Gut microbiota and fecal volatilome profile inspection in metabolically healthy and unhealthy obesity phenotypes.

BACKGROUND: People with metabolically healthy (MHO) and metabolically unhealthy obesity (MUO) differ for the presence or absence of cardio-metabolic complications, respectively. OBJECTIVE: Based on these differences, we are interested in deepening whether these obesity phenotypes could be linked to changes in microbiota and metabolome profiles. In this respect, the overt role of microbiota taxa composition and relative metabolic profiles is not completely understood. At this aim, biochemical and nutritional parameters, fecal microbiota, metabolome and SCFA compositions were inspected in patients with MHO and MUO under a restrictive diet regimen with a daily intake ranging from 800 to 1200 kcal. METHODS: Blood, fecal samples and food questionnaires were collected from healthy controls (HC), and an obese cohort composed of both MHO and MUO patients. Most impacting biochemical/anthropometric variables from an a priori sample stratification were detected by applying a robust statistics approach useful in lowering the background noise. Bacterial taxa and volatile metabolites were assessed by qPCR and gas chromatography coupled with mass spectrometry, respectively. A targeted GC-MS analyses on SCFAs was also performed. RESULTS: Instructed to follow a controlled and restricted daily calorie intake, MHO and MUO patients showed differences in metabolic, gut microbial and volatilome signatures. Our data revealed higher quantities of specific pro-inflammatory taxa (i.e., Desulfovibrio and Prevotella genera) and lower quantities of Clostridium coccoides group in MUO subset. Higher abundances in alkane, ketone, aldehyde, and indole VOC classes together with a lower amount of butanoic acid marked the faecal MUO metabolome. CONCLUSIONS: Compared to MHO, MUO subset symptom picture is featured by specific differences in gut pro-inflammatory taxa and metabolites that could have a role in the progression to metabolically unhealthy status and developing of obesity-related cardiometabolic diseases. The approach is suitable to better explain the crosstalk existing among dysmetabolism-related inflammation, nutrient intake, lifestyle, and gut dysbiosis.

GFAP serves as a structural element of tunneling nanotubes between glioblastoma cells and could play a role in the intercellular transfer of mitochondria.

Tunneling nanotubes (TNTs) are long F-actin-positive plasma membrane bridges connecting distant cells, allowing the intercellular transfer of cellular cargoes, and are found to be involved in glioblastoma (GBM) intercellular crosstalk. Glial fibrillary acid protein (GFAP) is a key intermediate filament protein of glial cells involved in cytoskeleton remodeling and linked to GBM progression. Whether GFAP plays a role in TNT structure and function in GBM is unknown. Here, analyzing F-actin and GFAP localization by laser-scan confocal microscopy followed by 3D reconstruction (3D-LSCM) and mitochondria dynamic by live-cell time-lapse fluorescence microscopy, we show the presence of GFAP in TNTs containing functional mitochondria connecting distant human GBM cells. Taking advantage of super-resolution 3D-LSCM, we show the presence of GFAP-positive TNT-like structures in resected human GBM as well. Using H2O2 or the pro-apoptotic toxin staurosporine (STS), we show that GFAP-positive TNTs strongly increase during oxidative stress and apoptosis in the GBM cell line. Culturing GBM cells with STS-treated GBM cells, we show that STS triggers the formation of GFAP-positive TNTs between them. Finally, we provide evidence that mitochondria co-localize with GFAP at the tip of close-ended GFAP-positive TNTs and inside receiving STS-GBM cells. Summarizing, here we found that GFAP is a structural component of TNTs generated by GBM cells, that GFAP-positive TNTs are upregulated in response to oxidative stress and pro-apoptotic stress, and that GFAP interacts with mitochondria during the intercellular transfer. These findings contribute to elucidate the molecular structure of TNTs generated by GBM cells, highlighting the structural role of GFAP in TNTs and suggesting a functional role of this intermediate filament component in the intercellular mitochondria transfer between GBM cells in response to pro-apoptotic stimuli.

Intercellular crosstalk mediated by tunneling nanotubes between central nervous system cells. What we need to advance.

Long-range intercellular communication between Central Nervous System (CNS) cells is an essential process for preserving CNS homeostasis. Paracrine signaling, extracellular vesicles, neurotransmitters and synapses are well-known mechanisms involved. A new form of intercellular crosstalk mechanism based on Tunneling Nanotubes (TNTs), suggests a new way to understand how neural cells interact with each other in controlling CNS functions. TNTs are long intercellular bridges that allow the intercellular transfer of cargoes and signals from one cell to another contributing to the control of tissue functionality. CNS cells communicate with each other via TNTs, through which ions, organelles and other signals are exchanged. Unfortunately, almost all these results were obtained through 2D in-vitro models, and fundamental mechanisms underlying TNTs-formation still remain elusive. Consequently, many questions remain open, and TNTs role in CNS remains largely unknown. In this review, we briefly discuss the state of the art regarding TNTs identification and function. We highlight the gaps in the knowledge of TNTs and discuss what is needed to accelerate TNTs-research in CNS-physiology. To this end, it is necessary to: 1) Develop an ad-hoc TNTs-imaging and software-assisted processing tool to improve TNTs-identification and quantification, 2) Identify specific molecular pathways involved into TNTs-formation, 3) Use in-vitro 3D-CNS and animal models to investigate TNTs-role in a more physiological context pushing the limit of live-microscopy techniques. Although there are still many steps to be taken, we believe that the study of TNTs is a new and fascinating frontier that could significantly contribute to deciphering CNS physiology.

Simvastatin increases AQP2 urinary excretion in hypercholesterolemic patients: A pleiotropic effect of interest for patients with impaired AQP2 trafficking.

We previously reported that statins improve the symptoms of X-linked nephrogenic diabetes insipidus (X-NDI) in animal models. The aim of this study was to verify whether the pleiotropic effect of statins on AQP2 trafficking and kidney-concentrating ability, observed in rodents, was attainable in humans at therapeutic doses. We enrolled 24 naïve hypercholesterolemic patients and measured urine excretion of AQP2 (uAQP2) at baseline and during 12 weeks of treatment with simvastatin 20 mg/day. Simvastatin induced a rapid and significant increase of uAQP2, reduced the 24-hour diuresis, and increased urine osmolality. These effects were also maintained in patients chronically treated with statins for at least 1 year. This study strongly suggests that statins may effectively enhance the efficacy of current pharmacological treatment of patients with urine-concentrating defects caused by defective AQP2 plasma membrane trafficking, like X-NDI.

Reduced hepatic aquaporin-9 and glycerol permeability are related to insulin resistance in non-alcoholic fatty liver disease.

BACKGROUND/OBJECTIVES: Glycerol represents an important metabolite for the control of lipid accumulation and hepatic gluconeogenesis. We investigated whether hepatic expression and functionality of aquaporin-9 (AQP9), a channel mediating glycerol influx into hepatocytes, is impaired in non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) in the context of insulin resistance. SUBJECTS/METHODS: Liver biopsies were obtained from 66 morbid obese patients undergoing bariatric surgery (66% women, mean body mass index (BMI) 46.1±1.0 kg m(-2)) with available liver echography and pathology analysis of the biopsies in this cross-sectional study. Subjects were classified according to normoglycemia (NG), impaired glucose tolerance (IGT) or type 2 diabetes (T2D). Hepatic expression of AQP9 was analyzed by real-time PCR, western blotting and immunohistochemistry, while glycerol permeability (P(gly)) was measured by stopped-flow light scattering. RESULTS: AQP9 was the most abundantly (P<0.0001) expressed aquaglyceroporin in human liver (AQP9>>>AQP3>AQP7>AQP10). Obese patients with T2D showed increased plasma glycerol as well as lower P(gly) and hepatic AQP9 expression. The prevalence of NAFLD and NASH in T2D patients was 100 and 65%, respectively. Interestingly, AQP9 expression was decreased in patients with NAFLD and NASH as compared with those without hepatosteatosis, in direct relation to the degree of steatosis and lobular inflammation, being further reduced in insulin-resistant individuals. The association of AQP9 with insulin sensitivity was independent of BMI and age. Consistent with these data, fasting insulin and C-reactive protein contributed independently to 33.1% of the hepatic AQP9 mRNA expression variance after controlling for the effects of age and BMI. CONCLUSIONS: AQP9 downregulation together with the subsequent reduction in hepatic glycerol permeability in insulin-resistant states emerges as a compensatory mechanism whereby the liver counteracts further triacylglycerol accumulation within its parenchyma as well as reduces hepatic gluconeogenesis in patients with NAFLD.

Cell culture models and animal models for studying the patho-physiological role of renal aquaporins.

Aquaporins (AQPs) are key players regulating urinary-concentrating ability. To date, eight aquaporins have been characterized and localized along the nephron, namely, AQP1 located in the proximal tubule, thin descending limb of Henle, and vasa recta; AQP2, AQP3 and AQP4 in collecting duct principal cells; AQP5 in intercalated cell type B; AQP6 in intercalated cells type A in the papilla; AQP7, AQP8 and AQP11 in the proximal tubule. AQP2, whose expression and cellular distribution is dependent on vasopressin stimulation, is involved in hereditary and acquired diseases affecting urine-concentrating mechanisms. Due to the lack of selective aquaporin inhibitors, the patho-physiological role of renal aquaporins has not yet been completely clarified, and despite extensive studies, several questions remain unanswered. Until the recent and large-scale development of genetic manipulation technology, which has led to the generation of transgenic mice models, our knowledge on renal aquaporin regulation was mainly based on in vitro studies with suitable renal cell models. Transgenic and knockout technology approaches are providing pivotal information on the role of aquaporins in health and disease. The main goal of this review is to update and summarize what we can learn from cell and animal models that will shed more light on our understanding of aquaporin-dependent renal water regulation.

D184E mutation in aquaporin-4 gene impairs water permeability and links to deafness.

Aquaporins (AQPs) play a physiological role in several organs and tissues, and their alteration is associated with disorders of water regulation. The identification of molecular interactions, which are crucial in determining the rate of water flux through the channel, is of pivotal role for the discovery of molecules able to target those interactions and therefore to be used for pathologies ascribable to an altered AQP-dependent water balance. In the present study, a mutational screening of human aquaporin-4 (AQP4) gene was performed on subjects with variable degrees of hearing loss. One heterozygous missense mutation was identified in a Spanish sporadic case, leading to an Asp/Glu amino acid substitution at position 184 (D184E). A BLAST analysis revealed that the amino acid D184 is conserved across species, consistently with a crucial role in the structure/function of AQP4 water channels. The mutation induces a significant reduction in water permeability as measured by the Xenopus laevis oocytes swelling assay and by the use of mammalian cells by total internal reflection microscopy. By Western blot, immunofluorescence and 2D Blue Native/SDS-PAGE we show that the reduction in water permeability is not ascribable to a reduced expression of AQP4 mutant protein or to its incorrect plasma membrane targeting and aggregation into orthogonal arrays of particles. Molecular dynamics simulation provided a molecular explanation of the mechanism whereby the mutation induces a loss of function of the channel. Substituting glutamate for aspartate affects the mobility of the D loop, which acquires a higher propensity to equilibrate in a "closed conformation", thus affecting the rate of water flux. We speculate that this mutation, combined with other genetic defects or concurrently with certain environmental stimuli, could confer a higher susceptibility to deafness.

Serum and CSF N-acetyl aspartate levels differ in multiple sclerosis and neuromyelitis optica.

BACKGROUND: The identification of biomarkers able to improve the differential diagnosis between multiple sclerosis (MS) and neuromyelitis optica (NMO) is challenging because of a different prognosis and response to treatment. Growing evidence indicates that brain and CSF N-acetyl aspartate (NAA) concentration is a useful marker for characterising different phases of axonal pathology in demyelinating diseases, and preliminary studies suggest that increased serum NAA levels may be a telltale sign of acute neuronal damage or defective NAA metabolism in oligodendrocytes. OBJECTIVE: To evaluate whether serum and CSF NAA concentration differs in patients with MS and NMO. DESIGN: Observational, multicentre, prospective, cross sectional study. METHODS: Serum samples were collected from 48 relapsing-remitting MS, 32 NMO and 76 age matched healthy controls. Coeval CSF samples were available for all MS and for 8/32 NMO patients. NAA was measured in serum and CSF by liquid chromatography-mass spectrometry. RESULTS: MS patients showed higher serum and CSF NAA levels than NMO patients, and higher serum NAA levels than healthy controls (p<0.001). High serum NAA values, exceeding the 95th percentile of serum NAA values in healthy controls, were found in 100% of patients with MS and in no patient with NMO. No differences in serum NAA levels were found between NMO and healthy controls. In MS, serum and CSF NAA levels correlated with disability score. CONCLUSIONS: Determination of serum and CSF NAA levels may represent a suitable tool in the diagnostic laboratory workup to differentiate MS and NMO.

Aquaporin-2 urinary excretion in preterm infants: relationship to diuresis and vasopressin.

AIMS: Few investigations have explored the urinary aquaporin-2 (u-AQP2) excretion pattern after birth in preterm infants with conflicting results regarding the correlation between u-AQP2, urinary osmolality and vasopressin. The aims of this study were to evaluate u-AQP2 excretion during the first week of life in preterm infants, to correlate u-AQP2 with other markers of renal function and to investigate the relationship between u-AQP2, urinary tonicity and arginine-vasopressin in the immature kidney. METHODS: In infants born less than 33 weeks daily diuresis, u-AQP2, urinary arginine-vasopressin, urine and plasma tonicity, creatinine and electrolytes were measured through the first 7 days of life. RESULTS: Fifty-five infants were evaluated. u-AQP2 excretion showed the following profile: the highest u-AQP2 levels were found on day 2 and values remained significantly higher until day 5 with respect to day 1. On day 6, u-AQP2 levels significantly decreased to values closer to those found on day 1. u-AQP2 excretion was not associated with arginine-vasopressin while significant, but weak association was found with urinary tonicity (r = -0.20; -0.32 < r < -0.11; P < 0.05). u-AQP2 excretion and creatinine clearance were significantly associated during the study period (r = 0.19; 0.08 < r < 0.29; P < 0.05). There was a strong association between totally u-AQP2 excretion and diuresis over the week (r = 0.72; 0.66 < r < 0.76; P < 0.0001). CONCLUSION: Significant variations occur in AQP2 expression levels during the first week of life in preterm infants. AQP2 does not seem to contribute to the urinary concentration ability after birth. Further investigations are required to elucidate the mechanisms underlying the strong association between diuresis and u-AQP2 excretion in early postnatal life.

Higher order structure of aquaporin-4.

Unlike other mammalian AQPs, multiple tetramers of AQP4 associate in the plasma membrane to form peculiar structures called Orthogonal Arrays of Particles (OAPs), that are observable by freeze-fracture electron microscopy (FFEM). However, FFEM cannot give information about the composition of OAPs of different sizes, and due to its technical complexity is not easily applicable as a routine technique. Recently, we employed the 2D gel electrophoresis BN-SDS/PAGE that for the first time enabled the biochemical isolation of AQP4-OAPs from several tissues. We found that AQP4 protein is present in several higher-order complexes (membrane pools of supra-structures) which contain different ratios of M1/M23 isoforms corresponding to AQP4-OAPs of different size. In this paper, we illustrate in detail the potentiality of 2D BN/SDS-PAGE for analyzing AQP4 supra-structures, their relationship with the dystrophin glycoprotein complex and other membrane proteins, and their role as a specific target of Neuromyelitis Optica autoantibodies.

Lovastatin-induced cholesterol depletion affects both apical sorting and endocytosis of aquaporin-2 in renal cells.

Vasopressin causes the redistribution of the water channel aquaporin-2 (AQP2) from cytoplasmic storage vesicles to the apical plasma membrane of collecting duct principal cells, leading to urine concentration. The molecular mechanisms regulating the selective apical sorting of AQP2 are only partially uncovered. In this work, we investigate whether AQP2 sorting/trafficking is regulated by its association with membrane rafts. In both MCD4 cells and rat kidney, AQP2 preferentially associated with Lubrol WX-insoluble membranes regardless of its presence in the storage compartment or at the apical membrane. Block-and-release experiments indicate that 1) AQP2 associates with detergent-resistant membranes early in the biosynthetic pathway; 2) strong cholesterol depletion delays the exit of AQP2 from the trans-Golgi network. Interestingly, mild cholesterol depletion promoted a dramatic accumulation of AQP2 at the apical plasma membrane in MCD4 cells in the absence of forskolin stimulation. An internalization assay showed that AQP2 endocytosis was clearly reduced under this experimental condition. Taken together, these data suggest that association with membrane rafts may regulate both AQP2 apical sorting and endocytosis.

Aquaporin 2 and apical calcium-sensing receptor: new players in polyuric disorders associated with hypercalciuria.

The kidney plays a critical role in regulating water homeostasis through specific proteins highly expressed in the kidney, called aquaporins, allowing water permeation at a high rate. This brief review focuses on some nephropathies associated with impaired urinary concentrating ability and in particular analyzes the role of aquaporin 2 in hypercalciuria, the most common metabolic abnormality in patients with nephrolithiasis. Specifically, this review discusses the relationship between hypercalciuria and impaired aquaporin 2-mediated water handling in both acquired and inherited disorders characterized by hypercalciuria, including those affecting the sensor of extracellular calcium concentration, the calcium-sensing receptor, which represents the principal target for extracellular calcium regulation of several tissues including parathyroid glands and kidney. In the kidney, the calcium-sensing receptor regulates renal calcium excretion and influences the transepithelial movement of water and other electrolytes. Understanding the molecular basis of alteration of kidney concentrating ability found in hypercalciuria will help for devising strategies for reducing the risk of nephrocalcinosis, nephrolithiasis, and renal insufficiency.

Aquaporins in the hepatobiliary tract. Which, where and what they do in health and disease.

The biological importance of the aquaporin family of water channels was recently acknowledged by the 2003 Nobel Prize for Chemistry awarded to the discovering scientist Peter Agre. Among the pleiotropic roles exerted by aquaporins in nature in both health and disease, the review addresses the latest acquisitions about the expression and regulation, as well as physiology and pathophysiology of aquaporins in the hepatobiliary tract. Of note, at least seven out of the thirteen mammalian aquaporins are expressed in the liver, bile ducts and gallbladder. Aquaporins are essential for bile water secretion and reabsorption, as well as for plasma glycerol uptake by the hepatocyte and its conversion to glucose during starvation. Novel data are emerging regarding the physio-pathological involvement of aquaporins in multiple diseases such as cholestases, liver cirrhosis, obesity and insulin resistance, fatty liver, gallstone formation and even microparasite invasion of intrahepatic bile ducts. This body of knowledge represents the mainstay of present and future research in a rapidly expanding field.

Effects of chronic treatment with statins and fenofibrate on rat skeletal muscle: a biochemical, histological and electrophysiological study.

BACKGROUND AND PURPOSE: Skeletal muscle injury by hypolipidemic drugs is not fully understood. An extensive analysis of the effect of chronic treatment with fluvastatin (5 mgkg(-1) and 20 mgkg(-1)), atorvastatin (10 mgkg(-1)) and fenofibrate (60 mgkg(-1)) on rat skeletal muscle was undertaken. EXPERIMENTAL APPROACH: Myoglobinemia as sign of muscle damage was measured by enzymatic assay. Histological and immunohistochemical techniques were used to estimate muscle integrity and the presence of aquaporin-4, a protein controlling water homeostasis. Electrophysiological evaluation of muscle Cl(-) conductance (gCl) and mechanical threshold (MT) for contraction, index of intracellular calcium homeostasis, was performed by the two-intracellular microelectrodes technique. KEY RESULTS: Fluvastatin (20 mgkg(-1)) increased myoglobinemia. The lower dose of fluvastatin did not modify myoglobinemia, but reduced urinary electrolytes, suggesting direct effects on renal function. Atorvastatin also increased myoglobinemia, with slight effects on urinary parameters. No treatment caused any histological damage to muscle or modification in the number of fibres expressing aquaporin-4. Either fluvastatin (at both doses) or atorvastatin reduced sarcolemma gCl and changed MT. Both statins produced slight effects on total cholesterol, suggesting that the observed modifications occur independently of HMGCoA-reductase inhibition. Fenofibrate increased myoglobinemia and decreased muscle gCl, whereas it did not change the MT, suggesting a different mechanism of action from the statins. CONCLUSIONS AND IMPLICATIONS: This study identifies muscle gCl and MT as early targets of drugs action that may contribute to milder symptoms of myotoxicity, such as muscle cramps, while the increase of myoglobinemia is a later phenomenon.

Aquaporin-2 excretion and renal function during the 1st week of life in preterm newborn infants.

In many preterm infants, a characteristic pattern of fluid and electrolyte homeostasis occurs during the 1st week of life, consisting of three phases: prediuretic, diuretic, and postdiuretic. In this study, we evaluated the possible role of aquaporin-2 (AQP2) in renal concentrating ability and correlated it with other markers of the renal function in healthy preterm infants. Daily urine and spot blood samples were collected from 9 healthy preterm (32 +/- 1 weeks) infants at postnatal ages 1, 3, and 7 days. Urine and serum osmolality, creatinine, electrolytes, and AQP2 excretion were measured. All infants showed a significant (about 7%) weight loss on day 3 associated with a more than threefold increase in urine output without a significant change in fluid intake (diuretic phase). The creatinine clearance increased on day 3, indicating an increase in glomerular filtration rate. Interestingly, on day 3, the level of total excreted AQP2 (pmol/h) was significantly higher when compared to day 1 and day 7, and the same tendency was observed for urine osmolality. To conclude, the observed increase in urine osmolality and creatinine clearance during the diuretic phase, paralleled by an increase in total AQP2 excretion, suggests that AQP2 can contribute to the urinary concentrating ability early in postnatal life.

Water immersion is associated with an increase in aquaporin-2 excretion in healthy volunteers.

Here, we report the alterations in renal water handling in healthy volunteers during a 6 h thermoneutral water immersion at 34 to 36 degrees C. We found that water immersion is associated with a reversible increase in total urinary AQP2 excretion.

In vivo silencing of aquaporin-1 by RNA interference inhibits angiogenesis in the chick embryo chorioallantoic membrane assay.

Aquaporin-1 (AQP1) is a water channel protein mainly expressed in endothelial and epithelial cells of many tissues, including the vasculature where it serves to increase cell membrane water permeability. Previous studies in active multiple myeloma patients and in AQP1 KO mice indicated an involvement of AQP1 in physiological and tumor angiogenesis. To understand the physiological role of AQP1 in angiogenesis, we used a 21-nucleotide small interfering RNA duplexes (siRNA) to knockdown AQP1 in the chick embryo chorioallantoic membrane (CAM), a commonly used in vivo assay to study both angiogenic and angiostatic molecules. Chicken AQP1 sequence was identified and utilized to synthesize a siRNA directed to the AQP1 sequence. We then tested the efficiency of the siRNA in vitro, using an AQP1 transfected cell line. The level of AQP1 protein reduction obtained using siRNA was 98 % and 92 % after 1 and 2 day transfection respectively. RNA interference experiments were then performed in vivo by using the CAM assay. Results showed that after 4 days of treatment, AQP1 siRNA was able to strongly inhibit angiogenesis. This is the first study showing the in vivo use of RNA interference technique in the CAM assay. Our results strongly support the hypothesis that AQP1 could have a key role in physiological and pathological angiogenesis.

Altered expression of aquaporin 4 and H(+)/K(+)-ATPase in the stomachs of peptide YY (PYY) transgenic mice.

BACKGROUND INFORMATION: The hormone PYY (peptide YY), synthesized by endocrine cells in the pancreas, ileum, colon and stomach has widespread inhibitory effects on gastrointestinal and pancreatic fluid secretion. Transgenic mice expressing a viral oncoprotein under the control of the PYY gene 5'-flanking region develop well-differentiated colonic endocrine tumours producing mainly PYY and enteroglucagon. In the present study, we investigated the expression of AQP4 (aquaporin 4) water channel and H(+)/K(+)-ATPase in stomachs from both control and transgenic mice. RESULTS: Semi-quantitative RT (reverse transcriptase)-PCR showed an increase in the AQP4 transcript compared with control mice. Quantitative Western-blot analysis of stomachs from control and transgenic mice confirmed a significant increase in the 30 kDa AQP4 protein in transgenic mice. In control mice, AQP4 is specifically expressed in the basolateral membrane of gastric parietal cells, located in the basal region of the fundic glands. This particular location suggests that parietal cells in the base region of gastric pits might have a major role in water transport when compared with the more superficial parietal cells. Interestingly, immunofluorescence studies on transgenic mice revealed that the quantitative increase of AQP4 expression was actually due to an increase in the number of AQP4-expressing epithelial cells rather than to a higher expression of AQP4 in parietal cells. In fact, immunofluorescence experiments using the specific antibody raised against the AE2 isoform of Cl(-)/HCO3- exchanger specifically expressed in parietal cells confirmed that the number of parietal cells was comparable in both PYY and control stomachs. Moreover, in transgenic mice, a parallel significant decrease in the expression of H(+)/K(+)-ATPase was observed, as revealed by RT-PCR, quantitative immunoblotting and immunofluorescence. CONCLUSIONS: In the present study, we demonstrate that the sustained inhibition of gastric secretion due to tumours producing PYY/enteroglucagon in transgenic mice is associated with an increase in AQP4 expression and a down-regulation of H(+)/K(+)-ATPase in parietal cells that acquire the characteristics of basal parietal cells. The absence of H2 receptors-mediated signalling due to the inhibition of histamine release from ECL (enterochromaffin-like) cells by PYY may be in part responsible for the observed increase in the number of parietal cells expressing AQP4.

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. RESULTS: In the present study, we investigate the presence and subcellular localization of AQP (aquaporin) water channels in the mouse gall-bladder epithelium. Considerable AQP8 mRNA was detected in the gall-bladder epithelium of mouse, calf, rabbit, guinea pig and man. Studies of subcellular localization were then addressed to the mouse gall-bladder where the transcript of a second AQP, AQP1, was also detected. Immunoblotting experiments confirmed the presence of AQP8 and AQP1 at a protein level. Immunohistochemistry showed intense expression of AQP8 and AQP1 in the gall-bladder epithelial cells where AQP8 was localized in the apical membrane, whereas AQP1 was seen both in the apical and basolateral membranes, and in vesicles located in the subapical cytoplasm. CONCLUSIONS: The pattern of subcellular distribution of AQP8 and AQP1 strongly corroborates the hypothesis of a transcellular route for the movement of water across the gall-bladder epithelium. Osmotic water would cross the apical membrane through AQP8 and AQP1, although AQP1 would be the facilitated pathway for the movement of water across the basolateral membrane. The presence of two distinct AQPs in the apical membrane is an unusual finding and may relate to the membrane's ability both to absorb and secrete fluid. It is tempting to hypothesize that AQP1 is hormonally translocated to the gall-bladder apical membrane to secrete water as in the bile duct epithelium, a functional homologue of the gall-bladder epithelium, whereas apical AQP8 may account for the absorption of water from gall-bladder bile.

The role of aquaporin-4 in the blood-brain barrier development and integrity: studies in animal and cell culture models.

Aquaporin-4 (AQP4) is the major water channel expressed in brain perivascular astrocyte processes. Although the role of AQP4 in brain edema has been extensively investigated, little information exists regarding its functional role at the blood-brain barrier (BBB). The purpose of this work is to integrate previous and recent data regarding AQP4 expression during BBB formation and depending on BBB integrity, using several experimental models. Results from studies on the chick optic tectum, a well-established model of BBB development, and the effect of lipopolysaccharide on the BBB integrity and on perivascular AQP4 expression have been analyzed and discussed. Moreover, data on the BBB structure and AQP4 expression in murine models of Duchenne muscular dystrophy are reviewed. In particular, published results obtained from mdx(3cv) mice have been analyzed together with new data obtained from mdx mice in which all the dystrophin isoforms including DP71 are strongly reduced. Finally, the role of the endothelial component on AQP4 cellular expression and distribution has been investigated using rat primary astrocytes and brain capillary endothelial cell co-cultures as an in vitro model of BBB.