Colon water transport in transgenic mice lacking aquaporin-4 water channels

Transgenic null mice were used to test the hypothesis that water channel aquaporin-4 (AQP4) is involved in colon water transport and fecal dehydration. AQP4 was immunolocalized to the basolateral membrane of colonic surface epithelium of wild-type (+/+) mice and was absent in AQP4 null (−/−) mice. The transepithelial osmotic water permeability coefficient ( P f) of in vivo perfused colon of +/+ mice, measured using the volume marker 14C-labeled polyethylene glycol, was 0.016 ± 0.002 cm/s. P f of proximal colon was greater than that of distal colon (0.020 ± 0.004 vs. 0.009 ± 0.003 cm/s, P < 0.01). P f was significantly lower in −/− mice when measured in full-length colon (0.009 ± 0.002 cm/s, P< 0.05) and proximal colon (0.013 ± 0.002 cm/s, P< 0.05) but not in distal colon. There was no difference in water content of cecal stool from +/+ vs. −/− mice (0.80 ± 0.01 vs. 0.81 ± 0.01), but there was a slightly higher water content in defecated stool from −/− mice (0.68 ± 0.01 vs. 0.65 ± 0.01, P < 0.05). Despite the differences in water permeability with AQP4 deletion, theophylline-induced secretion was not impaired (50 ± 9 vs. 51 ± 8 μl · min−1 · g−1). These results provide evidence that transcellular water transport through AQP4 water channels in colonic epithelium facilitates transepithelial osmotic water permeability but has little or no effect on colonic fluid secretion or fecal dehydration.

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Membrane organization and function of M1 and M23 isoforms of aquaporin-4 in epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.00439.2003 ◽

2004 ◽

Vol 287 (3) ◽

pp. F501-F511 ◽

Cited By ~ 105

Author(s):

Claudia Silberstein ◽

Richard Bouley ◽

Yan Huang ◽

Pingke Fang ◽

Nuria Pastor-Soler ◽

...

Keyword(s):

Water Permeability ◽

Single Channel ◽

Phosphorylation Site ◽

Freeze Fracture ◽

Parietal Cells ◽

Aquaporin 4 ◽

Osmotic Water Permeability ◽

Cellular Expression ◽

Water Permeability Coefficient ◽

Osmotic Water

Aquaporin-4 (AQP4) water channels exist as heterotetramers of M1 and M23 splice variants and appear to be present in orthogonal arrays of intramembraneous particles (OAPs) visualized by freeze-fracture microscopy. We report that AQP4 forms OAPs in rat gastric parietal cells but not in parietal cells from the mouse or kangaroo rat. Furthermore, the organization of principal cell OAPs in Brattleboro rat kidney is perturbed by vasopressin (arginine vasopressin). Membranes of LLC-PK1 cells expressing M23-AQP4 showed large, abundant OAPs, but none were detectable in cells expressing M1-AQP4. Measurements of osmotic swelling of transfected LLC-PK1 cells using videomicroscopy, gave osmotic water permeability coefficient ( Pf) values (in cm/s) of 0.018 (M1-AQP4), 0.019 (M23-AQP4), and 0.003 (control). Quantitative immunoblot and immunofluorescence showed an eightfold greater expression of M1- over M23-AQP4 in the cell lines, suggesting that single-channel pf (cm3/s) is much greater for the M23 variant. Somatic fusion of M1- and M23-AQP4 cells ( Pf = 0.028 cm/s) yielded OAPs that were fewer and smaller than in M23 cells alone, and M1-to-M23 expression ratios (∼1:4) normalized to AQP4 in M1 or M23 cells indicated a reduced single-channel pf for the M23 variant. Expression of an M23-AQP4-Ser111E mutant produced ∼1.5-fold greater single-channel pf and OAPs that were up to 2.5-fold larger than wild-type M23-AQP4 OAPs, suggesting that a putative PKA phosphorylation site Ser111 is involved in OAP formation. We conclude that the higher-order organization of AQP4 in OAPs increases single-channel osmotic water permeability by one order of magnitude and that differential cellular expression levels of the two isoforms could regulate this organization.

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Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney.

The Journal of General Physiology ◽

10.1085/jgp.101.6.827 ◽

1993 ◽

Vol 101 (6) ◽

pp. 827-841 ◽

Cited By ~ 38

Author(s):

M Echevarria ◽

G Frindt ◽

G M Preston ◽

S Milovanovic ◽

P Agre ◽

...

Keyword(s):

Water Permeability ◽

Antisense Oligonucleotide ◽

Xenopus Oocytes ◽

Rat Kidney ◽

Water Channel ◽

Water Channels ◽

Renal Tissue ◽

Osmotic Water Permeability ◽

Volume Increase ◽

Osmotic Water

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.

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Adaptation of inner medullary collecting duct to dehydration involves a paracellular pathway

AJP Renal Physiology ◽

10.1152/ajprenal.1995.268.1.f53 ◽

1995 ◽

Vol 268 (1) ◽

pp. F53-F63 ◽

Cited By ~ 3

Author(s):

B. Flamion ◽

K. R. Spring ◽

M. Abramow

Keyword(s):

Water Permeability ◽

Apical Membrane ◽

Collecting Duct ◽

Basolateral Membrane ◽

Osmotic Water Permeability ◽

Inner Medullary Collecting Duct ◽

Osmotic Water ◽

Medullary Collecting Duct ◽

Paracellular Pathways

Prolonged fluid restriction in rats is accompanied by functional modifications of the terminal part of the inner medullary collecting duct (IMCD) revealed by a sustained increase in arginine vasopressin (AVP)-independent transepithelial osmotic water permeability (PTE) in vitro. The cellular basis of this adaptation was explored in isolated and perfused terminal IMCDs of Sprague-Dawley rats using video and fluorescence microscopy. Basolateral membrane osmotic water permeability (Posm), transcellular Posm, and PTE were measured in quick sequence in every tubule. They were expressed per unit area of basolateral membrane corrected for infoldings, based on previous stereological studies and assuming no major change in membrane surface area between hydrated and dehydrated animals. Compared with IMCDs of rats with a high water intake, IMCDs of rats deprived of fluid for 36 h displayed a significantly higher basal PTE (24.9 +/- 5.1 vs. 6.1 +/- 0.6 microns/s), a similar basolateral Posm, and a higher transcellular Posm, implying a higher permeability of the apical membrane, despite the absence of exogenous AVP. However, when IMCDs of thirsted rats were exposed to AVP in vitro, their transcellular Posm (36.0 +/- 2.4 microns/s) was significantly smaller than their PTE determined simultaneously (51.8 +/- 7.1 microns/s), suggesting that part of the water flow may follow a paracellular route. A change in paracellular pathways was supported by higher apparent permeabilities to [14C]sucrose (0.85 +/- 0.27 vs. 0.28 +/- 0.04 x 10(-5) cm/s) and to [methoxy-3H]inulin (0.25 +/- 0.04 vs. 0.14 +/- 0.03 x 10(-5) cm/s) in IMCDs of thirsted rats. The nonelectrolyte permeabilities were affected neither by AVP nor by urea-rich bathing solutions. We conclude that in vivo factors related to dehydration produce a conditioning effect on terminal IMCD, which includes stabilization of the apical membrane in a state of high Posm and opening up of paracellular pathways revealed by a higher permeability to water and nonelectrolytes. The role of these adaptive phenomena remains unclear but may pertain to the sudden transitions between antidiuresis and diuresis.

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Role of multiple phosphorylation sites in the COOH-terminal tail of aquaporin-2 for water transport: evidence against channel gating

AJP Renal Physiology ◽

10.1152/ajprenal.90682.2008 ◽

2009 ◽

Vol 296 (3) ◽

pp. F649-F657 ◽

Cited By ~ 60

Author(s):

Hanne B. Moeller ◽

Nanna MacAulay ◽

Mark A. Knepper ◽

Robert A. Fenton

Keyword(s):

Plasma Membrane ◽

Water Transport ◽

Water Permeability ◽

Laser Scanning Microscopy ◽

Apical Plasma Membrane ◽

Phosphorylation Sites ◽

Osmotic Water Permeability ◽

Aquaporin 2 ◽

Osmotic Water

Arginine vasopressin (AVP)-regulated phosphorylation of the water channel aquaporin-2 (AQP2) at serine 256 (S256) is essential for its accumulation in the apical plasma membrane of collecting duct principal cells. In this study, we examined the role of additional AVP-regulated phosphorylation sites in the COOH-terminal tail of AQP2 on protein function. When expressed in Xenopus laevis oocytes, prevention of AQP2 phosphorylation at S256A (S256A-AQP2) reduced osmotic water permeability threefold compared with wild-type (WT) AQP2-injected oocytes. In contrast, prevention of AQP2 single phosphorylation at S261 (S261A), S264 (S264A), and S269 (S269A), or all three sites in combination had no significant effect on water permeability. Similarly, oocytes expressing S264D-AQP2 and S269D-AQP2, mimicking AQP2 phosphorylated at these residues, had similar water permeabilities to WT-AQP2-expressing oocytes. The use of high-resolution confocal laser-scanning microscopy, as well as biochemical analysis demonstrated that all AQP2 mutants, with the exception of S256A-AQP2, had equal abundance in the oocyte plasma membrane. Correlation of osmotic water permeability relative to plasma membrane abundance demonstrated that lack of phosphorylation at S256, S261, S264, or S269 had no effect on AQP2 unit water transport. Similarly, no effect on AQP2 unit water transport was observed for the 264D and 269D forms, indicating that phosphorylation of the COOH-terminal tail of AQP2 is not involved in gating of the channel. The use of phosphospecific antibodies demonstrated that AQP2 S256 phosphorylation is not dependent on any of the other phosphorylation sites, whereas S264 and S269 phosphorylation depend on prior phosphorylation of S256. In contrast, AQP2 S261 phosphorylation is independent of the phosphorylation status of S256.

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Sevenfold-reduced osmotic water permeability in primary astrocyte cultures from AQP-4-deficient mice, measured by a fluorescence quenching method

AJP Cell Physiology ◽

10.1152/ajpcell.00298.2003 ◽

2004 ◽

Vol 286 (2) ◽

pp. C426-C432 ◽

Cited By ~ 215

Author(s):

Eugen Solenov ◽

Hiroyuki Watanabe ◽

Geoffrey T. Manley ◽

A. S. Verkman

Keyword(s):

Fluorescence Quenching ◽

Water Transport ◽

Water Permeability ◽

Cell Swelling ◽

Osmotic Water Permeability ◽

Wild Type ◽

Osmotic Water ◽

Fluorescence Quenching Method ◽

Quenching Method ◽

Deficient Mice

A calcein fluorescence quenching method was applied to measure osmotic water permeability in highly differentiated primary cultures of brain astrocytes from wild-type and aquaporin-4 (AQP-4)-deficient mice. Cells grown on coverglasses were loaded with calcein for measurement of volume changes after osmotic challenge. Hypotonic shock producing twofold cell swelling resulted in a reversible ∼12% increase in calcein fluorescence, which was independent of cytosolic calcein concentration at levels well below where calcein self-quenching occurs. Calcein fluorescence was quenched in <200 ms in response to addition of cytosol in vitro, indicating that the fluorescence signal arises from changes in cytosol concentration. In astrocytes from wild-type CD1 mice, calcein fluorescence increased reversibly in response to hypotonic challenge with a half-time of 0.92 ± 0.05 s at 23°C, corresponding to an osmotic water permeability ( Pf) of ∼0.05 cm/s. Pf was reduced 7.1-fold in astrocytes from AQP-4-deficient mice. Temperature dependence studies indicated an increased Arrhenius activation energy for water transport in AQP-4-deficient astrocytes (11.3 ± 0.5 vs. 5.5 ± 0.4 kcal/mol). Our studies establish a calcein quenching method for measurement of cell membrane water permeability and indicate that AQP-4 provides the principal route for water transport in astrocytes.

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Basolateral Membrane Vesicle (BLMV) Osmotic Water Permeability (Pf) in Adult and Immature Rabbit Proximal Tubules

Pediatric Research ◽

10.1203/00006450-199904020-01998 ◽

1999 ◽

Vol 45 (4, Part 2 of 2) ◽

pp. 336A-336A

Author(s):

Raymond Quigley ◽

Amber D Lisec ◽

Neena Gupta ◽

Michel Baum

Keyword(s):

Water Permeability ◽

Membrane Vesicle ◽

Basolateral Membrane ◽

Proximal Tubules ◽

Osmotic Water Permeability ◽

Osmotic Water

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Importance of the mercury-sensitive cysteine on function and routing of AQP1 and AQP2 in oocytes

AJP Renal Physiology ◽

10.1152/ajprenal.1997.273.3.f451 ◽

1997 ◽

Vol 273 (3) ◽

pp. F451-F456 ◽

Cited By ~ 3

Author(s):

S. M. Mulders ◽

J. P. Rijss ◽

A. Hartog ◽

R. J. Bindels ◽

C. H. van Os ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Water Transport ◽

Water Permeability ◽

Nephrogenic Diabetes Insipidus ◽

Osmotic Water Permeability ◽

Wild Type ◽

Mutant Proteins ◽

Osmotic Water ◽

Structural Differences

To discriminate between water transport of of aquaporin-2 (AQP2) mutants in nephrogenic diabetes insipidus and that of an AQP2 molecule used to drag them to the oolemma, we investigated the mercury sensitivity of wild-type and AQP2 C181S proteins in oocytes. Incubation with HgCl2 inhibited the osmotic water permeability (Pf) of human (h) AQP2 by 40%, whereas inhibition of hAQP1 was 75%. Oocytes expressing hAQP1 C189S revealed a Pf comparable to wild-type hAQP1, but mercury sensitivity was lost. In contrast, no increase in Pf was obtained when hAQP2 C181S was expressed. Also, expression of rat AQP2 C181A and C181S mutants did not increase the Pf, which contrasts with published observations. Immunocytochemistry and immunoblotting revealed that only AQP1, AQP1 C189S, and AQP2 were targeted to the plasma membrane and that AQP2 mutant proteins are retarded in the endoplasmic reticulum. In conclusion, water transport through AQP2 is less sensitive to mercury inhibition than through AQP1. Furthermore, substitution of the mercury-sensitive cysteine for a serine results in an impaired routing of human and rat AQP2. Similar mutations have no effect on AQP1 function, which is indicative of structural differences between AQP1 and AQP2.

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