μkat/g of protein in sham operated rats, P〈0.05; In the cerebellum, the PAG activity was 2471.6±701.4 μkat/g of protein vs 1452.9 ±567.8 μkat/g of protein in the PCA and sham rats, respectively, P〈0.05; content of protein： cerebral cortex： 162% ±40% vs 100% ± 26%, P〈 0.009; and basal ganglia： 140% ±39% vs 100% ±14%, P〈0.05; but not in cerebellum： 100% ±25% vs 100% ± 16%, P= ns）. CONCLUSION： Increased PAG activity in kidney and duodenum could contribute significantly to the hyperammonaemia in PCA rats, animal model of encephalopathy. PAG is increased in non-synaptic mitochondria from the cortex and basal ganglia and could be implicated in the pathogenesis of hepatic encephalopathy. Therefore, PAG could be a possible target for the treatment of HE or liver dysfunction.
