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Background: The poor response to antiviral treatment of hepatitis C virus (HCV)-infected patients with genotype 1b has been associated with a higher prevalence of metabolic syndrome. However, the molecular link between these clinical entities is not clear. The goal of this study was to clarify the role of genotype 1b and 2 in the genetic expression of suppressor of cytokine signaling 3 (SOCS3) and insulin receptor substrate 1 (IRS-1)., Methods: We infected human hepatocellular carcinoma cell line (HepG2) cells with human HCV genotype 1b or 2 and measured the gene and protein expression of SOCS3 at various times. We also evaluated impairment in the insulin pathway by analysis of IRS-1 and phospho-AKT. For the control, we used HepG2 cell cultures treated with non-infectious serum. We also demonstrated the occurrence of HCV infection by the detection of both positive and negative strands in the cells and culture medium. To test infection of the HepG2 cells, we performed quantitative real-time polymerase chain reaction (qRT-PCR) of viral load at different time points. We analyzed the viral genotype in the pellet and supernatant., Results: At each time point, we found positive and negative strands in the infected cells, while in the medium we found positive, but no negative strands. We also detected the presence of the correct genotype in the medium. Two weeks following infection when the viral load was higher, we tested genotype 1b and 2 infected cells. SOCS3 gene expression was significantly higher in genotype 1b-infected cells (median 2.56; mean 2.82+/-0.59) compared with genotype 2 (median 1.34; mean 1.46+/-0.31) (p=0.04) and control cells (median 1.09; mean 1.02+/-0.11, p=0.02). There was no difference between cells exposed to genotype 2 and control cells. Conversely, IRS-1 was significantly lower in genotype 1b-infected cells (median 15.97; mean 15.45+/-0.67) compared with genotype 2-infected cells (median 16.45; mean 16.44+/-0.01, p=0.04). Statistically significant differences were seen when comparing the pAKT/AKT ratio in genotype 1b-infected cells (0.19+/-0.034) and not genotype 1b-infected (genotype 2-infected and non-infected) cells (0.253+/-0.004, p=0.03). This inverse regulation is compatible with interactions between the molecular expression of SOCS3, IRS-1 and phospho-AKT mediated by the genotype 1b virus., Conclusions: Up-regulation of the SOCS3 gene might be one of the mechanisms governing non-response to therapy and expression of insulin resistance mediated via a direct mechanism at this level of genotype 1b HCV.

Aquaporin-4 (AQP4) is constitutively concentrated in the plasma membrane of the perivascular glial processes, and its expression is altered in certain pathological conditions associated with brain edema or altered glial migration. When astrocytes are grown in culture, they lose their characteristic star-like shape and AQP4 continuous plasma membrane localization observed in vivo. In this study, we differentiated primary astrocyte cultures with cAMP and lovastatin, both able to induce glial stellation through a reorganization of F-actin cytoskeleton, and obtained AQP4 selectively localized on the cell plasma membrane associated with an increase in the plasma membrane water transport level, but only cAMP induced an increase in AQP4 total protein expression. Phosphorylation experiments indicated that AQP4 in astrocytes is neither phosphorylated nor a substrate of PKA. Depolymerization of F-actin cytoskeleton performed by cytochalasin-D suggested that F-actin cytoskeleton plays a primary role for AQP4 plasma membrane localization and during cell adhesion. Finally, AQP4 knockdown does not compromise the ability of astrocytes to stellate in the presence of cAMP, indicating that astrocyte stellation is independent of AQP4., (Copyright 2008 Wiley-Liss, Inc.)

Changes in gravitational force such as that experienced by astronauts during space flight induce a redistribution of fluids from the caudad to the cephalad portion of the body together with an elimination of normal head-to-foot hydrostatic pressure gradients. To assess brain gene profile changes associated with microgravity and fluid shift, a large-scale analysis of mRNA expression levels was performed in the brains of 2-week control and hindlimb-unloaded (HU) mice using cDNA microarrays. Although to different extents, all functional categories displayed significantly regulated genes indicating that considerable transcriptomic alterations are induced by HU. Interestingly, the TIC class (transport of small molecules and ions into the cells) had the highest percentage of up-regulated genes, while the most down-regulated genes were those of the JAE class (cell junction, adhesion, extracellular matrix). TIC genes comprised 16% of those whose expression was altered, including sodium channel, nonvoltage-gated 1 beta (Scnn1b), glutamate receptor (Grin1), voltage-dependent anion channel 1 (Vdac1), calcium channel beta 3 subunit (Cacnb3) and others. The analysis performed by GeneMAPP revealed several altered protein classes and functional pathways such as blood coagulation and immune response, learning and memory, ion channels and cell junction. In particular, data indicate that HU causes an alteration in hemostasis which resolves in a shift toward a more hyper-coagulative state with an increased risk of venous thrombosis. Furthermore, HU treatment seems to impact on key steps of synaptic plasticity and learning processes.

Aminoglycosides force read through of premature stop codon mutations and introduce new mutation-specific gene-corrective strategies in Duchenne muscular dystrophy. A chronic treatment with gentamicin (32 mg/kg/daily i.p., 8-12 weeks) was performed in exercised mdx mice with the dual aim to clarify the dependence on dystrophin of the functional, biochemical and histological alterations present in dystrophic muscle and to verify the long term efficiency of small molecule gene-corrective strategies in work-loaded dystrophic muscle. The treatment counteracted the exercise-induced impairment of in vivo forelimb strength after 6-8 weeks. We observed an increase in dystrophin expression level in all the fibers, although lower than that observed in normal fibers, and found a concomitant recovery of aquaporin-4 at sarcolemma. A significant reduction in centronucleated fibers, in the area of necrosis and in the percentage of nuclear factor-kB-positive nuclei was observed in gastrocnemious muscle of treated animals. Plasma creatine kinase was reduced by 70%. Ex vivo, gentamicin restored membrane ionic conductance in mdx diaphragm and limb muscle fibers. No effects were observed on the altered calcium homeostasis and sarcolemmal calcium permeability, detected by electrophysiological and microspectrofluorimetric approaches. Thus, the maintenance of a partial level of dystrophin is sufficient to reinforce sarcolemmal stability, reducing leakiness, inflammation and fiber damage, while correction of altered calcium homeostasis needs greater expression of dystrophin or direct interventions on the channels involved.

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.

Aquaporin-4 (AQP4) is the major water channel expressed in fast-twitch skeletal muscle fibers. AQP4 is reduced in Duchenne and Becker Muscular Dystrophies, but not in caveolinopathies, thus suggesting an interaction with dystrophin or with members of the dystrophin-glycoprotein complex (DGC) rather than a nonspecific effect due to muscle membrane damage. To establish the role of sarcoglycans in AQP4 decrease occurring in muscular dystrophy, AQP4 expression was analyzed in muscle biopsies from patients affected by Limb Girdle Muscular Dystrophies (LGMDs) 2C-F genetically confirmed. In all the LGMD 2C-F (2alpha-, 1beta-, 2gamma-, 1delta-deficiency), AQP4 was severely decreased. This effect was associated to a marked reduction in alpha1-syntrophin levels. In control muscle AQP4 did not show a direct interaction with any of the four sarcoglycans but, it co-immunoprecipitated with alpha1-syntrophin, indicating that this modular protein may link AQP4 levels with the DGC complex. To determine whether AQP4 expression could be affected in other LGMDs due to the defect of a membrane protein not associated to the dystrophin complex, we examined AQP4 expression in 6 patients affected by dysferlin deficiency genetically confirmed. All the patients displayed a reduction of the water channel, and AQP4 expression appeared to correlate with the severity of the muscle histopathological lesions. However, differently from what observed in the sarcoglycans, alpha1-syntrophin expression was normal or just slightly reduced. These results seem to indicate an additional mechanism of regulation of AQP4 levels in muscle cells. In accordance with a specific effect of membrane muscle disorders, AQP4 protein levels were not changed in 3 mitochondrial and 3 metabolic myopathies. In conclusion, AQP4 expression and membrane localization are markedly reduced in LGMD 2B-2F. The role of AQP4 in the degenerative mechanism occurring in these diseases will be the object of our future research.

Annexin-2 is required for the apical transport in epithelial cells. In this study, we investigated the involvement of annexin-2 in cAMP-induced aquaporin-2 (AQP2) translocation to the apical membrane in renal cells. We found that the cAMP-elevating agent forskolin increased annexin-2 abundance in the plasma membrane enriched fraction with a parallel decrease in the soluble fraction. Interestingly, forskolin stimulation resulted in annexin-2 enrichment in lipid rafts, suggesting that hormonal stimulation might be responsible for a new configuration of membrane interacting proteins involved in the fusion of AQP2 vesicles to the apical plasma membrane. To investigate the functional involvement of annexin-2 in AQP2 exocytosis, the fusion process between purified AQP2 membrane vesicles and plasma membranes was reconstructed in vitro and monitored by a fluorescence assay. An N-terminal peptide that comprises 14 residues of annexin-2 and that includes the binding site for the calcium binding protein p11 strongly inhibited the fusion process. Preincubation of cells with this annexin-2 peptide also failed to increase the osmotic water permeability in the presence of forskolin in intact cells. Altogether, these data demonstrate that annexin-2 is required for cAMP-induced AQP2 exocytosis in renal cells.

Vasopressin regulates the fusion of the water channel aquaporin 2 (AQP2) to the apical membrane of the renal collecting-duct principal cells and several lines of evidence indicate that SNARE proteins mediate this process. In this work MCD4 renal cells were used to investigate the functional role of a set of Q- and R-SNAREs, together with that of Munc18b as a negative regulator of the formation of the SNARE complex. Both VAMP2 and VAMP3 were associated with immunoisolated AQP2 vesicles, whereas syntaxin 3 (Stx3), SNAP23 and Munc18 were associated with the apical plasma membrane. Co-immunoprecipitation experiments indicated that Stx3 forms complexes with VAMP2, VAMP3, SNAP23 and Munc18b. Protein knockdown coupled to apical surface biotinylation demonstrated that reduced levels of the R-SNAREs VAMP2 and VAMP3, and the Q-SNAREs Stx3 and SNAP23 strongly inhibited AQP2 fusion at the apical membrane. In addition, knockdown of Munc18b promoted a sevenfold increase of AQP2 fused at the plasma membrane without forskolin stimulation. Taken together these findings propose VAMP2, VAMP3, Stx3 and SNAP23 as the complementary set of SNAREs responsible for AQP2-vesicle fusion into the apical membrane, and Munc18b as a negative regulator of SNARE-complex formation in renal collecting-duct principal cells.

In a recent study, we demonstrated that in the plasma membrane AQP4 is organized into several distinct large multisubunit complexes. In this study, we analysed whether these pools are similarly affected in dystrophin-deficient mice and immunolocalized the sites of dystrophin-dependent and -independent AQP4 pools. Western blot performed on two-dimensional Blue Native/SDS-PAGE membranes indicated that, among the AQP4 pools, it was mainly a large multisubunit complex that was specifically affected in dystrophin-deficient mice (DP71 and mdx3cv mice). This dystrophin-dependent AQP4 pool was immunolocalized in perivascular astrocytes, since it was found to be significantly altered in both types of dystrophin-deficient mice. Dystrophin-independent pools were immunolocalized in the granular cell layer of the cerebellum and in the subpial endfoot layer and ependymal cells in the brain. These data provide a better understanding on the association between AQP4 and the dystrophin-glycoprotein complex in the central nervous system., (Copyright (c) 2008 Wiley-Liss, Inc.)

Altered aquaporin-4 (AQP4) expression has been reported in brain edema, tumors, muscular dystrophy, and neuromyelitis optica. However, the plasma membrane organization of AQP4 and its interaction with proteins such as the dystrophin-associated protein complex are not well understood. In this study, we used sucrose density gradient ultracentrifugation and 2D blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed the expression of several AQP4 multi-subunit complexes (pools) of different sizes, ranging from >> 1 MDa to approximately 500 kDa and containing different ratios of the 30/32 kDa AQP4 isoforms, indicative of orthogonal arrays of particles of various sizes. A high molecular weight pool co-purified with dystrophin and beta-dystroglycan and was drastically reduced in the skeletal muscle of mdx3cv mice, which have no dystrophin. The number and size of the AQP4 pools were the same in the kidney where dystrophin is not expressed, suggesting the presence of dystrophin-like proteins for their expression. We found that AQP2 is expressed only in one major pool of approximately 500 kDa, indicating that the presence of different pools is a peculiarity of AQP4 rather than a widespread feature in the AQP family. Finally, in skeletal muscle caveolin-3 did not co-purify with any AQP4 pool, indicating the absence of interaction of the two proteins and confirming that caveolae and orthogonal arrays of particles are two independent plasma membrane microdomains. These results contribute to a better understanding of AQP4 membrane organization and raise the possibility that abnormal expression of specific AQP4 pools may be found in pathological states.

Unlabelled: The response to antiviral therapy is lower in hepatitis C virus (HCV) patients with genotype 1 than in those with genotype 2. Overexpression of the suppressor of cytokine signaling 3 (SOCS3) gene in liver tissue is associated with a poorer treatment outcome in patients with chronic hepatitis C viral genotype 1. Also, insulin resistance has been implicated in nonresponse to an anti-HCV treatment. To understand why HCV genotype 1 patients respond differently, we investigated SOCS3 gene expression, metabolic syndrome (MS), and the response to therapy in a cohort of patients with HCV-related hepatitis. A total of 198 patients (108 with genotype 1 and 90 with genotype 2) treated with pegylated interferon plus ribavirin were consecutively enrolled in the study. We measured SOCS3 expression in Epstein-Barr virus-transformed lymphoblastoid cell lines derived from peripheral lymphocytes of a subset of 130 patients. MS was more frequent in genotype 1 patients than in genotype 2 patients (P < 0.01). Nonresponders (P < 0.01), MS (P < 0.001), and genotype 1 (P < 0.001) were significantly related to SOCS3 overexpression. However, SOCS3 levels were higher in nonresponders also, regardless of the genotype (P < 0.01). In a univariate analysis, the genotype (P < 0.001), age (P < 0.001), SOCS3 (P < 0.001), and MS (P < 0.001) were significantly related to the response to therapy. However, in a multivariate analysis, SOCS3 was the only independent predictor of the response (odds ratio = 6.7; P < 0.005)., Conclusion: We speculate that SOCS3 expression per se may influence the response to antiviral therapy and that the genotype 1b virus might induce its up-regulation. This may account for the different responses to therapy between genotype 1-infected and genotype 2-infected patients.

Aquaporin-4 (AQP4) is the neuromuscular water channel expressed at the sarcolemma of mammalian fast-twitch fibers that mediates a high water transport rate, which is important during muscle activity. Clinical interest in the neuromuscular expression of AQP4 has increased as it is associated with the protein complex formed by dystrophin, the product of the gene affected in Duchenne muscular dystrophy. The expression of AQP4 during development has not been characterized. In this study, we analyzed the expression of AQP4 in extensor digitorum longus (EDL) and soleus, a fast- and slow-twitch muscle, respectively, during the first weeks after birth. The results show that AQP4 expression in both types of skeletal muscle occurs postnatally. The time course of expression of AQP4 in the two types of muscles was also different. Whereas the expression of AQP4 protein levels in the EDL showed a progressive increase during the first month after birth, reaching levels found in adults by day 24, the levels of the protein in the soleus showed a transient peak between day 12 and day 24 and declined thereafter, an effect that may be related to the transient high number of fast motor units innervating the soleus muscle during this time. The results suggest that AQP4 expression in skeletal muscle is under neuronal influence and contribute to the understanding of the molecular events of fiber differentiation during development.

Hypotonicity-induced cell swelling is characterized by a modification in cell architecture associated with actin cytoskeleton remodeling. The ezrin/radixin/moesin (ERM) family proteins are important signal transducers during actin reorganization regulated by the monomeric G proteins of the Rho family. We report here that in collecting duct CD8 cells hypotonicity-induced cell swelling resulted in deep actin reorganization, consisting of loss of stress fibers and formation of F-actin patches in membrane protrusions where the ERM protein moesin was recruited. Cell swelling increased the interaction between actin and moesin and induced the transition of moesin from an oligomeric to a monomeric functional conformation, characterized by both the COOH- and NH(2)-terminal domains being exposed. In this conformation, which is stabilized by phosphorylation of a conserved threonine in the COOH-terminal domain by PKC or Rho kinase, moesin can bind interacting proteins. Interestingly, hypotonic stress increased the amount of threonine-phosphorylated moesin, which was prevented by the PKC-alpha inhibitor Gö-6976 (50 nM). In contrast, the Rho kinase inhibitor Y-27632 (1 microM) did not affect the hypotonicity-induced increase in phosphorylated moesin. The present data represent the first evidence that hypotonicity-induced actin remodeling is associated with phosphorylated moesin recruitment at the cell border and interaction with actin.

Kidney collecting-duct cells swell in response to changes in medulla osmolality caused by the transition from antidiuresis to diuresis. Regulatory volume decrease (RVD) mechanisms must be activated to face this hypotonic stress. In Aquaporin-2 (AQP2)-expressing renal CD8 cells, hypotonicity decreased cell surface expression of AQP2 and increased the amount of AQP2 localized intracellularly, whereas the total amount of AQP2 phosphorylated at ser-256 decreased. Analysis of cAMP dynamics using fluorescence resonance energy transfer (FRET) showed that hypotonicity causes a reduction of cAMP, consistent with a decrease in phospho-AQP2. Moreover, hypotonicity caused a profound actin reorganization, associated with the loss of stress fibers and formation of F-actin patches (microspikes) at the cell border. Those changes were regulated by the monomeric GTPase Cdc42. Interestingly, expression of the dominant-negative Cdc42 (N17-Cdc42) prevented the hypotonicity-induced microspike formation and the generation of Cl(-) currents. Hypotonicity also caused the relocation from the cytosol to the plasma membrane and increase in interaction with actin of ICln (nucleotide-sensitive chloride current protein), which is essential for the generation of ion currents activated during RVD. Together, the profound actin remodeling, internalization of AQP2 and translocation of ICln to the plasma membrane during hypotonicity may contribute to RVD after cell swelling in renal medulla.

The intracellular hydric balance is an essential process of mammalian cells. The water movement across cell membranes is driven by osmotic and hydrostatic forces and the speed of this process is dependent on the presence of specific aquaporin water channels. Since the molecular identification of the first water channel, AQP1, by Peter Agre's group, 13 homologous members have been found in mammals with varying degree of homology. The fundamental importance of these proteins in all living cells is suggested by their genetic conservation in eukaryotic organisms through plants to mammals. A number of recent studies have revealed the importance of mammalian AQPs in both physiology and pathophysiology and have suggested that pharmacological modulation of aquaporins expression and activity may provide new tools for the treatment of variety of human disorders, such as brain edema, glaucoma, tumour growth, congestive heart failure and obesity in which water and small solute transport may be involved. This review will highlight the physiological role and the pathological involvement of AQPs in mammals and the potential use of some recent therapeutic approaches, such as RNAi and immunotherapy, for AQP-related diseases. Furthermore, strategies that can be developed for the discovery of selective AQP-drugs will be introduced and discussed.

The recent identification of aquaporin-8 (AQP8), an aquaporin (AQP) channel permeable to water and ammonia, in the inner membrane (IMM) of rat liver mitochondria suggested a role for such AQP in the hydration state and the metabolic function of mitochondria. Since thyroid hormone triiodothyronine (T3) is known to modulate both the shape and the metabolic activities of liver mitochondria, it was interesting to investigate the expression and distribution of AQP8 as well as the osmotic water permeability of the IMM in liver mitochondria from rats in different thyroid states. By semi-quantitative reverse transcriptase (RT)-PCR, when compared with the euthyroid counterpart, the levels of hepatic AQP8 mRNA significantly increased in the hypothyroid state, whereas they were strongly decreased after administration of T3. A similar pattern was seen at the protein level by immunoblotting mitochondrial membranes. The upregulation of mitochondrial AQP8 in the hypothyroid liver was confirmed by immunogold electron microscopy. Stopped-flow light scattering with IMM vesicles showed no significant differences in terms of osmotic water permeability among the IMMs in the various thyroid states. Overall, our data indicate that the T3 modulation of the AQP8 gene is a rapid downregulation of transcription. Modulation of hepatic AQP8 expression may be relevant to the regulation of mitochondrial metabolism by thyroid hormones.

In the brain, the astroglial syncytium is crucially involved in the regulation of water homeostasis. Accumulating evidence indicates that a dysregulation of the astrocytic processes controlling water homeostasis has a pathogenetic role in several brain injuries. Here, we have analysed by RNA interference technology the functional interactions occurring between the most abundant water channel in the brain, aquaporin-4 (AQP4), and the swelling-activated Cl(-) current expressed by cultured rat cortical astrocytes. We show that in primary cultured rat cortical astrocytes transfected with control small interfering RNA (siRNA), hypotonic shock promotes an increase in cellular volume accompanied by augmented membrane conductance mediated by volume-regulated anion channels (VRAC). Conversely, astroglia in which AQP4 was knocked down (AQP4 KD) by transfection with AQP4 siRNA changed their morphology from polygonal to process-bearing, and displayed normal cell swelling but reduced VRAC activity. Pharmacological manipulations of actin cytoskeleton in rat astrocytes, and functional analysis in mouse astroglial cells, which retain their morphology upon knockdown of AQP4, suggest that stellation of AQP4 KD rat cortical astrocytes was not causally linked to reduction of VRAC current. Molecular analysis of possible candidates of swelling-activated Cl(-) current provided evidence that in AQP4 KD astrocytes, there was a down-regulation of chloride channel-2 (CIC-2), which, however, was not involved in VRAC conductance. Inclusion of ATP in the intracellular saline restored VRAC activity upon hypotonicity. Collectively, these results support the view that in cultured astroglial cells, plasma membrane proteins involved in cell volume homeostasis are assembled in a functional platform.

Here, we report the aquaporin 2 (AQP2) mutational analysis of a patient with nephrogenic diabetes insipidus heterozygote due to two novel missense mutations. Direct sequencing of DNA in the male patient revealed that he was compound heterozygote for two mutations in the AQP2 gene: a thymine-to-adenine transversion at position 450 (c.450T>A) in exon 2 and a guanine-to-thymine at nucleotide position 643 (c.643G>T) in exon 4. The double heterozygous 450T>A and 643G>T transversion causes the amino acid substitution D150E and G215C. Direct sequencing of exons 2 and 4 of the AQP2 gene from each of the parents revealed that the c.450T>A mutation was inherited from the father while the c.643G>T mutation was inherited from the mother. Analysis of AQP2 excretion demonstrated that no AQP2 was detectable in the urine of the proband, whereas normal AQP2 levels were measured in both parents. When expressed in renal cells, both proteins were retarded in the endoplasmic reticulum and no redistribution was observed after forskolin stimulation. Of note, homology modeling revealed that the two mutations involve two highly conserved residues providing important clues about the role of the wt residues in AQP2 stability and function., (Copyright 2007 S. Karger AG, Basel.)

Background Information: AQP4 (aquaporin 4) internalization and a concomitant decrease in the osmotic water permeability coefficient (Pf) after histamine exposure has been reported in AQP4-transfected gastric HGT1 cells., Results: In the present study we report that AQP4 internalization is followed by an increase in AQP4 phosphorylation. Histamine treatment for 30 min resulted in an approx. 10-fold increase in AQP4 phosphorylation that was inhibited by 1 microM H89, a specific PKA (protein kinase A) inhibitor, but not by PKC (protein kinase C) and CK2 inhibitors. Moreover, measurement of PKA activity after 30 min of histamine treatment showed that PKA activity was approx. 3-fold higher compared with basal conditions. AQP4 phosphorylation was prevented in cells treated with histamine for 30 min after pre-incubation with PAO (phenylarsine oxide), an inhibitor of protein endocytosis. Using an endo-exocytosis assay we showed that, after histamine washed out, internalized AQP4 recycled back to the cell surface, even in cells in which de novo protein synthesis was inhibited by cycloheximide., Conclusions: Phosphorylation experiments, combined with immunolocalization studies, indicated that AQP4 phosphorylation is mediated by PKA and occurs subsequently to its internalization in late endosomes. We suggest that phosphorylation might be a mechanism involved in retaining AQP4 in a vesicle-recycling compartment.

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-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.

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.

The movement of water accompanying solutes between the cytoplasm and the mitochondrial spaces is central for mitochondrial volume homeostasis, an important function for mitochondrial activities and for preventing the deleterious effects of excess matrix swelling or contraction. While the discovery of aquaporin water channels in the inner mitochondrial membrane provided valuable insights into the basis of mitochondrial plasticity, questions regarding the identity of mitochondrial water permeability and its regulatory mechanism remain open. Here, we use a stopped flow light scattering approach to define the water permeability and Arrhenius activation energy of the rat liver whole intact mitochondrion and its membrane subcompartments. The water permeabilities of whole brain and testis mitochondria as well as liposome models of the lipid bilayer composing the liver inner mitochondrial membrane are also characterized. Besides finding remarkably high water permeabilities for both mitochondria and their membrane subcompartments, the existence of additional pathways of water movement other than aquaporins are suggested.

Background Information: Fluid homoeostasis is of critical importance in many functions of the CNS (central nervous system) as indicated by the fact that dysregulation of cell volume underlies clinical conditions such as brain oedema and hypoxia. Water balance is also important during neurogenesis as neural stem cells move considerable amounts of water into or out of the cell to rapidly change their volume during differentiation. Consistent with the relevance of water transport in CNS, multiple AQP (aquaporin) water channels have been recognized and partially characterized in brain cell function. However, the presence and distribution of AQPs in CNS stem cells has not yet been assessed., Results: In the present study, we investigate the expression and subcellular localization of AQPs in murine ANSCs (adult neural stem cells). Considerable AQP8 mRNAs were found in ANSCs where, as expected, the transcript of two additional AQPs, AQP4 and AQP9, was also detected. Immunoblotting with subcellular membrane fractions of ANSCs showed predominant expression of AQP8 in the mitochondria-enriched fraction. This result was consistent with the spotted immunoreactivity profile encountered within the ANSCs by confocal immunofluorescence., Conclusions: AQP8 may have a role in mitochondrial volume regulation during ANSC differentiation. Recognition of AQPs in ANSCs is a step forward in our knowledge of water homoeostasis in the CNS and provides useful information for the purposes of stem cell technology.

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 microl vs. 8.8 +/- 1.0 microl). On the diet, fasting volumes increased exclusively in C57L (28.6 +/- 2.9 microl 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.

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., (Copyright 2006 S. Karger AG, Basel.)

In the kidney aquaporin-2 (AQP2) provides a target for hormonal regulation of water transport by vasopressin. Short-term control of water permeability occurs via vesicular trafficking of AQP2 and long-term control through changes in the abundance of AQP2 and AQP3 water channels. Defective AQP2 trafficking causes nephrogenic diabetes insipidus, a condition characterized by the kidney inability to produce concentrated urine because of the insensitivity of the distal nephron to vasopressin. AQP2 is redistributed to the apical membrane of collecting duct cells through activation of a cAMP signaling cascade initiated by the binding of vasopressin to its V2-receptor. Protein kinase A-mediated phosphorylation of AQP2 has been proposed to be essential in regulating AQP2-containing vesicle exocytosis. Cessation of the stimulus is followed by endocytosis of the AQP2 proteins exposed on the plasma membrane and their recycling to the original stores, in which they are retained. Soluble N-ethylmaleimide sensitive fusion factor attachment protein receptors (SNARE) and actin cytoskeleton organization regulated by small GTPase of the Rho family were also proved to be essential for AQP2 trafficking. Data for functional involvement of the SNARE vesicle-associated membrane protein 2 in AQP2 targeting has recently been provided. Changes in AQP2 expression/trafficking are of particular importance in pathological conditions characterized by both dilutional and concentrating defects. One of these conditions, hypercalciuria, has shown to be associated with alteration of AQP2 urinary excretion. More precisely, recent data support the hypothesis that, in vivo external calcium, through activation of calcium-sensing receptors, modulates the expression/trafficking of AQP2. Together these findings underscore the importance of AQP2 in kidney pathophysiology.

Bradykinin (BK) is one of the most important peptides regulating vascular tone, water, and ionic balance in the body, playing a key role in controlling BP. It is interesting that patients with essential hypertension excrete less BK than normotensive individuals. For elucidating the mechanism by which BK regulates renal water transport that contributes to its antihypertensive effect, aquaporin 2 (AQP2)-transfected collecting duct CD8 cells, expressing the BK type II receptor (BK2R), were used as an experimental model. In CD8 cells, BK pretreatment impaired forskolin-induced AQP2 translocation to the apical plasma membrane. For clarifying the signal transduction cascade associated with this effect, whether BK induced an increase in cytosolic calcium, via the G protein Gq, known to be coupled to BK2R, first was investigated. Spectrofluorometry using fura-2-AM revealed that 100 nM BK elicited a significant increase in Ca(i), which was abolished by the receptor antagonist HOE-140. BK acts through BK2R coupled to both Gq and Galpha13, a known upstream effector of Rho protein. In CD8 cells, BK causes an increase in Rho activity, likely as a result of Galpha13 activation. This results in stabilization of the cortical F-actin network, thus impairing AQP2 trafficking. These effects counteract physiologic vasopressin stimulation, which instead has an opposite effect on actin network organization through Rho inactivation.

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.

This study provides the first evidence that actin reorganization during AQP2 vesicular trafficking to the plasma membrane requires the functional involvement of ERM (ezrin/radixin/moesin) proteins cross-linking actin filaments with plasma membrane proteins. We report that forskolin stimulation was associated with a redistribution of moesin from intracellular sites to the cell cortex and with a concomitant enrichment of moesin in the particulate fraction in renal cells. Introduction of a peptide reproducing a short sequence of moesin within the binding site for F-actin induced all the key effects of forskolin stimulation, including a decrease in F-actin, translocation of endogenous moesin, and AQP2 translocation. A straightforward explanation for these effects is the ability of the peptide to uncouple moesin from its putative effector. This modifies the balance between the active and inactive forms of moesin. Extraction with Triton X-100, which preserves cytoskeletal associated proteins, showed that forskolin stimulation or peptide introduction reduced the amount of phophorylated moesin, a molecular modification known to stabilize moesin in an active state. Our data point to a dual role of moesin in AQP2 trafficking: it might modulate actin depolymerization and it participates in the reorganization of F-actin-containing cytoskeletal structures close to the fusion sites of the AQP2-bearing vesicles.

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.

Formation of bile and generation of bile flow are driven by the active secretion of bile salts (BS), lipids and electrolytes into the canalicular and bile duct lumens followed by the osmotic movement of water. Although the transporting proteins involved in solute secretion have been cloned and their coordinated interplay defined both in health and disease, boosted by the discovery of the aquaporin water channels, only recently has considerable attention been addressed to the mechanism by which water, the major component of bile (> 95%), moves across the hepatobiliary epithelia. This review summarizes the novel acquisitions in liver membrane water transport and functional participation of aquaporin water channels in multiple aspects of hepatobiliary fluid balance. Emerging evidences suggesting involvement of aquaporins in the metabolic homeostasis of the hepatobiliary tract are also discussed.

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.

The hindlimb unloading (HU) rat is a model of muscle disuse characterized by atrophy and slow-to-fast phenotype transition of the postural muscles, such as the soleus. We previously found that the resting sarcolemmal chloride conductance (gCl) that is typically lower in slow-twitch myofibers than in fast ones increased in soleus fibers following 1 to 3 weeks of HU in accord with the slow-to-fast transition of myosin heavy chain (MHC) isoforms. Nevertheless, the gCl already raised after a 3-day HU, whereas no change in MHC expression was detected. The present work evaluates the ability of soleus muscle to recover on return to normal load after a short (3 days) or long (2 weeks) disuse period. The changes observed after a 2-week HU were slowly reversible, since 3-4 weeks of reloading were needed to completely recover gCl, fiber diameter, MHC expression pattern, as well as the mechanical threshold Rheobase, an index of calcium homeostasis. After 3-day HU, the gCl increased homogeneously in most of the soleus muscle fibers and gCl recovery was rapidly completed after 4-day reloading. These results suggest different induction mechanisms for gCl augmentation after the short and long HU periods, as well as a possible role for gCl in the slow muscle adaptation to disuse.

Chronic inflammation is a secondary reaction of Duchenne muscular dystrophy and may contribute to disease progression. To examine whether immunosuppressant therapies could benefit dystrophic patients, we analyzed the effects of cyclosporine A (CsA) on a dystrophic mouse model. Mdx mice were treated with 10 mg/kg of CsA for 4 to 8 weeks throughout a period of exercise on treadmill, a protocol that worsens the dystrophic condition. The CsA treatment fully prevented the 60% drop of forelimb strength induced by exercise. A significant amelioration (P < 0.05) was observed in histological profile of CsA-treated gastrocnemius muscle with reductions of nonmuscle area (20%), centronucleated fibers (12%), and degenerating area (50%) compared to untreated exercised mdx mice. Consequently, the percentage of normal fibers increased from 26 to 35% in CsA-treated mice. Decreases in creatine kinase and markers of fibrosis were also observed. By electrophysiological recordings ex vivo, we found that CsA counteracted the decrease in chloride conductance (gCl), a functional index of degeneration in diaphragm and extensor digitorum longus muscle fibers. However, electrophysiology and fura-2 calcium imaging did not show any amelioration of calcium homeostasis in extensor digitorum longus muscle fibers. No significant effect was observed on utrophin levels in diaphragm muscle. Our data show that the CsA treatment significantly normalized many functional, histological, and biochemical endpoints by acting on events that are independent or downstream of calcium homeostasis. The beneficial effect of CsA may involve different targets, reinforcing the usefulness of immunosuppressant drugs in muscular dystrophy.

Background: Urinary concentrating defects and polyuria are the most important renal manifestations of hypercalcemia and the resulting hypercalciuria. In this study, we tested the hypothesis that hypercalciuria-associated polyuria in kidney collecting duct occurs through an impairment of the vasopressin-dependent aquaporin 2 (AQP2) water channel targeting to the apical membrane possibly involving calcium-sensing receptor (CaR) signaling., Methods: AQP2-transfected collecting duct CD8 cells were used as experimental model. Quantitation of cell surface AQP2 immunoreactivity was performed using an antibody recognizing the extracellular AQP2 C loop. Intracellular cyclic adenosine monophosphate (cAMP) accumulation was measured in CD8 cells using a cAMP enzyme immunoassay kit. To study the translocation of protein kinase C (PKC), membranes or cytosol fractions from CD8 cells were subjected to Western blotting using anti-PKC isozymes antibodies. The amount of F-actin was determined by spectrofluorometric techniques. Intracellular calcium measurements were performed by spectrofluorometric analysis with Fura-2/AM., Results: We demonstrated that extracellular calcium (Ca2+ o) (5 mmol/L) strongly inhibited forskolin-stimulated increase in AQP2 expression in the apical plasma membrane. At least three intracellular pathways activated by extracellular calcium were found to contribute to this effect. Firstly, the increase in cAMP levels in response to forskolin stimulation was drastically reduced in cells pretreated with Ca2+ o compared to untreated cells. Second, Ca2+ o activated PKC, known to counteract vasopressin response. Third, quantification of F-actin demonstrated that Ca2+ o caused a nearly twofold increase in F-actin content compared with basal conditions. All these effects were mimicked by a nonmembrane permeable agonist of the extracellular CaR, Gd3+., Conclusion: Together, these data demonstrate that extracellular calcium, possibly acting through the endogenous CaR, antagonizes forskolin-induced AQP2 translocation to the apical plasma membrane in CD8 cells. In hypercalciuria, this mechanism might blunt water reabsorption and prevent further calcium concentration, thus protecting against a potential risk of urinary calcium-containing stone formation.

To investigate the role of Shc in IGF action and signaling in skeletal muscle cells, Shc protein levels were reduced in rat L6 myoblasts by stably overexpressing a Shc cDNA fragment in antisense orientation (L6/Shcas). L6/Shcas myoblasts showed marked reduction of the p66Shc protein isoform and no change in p52Shc or p46Shc proteins compared with control myoblasts transfected with the empty vector (L6/Neo). When compared with control, L6/Shcas myoblasts demonstrated 3-fold increase in Erk-1/2 phosphorylation under basal conditions and blunted Erk-1/2 stimulation by insulin-like growth factor I (IGF-I), in the absence of changes in total Erk-1/2 protein levels. Increased basal Erk-1/2 activation was paralleled by a greater proportion of phosphorylated Erk-1/2 in the nucleus of L6/Shcas myoblasts in the absence of IGF-I stimulation. The reduction of p66Shc in L6/Shcas myoblasts resulted in marked phenotypic abnormalities, such as rounded cell shape and clustering in islets or finger-like structures, and was associated with impaired DNA synthesis in response to IGF-I and lack of terminal differentiation into myotubes. In addition, L6/Shcas myoblasts were characterized by complete disruption of actin filaments and cell cytoskeleton. Treatment of L6/Shcas myoblasts with the MEK inhibitor PD98059 reduced the abnormal increase in Erk-1/2 activation to control levels and restored the actin cytoskeleton, re-establishing the normal cell morphology. Thus, the p66Shc isoform exerts an inhibitory effect on the mitogen-activated protein kinase signaling pathway in rodent myoblasts, which is necessary for maintenance of IGF responsiveness of the MEK/Erk pathway and normal cell phenotype.

The aim of this study was to investigate the impact of environmental pollution at different stations along the Taranto coastline (Ionian Sea, Puglia, Italy) using several biomarkers of exposure and the effect on mussels, Mytilus galloprovincialis, collected in October 2001 and October 2002. Five sampling sites were compared with a "cleaner" reference site in the Aeronautics Area. In this study we also investigated the differences between adduct levels in gills and digestive gland. This Taranto area is the most significant industrial settlement on the Ionian Sea known to be contaminated by polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, heavy metals, etc. Exposure to PAHs was evaluated by measuring DNA adduct levels and benzo(a)pyrene monooxygenase activity (B(a)PMO); DNA adducts were analyzed by 32P-postlabeling with nuclease P1 enhancement in both gills and digestive glands to evaluate differences between DNA adduct levels in the two tissues. B(a)PMO was assayed in the microsomal fraction of the digestive glands as a result of the high expression of P450-metabolizing enzymes in this tissue. Lysosomal membrane stability, a potential biomarker of anthropogenic stress, was also evaluated in the digestive glands of mussels, by measuring the latent activity of beta-N-acetylhexosaminidase. Induction of DNA adducts was evident in both tissues, although the results revealed large tissue differences in DNA adduct formation. In fact, gills showed higher DNA adduct levels than did digestive gland. No significant differences were found in DNA adduct levels over time, with both tissues providing similar results in both years. DNA adduct levels were correlated with B(a)PMO activity in digestive gland in both years (r = 0.60 in 2001; r = 0.73 in 2002). Increases were observed in B(a)PMO activity and DNA adduct levels at different stations; no statistical difference was observed in B(a)PMO activity over the two monitoring campaigns. The membrane labilization period in mussels from some stations was decreased in both years. No statistical differences were established in the membrane labilization times from 2001 to 2002. Our results suggest the existence of different sources and amounts of environmental contaminants at the stations investigated. The formation of DNA adducts confirms the existence of activation pathways in mussels and shows the importance of DNA adduct analysis in the gill tissue in addition to the more commonly used digestive gland; these results confirm the utility of lysosomal membrane stability as a biomarker of general stress. Overall, the integrated use of biomarkers of exposure and the effects of environmental contaminants on living marine organisms may help to better interpret the impact of pollutants in a marine coastal environment.

This study was undertaken to determine the role of aquaporin 2 (AQP2) in the impaired urinary concentrating capacity observed in patients who underwent pyeloplasty because of congenital unilateral hydronephrosis as a result of pyeloureteral junction disease. Twelve children (mean age, 8 +/- 2 mo) were examined in the study. From day 1 to day 5 after surgery, the urine was collected separately from pyelostomy draining only from the postobstructed kidney and from the bladder catheter draining mostly from the contralateral kidney used as internal control. After pyeloplasty, the postobstructed kidney was characterized by a reduced urinary excretion of AQP2 (approximately 54%) associated with polyuria that persisted from day 1 to day 5 (433 +/- 58 versus 310 +/- 74 ml/24 h at day 1; 326 +/- 44 versus 227 +/- 26 ml/24 h at day 5). In parallel, urine osmolality from the postobstructed kidney was significantly reduced compared with the contralateral kidney (111 +/- 12 versus 206 +/- 49 at day 1; 136 +/- 24 versus 235 +/- 65 mOsm/kg at day 5). Creatinine clearance from the postobstructed kidney was not significantly different compared with the contralateral kidney throughout the 4 d after surgery. However, on day 5, creatinine clearance from the postobstructed kidney became significantly lower. Prostaglandin E2 in the urine from postobstructed kidneys was found to be twofold higher than in the contralateral samples (26.0 +/- 6.7 versus 13.5 +/- 2.5 at day 5). It is concluded that (1) the selective downregulation of AQP2 in postobstructed kidney may account for the higher excretion of hypotonic urine, and (2) the local increase in prostaglandin E2 synthesis in postobstructed kidney may be involved in AQP2 downregulation and in maintaining a GFR similar to that of the contralateral kidney.

Aquaporin-4 (AQP4) is the major water channel of the neuromuscular system, but its physiological function in both perivascular astrocytes and skeletal muscle sarcolemma is unclear. The purpose of this study was to assess the following in skeletal muscle: a) the expression of all cloned water cannels; b) the functional role of AQP4 using sarcolemma vesicles purified by means of several fractionation methods, and c) the functional effect of AQP4 reduction in mdx mice, the animal model of Duchenne muscular dystrophy (DMD). Immunofluorescence and immunoblot experiments performed with affinity purified antibodies revealed that only AQP1 and AQP4 are expressed in mouse skeletal muscle: AQP1 in endothelial cells of continuous capillaries and AQP4 on the plasma membrane of muscle fiber. Plasma membrane vesicle purification was performed with a procedure extensively used to purify and characterize dystrophin-associated proteins (DAPs) from rabbit skeletal muscle. Western blot analysis showed strong co-enrichment of the analyzed DAPs and AQP4, indicating that the membrane vesicle preparation was highly enriched in sarcolemma. Stopped-flow light-scattering measurements showed high osmotic water permeability of sarcolemma vesicles (approximately 150 microm/s) compatible with the AQP-mediated pathway for water movement. Sarcolemma vesicles prepared from mdx mice revealed, in parallel with AQP4 disappearance from the plasma membrane, a strong reduction in water permeability compared with wild-type mice. Altogether, these results demonstrate high AQP4-mediated water permeability of the skeletal muscle sarcolemma. Expression of sarcolemmal AQP4 together with that of vascular AQP1 may be responsible for the fast water transfer from the blood into the muscle during intense activity. These data imply an important role for aquaporins in skeletal muscle physiology as well as an involvement of AQP4 in the molecular alterations that occur in the muscle of DMD patients.

In order to ascertain whether the alterations of the blood-brain barrier (BBB) seen in adult dystrophic mdx-mice [Glia 42 (2003) 235], a human model of Duchenne muscular dystrophy (DMD), are developmentally established and correlated with other dystrophin isoforms which are localized at the glial-vascular interface, we used immunocytochemistry to investigate the expression of dystrophin isoforms (Dp71) during BBB development in mdx fetuses and in adult mice. Parallelly, we used Western blot, immunocytochemistry and immunogold electron microscopy to analyze the expression of the zonula occludens (ZO-1), aquaporin-4 (AQP4) and glial fibrillary acidic (GFAP) proteins as endothelial and glial markers, and we evaluated the integrity of the mdx BBB by means of intravascular injection of horseradish peroxidase (HRP). The results show reduced dystrophin isoforms (Dp71) in the mdx mouse compared with the control, starting from early embryonic life. Endothelial ZO-1 expression was reduced, and the tight junctions were altered and unlabeled. AQP4 and GFAP glial proteins in mdx mice also showed modifications in developmental expression, the glial vascular processes being only lightly AQP4- and GFAP-labeled compared with the controls. Confocal microscopy and HRP assays confirmed the alteration in vessel glial investment, GFAP perivascular endfoot reactivity being strongly reduced and BBB permeability increasing. These results demonstrate that a reduction in dystrophin isoforms (Dp71) at glial endfeet leads to an altered development of the BBB, whose no-closure might contribute to the neurological dysfunctions associated with DMD.

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.

Aquaporin 2 (AQP2) phosphorylation at Ser-256 by protein kinase A (PKA) is a key signal for vasopressin-stimulated AQP2 insertion into the plasma membrane in renal cells. This study underscores the possible role of phosphorylation at Ser-256 in regulating AQP2 maturation. AQP2-transfected renal CD8 cells were incubated with brefeldin A (BFA) to accumulate newly synthesized AQP2 in the endoplasmic reticulum (ER), and AQP2 flow from ER to the vesicular compartment was analyzed after BFA washout. We found that a) in the ER, AQP2 is weakly phosphorylated; b) the amount of phosphorylated AQP2 (p-AQP2) at Ser-256 increased significantly during transit in the Golgi, even in the presence of the PKA inhibitor H89; and c) AQP2 transport from the Golgi to the vasopressin-regulated vesicular compartment occurred with a concomitant decrease in p-AQP2 at Ser-256. These results support the hypothesis that AQP2 transition in the Golgi apparatus is associated with a PKA-independent increase in AQP2 phosphorylation at Ser-256. Conversely, impaired constitutive phosphorylation in a Golgi-associated compartment occurring in cells expressing mutated S256A-AQP2 or E258K-AQP2 causes phosphorylation-defective AQP2 routing to lysosomes. This result might explain the molecular basis of the dominant form of nephrogenic diabetes insipidus caused by the mutation E258K-AQP2, in which the phenotype is caused by an impaired routing of AQP2.

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.

Recent studies indicate a key role of aquaporin (AQP) 4 in astrocyte swelling and brain edema and suggest that AQP4 inhibition may be a new therapeutic way for reducing cerebral water accumulation. To understand the physiological role of AQP4-mediated astroglial swelling, we used 21-nucleotide small interfering RNA duplexes (siRNA) to specifically suppress AQP4 expression in astrocyte primary cultures. Semiquantitative RT-PCR experiments and Western blot analysis showed that AQP4 silencing determined a progressive and parallel reduction in AQP4 mRNA and protein. AQP4 gene suppression determined the appearance of a new morphological cell phenotype associated with a strong reduction in cell growth. Water transport measurements showed that the rate of shrinkage of AQP4 knockdown astrocytes was one-half of that of controls. Finally, cDNA microarray analysis revealed that the gene expression pattern perturbed by AQP4 gene silencing concerned ischemia-related genes, such as GLUT1 and hexokinase. Taken together, these results indicate that 1) AQP4 seems to be the major factor responsible for the fast water transport of cultured astrocytes; 2) as in skeletal muscle, AQP4 is a protein involved in cell plasticity; 3) AQP4 alteration may be a primary factor in ischemia-induced cerebral edema; and 4) RNA interference could be a new potent tool for studying AQP pathophysiology in those organs and tissues where they are expressed.

Spermatogenesis, the maturation of spermatozoa and their concentration and storage in the seminiferous vessels are associated with considerable fluid secretion or absorption in the male reproductive tract. These fluid movements are in total agreement with the presence of multiple aquaporin (AQP) water channel proteins in germ cells and other tissues within the male reproductive tract. A series of functions of prime importance have already been hypothesized for aquaporins in the physiology of male reproduction. Aquaporins could be involved in the early stages of spermatogenesis, in the secretion of tubular liquid and in the concentration and storage of spermatozoa in the epididymis. In the male reproductive tract, alterations in the expression and functionality and/or regulation of aquaporins have already been demonstrated to be at the basis of forms of male infertility. Indeed, rats with reduced reabsorption of seminiferous fluid in the efferent ducts have been shown to be sub-fertile or infertile. Functions have also been suggested in the fertilization process, where aquaporins may play a role in maintaining osmotic homeostasis in gametes during fertilization. Aquaporins have also been suggested to mediate water movement into antral follicles and to be the pathway for transtrophectodermal water movement during cavitation. Aquaporins are the subject of considerable technological interest for cryopreservation used in medically assisted procreation, as they could be the molecular pathway by which water and/or solutes move across the plasma membrane during the process of freezing/thawing gametes and embryos. Indeed, artificial expression ofAQP3 has been showed to improve the survival of mouse oocytes after cryopreservation.

In this study, we investigated the involvement of the blood-brain barrier (BBB) in the brain of the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy (DMD). To this purpose, we used two tight junction markers, the Zonula occludens (ZO-1) and claudin-1 proteins, and a glial marker, the aquaporin-4 (AQP4) protein, whose expression is correlated with BBB differentiation and integrity. Results showed that most of the brain microvessels in mdx mice were lined by altered endothelial cells that showed open tight junctions and were surrounded by swollen glial processes. Moreover, 18% of the perivascular glial endfeet contained electron-dense cellular debris and were enveloped by degenerating microvessels. Western blot showed a 60% reduction in the ZO-1 protein content in mdx mice and a similar reduction in AQP4 content compared with the control brain. ZO-1 immunocytochemistry and claudin-1 immunofluorescence in mdx mice revealed a diffuse staining of microvessels as compared with the control ones, which displayed a banded staining pattern. ZO-1 immunogold electron microscopy showed unlabeled tight junctions and the presence of gold particles scattered in the endothelial cytoplasm in the mdx mice, whereas ZO-1 gold particles were exclusively located at the endothelial tight junctions in the controls. Dual immunofluorescence staining of alpha-actin and ZO-1 revealed colocalization of these proteins. As in ZO-1 staining, the pattern of immunolabeling with anti-alpha-actin antibody was diffuse in the mdx vessels and pointed or banded in the controls. alpha-actin immunogold electron microscopy showed gold particles in the cytoplasms of endothelial cells and pericytes in the mdx mice, whereas alpha-actin gold particles were revealed on the endothelial tight junctions and the cytoskeletal microfilaments of pericytes in the controls. Perivascular glial processes of the mdx mice appeared faintly stained by anti-AQP4 antibody, while in the controls a strong AQP4 labeling of glial processes was detected at light and electron microscope level. The vascular permeability of the mdx brain microvessels was investigated by means of the horseradish peroxidase (HRP). After HRP injection, extensive perivascular areas of marker escape were observed in mdx mice, whereas HRP was exclusively intravascularly localized in the controls. Inflammatory cells, CD4-, CD8-, CD20-, and CD68-positive cells, were not revealed in the perivascular stroma of the mdx brain. These findings indicate that dystrophin deficiency in the mdx brain leads to severe injury of the endothelial and glial cells with disturbance in alpha-actin cytoskeleton, ZO-1, claudin-1, and AQP4 assembly, as well as BBB breakdown. The BBB alterations suggest that changes in vascular permeability are involved in the pathogenesis of the neurological dysfunction associated with DMD., (Copyright 2003 Wiley-Liss, Inc.)

We have recently demonstrated that inhibition of Rho GTPase with Clostridium difficile toxin B, or with Clostridium botulinum C3 toxin, causes actin depolymerization and translocation of aquaporin 2 (AQP2) in renal CD8 cells in the absence of hormonal stimulation. Here we demonstrate that Rho inhibition is part of the signal transduction cascade activated by vasopressin leading to AQP2 insertion into the apical membrane. Quantitation of active RhoA (GTP-bound) by selective pull down experiments demonstrated that the amount of active RhoA decreased upon stimulation of CD8 cells with the cAMP-elevating agent forskolin. Consistent with this observation, forskolin treatment resulted in a decreased expression of membrane-associated (active) Rho, as assessed by cell fractionation followed by western blotting analysis. In addition, the abundance of the endogenous Rho GDP dissociation inhibitor (Rho-GDI) was found to have decreased in the membrane fraction after forskolin stimulation. Co-immunoprecipitation experiments revealed that, after forskolin stimulation, the amount of Rho-GDI complexed with RhoA increased, suggesting that Rho GTPase inhibition occurs through association of RhoA with Rho-GDI. Finally, forskolin stimulation was associated with an increase in Rho phosphorylation on a serine residue, a protein modification known to stabilize the inactive form of RhoA and to increase its interaction with Rho-GDI. Taken together, these data demonstrate that RhoA inhibition through Rho phosphorylation and interaction with Rho-GDI is a key event for cytoskeletal dynamics controlling cAMP-induced AQP2 translocation.

In this study, we analyzed the effect of a therapeutic intervention in 46 enuretic children, 26 (57%) of whom were hypercalciuric. All the patients (n = 46) were treated with DDAVP for 3-6 mo. The hypercalciuric patients (n = 26) received a low-calcium diet (approximately 500 mg/day) for the same period. After the therapy, the bed-wetting episodes stopped in 80% of the 46 patients tested. In those patients having low-AVP levels before the therapy, circulating AVP concentration returned to normal (>4 pg/ml), and the hypercalciuria was resolved in the hypercalciuric patients (calcium/creatinine ratio <0.2). Urinary aquaporin-2 (AQP2) levels were semiquantified by densitometric scanning and reported as a ratio between the intensity of the signal in the day vs. the night urine samples (day/night AQP2 ratio). In the hypercalciuric patients, the day/night AQP2 ratio returned to values close to those found in the healthy children (from 1.19 +/- 0.20 before to 0.69 +/- 0.10 after the treatment, n = 26, P = 0.03). In contrast, in the normocalciuric children we saw no significant modulation of AQP2 excretion (from 1.07 +/- 0.14 before to 0.99 +/- 0.14 after the treatment, n = 20). This study clearly demonstrates that urinary calcium levels modulate AQP2 excretion and is likely to be useful for treatment of children with enuresis.