We have shown recently, in a yeast expression system, that some aquaporins are permeable to ammonia. In the present study, we expressed the mammalian aquaporins AQP8, AQP9, AQP3, AQP1 and a plant aquaporin TIP2;1 in Xenopus oocytes to study the transport of ammonia (NH3) and ammonium (NH4+) under open-circuit and voltage-clamped conditions. TIP2;1 was tested as the wild-type and in a mutated version (tip2;1) in which the water permeability is intact. When AQP8-, AQP9-, AQP3- and TIP2;1-expressing oocytes were placed in a well-stirred bathing medium of low buffer capacity, NH3 permeability was evident from the acidification of the bathing medium; the effects observed with AQP1 and tip2;1 did not exceed that of native oocytes. AQP8, AQP9, AQP3, and TIP2;1 were permeable to larger amides, while AQP1 was not. Under voltage-clamp conditions, given sufficient NH3, AQP8, AQP9, AQP3, and TIP2;1 supported inwards currents carried by NH4+. This conductivity increased as a sigmoid function of external [NH3]: for AQP8 at a bath pH (pH(e)) of 6.5, the conductance was abolished, at pH(e) 7.4 it was half maximal and at pH(e) 7.8 it saturated. NH4+ influx was associated with oocyte swelling. In comparison, native oocytes as well as AQP1 and tip2;1-expressing oocytes showed small currents that were associated with small and even negative volume changes. We conclude that AQP8, AQP9, AQP3, and TIP2;1, apart from being water channels, also support significant fluxes of NH3. These aquaporins could support NH4+ transport and have physiological implications for liver and kidney function.

Mitochondria are remarkably plastic organelles constantly changing their shape to fulfil their various functional activities. Although the osmotic movement of water into and out of the mitochondrion is central for its morphology and activity, the molecular mechanisms and the pathways for water transport across the inner mitochondrial membrane (IMM), the main barrier for molecules moving into and out of the organelle, are completely unknown. Here, we show the presence of a member of the aquaporin family of water channels, AQP8, and demonstrate the strikingly high water permeability (P f ) characterizing the rat liver IMM. Immunoblotting, electron microscopy, and biophysical studies show that the largest mitochondria feature the highest AQP8 expression and IMM P f . AQP8 was also found in the mitochondria of other organs, whereas no other known aquaporins were seen. The osmotic water transport of liver IMM was partially inhibited by the aquaporin blocker Hg 2؉ , while the related activation energy remained low, suggesting the presence of a Hg 2؉ -insensitive facilitated pathway in addition to AQP8. It is suggested that AQP8-mediated water transport may be particularly important for rapid expansions of mitochondrial volume such as those occurring during active oxidative phosphorylation and those following apoptotic signals.Mitochondrial volume is of pivotal importance for the activity of the electron transport chain (1) and a control point of apoptosis (2). Changes in mitochondrial volume occur in many other physiological and patho-physiological conditions, including intracellular signal transduction, liver regeneration, ischemia/reperfusion-induced damage, and anoxia (3-5). Mitochondria are well behaved osmometers, and swelling and contraction of the mitochondrial matrix and related changes to mitochondrial morphology are the consequence of the water movement that osmotically accompanies the net transport of solutes into and out of the mitochondrion (6), respectively. Mitochondrial volume changes are modulated by the net movement of solutes including K ϩ and Ca 2ϩ ions across the IMM 1 (7,8). The inner mitochondrial membrane acts as a major barrier for the solutes and water moving between the cytoplasm and the mitochondrial matrix, the outer membrane being freely permeable to molecules of up to 1.5 kDa due to the presence of the exceedingly large pores formed by VDAC, the voltage-dependent anion channel (9). However, although a number of IMM transport systems have been cloned and characterized for their ability to transport solutes across the IMM (10), the molecular pathway for the movement of water remains obscure. Important clues for understanding the molecular bases of the mitochondrial osmotic properties were recently provided by the identification of an aquaporin water channel (11), AQP8, in rat hepatocyte mitochondria (12). AQP8 was also found in intracellular vesicles that are shuttled to the hepatocyte apical membrane under choleretic stimuli such as those brought about by glucagon (13). This led us to hyp...

Overall, these results experimentally prove major functional significance for AQP9 in maximising liver glycerol import during states requiring increased glucose production. If any, alternative facilitated pathways would be of minor importance in transporting glucogenetic glycerol into hepatocytes during starvation. Refining the understanding of liver AQP9 in metabolic and energy homeostasis may reveal helpful for therapeutic purposes.

C57L mice are susceptible and AKR mice are resistant to gallstone formation. We studied in male mice of both strains gallbladder histopathology, cholecystokinininduced emptying, and concentrating function at 0, 14, 28, and 56 days on a lithogenic diet. Gallbladder wall thickness increased on the diet, with stromal granulocyte infiltration, progressive fibrosis, edema, and epithelial cell indentation, particularly in C57L. Strong basal cholecystokinin octapeptide-induced gallbladder emptying (70% of fasting volumes) occurred in both strains, but fasting gallbladder volumes were significantly larger in C57L (14.8 6 2.2 ml vs. 8.8 6 1.0 ml). On the diet, fasting volumes increased exclusively in C57L (28.6 6 2.9 ml on day 56), with progressively decreased emptying (27% of fasting volumes on day 56). Gallbladder emptying remained normal in AKR. Gallbladder concentrating function decreased on the lithogenic diet (especially in C57L), coinciding with decreased aquaporin-1 (AQP1) and AQP8 expression at the mRNA and protein levels. In additional experiments, similar downregulation of AQP1 and AQP8 mRNA expression occurred in farnesoid X receptor (FXR)-deficient mice after 1 week on the lithogenic diet, without any difference from corresponding wild-type mice. In conclusion, during murine lithogenesis, altered gallbladder histology is associated with impaired motility, reduced concentrating function, and decreased AQP1 and AQP8 expression, the latter without the involvement of the FXR. Supplementary key words aquaporin . cholesterol . farnesoid X receptor . gallbladder emptying . water channel C57L and AKR inbred mice exhibit different susceptibilities to cholesterol gallstone formation, depending on Lith genes. Susceptibility is high in C57L males (gallstones in 80% of mice after 56 days on a lithogenic diet) and low in AKR males (gallstones in 15% after 56 days on a lithogenic diet) (1). Based on their time course during the earliest stages of lithogenesis, biliary cholesterol supersaturation, the hydrophobic bile salt deoxycholate, and high concentrations of crystallization-promoting mucin are thought to play crucial roles in murine gallstone formation (1, 2). In humans, the gallbladder is thought to be another key player in gallstone pathogenesis. Impaired postprandial and interdigestive gallbladder emptying are often found in gallstone patients, providing time for nucleation of cholesterol crystals and their aggregation into macroscopic stones (3). Also in animal models, gallbladder contractility is decreased in the earliest stages of gallstone formation, even before gallstones have formed (4). Furthermore, in the fasting gallbladder, hepatic bile is concentrated 4-to 5-fold by absorption of water, thereby enhancing cholesterol crystallization (5, 6). Aquaporins (AQP0 to AQP10) are a family of transmembrane channels mediating the movement of water through the lipid bilayer. AQP1 (7-12) and AQP8 (12) have recently been detected in gallbladder epithelial cells. Virtually no information is available about the g...

Aquaporins are channel proteins widely expressed in nature and known to facilitate the rapid movement of water across numerous cell membranes. A mammalian aquaporin, AQP8, was recently discovered and found to have a very distinct evolutionary pathway. To understand the reason for this divergence, here we define the ontogeny and exact subcellular localization of AQP8 in mouse liver, a representative organ transporting large volumes of water for secretion of bile. Northern blotting showed strong AQP8 expression between fetal day 17 and birth as well as at weaning and thereafter. Interestingly, this pattern was confirmed by immunohistochemistry and coincided both temporally and spatially with that of hepatic glycogen accumulation. As seen by reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, fasting was accompanied by remarkable down-regulation of hepatic AQP8 that paralleled the expected depletion of glycogen content. The level of hepatic AQP8 returned to be considerable after refeeding. Immunoelectron microscopy confirmed AQP8 in hepatocytes where labeling was over smooth endoplasmic reticulum (SER) membranes adjacent to glycogen granules and in canalicular membranes, subapical vesicles, and some mitochondria. In conclusion, in addition to supporting a role for AQP8 in canalicular water secretion, these findings also suggest an intracellular involvement of AQP8 in preserving cytoplasmic osmolality during glycogen metabolism and in maintaining mitochondrial volume. AQP8 may have evolved separately to feature these intracellular roles as no other known aquaporin shows this specialization.

Carreras FI, Lehmann GL, Ferri D, Tioni MF, Calamita G, Marinelli RA. Defective hepatocyte aquaporin-8 expression and reduced canalicular membrane water permeability in estrogeninduced cholestasis. Am J Physiol Gastrointest Liver Physiol 292: G905-G912, 2007. First published November 16, 2006; doi:10.1152/ajpgi.00386.2006.-Our previous work supports a role for aquaporin-8 (AQP8) water channels in rat hepatocyte bile formation mainly by facilitating the osmotically driven canalicular secretion of water. In this study, we tested whether a condition with compromised canalicular bile secretion, i.e., the estrogen-induced intrahepatic cholestasis, displays defective hepatocyte AQP8 functional expression. After 17␣-ethinylestradiol administration (5 mg ⅐ kg body wt Ϫ1 ⅐ day Ϫ1 for 5 days) to rats, the bile flow was reduced by 58% (P Ͻ 0.05). By subcellular fractionation and immunoblotting analysis, we found that 34 kDa AQP8 was significantly decreased by ϳ70% in plasma (canalicular) and intracellular (vesicular) liver membranes. However, 17␣-ethinylestradiol-induced cholestasis did not significantly affect the protein level or the subcellular localization of sinusoidal AQP9. Immunohistochemistry for liver AQPs confirmed these observations. Osmotic water permeability (P f) of canalicular membranes, measured by stopped-flow spectrophotometry, was significantly reduced (73 Ϯ 1 vs. 57 Ϯ 2 m/s) in cholestasis, consistent with defective canalicular AQP8 functional expression. By Northern blotting, we found that AQP8 mRNA expression was increased by 115% in cholestasis, suggesting a posttranscriptional mechanism of protein level reduction. Accordingly, studies in primary cultured rat hepatocytes indicated that the lysosomal protease inhibitor leupeptin prevented the estrogen-induced AQP8 downregulation. In conclusion, hepatocyte AQP8 protein expression is downregulated in estrogeninduced intrahepatic cholestasis, presumably by lysosomal-mediated degradation. Reduced canalicular membrane AQP8 expression is associated with impaired osmotic membrane water permeability. Our data support the novel notion that a defective expression of canalicular AQP8 contributes as a mechanism for bile secretory dysfunction of cholestatic hepatocytes.aquaporins; intrahepatic cholestasis; water transport; liver AQUAPORIN (AQP) water channels are a family of integral membrane proteins known to facilitate the osmotic water movement across the cellular membranes (3). Three members of the AQP family are expressed in rat hepatocytes: AQP8 (7, 13, 16, 21), AQP9 (14, 21, 40), and AQP0 (21). AQP8 is localized in the canalicular plasma membrane domain (7,13,16,21) as well as in intracellular vesicles (7,13,16,21) and mitochondria (6). Its trafficking from a vesicular compartment to the canalicular membrane can be induced by a choleretic stimulus, such as dibutyryl-cAMP (16,21) or the hormone glucagon (18,19,30). AQP9 resides exclusively on the sinusoidal plasma membranes of hepatocytes and thereby may facilitate the movement of water and certain small so...

Mitochondrial dysfunction and oxidative stress play a central role in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). This study aimed to elucidate the mechanism(s) responsible for mitochondrial dysfunction in nonalcoholic fatty liver. Fatty liver was induced in rats with a choline-deficient (CD) diet for 30 days. We examined the effect of CD diet on various parameters related to mitochondrial function such as complex I activity, oxygen consumption, reactive oxygen species (ROS) generation and cardiolipin content and oxidation. The activity of complex I was reduced by 35% in mitochondria isolated from CD livers compared with the controls. These changes in complex I activity were associated with parallel changes in state 3 respiration. Hydrogen peroxide (H(2)O(2)) generation was significantly increased in mitochondria isolated from CD livers. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, decreased by 38% as function of CD diet, while there was a significantly increase in the level of peroxidized cardiolipin. The lower complex I activity in mitochondria from CD livers could be completely restored to the level of control livers by exogenously added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids nor by peroxidized cardiolipin. It is concluded that CD diet causes mitochondrial complex I dysfunction which can be attributed to ROS-induced cardiolipin oxidation. These findings provide new insights into the alterations underlying mitochondrial dysfunction in NAFLD.

Aquaporins are channel proteins widely expressed in nature and known to facilitate the rapid movement of water across numerous cell membranes. A mammalian aquaporin, AQPS, was recently discovered and found to have a very distinct evolutionary pathway. To understand the reason for this divergence, here we define the ontogeny and exact subcellular localization of AQPS in mouse liver, a representative organ transporting large volumes of water for secretion of bile. Northern blotting showed strong AQPS expression between fetal day 17 and birth as well as at weaning and thereafter. Interestingly, this pattern was confirmed by immunohistochemistry and coincided both temporally and spatially with that of hepatic glycogen accumulation. As seen by reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, fasting was accompanied by remarkable down-regulation of hepatic AQPS that paralleled the expected depletion of glycogen content. The level of hepatic AQPS returned to be considerable after refeeding. Immunoelectron microscopy confirmed AQPS in hepatocytes where labeling was over smooth endoplasmic reticulum (SER) membranes adjacent to glycogen granules and in canalicular membranes, subapical vesicles, and some mitochondria. In conclusion, in addition to supporting a role for AQPS in canalicular water secretion, these findings also suggest an intracellular involvement of AQPS in preserving cytoplasmic osmolality during glycogen metabolism and in maintaining mitochondrial volume. AQPS may have evolved separately to feature these intracellular roles as no other known aquaporin shows this specialization. (HEPATOLOGY 2003;38:947-957.) quaporins are a family of proteinaceous channels widely distributed in a range of living organisms A from bacteria to humans, where they mediate the osmotic movement of water across biological membranes.' Aquaporins are being shown to be involved in physiologic processes of central importance, including se- cretion and absorption of fluids in the gastrointestinal and reproductive tracts, renal water handling, fluid balance in the lung and brain, maintenance of fluid and ionic homeostases in the inner ear,2 and preserving corneal transparency.3 Aquaporins also have pathophysiologic relevance because they have been found in humans to underlie severe forms of nephrogenic diabetes in~ipidus,~ defective urinary concentrating,5 and congenital cataracts,6 and their involvement in the pathogenesis of many other clinical conditions with altered fluid homeostasis is under intense investigation. Among the 1 1 aquaporins (AQPO-AQP 10) that have so far been recognized in mammals, AQP8 represents a very distinctive homologue as indicated by the striking divergence of its evolutionary pathway.' However, the reasons for this phylogenetic divergence are obscure. AQP8 was recently cloned from rat,8,9 mouse,1° and human' ' tissues. By heterologous expression in Xenopw hevis oocytes, the rat8,9 and human' ' AQP8 channels were found to be selectively permeable to water, whereas the mouse...