Hepatocyte mitochondrial ammonia detoxification via ureagenesis is critical for prevention of hyperammonemia and hepatic encephalopathy. Aquaporin-8 (AQP8) channels facilitate the membrane transport of ammonia. Since AQP8 is expressed in hepatocyte inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) plays a role in ureagenesis from ammonia. Primary cultured rat hepatocytes were transfected with siRNAs targeting two different regions of the rat AQP8 molecule or with scrambled control siRNA. After 48 h, the levels of mtAQP8 protein specifically decreased by around 80% (P < 0.05) without affecting cell viability. The mtAQP8-knockdown cells in the presence of ammonium chloride, showed a decrease in ureagenesis of around 30% (P < 0.05). Glucagon strongly stimulated ureagenesis in control hepatocytes (+120%, P < 0.05), whereas in mtAQP8-knockdown cells, it induced no significant stimulation. Contrarily, mtAQP8-silencing induced no significant change in basal and glucagoninduced ureagenesis when glutamine or alanine was used as source of nitrogen. NMR studies using 15N-labeled ammonia, confirmed that glucagon-induced 15N-labeled urea synthesis was markedly reduced in mtAQP8-knockdown hepatocytes (-90%, P < 0.05). In vivo studies in the rat showed that under glucagon-induced ureagenesis, hepatic mtAQP8 protein expression was markedly upregulated (+160% P < 0.05). Moreover, transport studies in liver inner mitochondrial membrane vesicles showed that glucagon increased the diffusional permeability to the ammonia analog [14C]methylamine (+80%, P < 0.05). Conclusion: Hepatocyte mtAQP8 channels facilitate the mitochondrial uptake of ammonia and its metabolism into urea, mainly under glucagon stimulation. This mechanism may be relevant to hepatic ammonia detoxification and in turn, avoid the deleterious effects of hyperammonemia.

Ammonia detoxification via ureagenesis in rat hepatocytes involves mitochondrial aquaporin-8 channels

CALAMITA, Giuseppe;
2013

Abstract

Hepatocyte mitochondrial ammonia detoxification via ureagenesis is critical for prevention of hyperammonemia and hepatic encephalopathy. Aquaporin-8 (AQP8) channels facilitate the membrane transport of ammonia. Since AQP8 is expressed in hepatocyte inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) plays a role in ureagenesis from ammonia. Primary cultured rat hepatocytes were transfected with siRNAs targeting two different regions of the rat AQP8 molecule or with scrambled control siRNA. After 48 h, the levels of mtAQP8 protein specifically decreased by around 80% (P < 0.05) without affecting cell viability. The mtAQP8-knockdown cells in the presence of ammonium chloride, showed a decrease in ureagenesis of around 30% (P < 0.05). Glucagon strongly stimulated ureagenesis in control hepatocytes (+120%, P < 0.05), whereas in mtAQP8-knockdown cells, it induced no significant stimulation. Contrarily, mtAQP8-silencing induced no significant change in basal and glucagoninduced ureagenesis when glutamine or alanine was used as source of nitrogen. NMR studies using 15N-labeled ammonia, confirmed that glucagon-induced 15N-labeled urea synthesis was markedly reduced in mtAQP8-knockdown hepatocytes (-90%, P < 0.05). In vivo studies in the rat showed that under glucagon-induced ureagenesis, hepatic mtAQP8 protein expression was markedly upregulated (+160% P < 0.05). Moreover, transport studies in liver inner mitochondrial membrane vesicles showed that glucagon increased the diffusional permeability to the ammonia analog [14C]methylamine (+80%, P < 0.05). Conclusion: Hepatocyte mtAQP8 channels facilitate the mitochondrial uptake of ammonia and its metabolism into urea, mainly under glucagon stimulation. This mechanism may be relevant to hepatic ammonia detoxification and in turn, avoid the deleterious effects of hyperammonemia.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11586/34192
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