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.

Effect of elevated intracellular cAMP levels on actomyosin contraction in bovine trabecular meshwork cells.

PURPOSE: Elevated cAMP in the trabecular meshwork (TM) cells increases the aqueous humor outflow facility. The authors investigated the mechanisms by which elevated cAMP opposes the RhoA-Rho kinase pathway, leading to the relaxation of the actomyosin system in bovine TM cells. METHODS: Forskolin (Fsk) and rolipram were used to elevate cAMP levels. Changes in the phosphorylation of RhoA at Ser188 (a putative inhibitory site), the regulatory light chain of myosin (pMLC), and the regulatory subunit of myosin phosphatase (MYPT1) were determined by Western blot analysis. The actomyosin contraction was measured by collagen gel contraction (CGC) assay. The impact of cAMP on cell-matrix adhesion was followed by immunostaining of focal adhesion proteins and by electric cell-substrate impedance sensing. RESULTS: Elevated cAMP led to an increase in the phosphorylation of RhoA at Ser188, to the inhibition of endothelin-1 (ET-1)-induced activation of RhoA, and to the formation of stress fibers. The loss of pMLC along the stress fibers was comparable to that induced by Y-27632 (Rho kinase inhibitor). A concomitant reduction in both MYPT1 phosphorylation and pMLC was observed. Elevated cAMP also reduced (ET-1)-induced CGC and the cell-substrate resistance by >50%. CONCLUSIONS: In TM cells, elevated cAMP leads to the phosphorylation of RhoA at Ser188. Consequent inhibition of RhoA activity reduces the phosphorylation of MYPT1 at Thr853, leading to a reduction in MLC phosphorylation and actomyosin contraction. These actions, similar to those of the Rho kinase inhibitors, possibly underlie the reported increase in outflow facility in response to Fsk perfusion ex vivo.

Effect of down-regulation of aquaporins in human corneal endothelial and epithelial cell lines.

PURPOSE: The purpose of this study was to determine the effects of down-regulation of Aquaporin 1 (AQP1) and Aquaporin 5 (AQP5) on cell proliferation and migration in human corneal endothelial (HCEC) and human corneal epithelial (CEPI17) cell lines, respectively. METHODS: AQP1 and AQP5 were down regulated using siRNA following lipofectamine-mediated transfection in corneal endothelial and epithelial cells, respectively. Down-regulation was confirmed using RT-PCR, indirect immunofluorescence, and immunoblot analysis. Total internal reflection fluorescence (TIRF) microscopy was used to detect cell surface aquaporin expression. Cell proliferation was determined by SRB (sulfrodamine B) assay. Cell migration was determined by in vitro wound healing and migration assay. RESULTS: In HCEC cells, AQP1 was localized to the cytosol as well as cell membrane and its down-regulation resulted in decreased cell proliferation and migration with a significant decrease in phosphorylated ERK (pERK). In CEPI17 cells AQP5 protein expression was also localized to cytosol as well as cell membrane. AQP5 down-regulation resulted in an increase in proliferation and cell migration with no significant difference in pERK. CONCLUSIONS: AQP1 plays a role in HCEC proliferation and migration via the ERK signaling pathway and therefore may have significant implications in corneal endothelial dysfunction whereas; AQP5 may play an indirect role in human corneal epithelial cell proliferation and migration.

Changes in ocular aquaporin expression following optic nerve crush.

PURPOSE: Changes in the expression of water channels (aquaporins; AQP) have been reported in several diseases. However, such changes and mechanisms remain to be evaluated for retinal injury after optic nerve crush (ONC). This study was designed to analyze changes in the expression of AQP4 (water selective channel) and AQP9 (water and lactate channel) following ONC in the rat. METHODS: Rat retinal ganglion cells (RGCs) were retrogradely labeled by applying FluoroGold onto the left superior colliculus 1 week before ONC. Retinal injuries were induced by ONC unilaterally. Real-time PCR was used to measure changes in AQP4, AQP9, thy-1, Kir4.1 (K(+) channel), and beta-actin messages. Changes in AQP4, AQP9, Kir4.1, B cell lymphoma-x (bcl-xl), and glial fibrillary acidic protein (GFAP) expression were measured in total retinal extracts using western blotting. RESULTS: The number of RGCs labeled retrogradely from the superior colliculus was 2,090+/-85 cells/mm(2) in rats without any treatment, which decreased to 1,091+/-78 (47% loss) and 497+/-87 cells/mm(2) (76% loss) on days 7 and 14, respectively. AQP4, Kir4.1, and thy-1 protein levels decreased at days 2, 7, and 14, which paralleled a similar reduction in mRNA levels, with the exception of Kir4.1 mRNA at day 2 showing an apparent upregulation. In contrast, AQP9 mRNA and protein levels showed opposite changes to those observed for the latter targets. Whereas AQP9 mRNA increased at days 2 and 14, protein levels decreased at both time points. AQP9 mRNA decreased at day 7, while protein levels increased. GFAP (a marker of astrogliosis) remained upregulated at days 2, 7, and 14, while bcl-xl (anti-apoptotic) decreased. CONCLUSIONS: The reduced expression of AQP4 and Kir4.1 suggests dysfunctional ion coupling in retina following ONC and likely impaired retinal function. The sustained increase in GFAP indicates astrogliosis, while the decreased bcl-xl protein level suggests a commitment to cellular death, as clearly shown by the reduction in the RGC population and decreased thy-1 expression. Changes in AQP9 expression suggest a contribution of the channel to retinal ganglion cell death and response of distinct amacrine cells known to express AQP9 following traumatic injuries.

Bestrophin 2 is expressed in human non-pigmented ciliary epithelium but not retinal pigment epithelium.

PURPOSE: Mice in which bestrophin 2 (Best2) is disrupted exhibit changes in aqueous flow and drainage, resulting in a reduction in intraocular pressure in comparison to wild-type mice. Best2 encodes a putative anion channel localized uniquely to the basolateral plasma membrane of non-pigmented epithelium cells in mice. In this study, we examine the localization of Best2 in the human eye. METHODS: Rabbit polyclonal antibodies recognizing human Best2 (hBest2) were generated and characterized for use in western blot, immunoprecipitation, and immunofluorescence assays. The expression of hBest2 using these antibodies was examined using human donor eye tissues. RESULTS: We could not detect hBest2 in human ciliary bodies or other ocular tissues by western blot. However, when enriched by immunoprecipitation, hBest2 was identified in ciliary bodies, but not in the retinal pigment epithelium. Using immunofluorescence, we located hBest2 in the basolateral plasma membrane of non-pigmented epithelial cells. CONCLUSIONS: We found expression of hBest2 similar to mice only in NPE cells. These data suggest that Best2 may play a functional role in the regulation of aqueous flow and drainage in humans. We conclude that Best2 represents a new potential target for glaucoma therapy.

Vasopressin-induced differential stimulation of AQP4 splice variants regulates the in-membrane assembly of orthogonal arrays.

Aquaporin-4 (AQP4) is a basolateral water channel in collecting duct principal cells and assembles into orthogonal array particles (OAPs), the size of which appears to depend on relative expression levels of AQP4 splice variants. Because the higher-order organization of AQP4 was perturbed by vasopressin in Brattleboro rats and phosphorylation sites have been identified on AQP4, we investigated whether vasopressin and forskolin (Fk) affect AQP4 assembly and/or expression in LLC-PK(1) cells stably transfected with the AQP4 splice variant M23, which is responsible for formation of OAPs, and/or the splice variant M1, which does not form OAPs. Our data show that [lys(8)]-vasopressin (LVP) and Fk treatment led to differential increases in expression levels of M23-AQP4 and M1-AQP4 that varied as a function of incubation time. At early time points (day 1) expression of M1 was significantly stimulated (4.5-fold), over that of M23 (1.6-fold), but after 3 days the expression of M23 became predominant (4.1-fold) over that of M1 (1.9-fold). This pattern of stimulation was dependent on an intact AQP4 residue serine 111 and required protein synthesis. In cells expressing both M1 and M23 (M1/M23 approximately 1), with small sized OAPs at the membrane, the LVP/Fk-induced stimulation of M23 was modified and mimicked that of M1 when expressed alone, suggesting a dominant role for M1. In Brattleboro kidney inner medulla, an 8-day chronic exposure to the vasopressin agonist (dDAVP) led to reduction in M1 and a significant increase in M23 immunoblot staining (M1/M23 = 2/3 --> 1/4). These results indicate that AQP4 organization and expression are regulated by vasopressin in vivo and in vitro and demonstrate that the dominant role for M1 is restricted to a one-to-one interaction between AQP4 splice variants that regulates the membrane expression of OAPs.

Induced autoimmunity to heat shock proteins elicits glaucomatous loss of retinal ganglion cell neurons via activated T-cell-derived fas-ligand.

Glaucomatous optic neuropathy causes blindness through the degeneration of retinal ganglion cells (RGCs) and their axons, which comprise the optic nerve. Glaucoma traditionally is associated with elevated intraocular pressure, but often occurs or may progress with intraocular pressure in the normal range. Like other diseases of the CNS, a subset of glaucoma has been proposed to involve an autoimmune component to help explain the loss of RGCs in the absence of elevated intraocular pressure. One hypothesis involves heat shock proteins (HSPs), because increased serum levels of HSP autoantibodies are prominent in some glaucoma patients with normal pressures. In the first direct support of this hypothesis, we found that HSP27 and HSP60 immunization in the Lewis rat induced RGC degeneration and axon loss 1-4 months later in vivo in a pattern with similarities to human glaucoma, including topographic specificity of cell loss. Infiltration of increased numbers of T-cells in the retina occurred much earlier, 14-21 d after HSP immunization, and appeared to be transient. In vitro studies found that T-cells activated by HSP immunization induced RGC apoptosis via the release of the inflammatory cytokine FasL, whereas HSP immunization induced activation of microglia cells and upregulation of the FasL receptor in RGCs. In summary, our results suggest that RGC degeneration in glaucoma for selected individuals likely involves failed immunoregulation of the T-cell-RGC axis and is thus a disturbance of both proapoptotic and protective pathways.

Interleukin-10 receptor signaling through STAT-3 regulates the apoptosis of retinal ganglion cells in response to stress.

PURPOSE: Interleukin (IL)-10 has recently been shown to promote survival of neurons and glia. The purpose of this report is to investigate whether IL-10 has any role in protecting retinal ganglion cells (RGCs) from death under conditions in which growth factors are removed, or in which oxidative stress is present. Signal transduction pathways that activate IL-10 signaling in RGCs were studied in both stress conditions. METHODS: Effects of various interleukins on the viability of the RGC cell line was determined, and apoptotic cells were quantified. Immunoblot analysis was preformed to identify the IL-10 receptor (IL-10R) and phosphorylated or nonphosphorylated Akt and STAT-3 proteins in RGC extracts. Immunohistochemistry was performed on the rat retinal sections to identify native IL-10R. RESULTS: Apoptosis of RGCs in the absence of growth factors with or without dexamethasone (1 microM) occurred in 68.5% +/- 3.4% and 53.4% +/- 2.6% of cells, respectively, after 96 hours. Addition of IL-10 at a concentration of 50 ng/mL significantly reduced the apoptotic population of RGCs to 28.2% +/- 2.3% in the absence of growth factors with dexamethasone and to 31% +/- 2.7% in the absence of growth factors alone. RGCs as well as native retina expressed functional IL-10R as determined by immunoblot analysis and by the ability of IL-10 to phosphorylate Stat-3. However, IL-10 failed to phosphorylate Akt in these cells. CONCLUSIONS: IL-10 caused a 59% and 42% reduction in the apoptotic population of serum-deprived cells with and without dexamethasone treatment, respectively. These observations establish that activation of IL-10R promotes survival of RGCs and this survival-promoting activity is due to IL-10 signaling through the Stat-3 pathway, which inhibits the cell death and not through the Akt cell survival pathway.

Mildly abnormal retinal function in transgenic mice without Müller cell aquaporin-4 water channels.

PURPOSE: Immunocytochemistry showed strong aquaporin (AQP)-4 water channel expression in Müller cells in mouse retina and fibrous astrocytes in optic nerve. This study was designed to test the hypothesis that AQP4 is required for vision by comparing electroretinograms and retinal morphology in wild-type mice and transgenic knockout mice with no AQP4. METHODS: Electroretinograms were recorded over a 10(5)-fold range of flash intensities in dark-adapted mice and analyzed for a- and b-wave amplitude and latency, a-wave normalized slope, and oscillatory potential amplitude and latency. AQP4 protein was localized in mouse retina by immunocytochemistry, and retinal morphology was studied by light and electron microscopy. RESULTS: Significantly reduced electroretinogram b-wave potentials were recorded in 10-month-old null mice with smaller changes in 1-month-old mice. Immunocytochemistry showed strong AQP4 protein expression in retina of wild-type mice. Morphologic analysis of retina by light and electron microscopy showed no differences in retinal ultrastructure. CONCLUSIONS: Retinal function is mildly impaired in AQP4-null mice, suggesting a role for AQP4 in Müller cell fluid balance. These results support the paradigm that AQP4 expression in supportive cells in the nervous system facilitates neural signal transduction in nearby electrically excitable cells.