cAMP has distinct acute and chronic effects on aquaporin-5 in lung epithelial cells

Aquaporin-5 (AQP5) is present on the apical membrane of epithelial cells in various secretory glands as well as on the apical membrane of the airway epithelium, airway submucosal glands, and type 1 pneumocytes, where it can participate in respiratory tract water homeostasis. We examined the effects of cAMP on AQP5 distribution and abundance. When AQP5-expressing mouse lung epithelial cells were treated with cAMP or the beta-adrenergic agonist terbutaline, a biphasic AQP5 response was observed. Short term (minutes) exposure to cAMP produced internalization of AQP5 off of the membrane and a decrease in protein abundance. Both of these responses were blocked by inhibition of protein kinase A and the decrease in abundance was blocked by chloroquine, indicating lysosome-mediated degradation. Sustained cAMP exposure (hours) produced an increase in membrane localization and increased abundance; these effects were also blocked by protein kinase A inhibition. The beta-adrenergic agonist terbutaline produced changes in AQP5 abundance in mouse trachea and lung, consistent with our findings in cultured epithelial cells. Purified AQP5 protein was phosphorylated by protein kinase A but not protein kinase C or casein kinase II, and aquaporin-5 was phosphorylated in cultured cells after long term (but not short term) exposure to cAMP. These studies indicate that cAMP and beta-adrenergic agonists produce distinct short and long term effects on AQP5 distribution and abundance that may contribute to regulation of lung water homeostasis.     

Subcellular redistribution of AQP5 by vasoactive intestinal polypeptide in the Brunner's gland of the rat duodenum

Aquaporin (AQP)5, an exocrine-type water channel, was detected in the rat duodenum by Western blot analysis, and was localized by immunohistochemistry in the secretory granule membranes as well as in the apical and lateral aspects of the plasma membrane of Brunner's gland cells. Incubation of duodenal slices with vasoactive intestinal polypeptide (VIP) in vitro significantly increased the amount of AQP5 in the apical membrane fraction in a dose- and time-dependent manner with the amount reaching a plateau at 100 nM VIP and becoming near maximal after a 30-s incubation. Protein kinase inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 50 muM), and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; PKA-specific, 1 muM) blocked this increase, but PKC-specific inhibitor calphostin C did not, implying the involvement of PKA but not PKC in this cellular event. Intravenous injection with VIP (40 mug/kg body wt) provoked dilation of the lumen of the Brunner's gland at 2 and 7 min and increased the staining intensity of AQP5 in the apical and lateral membranes. AQP1 (both nonglycosylated and glycosylated forms) was also found to localize in the apical and basolateral membranes of cells of Brunner's gland. VIP, however, did not provoke any significant change in the AQP1 level in the apical membrane, as judged from the results of the above in vitro and in vivo experiments. These results suggest that VIP induced the exocytosis of granule contents and simultaneously caused translocation of AQP5 but not of AQP1 to the apical membrane in Brunner's gland cells.     

Aquaporin-5 water channel in lipid rafts of rat parotid glands

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. The activation of M3 muscarinic acetylcholine receptors (mAChRs) or α1-adrenoceptors on the salivary glands induces salivary fluid secretion. AQP5 localizes in lipid rafts and activation of the M3 mAChRs or α1-adrenoceptors induced its translocation together with the lipid rafts to the APM in the interlobular ducts of rat parotid glands. This review focuses on the mechanisms of AQP5 translocation together with lipid rafts to the APM in the interlobular duct cells of parotid glands of normal rats and the impairment of AQP5 translocation in diabetes and senescence.     

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 water channel in lipid rafts of rat parotid glands

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. The activation of M3 muscarinic acetylcholine receptors (mAChRs) or alpha1-adrenoceptors on the salivary glands induces salivary fluid secretion. AQP5 localizes in lipid rafts and activation of the M3 mAChRs or alpha1-adrenoceptors induced its translocation together with the lipid rafts to the APM in the interlobular ducts of rat parotid glands. This review focuses on the mechanisms of AQP5 translocation together with lipid rafts to the APM in the interlobular duct cells of parotid glands of normal rats and the impairment of AQP5 translocation in diabetes and senescence.     

Aquaporin-5 (AQP5), a water channel protein, in the rat salivary and lacrimal glands: immunolocalization and effect of secretory stimulation

Aquaporin-5 (AQP5) is a water channel protein and is considered to play an important role in water movement across the plasma membrane. We raised anti-AQP5 antibody and examined the localization of AQP5 protein in rat salivary and lacrimal glands by immunofluorescence microscopy. AQP5 was found in secretory acinar cells of submandibular, parotid, and sublingual glands, where it was restricted to apical membranes including intercellular secretory canaliculi. In the submandibular gland, abundant AQP5 was also found additionally at the apical membrane of intercalated duct cells. Upon stimulation by isoproterenol, apical staining for AQP5 in parotid acinar cells tended to appear as clusters of dots. These results suggest that AQP5 is one of the candidate molecules responsible for the water movement in the salivary glands.     

All-trans retinoic acid increases expression of aquaporin-5 and plasma membrane water permeability via transactivation of Sp1 in mouse lung epithelial cells

Aquaporin-5 (AQP5) is a water-selective channel protein that is expressed in lacrimal glands, salivary glands, and distal lung. Several studies using AQP5 knockout mice have revealed that AQP5 plays an important role in maintaining water homeostasis in the lung. We report here that all-trans retinoic acid (atRA) increases plasma membrane water permeability, AQP5 mRNA and protein expression, and AQP5 promoter activity in MLE-12 cells. The promoter activation induced by atRA was diminished by mutation at the Sp1/Sp3 binding element (SBE), suggesting that the SBE mediates the effects of atRA. In addition, atRA increased the binding of Sp1 to the SBE without changing the levels of Sp1 in the nucleus. Taken together, our data indicate that atRA increases AQP5 expression through transactivation of Sp1, leading to an increase in plasma membrane water permeability.     

Membrane trafficking of AQP5 and cAMP dependent phosphorylation in bronchial epithelium

Phosphorylation pathway has been identified as an important step in membrane trafficking for AQP5. We generated stably transfected BEAS-2B human bronchial epithelial cells with various over-expression constructs on permeable support. In stable cells with wild-type AQP5 and S156A (AQP5 mutant targeting PKA consensus sequence), AQP5 expression was predominantly polarized to the apical membrane, whereas stable cells with N185D (AQP5 mutant targeting second NPA motif), mainly localized to the cytoplasm. Treatment with H89 and/or chlorophenylthio-cAMP (cpt-cAMP) did not affect membrane expression of AQP5 in any of three stable cells. In cells with wild-type AQP5 and N185D, AQP5s were phosphorylated by PKA, while phosphorylation of AQP5 was not detected in cells with S156A. These results indicate that, in AQP5, serine156 may be phosphorylated by PKA, but membrane expression of AQP5 may not be regulated by PKA phosphorylation. We conclude that AQP5 membrane targeting can include more than one mechanism besides cAMP dependent phosphorylation.     

Tumor necrosis factor-alpha inhibits aquaporin 5 expression in mouse lung epithelial cells

Aquaporin 5 (AQP5), the major water channel expressed in alveolar, tracheal, and upper bronchial epithelium, is significantly down-regulated during pulmonary inflammation and edema. The mechanisms that underlie this decrease in AQP5 levels are therefore of considerable interest. Here we show that AQP5 expression in cultured lung epithelial cells is decreased 2-fold at the mRNA level and 10-fold at the protein level by the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). Treatment of murine lung epithelial cells (MLE-12) with TNF-alpha results in a concentration- and time-dependent decrease in AQP5 mRNA and protein expression. Activation of the p55 TNF-alpha receptor (TNFR1) with an agonist antibody is sufficient to cause decreased AQP5 expression, demonstrating that the TNF-alpha effect is mediated through TNFR1. Inhibition of nuclear factor kappaB (NF-kappaB) translocation to the nucleus blocks the effect of TNF-alpha on AQP5 expression, indicating that activation of NF-kappaB is required, whereas inhibition of extracellular signal-regulated or p38 mitogen-activated protein kinases showed no effect. These data show that TNF-alpha decreases AQP5 mRNA and protein expression and that the molecular pathway for this effect involves TNFR1 and activated NF-kappaB. The ability of inflammatory cytokines to decrease aquaporin expression may help explain the connection between inflammation and edema.     

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.     

Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat

The molecular pathways for fluid transport in pulmonary, oral,and nasal tissues are still unresolved. Here we use immunocytochemistry and immunoelectron microscopy to define the sites of expression of fouraquaporins in the respiratory tract and glandular epithelia, where theyreside in distinct, nonoverlapping sites. Aquaporin-1 (AQP1) is presentin apical and basolateral membranes of bronchial, tracheal, andnasopharyngeal vascular endothelium and fibroblasts. AQP5 is localizedto the apical plasma membrane of type I pneumocytes and the apicalplasma membranes of secretory epithelium in upper airway and salivaryglands. In contrast, AQP3 is present in basal cells of tracheal andnasopharyngeal epithelium and is abundant in basolateral membranes ofsurface epithelial cells of nasal conchus. AQP4 resides in basolateralmembranes of columnar cells of bronchial, tracheal, and nasopharyngealepithelium; in nasal conchus AQP4 is restricted to basolateralmembranes of a subset of intra- and subepithelial glands. These sitesof expression suggest that transalveolar water movement, modulation ofairway surface liquid, air humidification, and generation ofnasopharyngeal secretions involve a coordinated network of aquaporinwater channels.

     

Hypertonicity-induced expression of aquaporin 3 in MDCK cells

Aquaporins (AQPs) are water channel proteins that participate in water transport. In the principal cells of the kidney collecting duct, water reabsorption is mediated by the combined action of AQP2 in the apical membrane and both AQP3 and AQP4 in the basolateral membrane, and the expression of AQP2 and AQP3 is regulated by antidiuretic hormone and water restriction. The effect of hypertonicity on AQP3 expression in Madin-Darby canine kidney (MDCK) epithelial cells was investigated by exposing the cells to hypertonic medium containing raffinose or NaCl. Northern blot and immunoblot analyses revealed that the amounts of AQP3 mRNA and AQP3 protein, respectively, were markedly increased by exposure of cells to hypertonicity. These effects were maximal at 12 and 24 h, respectively. Immunofluorescence and immunoelectron microscopy also demonstrated that the abundance of AQP3 protein was increased in cells incubated in hypertonic medium and that the protein was localized at the basolateral plasma membrane. These results indicate that the expression of AQP3 is upregulated by hypertonicity.     

The AQP2 water channel: Effect of vasopressin treatment,microtubule disruption,and distribution in neonatal rats

Aquaporin 2 is a collecting duct water channel that is located in apical vesicles and in the apical plasma membrane of collecting duct principal cells. It shares 42% identity with the proximal tubule/thin descending limb water channel, CHIP28. The present study was aimed at addressing three questions concerning the location and behavior of the AQP2 protein under different conditions. First, does the AQP2 channel relocate to the apical membrane after vasopressin treatment? Our results show that AQP2 is diffusely distributed in cytoplasmic vesicles in collecting duct principal cells of homozygous Brattleboro rats that lack vasopressin. In rats injected with exogenous vasopressin, however, AQP2 became concentrated in the apical plasma membrane of principal cells, as determined by immunofluorescence and immunogold electron microscopy. This behavior is consistent with the idea that AQP2 is the vasopressin-sensitive water channel. Second, is the cellular location of AQP2 modified by microtubule disruption? In normal rats, AQP2 has a mainly apical and subapical location in principal cells, but in colchicine-treated rats, it is distributed on vesicles that are scattered throughout the entire cytoplasm. This is consistent with the dependence on microtubules of apical protein targeting in many cell types, and explains the inhibitory effect of microtubule disruption on the hydroosmotic response to vasopressin in sensitive epithelia, including the collecting duct. Third, is AQP2 present in neonatal rat kidneys? We show that AQP2 is abundant in principal cells from neonatal rats at all days after birth. The detection of AQP2 in early neonatal kidneys indicates that a lack of this protein is not responsible for the relatively weak urinary concentrating response to vasopressin seen in neonatal rats.     

Differential localization of aquaporin-2 and glucose transporter 4 in polarized MDCK cells

Membrane water channel aquaporin-2 (AQP2) and glucose transporter 4 (GLUT4) exhibit a common feature in that they are stored in intracellular storage compartments and undergo translocation to the plasma membrane upon hormonal stimulation. We compared the intracellular localization and trafficking of AQP2 and GLUT4 in polarized Madin-Darby canine kidney cells stably transfected with human AQP2 (MDCK-hAQP2) by immunofluorescence microscopy. When expressed in MDCK-hAQP2 cells, GLUT4 and GLUT4—EGFP were predominantly localized in the perinuclear region close to and within the Golgi apparatus, similar to endogenous GLUT4 in adipocytes and myocytes. In addition, GLUT4 was occasionally seen in EEA1-positive early endosomes. AQP2, on the other hand, was sequestered in subapical Rab11-positive vesicles. In the basal state, the intracellular storage site of GLUT4 was distinct from that of AQP2. Forskolin induced translocation of AQP2 from the subapical storage vesicles to the apical plasma membrane, which did not affect GLUT4 localization. When forskolin was washed out, AQP2 was first retrieved to early endosomes from the apical plasma membrane, where it was partly colocalized with GLUT4. AQP2 was then transferred to Rab11-positive storage vesicles. These results show that AQP2 and GLUT4 share a common compartment after retrieval from the plasma membrane, but their storage compartments are distinct from each other in polarized MDCK-hAQP2 cells.     

Basolateral cholesterol depletion alters Aquaporin-2 post-translational modifications and disrupts apical plasma membrane targeting

Apical plasma membrane accumulation of the water channel Aquaporin-2 (AQP2) in kidney collecting duct principal cells is critical for body water homeostasis. Posttranslational modification (PTM) of AQP2 is important for regulating AQP2 trafficking. The aim of this study was to determine the role of cholesterol in regulation of AQP2 PTM and in apical plasma membrane targeting of AQP2. Cholesterol depletion from the basolateral plasma membrane of a collecting duct cell line (mpkCCD14) using methyl-beta-cyclodextrin (MBCD) increased AQP2 ubiquitylation. Forskolin, cAMP or dDAVP-mediated AQP2 phosphorylation at Ser269 (pS269-AQP2) was prevented by cholesterol depletion from the basolateral membrane. None of these effects on pS269-AQP2 were observed when cholesterol was depleted from the apical side of cells, or when MBCD was applied subsequent to dDAVP stimulation. Basolateral, but not apical, MBCD application prevented cAMP-induced apical plasma membrane accumulation of AQP2. These studies indicate that manipulation of the cholesterol content of the basolateral plasma membrane interferes with AQP2 PTM and subsequently regulated apical plasma membrane targeting of AQP2.     

Regulation of the immunoexpression of aquaporin 9 by ovarian hormones in the rat oviductal epithelium

The volume of oviductal fluid fluctuates during the estrous cycle, suggesting that water availability is under hormonal control. It has been postulated that sex-steroid hormones may regulate aquaporin (AQP) channels involved in water movement across cell membranes. Using a functional assay (oocytes of Xenopus laevis), we demonstrated that the rat oviductal epithelium contains mRNAs coding for water channels, and we identified by RT-PCR the mRNAs for AQP5, -8, and -9, but not for AQP2 and -3. The immunoreactivity for AQP5, -8, and -9 was localized only in epithelial cells of the oviduct. The distribution of AQP5 and -8 was mainly cytoplasmic, whereas we confirmed, by confocal microscopy, that AQP9 localized to the apical plasma membrane. Staining of AQP5, -8, and -9 was lost after ovariectomy, and only AQP9 immunoreactivity was restored after estradiol and/or progesterone treatments. The recovery of AQP9 reactivity after ovariectomy correlated with increased mRNA and protein levels after treatment with estradiol alone or progesterone administration after estradiol priming. Interestingly, progesterone administration after progesterone priming also induced AQP9 expression but without a change in mRNA levels. Levels of AQP9 varied along the estrous cycle with their highest levels during proestrus and estrus. These results indicate that steroid hormones regulate AQP9 expression at the mRNA and protein level and that other ovarian signals are involved in the expression of AQP5 and -8. Thus hormonal regulation of the type and quantity of water channels in this epithelium might control water transport in the oviductal lumen. water channels; epithelial cells; estradiol; progesterone     

Lung edema clearance: 20 years of progress: invited review: role of aquaporin water channels in fluid transport in lung and airways.

Water transport across epithelial and endothelial barriers in bronchopulmonary tissues occurs during airway hydration, alveolar fluid transport, and submucosal gland secretion. Many of the tissues involved in these processes are highly water permeable and express aquaporin (AQP) water channels. AQP1 is expressed in microvascular endothelia throughout the lung and airways, AQP3 in epithelia in large airways, AQP4 in epithelia throughout the airways, and AQP5 in type I alveolar epithelial cells and submucosal gland acinar cells. The expression of some of these AQPs increases near the time of birth and is regulated by growth factors, inflammation, and osmotic stress. Transgenic mouse models of AQP deletion have provided information about their physiological role. In lung, AQP1 and AQP5 provide the principal route for osmotically driven water transport; however, alveolar fluid clearance in the neonatal and adult lung is not affected by AQP deletion nor is lung CO(2) transport or fluid accumulation in experimental models of lung injury. In the airways, AQP3 and AQP4 facilitate water transport; however, airway hydration, regulation of the airway surface liquid layer, and isosmolar fluid absorption are not impaired by AQP deletion. In contrast to these negative findings, AQP5 deletion in submucosal glands in upper airways reduced fluid secretion and increased protein content by greater than twofold. Thus, although AQPs play a major physiological role outside of the airways and lung, AQPs appear to be important mainly in airway submucosal gland function. The substantially slower rates of fluid transport in airways, pleura, and lung compared with renal and some secretory epithelia may account for the apparent lack of functional significance of AQPs at these sites. However, the possibility remains that AQPs may play a role in lung physiology under conditions of stress and/or injury not yet tested or in functions unrelated to transepithelial fluid transport.     

Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways

Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren’s syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow.