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Abstract: Orthogonal arrays of particles (OAPs) have been visualized for many years by freeze-fracture electron microscopy. Our laboratory discovered that aquaporin-4 (AQP4) is the protein responsible for OAP formation by demonstrating OAPs in AQP4-transfected cells and absence of OAPs in AQP4 knockout mice. We recently developed live-cell, single-molecule imaging methods to study AQP4 diffusion and interactions in OAPs. The methods include single particle tracking of quantum-dot labeled AQP4, and total internal reflection fluorescence microscopy of green fluorescent protein (GFP) and small fluorophore-labeled AQP4. The full-length (M1) form of AQP4 diffuses freely in membranes and does not form OAPs, whereas the shorter (M23) form of AQP4 forms OAPs and is nearly immobile. Analysis of a series of AQP4 truncations, point mutants and chimeras revealed that OAP formation by AQP4-M23 is stabilized by hydrophobic tetramer-tetramer interactions involving N-terminus residues, and that absence of OAPs in AQP4-M1 results from blocking of this interaction by residues just upstream from Met23. These biophysical methods are being extended to identify the cellular site of AQP4 assembly, AQP4 isoform interactions, OAP size and dynamics, and the determinants of regulated OAP assembly. [Copyright &y& Elsevier] more...

Abstract: Inhibitors of brain glial water channel aquaporin-4 (AQP4) are of potential clinical utility, as they are predicted to modulate brain edema, neuroexcitation and glial scarring. Recently, Huber et al. (Bioorg. Med. Chem. 2007, 17, 1270–1273; in press) reported that a series of arylsulfonamides, antiepileptics, and related small molecules strongly inhibited AQP4 water transport with IC50s down to 1μM. We retested the compounds with greatest reported potencies, including acetylsulfanilamide, acetazolamide, 6-ethoxy-benzothiazole-2-sulfonamide, topiramate, zonisamide, phenytoin, lamotrigine, and sumatriptan, in AQP4-transfected mammalian cells and primary cultures of brain glial cells, using several sensitive assays of osmotic water permeability. Contrary to the findings of Huber et al., in our studies we found no significant inhibition of AQP4 water permeability by any of the compounds at concentrations up to 100μM. [Copyright &y& Elsevier] more...

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated C1- channel expressed at the apical plasma membrane. It has been proposed that the C-terminal PDZ binding motif of CFTR is required for its apical membrane targeting and that PDZ-domain interactions may tether CFTR to the actin cytoskeleton via soluble proteins including EBP50/ NHERF1 and ezrin. We measured the diffusional mobility of human CFTR in the plasma membrane of MadinDarby canine kidney cells by photobleaching of green fluorescent protein (GFP)-CFTR chimeras. After bleaching by a focused laser beam, GFP-CFTR fluorescence in the bleached membrane region recovered to ∼90% of its initial level, indicating that nearly all of the CFTR was mobile. The GFP-CFTR diffusion coefficient (D) was 0.99 ± 0.09 x l0-10 cm2/s at 37 °C, similar to that of other membrane proteins. GFP-CFTR diffusion was not altered by protein kinase A or C activators but was blocked by paraformaldehyde and filipin. CFTR mutants lacking functional PDZ-binding domains (GFPCFTR-ΔTRL and GFP-CFTR-ΔTRA) were also mobile with D significantly increased by ∼60% compared with GFP-CFTR. However, GFP-CFTR, GFP-CFTR-ATRL, and GFP-CFTR-ATRA had similar mobilities (D ∼12 × l0-10M cm2/s) at the endoplasmic reticulum in brefeldin A-treated cells. Agents that modulate the actin cytoskeleton (cytochalasin D and jasplakinolide) altered the plasma membrane mobility of CFTR but not CFTRATRL. EBP50 (NHERF1), a PDZ domain-containing protein that interacts with the C terminus of CFTR, diffused freely in the cytoplasm with a diffusion coefficient of 0.9 ± 0.1 × 10-7 cm2/s. EBP50 diffusion increased by ∼2-fold after deletion of its ezrin-binding domain. These results indicate that wild-type CFTR is not tethered statically at the plasma membrane hut that its diffusion is dependent on PDZ-domain interactions and an intact actin skeleton. PDZ-domain interactions of CFTR are thus dynamic and occur on a time scale of seconds or faster. [ABSTRACT FROM AUTHOR] more...

The "proton sponge hypothesis" postulates enhanced transgene delivery by cationic polymer. DNA complexes (polyplexes) containing H[sup +] buffering polyamines by enhanced endosomal Cl[sup -] accumulation and osmotic swelling/lysis. To test this hypothesis, we measured endosomai Cl[sup -] concentration, pH, and volume after internalization of polyplexes composed of plasmid DNA and polylysine (POL), a non-buffering polyamine, or the strongly buffering polyamines polyethylenimine (PEI) or polyamidoamine (PAM). [Cl[sup -]] and pH were measured by ratio imaging of fluorescently labeled polyplexes containing Cl[sup -] or pH indicators. [Cl[sup -]] increased from 41 to 80 mM over 60 min in endosomes-contained POL-polyplexes, whereas pH decreased from 6.8 to 5.3. Endosomal Cl[sup -] accumulation was enhanced (115 mM at 60 min) and acidification was slowed (pH 5.9 at 60 min) for PEI and PAM-polyplexes. Relative endosome volume increased 20% over 75 min for POL-polyplexes versus 140% for PEI-polyplexes. Endosome lysis was seen at >45 min for PEI but not POL-containing endosomes, and PEI-containing endosomes showed increased osmotic fragility in vitro. The slowed endosomal acidification and enhanced Cl[sup -] accumulation and swelling/lysis were accounted for by the greater H[sup +] buffering capacity of endosomes containing PEI or PAM versus POL (>90 mM versus 46 H[sup +]/pH unit). Our results provide direct support for the proton sponge hypothesis and thus a rational basis for the design of improved non-viral vectors for gene delivery. [ABSTRACT FROM AUTHOR] more...

The composition and depth of the airway surface liquid (ASL) are key parameters in airway physiology that are thought to be important in the pathophysiology of cystic fibrosis and other diseases of the airways. We reported novel fluorescent indicator and microscopy methods to measure [Na[sup +]], [Cl[sup -]], pH, and depth of the ASL in large airways (Jayaraman, S., Y. Song, L. Vetrivel, L. Shankar, and A.S. Verkman. 2001. J. Clin. Invest. 107:317324.). Here we report a stripped-lung preparation to measure ASL composition and depth in small distal airways. Distal ASL was stained with ion- or pH-sensitive fluorescent indicators by infusion into mouse trachea of a perfluorocarbon suspension of the indicator. After stripping the pleura and limited microdissection of the lung parenchyma, airways were exposed for measurement of ASL [Na[sup +]], [Cl[sup -]], and pH by ratio imaging microscopy, and depth by confocal microscopy. The stripped-lung preparation was validated in stability and tissue viability studies. ASL [Na[sup +]] was 122 ± 2 mM, [Cl[sup -]] was 123 ± 4 mM and pH was 7.28 ± 0.07, and not dependent on airway size (<100- to >250-µm diameter), ENaC inhibition by amiloride, or CFTR inhibition by the thiazolidinone CFTR[sub inh]172. ASL depth was 8-35 µm depending on airway size, substantially less than that in mouse trachea of ∼55 µm, and not altered significantly by amiloride. These results establish a novel lung preparation and fluorescence approach to study distal airway physiology and provide the first data on the composition and depth of distal ASL. [ABSTRACT FROM AUTHOR] more...

Develops methods to compare aqueous fluid dynamics in wild-type mice versus mice lacking aquaporin1 (AQP1) and AQP4. Importance of intraocular pressure to eye physiology; Measurement of the aqueous fluid production by in vivo confocal microscopy; Determination of intraocular pressure, outflow and anterior chamber compliance from pressure measurements in response to fluid infusions. more...

Examines the high-throughput screening of cystic fibrosis transmembrane conductance regulator (CFTR) chloride transport agonists. Restoration of cell chloride transport; Use of a conventional fluorescence plate reader; Significance of the fluorescence assay and cell lines in screening novel CFTR activators. more...

Characterizes airway surface liquid hydrogen-ion concentration (pH) airway epithelial cell cultures and mouse trachea. Methods in the conduct of the study; Evaporation of perfluorocarbon; Manifestation of fluorescence in the tracheal wall; Reduction of pH recovery rate by amiloride. more...

Aquaporin-4 (AQP4) is a water channel expressed in astrocyte end-feet lining the blood–brain barrier. AQP4 deletion in mice is associated with improved outcomes in global cerebral ischemia produced by transient carotid artery occlusion, and focal cerebral ischemia produced by permanent middle cerebral artery occlusion (MCAO). Here, we investigated the consequences of 1-h transient MCAO produced by intraluminal suture blockade followed by 23 h of reperfusion. In nine AQP4 +/+ and nine AQP4 −/− mice, infarct volume was significantly reduced by an average of 39 ± 4% at 24 h in AQP4 −/− mice, cerebral hemispheric edema was reduced by 23 ± 3%, and Evans Blue extravasation was reduced by 31 ± 2% (mean ± SEM). Diffusion-weighted magnetic resonance imaging showed greatest reduction in apparent diffusion coefficient around the occlusion site after reperfusion, with remarkably lesser reduction in AQP4 −/− mice. The reduced infarct volume in AQP4 −/− mice following transient MCAO supports the potential utility of therapeutic AQP4 inhibition in stroke. [ABSTRACT FROM AUTHOR] more...

The most common mutation causing cystic fibrosis (CF) is deletion of phenylalanine residue 508 in the cystic fibrosis transmembrane regulator conductance (CFTR) protein. Small molecules that are able to correct the misfolding of defective ΔF508-CFTR have considerable promise for therapy. Reported here are the design, preparation, and evaluation of five more hydrophilic bisazole analogs of previously identified bithiazole CF corrector 1 . Interestingly, bisazole ΔF508-CFTR corrector activity was not increased by incorporation of more H-bond acceptors (O or N), but correlated best with the overall bisazole molecular geometry. The structure activity data, together with molecular modeling, suggested that active bisazole correctors adopt a U-shaped conformation, and that corrector activity depends on the molecule’s ability to access this molecular geometry. [ABSTRACT FROM AUTHOR] more...

Abstract: Astrocyte water channel aquaporin-4 (AQP4) facilitates water movement across the blood–brain barrier and into injured astrocytes. We previously showed reduced cytotoxic brain edema with improved neurological outcome in AQP4 knockout mice in water intoxication, infection and cerebral ischemia. Here, we established a 4-vessel transient occlusion model to test the hypothesis that AQP4 deficiency in mice could improve neurological outcome following severe global cerebral ischemia as occurs in cardiac arrest/resuscitation. Mice were subjected to 10-min transient bilateral carotid artery occlusion at 24h after bilateral vertebral artery cauterization. Cerebral blood flow was reduced during occlusion by >94% in both AQP4+/+ and AQP4−/− mice. The primary outcome, neurological score, was remarkably better at 3 and 5 days after occlusion in AQP4−/− than in AQP4+/+ mice, and survival was significantly improved as well. Brain water content was increased by 2.8±0.4% in occluded AQP4+/+ mice, significantly greater than that of 0.3±0.6% in AQP4−/− mice. Histological examination and immunofluorescence of hippocampal sections at 5 days showed significantly greater neuronal loss in the CA1 region of hippocampus in AQP4+/+ than AQP4−/− mice. The neuroprotection in mice conferred by AQP4 deletion following severe global cerebral ischemia provides proof-of-concept for therapeutic AQP4 inhibition to improve neurological outcome in cardiac arrest. [Copyright &y& Elsevier] more...

Summary: Urea transporter (UT) proteins, including UT-A in kidney tubule epithelia and UT-B in vasa recta microvessels, facilitate urinary concentrating function. A screen for UT-A inhibitors was developed in MDCK cells expressing UT-A1, water channel aquaporin-1, and YFP-H148Q/V163S. An inwardly directed urea gradient produces cell shrinking followed by UT-A1-dependent swelling, which was monitored by YFP-H148Q/V163S fluorescence. Screening of ∼90,000 synthetic small molecules yielded four classes of UT-A1 inhibitors with low micromolar half-maximal inhibitory concentration that fully and reversibly inhibited urea transport by a noncompetitive mechanism. Structure-activity analysis of >400 analogs revealed UT-A1-selective and UT-A1/UT-B nonselective inhibitors. Docking computations based on homology models of UT-A1 suggested inhibitor binding sites. UT-A inhibitors may be useful as diuretics (“urearetics”) with a mechanism of action that may be effective in fluid-retaining conditions in which conventional salt transport-blocking diuretics have limited efficacy. [Copyright &y& Elsevier] more...

Abstract: The kidney urea transport protein UT-B is an attractive target for the development of small-molecule inhibitors with a novel diuretic (‘urearetic’) action. Previously, two compounds in the triazolothienopyrimidine scaffold (1a and 1c) were reported as UT-B inhibitors. Compound 1c incorporates a 1,1-difluoroethyl group, which affords improved microsomal stability when compared to the corresponding ethyl-substituted compound 1a. Here, a small focused library (4a–4f) was developed around lead inhibitor 1c to investigate the requirement of an amidine-linked thiophene in the inhibitor scaffold. Two compounds (4a and 4b) with nanomolar inhibitory potency (IC50 ≈40nM) were synthesized. Computational docking of lead structure 1c and 4a–4f into a homology model of the UT-B cytoplasmic surface suggested binding with the core heterocycle buried deep into the hydrophobic pore region of the protein. [Copyright &y& Elsevier] more...

Abstract: Copper catalyzed azide-alkyne cycloaddition (CuAAC) chemistry is reported for the construction of previously unknown 5-(1H-1,2,3-triazol-1-yl)-4,5′-bithiazoles from 2-bromo-1-(thiazol-5-yl)ethanones. These novel triazolobithiazoles are shown to have cystic fibrosis (CF) corrector activity and, compared to the benchmark bithiazole CF corrector corr-4a, improved log P values (4.5 vs 5.96). [Copyright &y& Elsevier] more...

Objective: Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system associated with pathogenic autoantibodies against the astrocyte water channel protein aquaporin-4 (AQP4). The presence of neutrophils is a characteristic feature in NMO lesions in humans. Neutrophils are not generally found in multiple sclerosis lesions. We evaluated the role of neutrophils in a mouse NMO model. Methods: NMO lesions were produced in mice by intracerebral injection of immunoglobulin G (IgG) isolated from NMO patient serum and human complement. We previously reported that this mouse model produces the characteristic histological features of NMO, including perivascular complement activation, inflammatory cell infiltration, and loss of myelin, AQP4, and glial fibrillary acidic protein. Lesions are absent when AQP4 null mice are used or when IgG from non-NMO patients is injected. Results: We found remarkably reduced neuroinflammation, myelin loss, and AQP4 loss in brains of neutropenic mice at 24 hours and 7 days, and increased severity of NMO lesions in mice made neutrophilic by granulocyte colony stimulating factor. NMO lesions were greatly reduced by intracerebral administration of the neutrophil protease inhibitors Sivelestat and cathepsin G inhibitor I or by intraperitoneal injection of Sivelestat alone. Immunostaining of human NMO lesions for neutrophil elastase revealed many degranulating perivascular neutrophils, with no equivalent perivascular neutrophils in human multiple sclerosis lesions. Interpretation: Our data implicate a central role of neutrophils in the pathogenesis of early NMO lesions and suggest the potential utility of neutrophil protease inhibitors such as Sivelestat in NMO therapy. ANN NEUROL 2012; [ABSTRACT FROM AUTHOR] more...

Abstract: 19F-modified bithiazole correctors and phenylglycine potentiators of the ΔF508-CFTR chloride channel were synthesized and their function assayed in cells expressing human ΔF508-CFTR and a halide-sensitive fluorescent protein. Fluorine was incorporated into each scaffold using prosthetic groups for future biodistribution imaging studies using positron emission tomography (PET). The ΔF508-CFTR corrector and potentiator potencies of the fluorinated analogs were comparable to or better than those of the original compounds. [Copyright &y& Elsevier] more...

Abstract: Pathogenic autoantibodies target aquaporin-4 (AQP4) water channels in individuals with neuromyelitis optica (NMO). Recently, allelic mutations were reported at residue 19 of AQP4 in three cases of NMO, and it was suggested that polymorphisms may influence disease by altering AQP4 supramolecular assembly into orthogonal arrays of particles (OAPs). We analyzed the determinants of OAP formation by human AQP4 to investigate the possible role of polymorphisms in NMO pathogenesis. NMO-associated mutations R19I and R19T in AQP4 did not affect OAP assembly, palmitoylation-dependent regulation of assembly, or NMO autoantibody binding. Residue-19 polymorphisms in AQP4 are thus unlikely to be disease relevant. [Copyright &y& Elsevier] more...

Abstract: We reported recently that intracerebral administration of NMO-IgG with human complement produces neuromyelitis optica (NMO) lesions in mice. We examined the role of T cells in the formation of NMO lesions by comparing brain histopathology in wildtype and nude mice. Brains were co-injected with IgG from NMO patients and human complement. At 24h and 5days, wildtype vs. nude mouse brains had comparable inflammation (CD45 immunoreactivity), loss of myelin (Luxol Fast Blue staining) and loss of AQP4 immunoreactivity. We conclude that T cells are not required for the formation of NMO lesions in this mouse model. [Copyright &y& Elsevier] more...

Abstract: Hydrocephalus is a pathological accumulation of cerebrospinal fluid (CSF) in the cerebral ventricles that constitutes a significant cause of neurological morbidity and mortality. Surgical treatment involving shunt placement is associated with a high failure rate and complications due to infection, motivating the development of alternative, non-surgical therapies. Here, we investigated the role in hydrocephalus of water channel aquaporin-1 (AQP1), which is expressed at the apical membrane of choroid plexus epithelium and is believed to facilitate CSF production. AQP1 expression and subcellular localization were studied in a kaolin-induced hydrocephalus model in mice and the effect AQP1 deficiency on the severity of hydrocephalus was determined. While total choroidal AQP1 protein was not significantly altered in hydrocephalus, ~50% of AQP1 protein was redistributed from the apical membrane to intracellular vesicles. We found that the ventricular size in AQP1-deficient mice was smaller than in wild-type mice, both at baseline and following hydrocephalus. The reduced plasma membrane AQP1 localization following kaolin-induced hydrocephalus, which involves endocytosis, may be a compensatory mechanism to reduce CSF secretion. The reduced ventricular size in AQP1-deficient mice following kaolin-induced hydrocephalus suggests AQP1 inhibition or down-regulation as a potential adjunctive treatment for hydrocephalus. [ABSTRACT FROM AUTHOR] more...

Abstract: We investigated the role of the glial water channel protein aquaporin-4 in brain edema in a mouse model of subarachnoid hemorrhage in which 30 μl of blood was injected into the basal cisterns. Brain water content, intracranial pressure and neurological score were compared in wildtype and aquaporin-4 null mice. We also measured blood-brain barrier permeability, and the osmotic permeability of the glia limitans, one of the routes of edema elimination. Wildtype and aquaporin-4 null mice had comparable baseline brain water content, intracranial pressure and neurological score. At 6 h after blood injection, aquaporin-4 null mice developed more brain swelling than wildtype mice. Brain water content increased by 1.5±0.1% vs. 0.5±0.2% (Mean±Standard Error, P<0.0005) and intracranial pressure by 36±5 vs. 21±3 mm Hg (P<0.05) above pre-injection baseline, and neurological score was worse at 18.0 vs. 24.5 (median, P<0.05), respectively. Although subarachnoid hemorrhage produced comparable increases in blood–brain barrier permeability in wildtype and aquaporin-4 null mice, aquaporin-4 null mice had a twofold reduction in glia limitans osmotic permeability. We conclude that aquaporin-4 null mice manifest increased brain edema following subarachnoid hemorrhage as a consequence of reduced elimination of excess brain water. [Copyright &y& Elsevier] more...

Abstract: A developing therapy of cystic fibrosis caused by the ΔF508 mutation in CFTR employs correction of defective CFTR chloride channel gating by a ‘potentiator’ and of defective CFTR protein folding by a ‘corrector’. Based on SAR data for phenylglycine-type potentiators and bithiazole correctors, we designed a hybrid molecule incorporating an enzymatic hydrolysable linker to deliver the potentiator (PG01) fragment 2 and the corrector (Corr-4a) fragment 13. The hybrid molecule 14 contained PG01-OH and Corr-4a–linker–CO2H moieties, linked with an ethylene glycol spacer through an ester bond. The potentiator 2 and corrector 13 fragments (after cleavage) had low micromolar potency for restoration of ΔF508-CFTR channel gating and cellular processing, respectively. Cleavage of hybrid molecule 14 by intestinal enzymes under physiological conditions produced the active potentiator 2 and corrector fragments 13, providing proof-of-concept for small-molecule potentiator–corrector hybrids as a single drug therapy for CF caused by the ΔF508 mutation. [Copyright &y& Elsevier] more...

Abstract: The glial cell water channel aquaporin-4 (AQP4) plays an important role in brain edema, astrocyte migration, and neuronal excitability. Zhou et al. [Zhou J, Kong H, Hua X, Xiao M, Ding J, Hu G (2008) Altered blood–brain barrier integrity in adult aquaporin-4 knockout mice. Neuroreport 19:1–5] recently reported that AQP4 deletion significantly altered blood–brain barrier integrity and glial fibrillary acidic protein (GFAP) immunoreactivity in their AQP4 null mice. Here we describe a detailed characterization of baseline brain properties in our AQP4 null mice, including gross appearance, neuronal, astrocyte and oligodendrocyte characteristics, and blood–brain barrier integrity. Gross anatomical measurements included estimates of brain and ventricle size. Neurons, astrocytes and oligodendrocytes were assessed using the neuronal nuclear marker NeuN, the astrocyte marker GFAP, and the myelin stain Luxol Fast Blue. The blood–brain barrier was studied by electron microscopy and the horseradish peroxidase extravasation technique. There were no differences in brain and ventricle sizes between wild type and AQP4 null mice, nor were there differences in the cerebral cortical density of NeuN positive nuclei, perimicrovessel and glia limitans GFAP immunoreactivity, or the thickness and myelination of the corpus callosum. The ultrastructure of microvessels in the frontal cortex and caudate nucleus of wild type vs. AQP4 null mice was indistinguishable, with features including intact endothelial tight junctions, absence of perimicrovessel astrocyte foot process edema, and absence of horseradish peroxidase extravasation. In contrast to the report by , our data show that AQP4 deletion in mice does not produce major structural abnormalities in the brain. [Copyright &y& Elsevier] more...

Summary: Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have potential application as antisecretory therapy in cholera. We synthesized mono- and divalent CFTR inhibitors consisting of a malonic acid hydrazide (MalH) coupled via a disulfonic stilbene linker to polyethylene glycols (PEGs; 0.2–100 kDa). IC50 values for CFTR inhibition were 10–15 μM for the monovalent MalH-PEGs, but substantially lower for divalent MalH-PEG-MalH compounds, decreasing from 1.5 to 0.3 μM with increasing PEG size and showing positive cooperativity. Whole-cell patch-clamp showed voltage-dependent CFTR block with inward rectification. Outside-out patch-clamp showed shortened single-channel openings, indicating CFTR pore block from the extracellular side. Luminally added MalH-PEG-MalH blocked by >90% cholera toxin-induced fluid secretion in mouse intestinal loops (IC50 ∼10 pmol/loop), and greatly reduced mortality in a suckling mouse cholera model. These conjugates may provide safe, inexpensive antisecretory therapy. [Copyright &y& Elsevier] more...

Abstract: The synthesis and ΔF508-CFTR corrector activity of a 148-member methylbithiazole-based library are reported. Synthetic routes were devised and optimized to generate methylbithiazole analogs in four steps. Corrector potency and efficacy were assayed using epithelial cells expressing human ΔF508-CFTR. These structure–activity data establish that the bithiazole substructure plays a critical function; eight novel methylbithiazole correctors were identified with low micromolar potencies. [Copyright &y& Elsevier] more...

Abstract: Aquaporin-1 (AQP1), a membrane water channel, is expressed in choroid plexus where it contributes to cerebrospinal fluid production. Here, we show that AQP1 is also expressed in the dorsal horn of the spinal cord and the trigeminal nucleus caudalis, regions that process pain information. Within the dorsal root and trigeminal sensory ganglia, AQP1 is concentrated in small diameter cell bodies, most of which give rise to unmyelinated C-fibers. To study the role of AQP1 in pain signaling, we compared acute pain responses in wild-type mice and in mice lacking AQP1. AQP1−/− mice had reduced responsiveness to thermal and capsaicin chemical stimuli, but not to mechanical stimuli or formalin. These results provide evidence for AQP1 expression in nociceptive neurons and suggest that AQP1 may play a role in pain signal transduction. [Copyright &y& Elsevier] more...

Abstract: To clarify the potential role Rh/RhAG and AQP1 proteins in erythrocyte gas transport, NH3 and CO2 transport was measured in erythrocyte ghost membrane vesicles from rare human variants (Rhnull, COnull,) and knockout mice (homozygous AQP1–/–, Rh–/– and Rhag–/–) exhibiting well-characterized protein defects. Transport was measured from intracellular pH (pHi) changes in a stopped-flow fluorimeter. NH3 transport was measured in chloride-free conditions with ghosts exposed to 20 mM inwardly directed gradients of gluconate salts of ammonium, hydrazine and methylammonium at 15 °C. Alkalinization rates of control samples were 6.5±0.3, 4.03±0.17, 0.95±0.08 s–1 for each solute, respectively, but were significantly reduced for Rhnull and COnull samples that are deficient in RhAG and AQP1 proteins, respectively. Alkalinization rates of Rhnull ghosts were about 60%, 83% and 94% lower than that in control ghosts, respectively, for each solute. In COnull ghosts, the lack of AQP1 resulted in about 30% reduction of the alkalinization rates as compared to controls, but the transport selectivity of RhAG for the three solutes was preserved. Similar observations were made with ghosts from KO mice Rhag–/– and AQP1–/–. These results confirm the major contribution of RhAG/Rhag in the NH3 conductance of erythrocytes and suggest that the reduction of transport rates in the absence of AQP1 would be better explained by a direct or indirect effect on RhAG/Rhag-mediated transport. When ghosts were preloaded with carbonic anhydrase and exposed to a 25 mM CO2/HCO3 – gradient at 6 °C, an extremely rapid kinetics of acidification corresponding to CO2 influx was observed. The rate constants were not significantly different between controls and human variants (125±6 s–1), or between wild-type and KO mice, suggesting no major role of RhAG or AQP1 in CO2 transport, at least in our experimental conditions. [Copyright &y& Elsevier] more...

Abstract: The synthesis of a small collection of sulfamoyl-4-oxoquinoline-3-carboxamides is described for use as correctors of defective gating of the ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Several compounds with submicromolar potency were obtained. N-Ethyl 6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (7b) was found to be the most effective sulfonamide corrector of defective ΔF508-CFTR gating. [Copyright &y& Elsevier] more...

Abstract: ClC-5 chloride channel deficiency causes proteinuria, hypercalciuria, and nephrolithiasis (Dent’s disease). Impaired endosomal acidification in proximal tubule caused by reduced chloride conductance is a proposed mechanism; however, functional analysis of ClC-5 in oocytes predicts low ClC-5 chloride conductance in endosomes because of their acid interior pH and positive potential. Here, endosomal pH and chloride concentration were measured in proximal tubule cell cultures from wildtype vs. ClC-5 deficient mice using fluorescent sensors coupled to transferrin (early/recycling endosomes) or α2-macroglobulin (late endosomes). Initial pH in transferrin-labeled endosomes was ∼7.2, decreasing at 15min to 6.0 vs. 6.5 in wildtype vs. ClC-5 deficient cells, respectively; corresponding endosomal chloride concentration increased from ∼16mM to 47 vs. 36mM. In contrast, acidification and chloride accumulation were not impaired in late endosomes or Golgi. Our results provide direct evidence for ClC-5 involvement in acidification of early endosomes in proximal tubule by a chloride shunt mechanism. [Copyright &y& Elsevier] more...

Despite their diverse histological types, most brain tumours cause brain oedema, which is a significant cause of patient morbidity and mortality. Brain tumour oedema occurs when plasma-like fluid enters the brain extracellular space through impaired capillary endothelial tight junctions in tumours. Under-expression of the tight junction proteins occludin, claudin-1 and claudin-5 are key molecular abnormalities responsible for the increased permeability of tumour endothelial tight junctions. Recent evidence suggests that the membrane water channel protein aquaporin-4 (AQP4) also plays a role in brain tumour oedema. AQP4-deficient mice show remarkably altered brain water balance after various insults, including brain tumour implantation. AQP4 expression is strongly upregulated around malignant human brain tumours in association with reduced extracellular volume, which may restrict the flow of extracellular fluid from the tumour bed into the brain parenchyma. Elimination of excess fluid leaking into brain parenchyma requires passage across three AQP4-rich barriers: a) the glia limitans externa, b) the glia limitans interna/ependyma, and c) the blood–brain barrier. Modulation of the expression and/or function of endothelial tight junction proteins and aquaporins may provide novel therapeutic options for reducing brain tumour oedema. [Copyright &y& Elsevier] more...

Abstract: Aquaporin-4 (AQP4) is the major water channel in the CNS. Its expression at fluid-tissue barriers (blood–brain and brain–cerebrospinal fluid barriers) throughout the brain and spinal cord suggests a role in water transport under normal and pathological conditions. Phenotype studies of transgenic mice lacking AQP4 have provided evidence for a role of AQP4 in cerebral water balance and neural signal transduction. Primary cultures of astrocytes from AQP4-null mice have greatly reduced osmotic water permeability compared with wild-type astrocytes, indicating that AQP4 is the principal water channel in these cells. AQP4-null mice have reduced brain swelling and improved neurological outcome following water intoxication and focal cerebral ischemia, establishing a role of AQP4 in the development of cytotoxic (cellular) cerebral edema. In contrast, brain swelling and clinical outcome are worse in AQP4-null mice in models of vasogenic (fluid leak) edema caused by freeze-injury and brain tumor, probably due to impaired AQP4-dependent brain water clearance. AQP4-null mice also have markedly reduced acoustic brainstem response potentials and significantly increased seizure threshold in response to chemical convulsants, implicating AQP4 in modulation of neural signal transduction. Pharmacological modulation of AQP4 function may thus provide a novel therapeutic strategy for the treatment of stroke, tumor-associated edema, epilepsy, traumatic brain injury, and other disorders of the CNS associated with altered brain water balance. [Copyright &y& Elsevier] more...

We investigated the involvement of ClC-3 chloride channels in endosomal acidification by measurement of endosomal pH and chloride concentration [Cl-] in control versus ClC-3-deficient hepatocytes and in control versus ClC-3-transfected Chinese hamster ovary cells. Endosomes were labeled with pH or [Cl-]-sensing fluorescent transferrin (Tf), which targets to early/recycling endosomes, or α2-macroglobulin (α2M), which targets to late endosomes. In pulse label-chase experiments, [Cl-] was 19 mM just after internalization in α2M-labeled endosomes in primary cultures of hepatocytes from wildtype mice, increasing to 58 mM over 45 min, whereas pH decreased from 7.1 to 5.4. Endosomal acidification and [Cl-] accumulation were significantly impaired in hepatocytes from ClC-3 knock-out mice, with [Cl-] increasing from 16 to 43 mM and pH decreasing from 7.1 to 6.0. Acidification and Cl- accumulation were blocked by bafilomycin. In Tf-labeled endosomes, [Cl-] was 46 mM in wild-type versus 35 mM in ClC-3-deficient hepatocytes at 15 min after internalization, with corresponding pH of 6.1 versus 6.5. Approximately 4-fold increased Cl- conductance was found in α2M-labeled endosomes isolated from hepatocytes of wild-type versus ClC-3 null mice. In contrast, Golgi acidification was not impaired in ClC-3-deficient hepatocytes. In transfected Chinese hamster ovary cells expressing ClC-3A, endosomal acidification and [Cl-] accumulation were enhanced. [Cl-] in α2M-labeled endosomes was 42 mM (control) versus 53 mM (ClC-3A) at 45 min, with corresponding pH 5.8 versus 5.2; [Cl-] in Tf-labeled endosomes at 15 min was 37 mM (control) versus 49 mM (ClC-3A) with pH 6.3 versus 5.9. Our results provide direct evidence for involvement of ClC-3 in endosomal acidification by Cl- shunting of the interior-positive membrane potential created by the vacuolar H+ pump. [ABSTRACT FROM AUTHOR] more...

Aquaporins (AQPs) are membrane proteins involved in water transport in many fluid-transporting tissues. Aquaporins AQP1, AQP4, and AQP9 have been identified in brain and hypothesized to participate in brain water homeostasis. Here we use reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry to describe the expression and immunolocalization of AQPs in adult mouse spinal cord. AQP4 was expressed in glial cells, predominantly in gray matter, and in astrocytic end-feet surrounding capillaries in spinal cord white matter. AQP9 expression extensively co-localized with glial fibrillary acidic protein-immunoreactive astrocytes, located predominantly in the white matter. AQP5 was detected by RT-PCR but not by immunohistochemical analysis. Interestingly, AQP8 was detected primarily in ependymal cells lining the fluid-filled central canal. The aquaporin expression pattern in spinal cord suggests involvement in water homeostasis and diseases associated with abnormal water fluxes such as spinal cord injury and syringomyelia. [Copyright &y& Elsevier] more...

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated epithelial Gl- channel that, when defective, causes cystic fibrosis. Screening of a collection of 100,000 diverse small molecules revealed four novel chemical classes of CFTR inhibitors with Ki < 10 µM, one of which (glycine hydrazides) had many active structural analogues. Analysis of a series of synthesized glycine hydrazide analogues revealed maximal inhibitory potency for N-(2-naphthalenyl) and 3,5-dibromo-2,4-dihydroxyphenyl substituents. The compound N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl) methylene] glycine hydrazide (GlyH-101) reversibly inhibited CFTR Cl- conductance in <1 min. Whole-cell current measurements revealed voltage-dependent CFTR block by GlyH-101 with strong inward rectification, producing an increase in apparent inhibitory constant Ki from 1.4 µM at +60 mV to 5.6 µM at -60 mV. Apparent potency was reduced by lowering extracellular Cl- concentration. Patch-clamp experiments indicated fast channel closures within bursts of channel openings, reducing mean channel open time from 264 to 13 ms (-60 mV holding potential, 5 µM GlyH-101). GlyH-101 inhibitory potency was independent of pH from 6.5-8.0, where it exists predominantly as a monovalent anion with solubility ∼1 mM in water. Topical GlyH-101 (10 µM) in mice rapidly and reversibly inhibited forskolininduced hyperpolarization in nasal potential differences. In a closed-loop model of cholera, intraluminal GlyH-101 (2.5 µg) reduced by ∼80% cholera toxin-induced intestinal fluid secretion. Compared with the thiazolidinone CFTR inhibitor GFTRinh-172, GlyH-101 has substantially greater water solubility and rapidity of action, and a novel inhibition mechanism involving occlusion near the external pore entrance. Glycine hydrazides may be useful as probes of GFTR pore structure, in creating animal models of GF, and as antidiarrheals in enterotoxicmediated secretory diarrheas. [ABSTRACT FROM AUTHOR] more...

We explored the role of AQP1, the only known aquaporin in corneal endothelium, on active fluid transport and passive osmotic water movements across corneal endothelial layers cultured from AQP1 null mice and wildtype mice. AQP1 null mice had grossly transparent corneas, just as wildtype mice. Endothelial cell layers grown on permeable supports transported fluid at rates of (in μl h−1 cm−2, n=9, mean±s.e.): 4·3±0·6, wildtype mice (MCE); 3·5±0·6, AQP1 null mice (KMCE; difference not significant). The osmotic water flow (also in μl h−1 cm−2) induced by a 100 mOsm sucrose gradient across MCE cell layers (8·7±0·6, n=8) was significantly greater than that across KMCE (5·7±0·7, n=6, p=0·007). When plated on glass coverslips, plasma membrane osmotic water permeability determined by light scattering was significantly higher for cells from wildtype vs. AQP1 null mice (in μm sec−1: 74±4, n=19 vs. 44±4 μm sec−1, n=11, p<0·001). Unexpectedly, after 10% hypo-osmotic challenge, the extent of the regulatory volume recovery was significantly reduced for AQP1 null mice cells (in%: MCE controls, 99±1, n=19, vs. KMCE: 64±5, n=11, p<0·001). Thus, as in other ‘low rate’ fluid transporting epithelia, deletion of AQP1 in mice corneal endothelium reduces osmotic water permeability but not active transendothelial fluid transport. However, that deletion impaired the extent of regulatory volume decrease after a hypo-osmotic challenge, suggesting a novel role for AQP1 in corneal endothelium. [Copyright &y& Elsevier] more...

The thiazolidinone CFTRinh-172 was identified recently as a potent and selective blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Here, we characterized the CFTRinh-172 inhibition mechanism by patch-clamp and short-circuit analysis using cells stably expressing wild-type and mutant CFTRs. CFTRinh-172 did not alter CFTR unitary conductance (8 pS), but reduced open probability by >90% with Ki&ap;0.6 μM. This effect was due to increased mean channel closed time without changing mean channel open time. Short-circuit current experiments indicated similar CFTRinh-172 inhibitory potency (Ki&ap;0.5 μM) for inhibition of Cl− current in wild-type, G551D, and G1349D CFTR; however, Ki was significantly reduced to 0.2 μM for ΔF508 CFTR. Our studies provide evidence for CFTR inhibition by CFTRinh-172 by a mechanism involving altered CFTR gating. [Copyright &y& Elsevier] more...

Deletion of Phe-508 (ΔF508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. ΔF508CFTR has defects in both channel gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective ΔF508-CFTR Cl[sup -] channel gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing ΔF508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which ΔF508-CFTR was targeted to the plasma membrane by culture at 27 °C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360-480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated ΔF508-CFTR Cl[sup -] conductance reversibly with K[sub d] < 100 nM. Single-cell voltage clamp analysis showed characteristic CFTR currents after ΔF508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normai physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physicai character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective ΔF508-CFTR gating was also demonstrated in human bronchial epithelial cells from a ΔF508 cystic fibrosis subject after 27 °C temperature rescue. In conjunction with correctors of defective ΔF508-CFTR processing, small molecule potentiators of defective ΔF508CFTR gating may be useful for therapy of cystic fibrosis caused by the ΔF508 mutation. [ABSTRACT FROM AUTHOR] more...

Presents a study that defined the role of chloride transport in fluid clearance in the distal airspaces of the intact mouse lung using wild-type and cystic fibrosis deltaF508 mice. Examination of role of transcellular sodium and chloride transport; Role of cystic fibrosis transmembrane conductance regulator in cyclic adenosine monophosphate-stimulated fluid absorption; Analysis of [sup22]sodium and [sup36]chlorine in mouse lung under isotopic conditions. more...

Measures the sodium concentrations by in vivo fluorescence in the pericryptal space of mouse descending colon. Application of surgical exposure of colonic mucosa; Visualization by confocal microscopy; Use of low-affinity sodium to indicate fluorescent sensitivity; Evidence of accumulated hypertonic absorbate in the periphery. more...

Investigates the role of the four major lung aquaporins in two key airway functions related to fluid transport. Measurement of airway surface liquid depth and salt concentration; Role of aquaporins in lower airway humidification; Upper airway osmotic water permeability and isosmolar fluid absorption. more...

Cerebral edema contributes significantly to morbidity and death associated with many common neurological disorders. However, current treatment options are limited to hyperosmolar agents and surgical decompression, therapies introduced more than 70 years ago. Here we show that mice deficient in aquaporin-4 (AQP4), a glial membrane water channel, have much better survival than wild-type mice in a model of brain edema caused by acute water intoxication. Brain tissue water content and swelling of pericapillary astrocytic foot processes in AQP4-deficient mice were significantly reduced. In another model of brain edema, focal ischemic stroke produced by middle cerebral artery occlusion, AQP4-deficient mice had improved neurological outcome. Cerebral edema, as measured by percentage of hemispheric enlargement at 24 h, was decreased by 35% in AQP4-deficient mice. These results implicate a key role for AQP4 in modulating brain water transport, and suggest that AQP4 inhibition may provide a new therapeutic option for reducing brain edema in a wide variety of cerebral disorders. [ABSTRACT FROM AUTHOR] more...

Provides information on a study measuring the diffusion of green fluorescent protein (GFP) expressed in the mitochondrial matrix of fibroblast, liver, skeletal muscle, and epithelial cell lines to test the protein density in mitochondrial matrix. Methodology and materials used to conduct the study; Information on the isolation and labeling of mitochondria; Results of the study; Discussion on the results. more...