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

Abstract

Hepatocyte mitochondrial ammonia detoxification via ureagenesis is critical for the prevention of hyperammonemia and hepatic encephalopathy. Aquaporin-8 (AQP8) channels facilitate the membrane transport of ammonia. Because AQP8 is expressed in hepatocyte inner mitochondrial membranes (IMMs), we studied whether mitochondrial AQP8 (mtAQP8) plays a role in ureagenesis from ammonia. Primary cultured rat hepatocytes were transfected with small interfering RNAs (siRNAs) targeting two different regions of the rat AQP8 molecule or with scrambled control siRNA. After 48 hours, the levels of mtAQP8 protein decreased by approximately 80% (P < 0.05) without affecting cell viability. mtAQP8 knockdown cells in the presence of ammonium chloride showed a decrease in ureagenesis of approximately 30% (P < 0.05). Glucagon strongly stimulated ureagenesis in control hepatocytes (1120%, P < 0.05) but induced no significant stimulation in mtAQP8 knockdown cells. Contrarily, mtAQP8 silencing induced no significant change in basal and glucagon-induced ureagenesis when glutamine or alanine was used as a source of nitrogen. Nuclear magnetic resonance studies using 15N-labeled ammonia confirmed that glucagon-induced 15N-labeled urea synthesis was markedly reduced in mtAQP8 knockdown hepatocytes (290%, P < 0.05). In vivo studies in rats showed that under glucagoninduced ureagenesis, hepatic mtAQP8 protein expression was markedly up-regulated (1160%, P < 0.05). Moreover, transport studies in liver IMM vesicles showed that glucagon increased the diffusional permeability to the ammonia analog [ 14 C]methylamine (180%, 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. (HEPATOLOGY 2013;57:2061-2071 B y means of the urea cycle, hepatocytes metabolize waste nitrogen and prevent the deleterious consequences of hyperammonemia and hepatic encephalopathy. The initial and rate-controlling step in the urea cycle is the mitochondrial synthesis of carbamoyl-phosphate from ammonia by carbamyl phosphate synthetase 1 (CPS1), 1 a process that involves the mitochondrial uptake of ammonia and the metabolization of ammonia from amino acids such as glutamine and alanine. 2 However, the mechanisms involved in the mitochondrial entry of ammonia are still unknown (Fig. 1). Aquaporin-8 (AQP8) is a member of a family of homologous membrane channel proteins that facilitates the movement of water and certain small solutes across biological membranes. 3 Hepatocyte AQP8 is localized as a glycosylated 34-kDa protein in the canalicular plasma membrane domain and intracellular vesicles. 4-7 Canalicular AQP8 works as a water channel facilitating the movement of water molecules coupled to osmotic gradients, 8,9 a process that may play a role in hepatocyte bile formation and cholestasis. 7,10-13 A nonglycosylated 28-kDa form of the AQP8 protei...

Lipopolysaccharide impairs hepatocyte ureagenesis from ammonia: Involvement of mitochondrial aquaporin‐8

“…The lyophilizate was resuspended in 0.5 ml of dimethyl sulfoxide-d 6 (99.9 atom% D) (Cambridge Isotope Laboratories) containing 0.02 ml of formamide [natural abundance (0.365%) in 15 N] (Promega), that was used as internal standard. 15 N-labeled urea synthesis was determined by nuclear magnetic resonance spectroscopy, as previously described [17].…”

“…The transport of ammonia in these vesicles was measured using the ammonia analog [ 14 C]methylamine, by the rapid filtration technique as previously described [17]. Methylamine is an analog structurally similar to ammonia but of larger size.…”

“…After blocking and washing, blots were incubated overnight at 4°C with rabbit affinity purified antibody directed against an N-terminal peptide of rat AQP8 [14,17] or rabbit antibodies against Carbamyl phosphate synthetase 1 (CPS1) and Ornithine transcarbamylase (OTC) (Sigma). As loading control, membranes were also incubated with rabbit antibodies against Prohibitin (Abcam, Cambridge, UK) all at a final concentration of 1 lg/ml.…”

“…There is experimental evidence suggesting that mtAQP8 facilitates the ammonia transport across the IMM [15][16][17]. We recently reported that hepatocyte mtAQP8 is involved in the mitochondrial uptake of ammonia and its metabolism into urea [17]. Hence, in this study, we investigated whether mtAQP8 is involved in the molecular pathogenesis of the LPS-induced defective hepatic ammonia detoxification.…”

“…The expression of mitochondrial AQP8 (mtAQP8) seems to be greater in the periportal area of the hepatic lobule [13,14]. There is experimental evidence suggesting that mtAQP8 facilitates the ammonia transport across the IMM [15][16][17]. We recently reported that hepatocyte mtAQP8 is involved in the mitochondrial uptake of ammonia and its metabolism into urea [17].…”