Domenico Ferri

The Inner Mitochondrial Membrane Has Aquaporin-8 Water Channels and Is Highly Permeable to Water

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 (Pf) characterizing the rat liver IMM. Immunoblotting, electron microscopy, and biophysical studies show that the largest mitochondria feature the highest AQP8 expression and IMM Pf. 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 Hg2+, while the related activation energy remained low, suggesting the presence of a Hg2+-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.

Gallbladder histopathology during murine gallstone formation: relation to motility and concentrating function.

C57L mice are susceptible and AKR mice are resistant to gallstone formation. We studied in male mice of both strains gallbladder histopathology, cholecystokinin-induced 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 ± 2.2 μl vs. 8.8 ± 1.0 μl). On the diet, fasting volumes increased exclusively in C57L (28.6 ± 2.9 μl 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.

Biophysical assessment of aquaporin‐9 as principal facilitative pathway in mouse liver import of glucogenetic glycerol

Lipolytic glycerol, released from adipocytes, flows through the bloodstream to the liver, where its utilisation in supplying hepatocyte gluconeogenesis is rate‐limited by the permeation step. An aquaglyceroporin expressed in hepatocytes, aquaporin‐9 (AQP9), has been often linked to liver uptake of glycerol. However, the truthfulness of this postulation and the potential existence of additional pathways of glycerol import by hepatocytes have never been assessed directly. Here, we define the identity and extent of liver glycerol transport and evaluate the correlation between hepatic AQP9 expression and glycerol permeability (Pgly) in AQP9+/+ wild‐type mice in different nutritional states and circulating insulin levels. The liver Pgly of AQP9 null mice is also assessed.

Severe liver steatosis correlates with nitrosative and oxidative stress in rats

Background   Little is known about nitric oxide (NO) metabolism and redox changes with hepatocyte adipocytic transformation. The aims of this study were to investigate the changes occurring in plasma and hepatic NO metabolites and redox balance in a rat experimental model of simple fatty liver, and to relate plasma with hepatic and mitochondrial changes at different degrees of steatosis.

Expression and subcellular localization of the AQP8 and AQP1 water channels in the mouse gall-bladder epithelium

Background information. Transepithelial transport of water is one of the most distinctive functions by which the gall‐bladder rearranges its bile content. Water is reabsorbed from the gall‐bladder lumen during fasting, whereas it is secreted into the lumen following meal ingestion. Nevertheless, the molecular mechanism by which water is transported across the gall‐bladder epithelium remains mostly unclear.

Altered expression and distribution of aquaporin-9 in the liver of rat with obstructive extrahepatic cholestasis

Rat hepatocytes express aquaporin-9 (AQP9), a basolateral channel permeable to water, glycerol, and other small neutral solutes. Although liver AQP9 is known for mediating the uptake of sinusoidal blood glycerol, its relevance in bile secretion physiology and pathophysiology remains elusive. Here, we evaluated whether defective expression of AQP9 is associated to secretory dysfunction of rat hepatocytes following bile duct ligation (BDL). By immunoblotting, 1-day BDL resulted in a slight decrease of AQP9 protein in basolateral membranes and a simultaneous increase of AQP9 in intracellular membranes. This pattern was steadily accentuated in the subsequent days of BDL since at 7 days BDL basolateral membrane AQP9 decreased by 85% whereas intracellular AQP9 increased by 115%. However, the AQP9 immunoreactivity of the total liver membranes from day 7 of BDL rats was reduced by 49% compared with the sham counterpart. Results were confirmed by immunofluorescence and immunogold electron microscopy and consistent with biophysical studies showing considerable decrease of the basolateral membrane water and glycerol permeabilities of cholestatic hepatocytes. The AQP9 mRNA was slightly reduced only at day 7 of BDL, indicating that the dysregulation was mainly occurring at a posttranslational level. The altered expression of liver AQP9 during BDL was not dependent on insulin, a hormone known to negatively regulate AQP9 at a transcriptional level, since insulinemia was unchanged in 7-day BDL rats. Overall, these results suggest that extrahepatic cholestasis leads to downregulation of AQP9 in the hepatocyte basolateral plasma membrane and dysregulated aquaporin channels contribute to bile flow dysfunction of cholestatic hepatocyte.

Ultrastructural zonal heterogeneity of hepatocytes and mitochondria within the hepatic acinus during liver regeneration after partial hepatectomy.

Background information. Partial hepatectomy (70%) induces cell proliferation until the original mass of the liver is restored. In the first 24 h after partial hepatectomy, drastic changes in the metabolism of the remaining liver have been shown to occur. To evaluate changes in hepatocyte ultrastructure within the hepatic acinus during the liver regenerative process, we investigated, by light and electron microscopy observations on specimens taken 0 h, 24 h and 96 h after partial hepatectomy, the hepatocyte structure and ultrastructure in the periportal and pericentral area of the hepatic acinus, with a particular emphasis on mitochondria ultrastructure. Moreover, some biochemical events that could affect the mitochondria ultrastructure and function were investigated.

Glycoconjugate histochemistry of the digestive tract of Triturus carnifex (Amphibia, Caudata)

SummaryIn this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4)n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4)n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4)noligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4)noligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H+K+-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4)noligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.

Morphological and histochemical variations of mucous and oxynticopeptic cells in the stomach of the seps, Chalcides chalcides.

Mucous and oxynticopeptic cells in the gastric mucosa of the seps, Chalcides chalcides (Linnaeus, 1758) were examined by standard histochemical staining methods and by lectin histochemistry. The epithelial mucous cells lining the surface of the stomach and the mucous cells of the fundic glands elaborated mainly neutral glycoproteins with β(1,4)GlcNAc oligomers, GalNAc glycosidic residues and Galβ1,3GalNAc terminal sequences. The mucous cells of the fundic glands were stained specifically with the Paradoxical Con A method. The mucosecreting cells of the pyloric glands produced neutral glycoproteins, with β(1,4)GlcNAc oligomers, GalNAc residues and Galβ1,3GalNAc terminal sequences. Terminal L‐fucose bound to the penultimate GlcNAc residues, and/or difucosylated oligosaccharides were also present. The pyloric glands did not stain with the Paradoxical Con A procedure. The morphology of the oxynticopeptic cells changes from the oral to the aboral region of the fundic mucosa. In the oral fundic tract the oxynticopeptic cells showed cytoplasm filled with zymogen granules, while in the aboral fundic region these cells contained few zymogen granules and showed cytoplasm full of empty vesicles, typical of the acid secreting cells. A secretion gradient of proteolytic enzymes and hydrochloric acid along the fundic mucosa of the seps can be hypothesised.

Altered distribution of caveolin-1 in early liver steatosis

Eur J Clin Invest 2011; 41 (6): 642–651