Published papers

Background: Cellular quiescence is a state of reversible proliferation arrest that is induced by anti-mitogenic signals. The endogenous cardiac glycoside ouabain is a specific ligand of the ubiquitous sodium pump, Na,K-ATPase, also known to regulate cell growth through unknown signalling pathways. Methods: To investigate the role of ouabain/Na,K-ATPase in uncontrolled neuroblastoma growth we used xenografts, flow cytometry, immunostaining, comet assay, real-time PCR, and electrophysiology after various treatment strategies. Results: The ouabain/Na,K-ATPase complex induced quiescence in malignant neuroblastoma. Tumour growth was reduced by >50% when neuroblastoma cells were xenografted into immune-deficient mice that were fed with ouabain. Ouabain-induced S-G2 phase arrest, activated the DNA-damage response (DDR) pathway marker γH2AX, increased the cell cycle regulator p21 Waf1/Cip1 and upregulated the quiescence-specific transcription factor hairy and enhancer of split1 (HES1), causing neuroblastoma cells to ultimately enter G0. Cells re-entered the cell cycle and resumed proliferation, without showing DNA damage, when ouabain was removed. Conclusion: These findings demonstrate a novel action of ouabain/Na,K-ATPase as a regulator of quiescence in neuroblastoma, suggesting that ouabain can be used in chemotherapies to suppress tumour growth and/or arrest cells to increase the therapeutic index in combination therapies. © 2012 Cancer Research UK.

Aquaporin-4, a predominant water channel in the brain, is specifically expressed in astrocyte endfeet and plays a central role in water homeostasis, neuronal activity, and cell migration in the brain. It has two dominant isoforms called M1 and M23, whose mRNA is driven by distinct promoters located upstream of exons 0 and 1 of the aquaporin-4 gene, respectively. To identify cis-acting elements responsible for the astrocyte-specific transcription of M1 mRNA, the promoter activity of the 5′-flanking region upstream of exon 0 in primary cultured mouse astrocytes was examined by luciferase assay, and sequences, where nuclear factors bind, were identified by electrophoretic mobility shift assay. An astrocyte-specific activity enhancing transcription from the M1 promoter was observed within ∼2 kb from the transcriptional start sites of M1 mRNA. At least five elements clustered within the 286-bp region were found to function as a novel astrocyte-specific enhancer. Among the five elements, a consensus sequence of Pit-1/Oct/Unc-86 (POU) transcription factors was indispensable to the astrocyte-specific enhancer since disruption of the POU motif completely abolished the enhancer activity in astrocytes. However, the POU motif alone had little activity, indicating the requirement for cooperation with other upstream elements to exert full enhancer activity. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

The Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel plays an essential role in salt and water transport across epithelia and mutations of CFTR causing its dysfunction result in the genetic disease cystic fibrosis (CF). Various mutations induce CFTR channel dysfunction and various pharmacological approaches are currently being explored. The G551D mutation in CFTR is a common cause of CF, characterized by an extremely low open probability despite its normal trafficking to the plasma membrane. Recently we investigated the potentiation effect of curcumin, especially its combined effect with a flavonoid genistein, on G551D-CFTR activity using the patch-clamp technique. Curcumin potentiated G551D-CFTR less than genistein at their maximally effective concentrations. However, curcumin showed an additive effect with genistein and, more importantly, a significant synergistic effect with genistein in a low concentration range. This does not only suggest that multiple mechanisms are involved in action of the CFTR potentiators, but also pose pharmaceutical implications of curcumin in the development of drugs for CF pharmacotherpy. © 2012 Nova Science Publishers, Inc. All rights reserved.

Water can pass through biological membranes via two pathways: simple diffusion through the lipid bilayer, or water-selective facilitated diffusion through aquaporins (AQPs). Although AQPs play an important role in osmotic water permeability (Pf), the role of AQPs in diffusional water permeability remains unclear because of the difficulty of measuring diffusional water permeability (Pd). Here, we report an accurate and instantaneous method for measuring the Pd of a single HeLa S3 cell using coherent anti-Stokes Raman scattering (CARS) microscopy with a quick perfusion device for H2O/D2O exchange. Ultra-high-speed line-scan CARS images were obtained every 0.488 ms. The average decay time constant of CARS intensities (τCARS) for the external solution H2O/D2O exchange was 16.1 ms, whereas the intracellular H2O/D2O exchange was 100.7 ± 19.6 ms. To evaluate the roles of AQP in diffusional water permeability, AQP4 fused with enhanced green fluorescent protein (AQP4-EGFP) was transiently expressed in HeLa S3 cells. The average τCARS for the intracellular H 2O/D2O exchange in the AQP4-EGFP-HeLa S3 cells was 43.1 ± 15.8 ms. We also assessed the cell volume and the cell surface area to calculate Pd. The average Pd values for the AQP4-EGFP-HeLa S3 cells and the control EGFP-HeLa S3 cells were 2.7 ± 1.0 × 10-3 and 8.3 ± 2.6 × 10-4 cm/s, respectively. AQP4-mediated water diffusion was independent of the temperature but was dependent on the expression level of the protein at the plasma membrane. These results suggest the possibility of using CARS imaging to investigate the hydrodynamics of single mammalian cells as well as the regulation of AQPs. © 2011 Biophysical Society.

Anomalous diffusion in lipid bilayers is usually attributed to viscoelastic behavior. We compute the scaling exponent of relative fluctuations of the time-averaged mean square displacement in a lipid bilayer, by using a molecular dynamics simulation. According to the continuous time random walk theory, this exponent indicates non-Gaussian behavior caused by a power-law trapping time. Our results provide the first evidence that a lipid bilayer has not only viscoelastic properties but also trapping times distributed according to a power law. © 2011 American Physical Society.

Background: The G551D mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is a common cause of cystic fibrosis (CF). G551D-CFTR is characterized by an extremely low open probability despite its normal trafficking to the plasma membrane. Numerous small molecules have been shown to increase the activity of G551D-CFTR presumably by binding to the CFTR protein. Methods: We investigated the effect of curcumin, genistein and their combined application on G551D-CFTR activity using the patch clamp technique. Results: Curcumin increased G551D-CFTR whole-cell and single-channel currents less than genistein did at their maximally effective concentrations. However, curcumin further increased the channel activity of G551D-CFTR that had been already maximally potentiated by genistein, up to ~. 50% of the WT-CFTR level. In addition, the combined application of genistein and curcumin over a lower concentration range synergistically rescued the gating defect of G551D-CFTR. Conclusions: The additive effects between curcumin and genistein not only support the hypothesis that multiple mechanisms are involved in the action of CFTR potentiators, but also pose pharmaceutical implications in the development of drugs for CF pharmacotherapy. © 2011 European Cystic Fibrosis Society.

The genome of Synechocystis PCC 6803 contains a single gene encoding an aquaporin, aqpZ. The AqpZ protein functioned as a water-permeable channel in the plasma membrane. However, the physiological importance of AqpZ in Synechocystis remains unclear. We found that growth in glucose-containing medium inhibited proper division of ΔaqpZ cells and led to cell death. Deletion of a gene encoding a glucose transporter in the ΔaqpZ background alleviated the glucose-mediated growth inhibition of the ΔaqpZ cells. The ΔaqpZ cells swelled more than the wild type after the addition of glucose, suggesting an increase in cytosolic osmolarity. This was accompanied by a down-regulation of the pentose phosphate pathway and concurrent glycogen accumulation. Metabolite profiling by GC/TOF-MS of wild-type and ΔaqpZ cells revealed a relative decrease of intermediates of the tricarboxylic acid cycle and certain amino acids in the mutant. The changed levels of metabolites may have been the cause for the observed decrease in growth rate of the ΔaqpZ cells along with decreased PSII activity atpHvalues ranging from 7.5 to 8.5. A mutant in sll1961, encoding a putative transcription factor, and a Δhik31 mutant, lacking a putative glucose-sensing kinase, both exhibited higher glucose sensitivity than the ΔaqpZ cells. Examination of protein expression indicated that sll1961 functioned as a positive regulator of aqpZ gene expression but not as the only regulator. Overall, the ΔaqpZ cells showed defects in macronutrient metabolism, pH homeostasis, and cell division under photomixotrophic conditions, consistent with an essential role of AqpZ in glucose metabolism. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

The amniotic epithelium is in direct contact with the amniotic fluid and restricts fluid flux via the paracellular pathway by means of tight junctions (TJs). Several factors affect TJs to modulate the paracellular flux. Progesterone contributes to the antenatal formation and disappearance of TJs in uterine and mammary epithelial tissues. In this study, we investigated whether progesterone positively or negatively influences amniotic TJs. The administration of RU-486, a progesterone receptor (PR) antagonist, into pregnant mice adversely affects the localization and expression of claudin-3 and claudin-4 in the amniotic epithelium. RU-486 administration also increased the permeability of the amniotic membrane. In organ-cultured amniotic membranes, progesterone induced increases in claudin-3 and claudin-4 expression in a dose-dependent manner but did not influence their localization. PRs were also present in the amniotic epithelium during midpregnancy but they disappeared during late pregnancy. These results indicate that the progesterone/PR pathway maintains TJs in the amniotic epithelium during midpregnancy. © 2011 Elsevier Ireland Ltd.

The amniotic epithelium is in direct contact with the amniotic fluid and has tight junctions. The amniotic tight junctions function as a barrier to restrict fluid flux via the amniotic membrane during midpregnancy in the mouse. However, during late pregnancy, amniotic fluid volume significantly decreases in association with the disruption of amniotic tight junctions. The disruption of amniotic tight junctions is caused by apoptosis in the amniotic epithelium on Embryonic Day 17 (E17). In this study, we examine the molecular mechanisms underlying apoptosis of the amniotic epithelium of the mouse. We found that from E16, the number of activated macrophages that express high levels of NOS2 and tumor necrosis factor (TNF) increase in amniotic fluid. TNF receptor type 1 (TNFR1) was detectable from E16 onward. On E17, amniotic epithelial cells expressing TNFR1 became TUNEL positive, suggesting that TNF/TNFR1 signaling may initiate apoptosis. To further confirm the role of TNF/TNFR1 signaling, WP9QY, a TNFR1 antagonist, was injected into the amniotic cavity and was found to significantly reduce the numbers of apoptotic cells in the E17 amniotic epithelium. Furthermore, dehydroxymethylepoxyquinomicin, a specific nuclear factor-kappa B inhibitor, was found to inhibit TNF production in macrophages and amniotic apoptosis in vivo. Finally, we showed that injection of TNF into the amniotic cavity induces early onset of apoptosis. These results indicate that amniotic apoptosis is induced by the TNF pathway via TNFR1 expressed in the amniotic epithelial cells and that activation of macrophages may trigger amniotic apoptosis. © 2011 by the Society for the Study of Reproduction, Inc.

The electrical properties of axons critically influence the nature of communication between neurons. However, due to their small size, direct measurement of membrane potential dynamics in intact and complex mammalian axons has been a challenge. Furthermore, quantitative optical measurements of axonal membrane potential dynamics have not been available. To characterize the basic principles of somatic voltage signal propagation in intact axonal arbors, second-harmonic-generation (SHG) imaging is applied to cultured mouse hippocampal neurons. When FM4-64 is applied extracellularly to dissociated neurons, whole axonal arbors are visualized by SHG imaging. Upon action potential generation by somatic current injection, nonattenuating action potentials are recorded in intact axonal arbors. Interestingly, however, both current-and voltage-clamp recordings suggest that nonregenerative subthreshold somatic voltage changes at the soma are poorly conveyed to these axonal sites. These results reveal the nature of membrane potential dynamics of cultured hippocampal neurons, and further show the possibility of SHG imaging in physiological investigations of axons. © 2010 Society of Photo-Optical Instrumentation Engineers.

Background: The widespread use of proton pump inhibitors (PPIs) is known to cause sporadic gastric fundic gland polyps (FGPs). Altered expression and localization of the water or ion transport proteins might contribute to the excess fluid secretion into the cystic lumen for the development of FGPs. Aims: We investigated the alteration of the murine gastric fundic mucosa after PPI treatment, and examined the expression of water channel aquaporin-4 (AQP4) and potassium channel KCNQ1, which are expressed only in the parietal cells in the gastric mucosa. Methods: Male 5-week-old C57BL/6J mice were administered lansoprazole (LPZ) by subcutaneous injection for 8 weeks. The expression of AQP4 and KCNQ1 were investigated by Western blotting, quantitative RT-PCR, and immunohistochemistry. The expression of mucin-6 (Muc6), pepsinogen, and sonic hedgehog (Shh) were also investigated as mucosal cell lineage markers. Results: Gastric mucosal hyperplasia with multiple cystic dilatations, exhibiting similar histological findings to the FGPs, was observed in the LPZ-treated mice. An increase in the number of AQP4-positive parietal cells and KCNQ1-positive parietal cells was observed. The extension of the distribution of AQP4-positive cells toward the surface of the fundic glands was also observed. The expression levels of AQP4 mRNA and protein were significantly enhanced. The expression of KCNQ1 mRNA was correlated with that of AQP4 mRNA in the LPZ-treated mice. Mucous neck-to-zymogenic cell lineage differentiation was delayed in association with decreased expression of Shh in the LPZ-treated mice. Conclusions: PPI administration increased the number of parietal cells with enhanced expression of AQP4 and KCNQ1. © 2010 Springer Science+Business Media, LLC.

In chorioamnionitis, intra-amniotic infections render the amniotic fluid an adverse environment for the fetus and increase the risk of fetal mortality and morbidity. It remains unclear how infection crosses the amniotic barrier, which is made up of tight junctions (TJs). In this study, we investigated whether amniotic TJs are disrupted in inflammatory conditions such as chorioamnionitis. Amniotic TJs were disrupted by single applications of interleukin (IL)-1β, IL-6, tumour necrosis factor-α(TNF-α), and prostaglandin E2. In organ-cultured amniotic membranes, these inflammatory mediators decreased the claudin-3 and claudin-4 levels at the apical junction at different times. Injecting IL-6 into the amniotic cavity concurrently induced the disruption of amniotic TJs by decreasing the claudin-3 and claudin-4 levels at the apical junction, and the dysfunction of the amniotic barrier; in contrast, injecting TNF-α weakened the amniotic barrier by inducing apoptosis of the amniotic epithelial cells, with no decrease in claudin-3 and claudin-4 at the apical junction. Furthermore, inflammation in the amniotic membrane, which was induced by the administration of lipopolysaccharide to pregnant mice, concurrently caused dysfunction of the amniotic barrier and disruption of TJs, involving the decrease of claudin-3 and claudin-4 levels at the apical junction and apoptosis in the amniotic epithelium. These results indicate that the adverse effects of the inflammatory mediators on amniotic TJs cause severe dysfunction of the amniotic barrier.Chorioamnionitis increases the risk of mortality and morbidity in newborn infants because of amniotic fluid infection. However, the mechanism by which this infection crosses the amniotic barrier and spreads into the amniotic fluid remains unclear. In this study, we have shown that inflammation weakens the amniotic barrier and that this effect is induced by inflammatory mediators, such as interleukin-1β, interleukin-6, tumour necrosis factor-α, and prostaglandin E2, in distinctly different ways. Knowledge of the mechanism underlying the weakening of the amniotic barrier aids in increasing our understanding of the mechanism underlying antenatal infection. © 2010 The Authors. Journal compilation © 2010 The Physiological Society.

The amniotic membrane encloses the amniotic fluid and plays roles in the regulation of amniotic fluid flux through the intramembranous pathway during pregnancy. Aquaporins (AQPs) 1, 3, 8, and 9 are expressed in amniotic membranes. AQPs are water channel proteins that facilitate the rapid flux of water or small molecules across the plasma membrane. Recently, additional roles of AQPs in facilitating cell migration, proliferation, and apoptosis have been suggested, with AQPs being distributed in the appropriate subcellular regions for their functions. The cellular and subcellular distributions of AQPs in the amniotic membrane however remain unclear. We have examined the cellular and subcellular localization of AQPs in amniotic membranes during pregnancy in mice. After embryonic day 12 (E12), AQP1 was distributed in the plasma membrane of finely branched cell processes in the amniotic fibroblasts. AQP3 was present in both epithelial cells and fibroblasts between E10 and E12. The distribution of AQP3 in the epithelial cells dynamically changed as follows: at E14 in the lateral membrane and apical junction; at E16 in the lateral membrane alone; at E17 in the lateral membrane and cytoplasm. AQP8 was expressed in the epithelial cells and complementarily localized in the apical junction and the lateral membrane. AQP9 was detected only in the apoptotic cells of the epithelium. These cellular and subcellular localizations of amniotic AQPs indicate that each AQP plays distinct functional roles, such as in water and urea transport, cell migration, cell proliferation, and apoptosis, for amniotic fluid homeostasis or tissue remodeling of amniotic membranes. © 2010 Springer-Verlag.

Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2. © 2010 The Physiological Society of Japan and Springer.

Aquaporin-4 (AQP4) is a predominant water channel protein in mammalian brains that is distributed with the highest density in the perivascular and subpial astrocyte end-feet. AQP4 is a critical component of an integrated water and potassium homeostasis. Expression and regulation of AQP4 have been studied to understand the roles of AQP4 in physiology and several pathological conditions. Indeed, AQP4 has been implicated in several neurological conditions, such as brain edema and seizure. AQP4 is dynamically regulated at different levels: channel gating, subcellular distribution, phosphorylation, protein-protein interactions and orthogonal array formation. In this review, we focus on the short-term regulation of AQP4. Phosphorylation of AQP4 is important; AQP4 is inhibited when Ser180 is phosphorylated and activated when Ser111 is phosphorylated. AQP4 is also regulated by several metal ions. These metal ions inhibit AQP4 by interacting with the Cys178 residue located in the cytoplasmic loop D, suggesting that AQP4 is regulated by intracellular signaling pathways in response to extracellular stimuli. Recently, it was demonstrated that AQP4 may be inhibited by arylsulfonamides, antiepileptic drugs and other related chemical compounds. Structural analysis of AQP4 may guide a drug design for AQP4. © 2010 IBRO.

Conditions such as hypoxia and anoxia inflict serious damage to the brain and continue to be major medical problems. However, the molecular mechanisms that give rise to such damage are not well understood. To elucidate these mechanisms, we established a clinically relevant rodent model of anoxia/recovery by monitoring blood gas levels after oxygen deprivation. Using this animal model, we examined the regulation of Kv2.1, a voltage-gated potassium channel that plays pivotal roles in the homeostasis and survival of neurons. We found that exposure to anoxia induces rapid dephosphorylation of Kv2.1 in the brain, which can be blocked by pre-administration of a NMDA-type glutamate receptor antagonist, memantine. Furthermore, this change is rapidly reversed as the animal recovers from anoxic stress. These results suggest that Kv2.1 is tightly regulated in a clinically relevant animal model of anoxia and further implicate its role in the homeostasis of neurons during anoxic stress. © 2010 Elsevier Inc. All rights reserved.

Aquaporin (AQP) functions as a water-conducting pore. Mercury inhibits the water permeation through AQP. Although site-directed mutagenesis has shown that mercury binds to Cys189 during the inhibition process, it is not fully understood how this inhibits the water permeation through AQP1. We carried out 40 ns molecular dynamics simulations of bovine AQP1 tetramer with mercury (Hg-AQPI) or without mercury (Free AQP1). In Hg-AQPI, Cys191 (Cys189 In human AQP1) is converted to CyS-SHg+ in each monomer. During each last 10 ns, we observed water permeation events occurred 23 times in Free AQP1 and never In Hg-AQPL Mercury binding did not influence the whole structure, but did induce a collapse in the orientation of several residues at the ar/R region. In Free AQP1, backbone oxygen atoms of Gly 190, Cys191, and Gly192 lined, and were oriented to, the surface of the water pore channel. In Hg-AQP1, however, the SHg+ of Cys191-SHg+ was oriented toward the outside of the water pore. As a result, the backbone oxygen atoms of Gly190, Cys191, and Gly192 became disorganized and the ar/R region collapsed, thereby obstructing the permeation of water. We suggest that mercury disrupts the water pore of AQP1 through local conformational changes in the ar/R region. © 2010 by the Biophysical Society.

Amnion is the innermost layer of the fetal membrane and has been suggested to regulate the volume of amniotic fluid via the amniotic epithelium. The transepithelial pathway is generally restricted by tight junctions (TJs). Thus far, human amniotic TJs have not been identified. In this study, we determined whether the human amniotic epithelium contains TJs. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting analyses showed that the human amniotic epithelium has TJ components, such as occludin, ZO-1, and at least 2 types of claudins, i.e., claudin-4 and claudin-7. The TJ components were found to localize in the lateral membranes and cytoplasm at 35 weeks of gestation; these components disappeared from the lateral membrane at 37 weeks of gestation. Organ culturing of the amnion at 37 weeks gestation induced the relocalization of the TJ proteins from the cytoplasm to the lateral membranes. Furthermore, in cultured amniotic epithelial cells, dexamethasone induced the downregulation of the protein expression of TJs. These findings suggest that the human amniotic epithelium has TJs that disrupt during late pregnancy. The disruption may be induced by several factors such as glucocorticoids present in the amniotic fluid during late pregnancy. © 2009 Elsevier Ltd. All rights reserved.

The kidney is a model organ for transport physiology (Nielsen 1996). AQPs are well-characterized in mammalian kidneys, where they facilitate transep-ithelial water reabsorption. Most renal AQPs are expressed either in proximal tubule cells or in collecting duct principal cells, which are known as sites for water reab-sorption. AQP1 is present in both apical and basolateral membranes of proximal tubules, and in descending limbs of Henle's loop where 70% of filtrated water is isoosmotically reabsorbed (King and Agre 1996). AQP2 is expressed in principal cells of the collecting duct; in response to vasopressin, AQP2 translocates from in-tracellular vesicles to the apical plasma membranes, thereby increasing water permeability to concentrate urine (Nielsen et al. 1993, 1995; Knepper 1997; Schrier 2006). AQP3 and AQP4 reside in the basolateral membranes of collecting duct principal cells, where they may provide the exit pathways for urine. AQP7, AQP8, and AQP11 are also present in the proximal tubules (Nielsen et al. 1998). A rat cDNA clone encoding AQP6 was isolated by PCR-based homologous cloning from a rat kidney cDNA library (Ma et al. 1993; Yasui et al. 1999). AQP6 has high sequence homology to AQP0, AQP2, and AQP5. A human AQP6 was also cloned (Ma et al. 1996). Interestingly, the genes encoding AQP2, AQP5, and AQP6 are mapped to chromosome band 12q13 as a family gene cluster at this locus (Ma et al. 1997). Nevertheless, AQP6 is distinct from AQP0, AQP2, and AQP5 in terms of function. Among the renal aquaporins mentioned above, AQP6 has a unique distribution and a distinct function. © 2009 Springer Berlin Heidelberg.

Background. Several aquaporins (a family of integral membrane proteins) have been recently identified in the mammalian gastrointestinal tract, and their involvement in the movement of fluid and small solutes has been suggested. In this direction we investigated, in some regions of the rat gastrointestinal tract, the presence and localization of aquaporin-6, given its peculiar function as an ion selective channel. Results. RT-PCR and immunoblotting experiments showed that aquaporin-6 was expressed in all the investigated portions of the rat gastrointestinal tract. The RT-PCR experiments showed that aquaporin-6 transcript was highly expressed in small intestine and rectum, and less in stomach, caecum and colon. In addition, jejunal mRNA expression was specifically stimulated by feeding. Immunoblotting analysis showed a major band with a molecular weight of about 55 kDa corresponding to the aquaporin-6 protein dimer; this band was stronger in the stomach and large intestine than in the small intestine. Immunoblotting analysis of brush border membrane vesicle preparations showed an intense signal for aquaporin-6 protein. The results of in situ hybridization experiments demonstrate that aquaporin-6 transcript is present in the isthmus, neck and basal regions of the stomach lining, and throughout the crypt-villus axis in both small and large intestine. In the latter regions, immunohistochemistry revealed strong aquaporin-6 labelling in the apical membrane of the surface epithelial cells, while weak or no labelling was observed in the crypt cells. In the stomach, an intense staining was observed in mucous neck cells and lower signal in principal cells and some parietal cells. Conclusion. The results indicate that aquaporin-6 is distributed throughout the gastrointestinal tract. Aquaporin-6 localization at the apical pole of the superficial epithelial cells and its upregulation by feeding suggest that it may be involved in movements of water and anions through the epithelium of the villi. © 2009 Laforenza et al.