The role of aquaporin water channels in fluid secretion by the exocrine pancreas

The mammalian exocrine pancreas secretes a near-isosmotic fluid over a wide osmolarity range. The role of aquaporin (AQP) water channels in this process is now becoming clearer. AQP8 water channels, which were initially cloned from rat pancreas, are expressed at the apical membrane of pancreatic acinar cells and contribute to their osmotic permeability. However, the acinar cells secrete relatively little fluid and there is no obvious defect in pancreatic function in AQP8 knockout mice. Most of the fluid secreted by the pancreas is generated by ductal epithelial cells, which comprise only a small fraction of the gland mass. In the human pancreas, secretion occurs mainly in the intercalated ducts, where the epithelial cells express abundant AQP1 and AQP5 at the apical membrane and AQP1 alone at the basolateral membrane. In the rat and mouse, fluid secretion occurs mainly in the interlobular ducts where AQP1 and AQP5 are again co-localized at the apical membrane but appear to be expressed at relatively low levels. Nonetheless, the transepithelial osmotic permeability of rat interlobular ducts is sufficient to support near-isosmotic fluid secretion at observed rates. Furthermore, apical, but not basolateral, application of Hg²⁺ significantly reduces the transepithelial osmotic permeability, suggesting that apical AQP1 and AQP5 may contribute significantly to fluid secretion. The apparently normal fluid output of the pancreas in AQP1 knockout mice may reflect the presence of AQP5 at the apical membrane.     

Salivary acinar cells from aquaporin 5-deficient mice have decreased membrane water permeability and altered cell volume regulation

Aquaporins (AQPs) are channel proteins that regulate the movement of water through the plasma membrane of secretory and absorptive cells in response to osmotic gradients. In the salivary gland, AQP5 is the major aquaporin expressed on the apical membrane of acinar cells. Previous studies have shown that the volume of saliva secreted by AQP5-deficient mice is decreased, indicating a role for AQP5 in saliva secretion; however, the mechanism by which AQP5 regulates water transport in salivary acinar cells remains to be determined. Here we show that the decreased salivary flow rate and increased tonicity of the saliva secreted by Aqp5(-)/- mice in response to pilocarpine stimulation are not caused by changes in whole body fluid homeostasis, indicated by similar blood gas and electrolyte concentrations in urine and blood in wild-type and AQP5-deficient mice. In contrast, the water permeability in parotid and sublingual acinar cells isolated from Aqp5(-)/- mice is decreased significantly. Water permeability decreased by 65% in parotid and 77% in sublingual acinar cells from Aqp5(-)/- mice in response to hypertonicity-induced cell shrinkage and hypotonicity-induced cell swelling. These data show that AQP5 is the major pathway for regulating the water permeability in acinar cells, a critical property of the plasma membrane which determines the flow rate and ionic composition of secreted saliva.     

Hypertonic induction of aquaporin-5 expression through an ERK-dependent pathway

Aquaporin-5 (AQP5) is a water channel protein expressed in lung, salivary gland, and lacrimal gland epithelia. Each of these sites may experience fluctuations in surface liquid osmolarity; however, osmotic regulation of AQP5 expression has not been reported. This study demonstrates that AQP5 is induced by hypertonic stress and that induction requires activation of extracellular signal-regulated kinase (ERK). Incubation of mouse lung epithelial cells (MLE-15) in hypertonic medium produced a dose-dependent increase in AQP5 expression; AQP5 protein peaked by 24 h and returned to baseline levels within hours of returning cells to isotonic medium. AQP5 induction was observed only with relatively impermeable solutes, suggesting an osmotic pressure gradient is required for induction. ERK was selectively activated in MLE-15 cells by hypertonic stress, and inhibition of ERK activation with two distinct mitogen-activated extracellular regulated kinase kinase (MEK) inhibitors, U0126 and PD98059, blocked AQP5 induction. AQP5 induction was also observed in the lung, salivary, and lacrimal glands of hyperosmolar rats, suggesting potential physiologic relevance for osmotic regulation of AQP5 expression. This report provides the first example of hypertonic induction of an extrarenal aquaporin, as well as the first association between mitogen-activated protein kinase signaling and aquaporin expression.     

Water channels and their roles in some ocular tissues

Water is a major component of the eye, and water channels (aquaporins) are ubiquitous in ocular tissues, and quite abundant at their different locations. AQP1 is expressed in corneal endothelium, lens epithelium, ciliary epithelium, and retinal pigment epithelium. AQP3 is expressed in corneal epithelium, and in conjunctival epithelium. AQP4 is expressed in ciliary epithelium and retinal Muller cells. AQP5 is expressed in corneal epithelium, and conjunctival epithelium. AQP0 is expressed in lens fiber cells. It is known that five ocular tissues transport fluid, namely: (1) Corneal endothelium; (2) Conjunctival epithelium; (3) Lens epithelium; (4) Ciliary epithelium; (5) Retinal pigment epithelium. For the corneal endothelium, aquaporins are not the main route for trans-tissue water movement, which is paracellular. Instead, we propose that aquaporins allow fast osmotic equilibration of the cell, which is necessary to maintain optimal rates of fluid movement since the cyclic paracellular water transfer mechanism operates separately and tends to create periodic osmotic imbalances (τ～5s).     

Involvement of AQP6 in the Mercury-Sensitive Osmotic Lysis of Rat Parotid Secretory Granules

In secretory granules and vesicles, membrane transporters have been predicted to permeate water molecules, ions and/or small solutes to swell the granules and promote membrane fusion. We have previously demonstrated that aquaporin-6 (AQP6), a water channel protein, which permeates anions, is localized in rat parotid secretory granules (Matsuki-Fukushima et al., Cell Tissue Res 332:73–80, 2008). Because the localization of AQP6 in other organs is restricted to cytosolic vesicles, the native function or functions of AQP6 in vivo has not been well determined. To characterize the channel property in granule membranes, the solute permeation-induced lysis of purified secretory granules is a useful marker. To analyze the role of AQP6 in secretory granule membranes, we used Hg²⁺, which is known to activate AQP6, and investigated the characteristics of solute permeability in rat parotid secretory granule lysis induced by Hg²⁺ (Hg lysis). The kinetics of osmotic secretory granule lysis in an iso-osmotic KCl solution was monitored by the decay of optical density at 540 nm using a spectrophotometer. Osmotic secretory granule lysis was markedly facilitated in the presence of 0.5–2.0 μM Hg²⁺, concentrations that activate AQP6. The Hg lysis was completely blocked by β-mercaptoethanol which disrupts Hg²⁺-binding, or by removal of chloride ions from the reaction medium. An anion channel blocker, DIDS, which does not affect AQP6, discriminated between DIDS-insensitive and sensitive components in Hg lysis. These results suggest that Hg lysis is required for anion permeability through the protein transporter. Hg lysis depended on anion conductance with a sequence of NO₃ ^? > Br^? > I^? > Cl^? and was facilitated by acidic pH. The anion selectivity for NO₃ ^? and the acidic pH sensitivity were similar to the channel properties of AQP6. Taken together, it is likely that AQP6 permeates halide group anions as a Hg²⁺-sensitive anion channel in rat parotid secretory granules.     

Hypertonic induction of aquaporin-1 water channel independent of transcellular osmotic gradient

Aquaporin-1 (AQP1) water channel plays a critical role for water reabsorption in the urinary concentrating mechanism. AQP1 expression in renal cells is upregulated by hypertonicity, but not urea, suggesting the requirement of an osmotic gradient. To investigate whether AQP1 expression is regulated by apical and/or basolateral hypertonicity, murine renal medullary mIMCD-K2 cells grown on permeable support were exposed to hypertonic medium. When the medium on the apical or basolateral membrane side was switched to hypertonic, the transcellular osmotic gradient was dissipated within 8h. Basolateral hypertonicity increased AQP1 expression more than apical hypertonicity. Comparable apical and basolateral hypertonicity without a transcellular hypertonic gradient, however, increased AQP1 expression. Cell surface biotinylation experiments revealed that hypertonicity promoted AQP1 trafficking to both plasma cell membranes. These results indicate that AQP1 expression is predominantly mediated by basolateral hypertonicity but a transcellular osmotic gradient is not necessary for its induction.     

Expression and immunolocalization of aquaporin water channels in rat exocrine pancreas

Both the acinar and ductal cells of the pancreas secrete a near-isotonic fluid and may thus be sites of aquaporin (AQP) water channel expression. Northern blot analysis of mRNA from whole rat pancreas revealed high levels of AQP1 and AQP8 expression, whereas lower levels of AQP4 and AQP5 expression were just detectable by RT-PCR Southern blot analysis. Immunohistochemistry showed that AQP1 is localized in the microvasculature, whereas AQP8 is confined to the apical pole of the acinar cells. No labeling of acinar, ductal, or vascular tissue was detected with antibodies to AQP2-7. With immunoelectron microscopy, AQP8 labeling was observed not only at the apical membrane of the acinar cells but also among small intracellular vesicles in the subapical cytoplasm, suggesting that there may be regulated trafficking of AQP8 to the apical plasma membrane. To evaluate the contribution of AQPs to the membrane water permeability, video microscopy was used to measure the swelling of acinar cells in response to hypotonic stress. Osmotic water permeability was reduced by 90% following exposure to Hg(2+). Since AQP8 is confined to the apical membrane, the marked effect of Hg(2+) suggests that other water channels may be expressed in the basolateral membrane.     

Effects of naturally occurring G103D point mutation of AQP5 on its water permeability, trafficking and cellular localization in the submandibular gland of rats

Background information. AQPs (aquaporins) are water channel proteins that are expressed in almost all living things. In mammalians, 13 members of AQPs (AQP0–12) have been identified so far. AQP5 is known to be expressed mostly in the exocrine cells, including the salivary gland acinar cells. A naturally occurring point mutation (G308A, Gly¹⁰³ > Asp¹⁰³) was earlier found in the rat AQP5 gene [Murdiastuti, Purwanti, Karabasil, Li, Yao, Akamatsu, Kanamori and Hosoi (2006) Am. J. Physiol. 291 , G1081–G1088]; in this mutant, the rate of initial saliva secretion under stimulated and unstimulated conditions is less than that for the wt (wild‐type) animals. Results. Here the mutant molecule was characterized in detail. Using the Xenopus oocyte system, we demonstrated the mutant AQP5 to have water permeability almost the same as that of the wt molecule. Mutant and wt AQP5s, tagged with GFP (green fluorescent protein; GFP‐AQP5s) and expressed in polarized MDCK‐II (Madin—Darby canine kidney II) cells, first appeared in the vesicular structure(s) in the cytoplasm, and were translocated to the upper plasma membrane or apical membrane during cultivation, with the mutant GFP‐AQP5 being translocated less efficiently. Thapsigargin and H‐89 both induced translocation in vitro of either molecule, whereas colchicine inhibited this activity; the fraction of cells showing apical localization of mutant GFP‐AQP5 was less than that showing that of the wt molecule under any of the experimental conditions used. In the mutant SMG (submandibular gland) tissue, localization of AQP5 in the apical membrane of acinar cells was extremely reduced. Vesicular structures positive for AQP5 and present in the cytoplasm of the acinar cells were co‐localized with LAMP2 (lysosome‐associated membrane protein 2) or cathepsin D in the mutant gland, whereas such co‐localizations were very rare in the wt gland, suggesting that the mutant molecules largely entered lysosomes for degradation. Conclusion. Replacement of highly conserved hydrophobic Gly¹⁰³ with strongly hydrophilic Asp¹⁰³ in rat AQP5, though it did not affect water permeability, may possibly have resulted in less efficient membrane trafficking and increased lysosomal degradation, leading to its lower expression in the apical membrane of the acinar cells in the SMG.     

Localization of AQP5 during development of the mouse submandibular salivary gland

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5–18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs.     

Novel Endothelial Cell-Specific AQP1 Knockout Mice Confirm the Crucial Role of Endothelial AQP1 in Ultrafiltration during Peritoneal Dialysis

The water channel aquaporin-1 (AQP1) mediates about 50% ultrafiltration during a 2-hour hypertonic dwell in global AQP1 knockout (AQP1^-/-) mice. Although AQP1 is widely expressed in various cell types including mesothelial cells, the ultrafiltration has been assumed to be mediated via endothelial AQP1 of the peritoneum. The partial embryonic lethality and reduced body weight in AQP1^-/- mice may reflect potential confounding phenotypic effects evoked by ubiquitous AQP1 deletion, which may interfere with functional analysis of endothelial AQP1. Using a Cre/loxP approach, we generated and characterised endothelial cell- and time-specific AQP1 knockout (AQP1^fl/fl; Cdh5-Cre⁺) mice. Compared to controls, AQP1^fl/fl; Cdh5-Cre⁺ mice showed no difference in an initial clinical and biological analysis at baseline, including body weight and survival. During a 1-hour 3.86% mini-peritoneal equilibration test (mini-PET), AQP1^fl/fl; Cdh5-Cre⁺ mice exhibited strongly decreased indices for AQP1-related transcellular water transport (43.0% in net ultrafiltration, 93.0% in sodium sieving and 57.9% in free water transport) compared to controls. The transport rates for small solutes of urea and glucose were not significantly altered. Our data provide the first direct experimental evidence for the functional relevance of endothelial AQP1 to the fluid transport in peritoneal dialysis and thereby further validate essential predictions of the three-pore model of peritoneal transport.     

The Role of the Tight Junction in Paracellular Fluid Transport across Corneal Endothelium. Electro-osmosis as a Driving Force

The mechanism of epithelial fluid transport is controversial and remains unsolved. Experimental difficulties pose obstacles for work on a complex phenomenon in delicate tissues. However, the corneal endothelium is a relatively simple system to which powerful experimental tools can be applied. In recent years our laboratory has developed experimental evidence and theoretical insights that illuminate the mechanism of fluid transport across this leaky epithelium. Our evidence points to fluid being transported via the paracellular route by a mechanism requiring junctional integrity, which we attribute to electro-osmotic coupling at the junctions. Fluid movements can be produced by electrical currents. The direction of the movement can be reversed by current reversal or by changing junctional electrical charges by polylysine. Aquaporin 1 (AQP1) is the only AQP present in these cells, and its deletion in AQP1 null mice significantly affects cell osmotic permeability but not fluid transport, which militates against the presence of sizable water movements across the cell. By contrast, AQP1 null mice cells have reduced regulatory volume decrease (only 60% of control), which suggests a possible involvement of AQP1 in either the function or the expression of volume-sensitive membrane channels/transporters. A mathematical model of corneal endothelium predicts experimental results only when based on paracellular electro-osmosis, and not when transcellular local osmosis is assumed instead. Our experimental findings in corneal endothelium have allowed us to develop a novel paradigm for this preparation that includes: (1) paracellular fluid flow; (2) a crucial role for the junctions; (3) hypotonicity of the primary secretion; (4) an AQP role in regulation and not as a significant water pathway. These elements are remarkably similar to those proposed by the Hill laboratory for leaky epithelia.     

Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes

Background information. Mercurials inhibit AQPs (aquaporins), and site‐directed mutagenesis has identified Cys¹⁸⁹ as a site of the mercurial inhibition of AQP1. On the other hand, AQP4 has been considered to be a mercury‐insensitive water channel because it does not have the reactive cysteine residue corresponding to Cys¹⁸⁹ of AQP1. Indeed, the osmotic water permeability (P_f) of AQP4 expressed in various types of cells, including Xenopus oocytes, is not inhibited by HgCl₂. To examine the direct effects of mercurials on AQP4 in a proteoliposome reconstitution system, His‐tagged rAPR4 (rat AQP4) M23 was expressed in Saccharomyces cerevisiae, purified with an Ni²⁺‐nitrilotriacetate affinity column, and reconstituted into liposomes with the dilution method. Results. The water permeability of AQP4 proteoliposomes with or without HgCl₂ was measured with a stopped‐flow apparatus. Surprisingly, the P_f of AQP4 proteoliposomes was significantly decreased by 5 μM HgCl₂ within 30 s, and this effect was completely reversed by 2‐mercaptoethanol. The dose‐ and time‐dependent inhibitory effects of Hg²⁺ suggest that the sensitivity to mercury of AQP4 is different from that of AQP1. Site‐directed mutagenesis of six cysteine residues of AQP4 demonstrated that Cys¹⁷⁸, which is located at loop D facing the intracellular side, is a target responding to Hg²⁺. We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. Conclusions. Our results suggest that mercury inhibits the P_f of AQP4 by mechanisms different from those for AQP1 and that AQP4 may be gated by modification of a cysteine residue in cytoplasmic loop D.     

A New Approach to Epithelial Isotonic Fluid Transport: An Osmosensor Feedback Model

A model for control of the transport rate and osmolarity of epithelial fluid (isotonic transport) is presented by using an analogy with the control of temperature and flow rate in a shower. The model brings recent findings and theory concerning the role of aquaporins in epithelia together with measurements of epithelial paracellular flow into a single scheme. It is not based upon osmotic equilibration across the epithelium but rather on the function of aquaporins as osmotic sensors that control the tonicity of the transported fluid by mixing cellular and paracellular flows, which may be regarded individually as hyper- and hypo-tonic fluids, to achieve near-isotonicity. The system is built on a simple feedback loop and the quasi-isotonic behavior is robust to the precise values of most parameters. Although the two flows are separate, the overall fluid transport rate is governed by the rate of salt pumping through the cell. The model explains many things: how cell pumping and paracellular flow can be coupled via control of the tight junctions; how osmolarity is controlled without depending upon the precise magnitude of membrane osmotic permeability; and why many epithelia have different aquaporins at the two membranes.The model reproduces all the salient features of epithelial fluid transport seen over many years but also indicates novel behavior that may provide a subject for future research and serve to distinguish it from other schemes such as simple osmotic equilibration. Isotonic transport is freed from constraints due to limited permeability of the membranes and the precise geometry of the system. It achieves near-isotonicity in epithelia in which partial water transport by co-transporters may be present, and shows apparent electro-osmotic effects. The model has been developed with a minimum of parameters, some of which require measurement, but the model is flexible enough for the basic idea to be extended both to complex systems of water and salt transport that still await a clear explanation, such as intestine and airway, and to systems that may lack aquaporins or use other sensors.     

TNF-α inhibits aquaporin 5 expression in human salivary gland acinar cells via suppression of histone H4 acetylation

Sjögren's syndrome is a systemic autoimmune disease characterized by reductions in salivary and lacrimal secretions. The mechanisms underlying these reductions remain unclear. We have previously shown that TNF‐α plays an important role in the destruction of acinar structures. Here we examined TNF‐α's function in the expression of aquaporin (AQP) 5 in human salivary gland acinar cells. Immortalized human salivary gland acinar (NS‐SV‐AC) cells were treated with TNF‐α, and then the expression levels of AQP5 mRNA and protein were analysed. In addition, the mechanisms underlying the reduction of AQP5 expression by TNF‐α treatment were investigated. TNF‐α‐treatment of NS‐SV‐AC cells significantly suppressed the expression levels of AQP5 mRNA and protein, and reduced the net fluid secretion rate. We examined the expression and activation levels of DNA methyltransferases (Dnmts) in NS‐SV‐AC cells treated with TNF‐α. However, no significant changes were observed in the expression or activation levels of Dnmt1, Dnmt3a or Dnmt3b. Although we also investigated the role of NF‐κB activity in the TNF‐α‐induced suppression of AQP5 expression in NS‐SV‐AC cells, we detected similar TNF‐α suppression of AQP5 expression in non‐transfected cells and in a super‐repressor form of IκBα cDNA‐transfected cell clones. However, interestingly, chromatin immunoprecipitation analysis demonstrated a remarkable decrease in levels of acetylated histone H4 associated with the AQP5 gene promoter after treatment with TNF‐α in NS‐SV‐AC cells. Therefore, our results may indicate that TNF‐α inhibition of AQP5 expression in human salivary gland acinar cells is due to the epigenetic mechanism by suppression of acetylation of histone H4.     

A novel role for aquaporin-5 in enhancing microtubule organization and stability

Aquaporin-5 (AQP5) is a water-specific channel located on the apical surface of airway epithelial cells. In addition to regulating transcellular water permeability, AQP5 can regulate paracellular permeability, though the mechanisms by which this occurs have not been determined. Microtubules also regulate paracellular permeability. Here, we report that AQP5 promotes microtubule assembly and helps maintain the assembled microtubule steady state levels with slower turnover dynamics in cells. Specifically, reduced levels of AQP5 correlated with lower levels of assembled microtubules and decreased paracellular permeability. In contrast, overexpression of AQP5 increased assembly of microtubules, with evidence of increased MT stability, and promoted the formation of long straight microtubules in the apical domain of the epithelial cells. These findings indicate that AQP5-mediated regulation of microtubule dynamics modulates airway epithelial barrier properties and epithelial function.     

Lipopolysaccharide changes the subcellular distribution of aquaporin 5 and increases plasma membrane water permeability in mouse lung epithelial cells

Aquaporin-5 (AQP5), a major water channel in lung epithelial cells, plays an important role in maintaining water homeostasis in the lungs. Cell surface expression of AQP5 is regulated by not only mRNA and protein synthesis but also changes in subcellular distribution. We investigated the effect of lipopolysaccharide (LPS) on the subcellular distribution of AQP5 in a mouse lung epithelial cell line (MLE-12). LPS caused significant increases in AQP5 in the plasma membrane at 0.5-2 h. Immunofluorescence and Western blotting strongly suggested that LPS altered AQP5 subcellular distribution from an intracellular vesicular compartment to the plasma membrane. The specific p38 MAP kinase inhibitor SB 203580 apparently prevented LPS-induced changes in AQP5 distribution. Furthermore, LPS increased the osmotic water permeability of MLE-12 cells. These findings demonstrate that LPS increases cell surface AQP5 expression by changing its subcellular distribution and increases membrane osmotic water permeability through activation of p38 MAP kinase.     

Aquaporin 5 Interacts with Fluoride and Possibly Protects against Caries

Aquaporins (AQP) are water channel proteins and the genes coding for AQP2, AQP5, and AQP6 are clustered in 12q13. Since AQP5 is expressed in serous acinar cells of salivary glands, we investigated its involvement in caries. DNA samples from 1,383 individuals from six groups were studied. Genotypes of eight single nucleotide polymorphisms covering the aquaporin locus were tested for association with caries experience. Interaction with genes involved in enamel formation was tested. The association between enamel microhardness at baseline, after creation of artificial caries lesion, and after exposure to fluoride and the genetic markers in AQP5 was tested. Finally, AQP5 expression in human whole saliva, after exposure to fluoride in a mammary gland cell line, which is known to express AQP5, and in Wistar rats was also verified. Nominal associations were found between caries experience and markers in the AQP5 locus. Since these associations suggested that AQP5 may be inhibited by levels of fluoride in the drinking water that cause fluorosis, we showed that fluoride levels above optimal levels change AQP5 expression in humans, cell lines, and rats. We have shown that AQP5 is involved in the pathogenesis of caries and likely interacts with fluoride.