S. Sindhu Kumari

Intact AQP0 performs cell-to-cell adhesion

Aquaporins (AQPs) constitute a major conduit for movement of water across plasma membranes. AQP0 is expressed in the fiber cells and is critical for lens transparency and homeostasis as mutations and knockout have resulted in dominant lens cataract. Several functions have been attributed for AQP0. In vitro and ex vivo experiments from several laboratories have confirmed the water permeability function of AQP0. However, this function seems paradoxical when the lens switches protein expression from AQP1 in the equatorial epithelial cells to 40 times less efficient AQP0 in the differentiating fiber cells. A possible explanation is AQP0 may perform unique function/s besides being a water pore. Indirect evidences including those from structural studies indicate a cell-to-cell adhesion role for AQP0. However, there is a lack of experimental evidence directly demonstrating the cell-to-cell adhesion capability of AQP0. We studied the adhesion property of human intact AQP0 by expressing it in adhesion-deficient mouse fibroblast L-cells using a newly devised method as well as a traditional assay. Our results reveal that AQP0 indeed can perform cell-to-cell adhesion. AQP1, two alternate splice variants of AQP4 (AQP4-M1and AQP4-M23) and E-cadherin were also tested to validate the results. Cell-to-cell adhesion and cell aggregation properties of AQP0 expressing L-cells were less than those of the positive control L-cells expressing mouse E-cadherin and greater than those of AQP4-M23. AQP1 or AQP4-M1 expressing cells did not show cell-to-cell adhesion or cell aggregation. To our knowledge, this is the first report validating the possible structural role of intact AQP0 as a cell-to-cell adhesion protein, using an in vitro expression system.

Functional characterization of a human aquaporin 0 mutation that leads to a congenital dominant lens cataract.

The aquaporin (AQP) transmembrane proteins facilitate the movement of water across the plasma membrane. In the lens, AQP0 is expressed in fiber cells and AQP1 in the epithelium. Recently, two individuals were identified with congenital polymorphic autosomal dominant cataract, due to a single nucleotide base deletion mutation in the lens AQP0. The deletion modified the reading frame resulting in the addition of a premature stop codon. In the present study, we examined the water permeability properties, trafficking and dominant negative effects as well as cytotoxicity due to the mutant AQP0 (Delta213-AQP0) protein. The membrane water permeability (P(w)) of Delta213-AQP0 expressing oocytes (14+/-1 microm/s) was significantly lower than those expressing WT-AQP0 (25+/-3 microm/s). P(w) of water injected control oocytes was 13+/-2 microm/s. Co-expression of WT-AQP0 with Delta213-AQP0 significantly lowered the P(w) (18+/-3 microm/s) compared to WT-AQP0. With or without the EGFP tag, WT-AQP0 protein localized in the plasma membranes of oocytes and cultured cells whereas Delta213-AQP0 was retained in the ER. Forster Resonance Energy Transfer (FRET) showed that WT-AQP0 partly localized with the co-expressed Delta213-AQP0. Co-localization studies suggest that the mutant AQP0 gained its dominant function by trapping the WT-AQP0 in the ER through hetero-oligomerization. Incubating the cells with chemical chaperones, namely, TMAO and DMSO, did not correct the folding/trafficking defects. Cell death in the Delta213-AQP0 expressing cells was due to necrosis caused by the accumulation of Delta213-AQP0 protein in the ER in cytotoxic proportions. The data show that replacement of the distal end of the 6th TM domain and the C-terminal domain of AQP0 due to the deletion mutation resulted in the impairment of cell membrane P(w), localization of the mutant protein in the ER without trafficking to the plasma membrane, and cytotoxicity due to the accumulation of the mutant protein. Cataracts in patients with this mutation might have resulted from the above mentioned consequences.

Cannabinoid receptor 1 suppresses transient receptor potential vanilloid 1-induced inflammatory responses to corneal injury.

Cannabinoid receptor type 1 (CB1)-induced suppression of transient receptor potential vanilloid type 1 (TRPV1) activation provides a therapeutic option to reduce inflammation and pain in different animal disease models through mechanisms involving dampening of TRPV1 activation and signaling events. As we found in both mouse corneal epithelium and human corneal epithelial cells (HCEC) that there is CB1 and TRPV1 expression colocalization based on overlap of coimmunostaining, we determined in mouse corneal wound healing models and in human corneal epithelial cells (HCEC) if they interact with one another to reduce TRPV1-induced inflammatory and scarring responses. Corneal epithelial debridement elicited in vivo a more rapid wound healing response in wildtype (WT) than in CB1(-/-) mice suggesting functional interaction between CB1 and TRPV1. CB1 activation by injury is tenable based on the identification in mouse corneas of 2-arachidonylglycerol (2-AG) with tandem LC-MS/MS, a selective endocannabinoid CB1 ligand. Suppression of corneal TRPV1 activation by CB1 is indicated since following alkali burning, CB1 activation with WIN55,212-2 (WIN) reduced immune cell stromal infiltration and scarring. Western blot analysis of coimmunoprecipitates identified protein-protein interaction between CB1 and TRPV1. Other immunocomplexes were also identified containing transforming growth factor kinase 1 (TAK1), TRPV1 and CB1. CB1 siRNA gene silencing prevented suppression by WIN of TRPV1-induced TAK1-JNK1 signaling. WIN reduced TRPV1-induced Ca(2+) transients in fura2-loaded HCEC whereas pertussis toxin (PTX) preincubation obviated suppression by WIN of such rises caused by capsaicin (CAP). Whole cell patch clamp analysis of HCEC showed that WIN blocked subsequent CAP-induced increases in nonselective outward currents. Taken together, CB1 activation by injury-induced release of endocannabinoids such as 2-AG downregulates TRPV1 mediated inflammation and corneal opacification. Such suppression occurs through protein-protein interaction between TRPV1 and CB1 leading to declines in TRPV1 phosphorylation status. CB1 activation of the GTP binding protein, G(i/o) contributes to CB1 mediated TRPV1 dephosphorylation leading to TRPV1 desensitization, declines in TRPV1-induced increases in currents and pro-inflammatory signaling events.

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. Developmental Dynamics 236:1319–1328, 2007.

Comparison of Conditions for Hormonal Sex Reversal of Mozambique Tilapias

Abstract Roles of selected factors on sex reversal of fry of Mozambique tilapia (Tilapia mossambica) administered 17∝-methyltestosterone (MT) through the diet were investigated. The tested factors were temperature (22–38°C), photoperiod (darkness: light, 0 h:24 h to 24 h:0 h), stocking density (100–2,000 fry/m2), feeding rate (10 or 25% body weight!d), supplementation of the medium with a growth promotor (thyroxine), and storage conditions of MT and MT-treated feed. Survival, growth, and sex-reversing potency of MT were significantly influenced directly or indirectly by temperature, feeding rate, photoperiod, and storage conditions of both MT and MT-treated feed. Stocking density and supplementation of thyroxine in the rearing medium influenced survival and growth. without modifying the sex-reversing potency of MT, when the treated fry were fed ad libitum. Photoperiod and low rearing temperature did not directly alter MT-induced sex reversal but did affect sex reversal indirectly by inhibiting MT-treated ...

Transgenic expression of AQP1 in the fiber cells of AQP0 knockout mouse: effects on lens transparency.

Mutations and knockout of aquaporin 0 (AQP0) result in dominant lens cataract. To date, several functions have been proposed for AQP0; however, two functions, water permeability and cell-to-cell adhesion have been supported by several investigators and only water channel function has been readily authenticated by in vitro and ex vivo studies. Lens shifts protein expression from the more efficient AQP1 in the equatorial epithelial cells to the less efficient water channel, AQP0, in the differentiating secondary fiber cells; perhaps, AQP0 performs a distinctive function. If AQP0 has only water permeability function, can the more efficient water channel AQP1 transgenically expressed in the fiber cells compensate and restore lens transparency in the AQP0 knockout (AQP0(-/-)) mouse? To investigate, we generated a transgenic wild-type mouse line expressing AQP1 in the fiber cells using alphaA-crystallin promoter. These transgenic mice (TgAQP1(+/+)) showed increase in fiber cell membrane water permeability without any morphological, anatomical or physiological defects compared to the wild type indicating that the main purpose of the shift in expression from AQP1 to AQP0 may not be to lessen the membrane water permeability. Further, we transgenically expressed AQP1 in the lens fiber cells of AQP0 knockout mouse (TgAQP1(+/+)/AQP0(-/-)) to determine whether AQP1 could restore AQP0 water channel function and regain lens transparency. Fiber cells of these mice showed 2.6 times more water permeability than the wild type. Transgene AQP1 reduced the severity of lens cataract and prevented dramatic acceleration of cataractogenesis. However, lens fiber cells showed deformities and lack of compact cellular architecture. Loss of lens transparency due to the absence of AQP0 was not completely restored indicating an additional function for AQP0. In vitro studies showed that AQP0 is capable of cell-to-cell adhesion while AQP1 is not. To our knowledge, this is the first report which uses an animal model to demonstrate that AQP0 may have an additional function, possibly cell-to-cell adhesion.

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.

Aquaporin 0 plays a pivotal role in refractive index gradient development in mammalian eye lens to prevent spherical aberration

Aquaporin 0 (AQP0) is a transmembrane channel that constitutes ∼45% of the total membrane protein of the fiber cells in mammalian lens. It is critical for lens transparency and homeostasis as mutations and knockout cause autosomal dominant lens cataract. AQP0 functions as a water channel and as a cell-to-cell adhesion (CTCA) molecule in the lens. Our recent in vitro studies showed that the CTCA function of AQP0 could be crucial to establish lens refractive index gradient (RING). However, there is a lack of in vivo data to corroborate the role of AQP0 as a fiber CTCA molecule which is critical for creating lens RING. The present investigation is undertaken to gather in vivo evidence for the involvement of AQP0 in developing lens RING. Lenses of wild type (WT) mouse, AQP0 knockout (heterozygous, AQP0(+/-)) and AQP0 knockout lens transgenically expressing AQP1 (heterozygous AQP0(+/)(-)/AQP1(+/)(-)) mouse models were used for the study. Data on AQP0 protein profile of intact and N- and/or C-terminal cleaved AQP0 in the lens by MALDI-TOF mass spectrometry and SDS-PAGE revealed that outer cortex fiber cells have only intact AQP0 of ∼28kDa, inner cortical and outer nuclear fiber cells have both intact and cleaved forms, and inner nuclear fiber cells have only cleaved forms (∼26-24kDa). Knocking out of 50% of AQP0 protein caused light scattering, spherical aberration (SA) and cataract. Restoring the lost fiber cell membrane water permeability (Pf) by transgene AQP1 did not reinstate complete lens transparency and the mouse lenses showed light scattering and SA. Transmission and scanning electron micrographs of lenses of both mouse models showed increased extracellular space between fiber cells. Water content determination study showed increase in water in the lenses of these mouse models. In summary, lens transparency, CTCA and compact packing of fiber cells were affected due to the loss of 50% AQP0 leading to larger extracellular space, more water content and SA, possibly due to alteration in RING. To our knowledge, this is the first report identifying the role of AQP0 in RING development to ward off lens SA during focusing.

Aquaporin 5 knockout mouse lens develops hyperglycemic cataract.

The scope of this investigation was to understand the role of aquaporin 5 (AQP5) for maintaining lens transparency and homeostasis. Studies were conducted using lenses of wild-type (WT) and AQP5 knockout (AQP5-KO) mice. Immunofluorescent staining verified AQP5 expression in WT lens sections and lack of expression in the knockout. In vivo and ex vivo, AQP5-KO lenses resembled WT lenses in morphology and transparency. Therefore, we subjected the lenses ex vivo under normal (5.6mM glucose) and hyperglycemic (55.6mM glucose) conditions to test for cataract formation. Twenty-four hours after incubation in hyperglycemic culture medium, AQP5-KO lenses showed mild opacification which was accelerated several fold at 48 h; in contrast, WT lenses remained clear even after 48 h of hyperglycemic treatment. AQP5-KO lenses displayed osmotic swelling due to increase in water content. Cellular contents began to leak into the culture medium after 48 h. We reason that water influx through glucose transporters and glucose cotransporters into the cells could mainly be responsible for creating hyperglycemic osmotic swelling; absence of AQP5 in fiber cells appears to cause lack of required water efflux, challenging cell volume regulation and adding to osmotic swelling. This study reveals that AQP5 could play a critical role in lens microcirculation for maintaining transparency and homeostasis, especially by providing protection under stressful conditions. To the best of our knowledge, this is the first report providing evidence that AQP5 facilitates maintenance of lens transparency and homeostasis by regulating osmotic swelling caused by glucose transporters and cotransporters under hyperglycemic stressful conditions.

Intact and N- or C-terminal end truncated AQP0 function as open water channels and cell-to-cell adhesion proteins: End truncation could be a prelude for adjusting the refractive index of the lens to prevent spherical aberration

BACKGROUND Investigate the impact of natural N- or C-terminal post-translational truncations of lens mature fiber cell Aquaporin 0 (AQP0) on water permeability (Pw) and cell-to-cell adhesion (CTCA) functions. METHODS The following deletions/truncations were created by site-directed mutagenesis (designations in parentheses): Amino acid residues (AA) 2-6 (AQP0-N-del-2-6), AA235-263 (AQP0-1-234), AA239-263 (AQP0-1-238), AA244-263 (AQP0-1-243), AA247-263 (AQP0-1-246), AA250-263 (AQP0-1-249) and AA260-263 (AQP0-1-259). Protein expression was studied using immunostaining, fluorescent tags and organelle-specific markers. Pw was tested by expressing the respective complementary ribonucleic acid (cRNA) in Xenopus oocytes and conducting osmotic swelling assay. CTCA was assessed by transfecting intact or mutant AQP0 into adhesion-deficient L-cells and performing cell aggregation and adhesion assays. RESULTS AQP0-1-234 and AQP0-1-238 did not traffic to the plasma membrane. Trafficking of AQP0-N-del-2-6 and AQP0-1-243 was reduced causing decreased membrane Pw and CTCA. AQP0-1-246, AQP0-1-249 and AQP0-1-259 mutants trafficked properly and functioned normally. Pw and CTCA functions of the mutants were directly proportional to the respective amount of AQP0 expressed at the plasma membrane and remained comparable to those of intact AQP0 (AQP0-1-263). CONCLUSIONS Post-translational truncation of N- or C-terminal end amino acids does not alter the basal water permeability of AQP0 or its adhesive functions. AQP0 may play a role in adjusting the refractive index to prevent spherical aberration in the constantly growing lens. GENERAL SIGNIFICANCE Similar studies can be extended to other lens proteins which undergo post-translational truncations to find out how they assist the lens to maintain transparency and homeostasis for proper focusing of objects on to the retina.