Effect of hydrogen peroxide on lens transparency, intracellular pH, gap junction coupling, hydrostatic pressure and membrane water permeability.

Clouding of the eye lens or cataract is an age-related anomaly that affects middle-aged humans. Exploration of the etiology points to a great extent to oxidative stress due to different forms of reactive oxygen species/metabolites such as Hydrogen peroxide (H2O2) that are generated due to intracellular metabolism and environmental factors like radiation. If accumulated and left unchecked, the imbalance between the production and degradation of H2O2 in the lens could lead to cataracts. Our objective was to explore ex vivo the effects of H2O2 on lens physiology. We investigated transparency, intracellular pH (pHi), intercellular gap junction coupling (GJC), hydrostatic pressure (HP) and membrane water permeability after subjecting two-month-old C57 wild-type (WT) mouse lenses for 3 h or 8 h in lens saline containing 50 µM H2O2; the results were compared with control lenses incubated in the saline without H2O2. There was a significant decrease in lens transparency in H2O2-treated lenses. In control lenses, pHi decreases from ∼7.34 in the surface fiber cells to 6.64 in the center. Experimental lenses exposed to H2O2 for 8 h showed a significant decrease in surface pH (from 7.34 to 6.86) and central pH (from 6.64 to 6.56), compared to the controls. There was a significant increase in GJC resistance in the differentiating (12-fold) and mature (1.4-fold) fiber cells compared to the control. Experimental lenses also showed a significant increase in HP which was ∼2-fold higher at the junction between the differentiating and mature fiber cells and ∼1.5-fold higher at the center compared to these locations in control lenses; HP at the surface was 0 mm Hg in either type lens. Fiber cell membrane water permeability significantly increased in H2O2-exposed lenses compared to controls. Our data demonstrate that elevated levels of lens intracellular H2O2 caused a decrease in intracellular pH and led to acidosis which most likely uncoupled GJs, and increased AQP0-dependent membrane water permeability causing a consequent rise in HP. We infer that an abnormal increase in intracellular H2O2 could induce acidosis, cause oxidative stress, alter lens microcirculation, and lead to the development of accelerated lens opacity and age-related cataracts.

Positively charged amino acid residues in the extracellular loops A and C of lens aquaporin 0 interact with the negative charges in the plasma membrane to facilitate cell-to-cell adhesion.

This investigation was undertaken to find out whether the positive charges in the Extracellular Loops A (ELA) and C (ELC) of Aquaporin 0 (AQP0) are involved in lens fiber cell-to-cell adhesion (CTCA), and the possible mechanism of CTCA. AQP0 ELA or ELC was substituted with the corresponding AQP1 loop via Polymerase Chain Reaction. Positively charged arginine (R) and histidine (H) of mouse AQP0 ELA and ELC were substituted individually with glutamine (Q) to create R33Q, H40Q, R113Q and H122Q by mutagenesis. cRNA expression, immunostaining, Förster Resonance Energy Transfer (FRET) studies and protein analyses showed localization of all mutants except AQP0-AQP1ELC chimera (AQP0 ELC substituted with AQP1 ELC) at the plasma membrane. Osmotic Swelling Assay revealed comparable water permeability (Pf) among AQP0-AQP1ELA, R33Q, R113Q, and WT. CTCA assay demonstrated a significant reduction in adhesion in all mutants compared to the WT (14-73%) suggesting the importance of the conserved positively charged residues of ELA and ELC for adhesion. Studies involving AQP0-transfected L-cells, and lipid vesicles indicated that CTCA was due to the electrostatic interaction between the positively charged amino acids of AQP0 extracellular loops and the negative charges of the plasma membrane. Schematic models are provided to illustrate the mechanism.

C-Terminal End of Aquaporin 0 Regulates Lens Gap Junction Channel Function.

Purpose: We reported previously that aquaporin 0 (AQP0) modulates lens fiber cell gap junction (GJ) channel function. The present study was conducted to find out whether the C-terminal end of AQP0 is involved in this regulation. Methods: A mouse model, AQP0ΔC/ΔC, was genetically engineered to express AQP0 with 1-246 amino acids, without the normal intact AQP0 (1-263 amino acids) in the lens. Transparency and focusing of the lens were assessed. Intracellular impedance was measured to determine GJ coupling resistance. Intracellular hydrostatic pressure (HP) was also determined. Western blotting was performed to determine connexin (Cx46 and Cx50) expression levels. Results: At postnatal day 10, AQP0ΔC/ΔC mouse lenses relative to age-matched wild-type lenses showed loss of transparency and abnormal optical distortion; GJ coupling resistance increased in the differentiating (1.6-fold) and mature (8-fold) fiber cells; lens HP increased approximately 1.5-fold at the junction between the differentiating and mature fiber cells and approximately 2.0-fold in the center; there was no significant change (P > 0.05) in expression levels of Cx46 or Cx50. Conclusions: The increase in GJ coupling resistance was not associated with reduced connexin expression, suggesting either a reduction in the open probability or some physical change in plaque location. The increase in resistance was significantly greater than the increase in HP, suggesting less pressure-driven water flow through each open GJ channel. These changes may lead to a loss of transparency and abnormal optical distortion. Overall, our data demonstrate the C-terminal end of AQP0 is involved in modulating GJ coupling to maintain lens transparency and homeostasis.

Deletion of Seventeen Amino Acids at the C-Terminal End of Aquaporin 0 Causes Distortion Aberration and Cataract in the Lenses of AQP0ΔC/ΔC Mice.

Purpose: Investigate the effects of the absence of 17 amino acids at the C-terminal end of Aquaporin 0 (AQP0) on lens transparency, focusing property, and homeostasis. Methods: A knockin (KI) mouse model (AQP0ΔC/ΔC) was developed to express AQP0 only as the end-cleaved form in the lens. For this, AQP0 was genetically engineered as C-terminally end-cleaved with amino acids 1 to 246, instead of the full length 1 to 263 of the wild type (WT). After verifying the KI integration into the genome and its expression, the mouse model was bred for several generations. AQP0 KI homozygous (AQP0ΔC/ΔC) and heterozygous (AQP0+/ΔC) lenses were imaged and analyzed at different developmental stages for transparency. Correspondingly, aberrations in the lens were characterized using the standard metal grid focusing method. Data were compared with age-matched WT, AQP0 knockout (AQP0-/-), and AQP0 heterozygous (AQP0+/-) lenses. Results: AQP0ΔC/ΔC lenses were transparent throughout the embryonic development and until postnatal day 15 (P15) in contrast to age-matched AQP0-/- lenses, which developed cataract at embryonic stage itself. However, there was distortion aberration in AQP0ΔC/ΔC lens at P5; after P15, cataract began to develop and progressed faster surpassing that of age-matched AQP0-/- lenses. AQP0+/ΔC lenses were transparent even at the age of 1 year in contrast to AQP0+/- lenses; however, there was distortion aberration starting at P15. Conclusions: A specific distribution profile of intact and end-cleaved AQP0 from the outer cortex to the inner nucleus is required in the lens for establishing refractive index gradient to enable proper focusing without aberrations and for maintaining transparency.

Aquaporin 0 Modulates Lens Gap Junctions in the Presence of Lens-Specific Beaded Filament Proteins.

Purpose: The objective of this study was to understand the molecular and physiologic mechanisms behind the lens cataract differences in Aquaporin 0-knockout-Heterozygous (AQP0-Htz) mice developed in C57 and FVB (lacks beaded filaments [BFs]) strains. Methods: Lens transparency was studied using dark field light microscopy. Water permeability (Pf) was measured in fiber cell membrane vesicles. Western blotting/immunostaining was performed to verify expression of BF proteins and connexins. Microelectrode-based intact lens intracellular impedance was measured to determine gap junction (GJ) coupling resistance. Lens intracellular hydrostatic pressure (HP) was determined using a microelectrode/manometer system. Results: Lens opacity and spherical aberration were more distinct in AQP0-Htz lenses from FVB than C57 strains. In either background, compared to wild type (WT), AQP0-Htz lenses showed decreased Pf (approximately 50%), which was restored by transgenic expression of AQP1 (TgAQP1/AQP0-Htz), but the opacities and differences between FVB and C57 persisted. Western blotting revealed no change in connexin expression levels. However, in C57 AQP0-Htz and TgAQP1/AQP0-Htz lenses, GJ coupling resistance decreased approximately 2.8-fold and the HP gradient decreased approximately 1.9-fold. Increased Pf in TgAQP1/AQP0-Htz did not alter GJ coupling resistance or HP. Conclusions: In C57 AQP0-Htz lenses, GJ coupling resistance decreased. HP reduction was smaller than the coupling resistance reduction, a reflection of an increase in fluid circulation, which is one reason for the less severe cataract in C57 than FVB. Overall, our results suggest that AQP0 modulates GJs in the presence of BF proteins to maintain lens transparency and homeostasis.

Rapid Identification of Novel Inhibitors of the Human Aquaporin-1 Water Channel.

Aquaporins (AQPs) are a family of membrane proteins that function as channels facilitating water transport in response to osmotic gradients. These play critical roles in several normal physiological and pathological states and are targets for drug discovery. Selective inhibition of the AQP1 water channel may provide a new approach for the treatment of several disorders including ocular hypertension/glaucoma, congestive heart failure, brain swelling associated with a stroke, corneal and macular edema, pulmonary edema, and otic disorders such as hearing loss and vertigo. We developed a high-throughput assay to screen a library of compounds as potential AQP1 modulators by monitoring the fluorescence dequenching of entrapped calcein in a confluent layer of AQP1-overexpressing CHO cells that were exposed to a hypotonic shock. Promising candidates were tested in a Xenopus oocyte-swelling assay, which confirmed the identification of two lead classes of compounds belonging to aromatic sulfonamides and dihydrobenzofurans with IC50 s in the low micromolar range. These selected compounds directly inhibited water transport in AQP1-enriched stripped erythrocyte ghosts and in proteoliposomes reconstituted with purified AQP1. Validation of these lead compounds, by the three independent assays, establishes a set of attractive AQP1 blockers for developing novel, small-molecule functional modulators of human AQP1.

Functional characterization of an AQP0 missense mutation, R33C, that causes dominant congenital lens cataract, reveals impaired cell-to-cell adhesion.

Aquaporin 0 (AQP0) performs dual functions in the lens fiber cells, as a water pore and as a cell-to-cell adhesion molecule. Mutations in AQP0 cause severe lens cataract in both humans and mice. An arginine to cysteine missense mutation at amino acid 33 (R33C) produced congenital autosomal dominant cataract in a Chinese family for five generations. We re-created this mutation in wild type human AQP0 (WT-AQP0) cDNA by site-directed mutagenesis, and cloned and expressed the mutant AQP0 (AQP0-R33C) in heterologous expression systems. Mutant AQP0-R33C showed proper trafficking and membrane localization like WT-AQP0. Functional studies conducted in Xenopus oocytes showed no significant difference (P > 0.05) in water permeability between AQP0-R33C and WT-AQP0. However, the cell-to-cell adhesion property of AQP0-R33C was significantly reduced (P < 0.001) compared to that of WT-AQP0, indicated by cell aggregation and cell-to-cell adhesion assays. Scrape-loading assay using Lucifer Yellow dye showed reduction in cell-to-cell adhesion affecting gap junction coupling (P < 0.001). The data provided suggest that this mutation might not have caused significant alterations in protein folding since there was no obstruction in protein trafficking or water permeation. Reduction in cell-to-cell adhesion and development of cataract suggest that the conserved positive charge of Extracellular Loop A may play an important role in bringing fiber cells closer. The proposed schematic models illustrate that cell-to-cell adhesion elicited by AQP0 is vital for lens transparency and homeostasis.

Expression of skeletal but not cardiac Na+ channel isoform preserves normal conduction in a depolarized cardiac syncytium.

AIMS: Reentrant arrhythmias often develop in the setting of myocardial infarction and ensuing slow propagation. Increased Na(+) channel expression could prevent or disrupt reentrant circuits by speeding conduction if channel availability is not limited by membrane depolarization within the diseased myocardium. We therefore asked if, in the setting of membrane depolarization, action potential (AP) upstroke and normal conduction can be better preserved by the expression of a Na(+) channel isoform with altered biophysical properties compared to the native cardiac Na(+) channel isoform, namely having a positively shifted, voltage-dependent inactivation. METHODS AND RESULTS: The skeletal Na(+) channel isoform (SkM1) and the cardiac Na(+) channel isoform (Nav1.5) were expressed in newborn rat ventricular myocyte cultures with a point mutation introduced in Nav1.5 to increase tetrodotoxin (TTX) sensitivity so native and expressed currents could be distinguished. External K(+) was increased from 5.4 to 10 mmol/L to induce membrane depolarization. APs, Na(+) currents, and conduction velocity (CV) were measured. In control cultures, elevated K(+) significantly reduced AP upstroke ( approximately 75%) and CV ( approximately 25%). Expression of Nav1.5 did not protect AP upstroke from K(+) depolarization. In contrast, in SkM1 expressing cultures, high K(+) reduced AP upstroke <50% and conduction was not significantly reduced. In a simulated anatomical reentry setting (using a void), the angular velocity (AV) of induced reentry was faster and the excitable gap shorter in SkM1 cultures compared to control for both normal and high K(+). CONCLUSION: Expression of SkM1 but not Nav1.5 preserves AP upstroke and CV in a K(+)-depolarized syncytium. The higher AV and shorter excitable gap observed during reentry excitation around a void in SkM1 cultures would be expected to facilitate reentry self-termination. SkM1 Na(+) channel expression represents a novel gene therapy for the treatment of reentrant arrhythmias.

Functional expression of aquaporins in embryonic, postnatal, and adult mouse lenses.

Aquaporin 0 (AQP0) and AQP1 are expressed in the lens, each in a different cell type, and their functional roles are not thoroughly understood. Our previous study showed that these two AQPs function as water transporters. In order to further understand the functional significance of these two different aquaporins in the lens, we investigated their initiation and continued expression. AQP0 transcript and protein were first detected at embryonic stage (E) 11.25 in the differentiating primary fiber cells of the developing lens; its synthesis continued through the adult stage in the secondary fiber cells. Low levels of AQP1 expression were first seen in lens anterior epithelial cells at E17.5; following postnatal day (P) 6.5, the expression gradually progressed towards the equatorial epithelial cells. In the postnatal lens, the increase in membrane water permeability of epithelial cells and lens transparency coincides with the increase in AQP1 expression. AQP1 expression reaches its peak at P30 and continues through the adult stage both in the anterior and equatorial epithelial cells. The enhancement in AQP1 expression concomitant with the increase in the size of the lens suggests the progression in the establishment of the lens microcirculatory system. In vitro and in vivo studies show that both aquaporins share at least one important function, which is water transport in the lens microcirculatory system. However, the temporal expression of these two AQPs suggests an apparently unique role/s in lens development and transparency. To our knowledge, this is the first report on the expression patterns of AQP0 and AQP1 during lens development and differentiation and their relation to lens transparency.

Connexin43 with a cytoplasmic loop deletion inhibits the function of several connexins.

Connexins (Cx) form gap junction channels mediating direct intercellular communication. To study the role of amino acids within the cytoplasmic loop, we produced a recombinant adenovirus containing Cx43 with a deletion of amino acids 130-136 (Cx43del(130-136)). Cx43del(130-136) expressed alone in HeLa cells localized within the cytoplasm and did not allow transfer of ions, neurobiotin or Lucifer yellow. When co-expressed with wild type Cx43, Cx43del(130-136) blocked electrical coupling and transfer of neurobiotin or Lucifer yellow. Cx43del(130-136) and Cx43 co-localized by immunofluorescence and were co-purified from Triton X-100-solubilized cell extracts. Intercellular transfer mediated by Cx37 and Cx45 (but not Cx26 or Cx40) was inhibited when co-expressed with Cx43del(130-136). Cx43del(130-136) co-localized with Cx37, Cx40, or Cx45, but not Cx26. These data suggest that Cx43del(130-136) produces connexin-specific inhibition of intercellular communication through formation of heteromeric connexons that are non-functional and/or retained in the cytoplasm.

Regulation of aquaporin water permeability in the lens.

PURPOSE: To examine Ca(2+)- and pH-mediated regulation of water permeability of endogenously expressed aquaporin (AQP)0 in lens fiber cells and AQP1 in lens epithelial cells. METHODS: Large, right-side-out membrane vesicles were formed from freshly isolated groups of lens fiber cells. Osmotic shrinking or swelling of these vesicles was used to determine the water permeability of endogenously expressed AQP0. The results were compared with those in similar studies of freshly isolated lens epithelial cells, which endogenously expressed AQP1, and of oocytes, which exogenously expressed AQP0. RESULTS: In the lens or in oocytes, decreasing external pH from 7.5 to 6.5 caused a two- to fourfold increase in the water permeability of mammalian AQP0. Several lines of evidence suggest that this effect is mediated by the binding of H(+) to a histidine in the first extracellular loop (His40). Lens AQP1 lacks His40 and also lacks pH sensitivity. Increasing Ca(2+) caused a two- to fourfold increase in the water permeability of endogenous AQP0. The Ca(2+) effect on mouse AQP0 was a 2.5-fold increase in the lens, whereas in oocytes, it was a 4-fold decrease. In either environment, the effect was mediated through calmodulin, most likely through its binding to the proximal domain of the C terminus. Lens AQP1 does not have a similar domain and does not have calcium sensitivity. CONCLUSIONS: In either the lens or oocytes, Ca(2+) and H(+) appear to affect the same mechanism, probably either the open probability of the water channel, or open-channel permeability. The difference between calcium's effects in lens versus oocytes was remarkable and is not understood. However, in the lens, Ca(2+) and H(+) are both increased in inner fiber cells, and so in the physiologically relevant environment, both may act to increase the water permeability of AQP0.