In Situ Measurement of Airway Surface Liquid [K+] Using a Ratioable K+-sensitive Fluorescent Dye

The airway surface liquid (ASL) is the thin fluid layer lining airway surface epithelial cells, whose volume and composition are tightly regulated and may be abnormal in cystic fibrosis (CF). We synthesized a two-color fluorescent dextran to measure ASL [K⁺], TAC-Lime-dextran-TMR, consisting of a green-fluorescing triazacryptand K⁺ ionophore-Bodipy conjugate, coupled to dextran, together with a red fluorescing tetramethylrhodamine reference chromophore. TAC-Lime-dextran-TMR fluorescence was K⁺-selective, increasing >4-fold with increasing [K⁺] from 0 to 40 mm. In well differentiated human airway epithelial cells, ASL [K⁺] was 20.8 ± 0.3 mm and decreased by inhibition of the Na⁺/K⁺ pump (ouabain), ENaC (amiloride), CF transmembrane conductance regulator (CFTR_inh-172), or K⁺ channels (TEA or XE991). ASL [K⁺] was increased by forskolin but not affected by Na⁺/K⁺/2Cl⁻ cotransporter inhibition (bumetanide). Functional and expression studies indicated the involvement of [K⁺] channels KCNQ1, KCNQ3, and KCNQ5 as determinants of ASL [K⁺]. [K⁺] in CF cultures was similar to that in non-CF cultures, suggesting that abnormal ASL [K⁺] is not a factor in CF lung disease. In intact airways, ASL [K⁺] was also well above extracellular [K⁺]: 22 ± 1 mm in pig trachea ex vivo and 16 ± 1 mm in mouse trachea in vivo. Our results provide the first noninvasive measurements of [K⁺] in the ASL and indicate the involvement of apical and basolateral membrane ion transporters in maintaining a high ASL [K⁺].The airway surface liquid (ASL)2 is the thin layer of aqueous fluid that lines the mucosal surface of the airways, forming the interface between airway epithelial cells and the gas phase. The ASL contains water, ions, and macromolecules. It is believed that ASL volume and composition are tightly regulated to maintain a nonviscous fluid layer for mucociliary clearance by underlying epithelial cells and to support the intrinsic antimicrobial function of defensins and other macromolecules (1, 2). Abnormalities in ASL volume and/or composition are proposed to be important in the pathogenesis of cystic fibrosis (CF) lung disease (3). The ASL is formed by a combination of fluid secretion by airway submucosal glands, convective fluid transport up the airway tree, and water/ion transport by airway surface epithelial cells, the latter likely playing a key role in active regulation of ASL volume and ionic composition.Although early data suggested that salt concentration in the ASL is low in normal airways (4), the current view is that the ASL is approximately isotonic in both normal and CF airways (5–7). A concern with older measurements of ASL composition involving fluid collection by filter paper or microcapillaries is perturbation of the airway surface and the sampling, by capillary forces, of more fluid than contained in the very thin (tens of microns) ASL layer. Studies using ion-sensitive microelectrodes, although technically demanding and requiring direct contact with the ASL, provided evidence for a nearly isotonic ASL (5). Our laboratory developed ratioable fluorescent dyes to measure ASL [Na⁺] and [Cl⁻], in which the ASL was fluorescently stained for determination of ion concentrations by ratio imaging microscopy (7). ASL salt concentration ([Na⁺] and [Cl⁻]) was found to be approximately isotonic in airway epithelial cell cultures, mouse trachea and small airways, and ex vivo human airways, without differences in CFTR deficiency (7, 8). We also found the ASL to be approximately isosmolar with serum using fluorescent, osmotically sensitive liposomes (9).Relatively little is known about ASL potassium concentration ([K⁺]) or its regulation. As diagrammed in Fig. 1A, transcellular transport of K⁺ is believed to involve the coordinated activities of a Na⁺/K⁺ pump, Na⁺/K⁺/2Cl⁻ cotransporter, and K⁺ channel(s) at the basolateral membrane and a H⁺/K⁺ pump and K⁺ channel(s) at the apical membrane. The airway epithelium also has significant paracellular ion permeability. There is evidence for functional expression of several types of K⁺ channels in cell lines derived from airways/lung, including Ca²⁺-activated, cAMP-activated, and voltage-activated K⁺ channels (10–13). Steady state [K⁺] in a stationary ASL (without fluid convection) should depend on the activities of cell membrane K⁺ pumps and ion transporters, as well as non-K⁺ ion channels, such as CFTR and ENaC, which are involved in establishing membrane potentials and thus the electrochemical driving forces for K⁺ transport.Open in a separate windowFIGURE 1.Cell model and perfusion chamber for measurements of ASL K⁺ concentration. A, schematic of airway epithelium showing principal ion transporters on the apical and basolateral plasma membranes, and paracellular pathway. B, schematic of perfusion chamber. Cells on a porous filter, facing upward, are imaged from above after fluorescent dye staining of the ASL. The under surface of the porous filter is perfused continuously. C, short circuit current (I_sc) in HBE cell monolayers in response to the additions of amiloride (10 μm), forskolin (10 μm), CFTR_inh-172 (10 μm), ATP (100 μm), and CaCC_inh-A01 (30 μm), a CaCC-specific inhibitor. D, transepithelial PD in response to amiloride, forskolin, CFTR_inh-172, ATP, and CaCC_inh-A01. The data in C and D are representative of four sets of measurements.The purpose of this study was to develop a noninvasive fluorescence method to measure [K⁺] in the ASL and to establish the major determinants of [K⁺] regulation. Following several years of synthetic chemistry, we developed a series of water-soluble K⁺ sensors, the first being TAC-Red, in which K⁺ binding to a triazacryptand (TAC) K⁺ ionophore results in fluorescence enhancement of a conjugated xanthylium chromophore by a charge transfer quenching mechanism (14). TAC-Red was used to follow K⁺ waves in the extracellular space in brain in a neuroexcitation model of cortical spreading depression. Second generation K⁺ sensors of different colors, TAC-Crimson (15) and TAC-Lime (16), work by a similar K⁺-sensing mechanism but utilize different chromophores. These indicators are selective for K⁺ under physiological conditions and respond to changes in [K⁺] in milliseconds or less (15). For the measurements here, we synthesized a dextran conjugate containing TAC-Lime, which has K⁺-sensitive green fluorescence, and tetramethylrhodamine, a reference chromophore with K⁺-insensitive red fluorescence. The indicator allowed technically straightforward determination of ASL [K⁺] in cell culture and in vivo models by fluorescence ratio imaging.     

Effects of nestboxes on the breeding biology of Southern House Wrens Troglodytes aedon bonariae in the southern temperate zone

Nestboxes are known to increase clutch size, enhance breeding success and affect the social mating system of several cavity nesters. Although in recent years various cavity nesters have been studied in nestboxes in South America, the effects of boxes on the biology of the study species are unknown. We evaluated the effects of nestboxes on the breeding biology and social mating system of Southern House Wrens Troglodytes aedon bonariae by comparing birds breeding in nestboxes and tree cavities in two cattle ranches in Buenos Aires Province, Argentina. Southern House Wrens nesting in boxes had higher breeding success but, contrary to studies on the temperate zone, we did not find differences in clutch size between Wrens breeding in nestboxes and tree cavities. The main causes of nest failure in tree cavities were nest predation and flooding of the cavity (70 and 23% of the failures, respectively) while in nestboxes predation and desertion were the most important causes of failure (38 and 34% of the failures, respectively). The social mating system of Southern House Wrens is monogamy with biparental care, and neither was affected by the boxes. Males did not attract secondary females to additional nestboxes; however, nestboxes are safer breeding sites than tree cavities, and females seemed to prefer males with nestboxes on their territory. These results suggest that nest quality alone might be not enough for secondary females to accept polygyny.     

Predicting deleterious nsSNPs: an analysis of sequence and structural attributes

Background  

There has been an explosion in the number of single nucleotide polymorphisms (SNPs) within public databases. In this study we focused on non-synonymous protein coding single nucleotide polymorphisms (nsSNPs), some associated with disease and others which are thought to be neutral. We describe the distribution of both types of nsSNPs using structural and sequence based features and assess the relative value of these attributes as predictors of function using machine learning methods. We also address the common problem of balance within machine learning methods and show the effect of imbalance on nsSNP function prediction. We show that nsSNP function prediction can be significantly improved by 100% undersampling of the majority class. The learnt rules were then applied to make predictions of function on all nsSNPs within Ensembl.     

Tracking of quantum dot-labeled CFTR shows near immobilization by C-terminal PDZ interactions

Mutations in cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, cause cystic fibrosis. To investigate interactions of CFTR in living cells, we measured the diffusion of quantum dot-labeled CFTR molecules by single particle tracking. In multiple cell lines, including airway epithelia, CFTR diffused little in the plasma membrane, generally not moving beyond 100-200 nm. However, CFTR became mobile over micrometer distances after 1) truncations of the carboxy terminus, which contains a C-terminal PDZ (PSD95/Dlg/ZO-1) binding motif; 2) blocking PDZ binding by C-terminal green fluorescent protein fusion; 3) disrupting CFTR association with actin by expression of a mutant EBP50/NHERF1 lacking its ezrin binding domain; or 4) skeletal disruption by latrunculin. CFTR also became mobile when the cytoskeletal adaptor protein binding capacity was saturated by overexpressing CFTR or its C terminus. Our data demonstrate remarkable and previously unrecognized immobilization of CFTR in the plasma membrane and provide direct evidence that C-terminal coupling to the actin skeleton via EBP50/ezrin is responsible for its immobility.     

<Emphasis Type="Italic">In vivo</Emphasis> assessment of the impedance ratio method used in electronic foramen locators

Background  

The results of an in vivo study on the "ratio method" used in electronic foramen locators (EFL) are presented. EFLs are becoming widely used in the determination of the working length (WL) during the root canal treatment. The WL is the distance from a coronal reference point to the point at which canal preparation and filling should terminate. The "ratio method" was assessed by many clinicians with the aim of determining its ability to locate the apical foramen (AF). Nevertheless, in vivo studies to assess the method itself and to explain why the "ratio method" is able to locate the apical foramen and is unable to determine intermediate distances were not published so far.     

Slowed diffusion in tumors revealed by microfiberoptic epifluorescence photobleaching

It has not been possible to measure diffusion deep in solid tissues such as tumors because of the limited light penetration of conventional optical techniques. Here we report a microfiberoptic epifluorescence photobleaching (MFEP) method in which photobleaching is done by laser epi-illumination through a multimode fiberoptic whose micron-sized tip can be introduced deep into tissues. We applied MFEP to measure the diffusion of fluorescent macromolecules in tumors in living mice, at depths well beyond those accessible by surface optical measurements. Macromolecule diffusion was slowed about twofold within 200 microm of the surface of a solid tumor, but was slowed greater than tenfold beyond 500 microm. Our results reveal a remarkable and previously unrecognized slowing of diffusion deep in tumors, which correlated with the differing tissue architectures of tumor periphery versus core, and with altered tumor vasculature produced by aquaporin-1 deletion. MFEP should have wide applications for measuring diffusion in organs, solid tumors and other light-inaccessible tissue masses.     

Altered gravity downregulates aquaporin-1 protein expression in choroid plexus.

Aquaporin-1 (AQP1) is a water channel expressed abundantly at the apical pole of choroidal epithelial cells. The protein expression was quantified by immunocytochemistry and confocal microscopy in adult rats adapted to altered gravity. AQP1 expression was decreased by 64% at the apical pole of choroidal cells in rats dissected 5.5-8 h after a 14-day spaceflight. AQP1 was significantly overexpressed in rats readapted for 2 days to Earth's gravity after an 11-day flight (48% overshoot, when compared with the value measured in control rats). In a ground-based model that simulates some effects of weightlessness and alters choroidal structures and functions, apical AQP1 expression was reduced by 44% in choroid plexus from rats suspended head down for 14 days and by 69% in rats suspended for 28 days. Apical AQP1 was rapidly enhanced in choroid plexus of rats dissected 6 h after a 14-day suspension (57% overshoot, in comparison with control rats) and restored to the control level when rats were dissected 2 days after the end of a 14-day suspension. Decreases in the apical expression of choroidal AQP1 were also noted in rats adapted to hypergravity in the NASA 24-ft centrifuge: AQP1 expression was reduced by 47% and 85% in rats adapted for 14 days to 2 G and 3 G, respectively. AQP1 is downregulated in the apical membrane of choroidal cells in response to altered gravity and is rapidly restored after readaptation to normal gravity. This suggests that water transport, which is partly involved in the choroidal production of cerebrospinal fluid, might be decreased during spaceflight and after chronic hypergravity.     

Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels.

Aquaporin-5 (AQP5) is a water-selective transporting protein expressed in epithelial cells of serous acini in salivary gland. We generated AQP5 null mice by targeted gene disruption. The genotype distribution from intercross of founder AQP5 heterozygous mice was 70:69:29 wild-type:heterozygote:knockout, indicating impaired prenatal survival of the null mice. The knockout mice had grossly normal appearance, but grew approximately 20% slower than litter-matched wild-type mice when placed on solid food after weaning. Pilocarpine-stimulated saliva production was reduced by more than 60% in AQP5 knockout mice. Compared with the saliva from wild-type mice, the saliva from knockout mice was hypertonic (420 mosM) and dramatically more viscous. Amylase and protein secretion, functions of salivary mucous cells, were not affected by AQP5 deletion. Water channels AQP1 and AQP4 have also been localized to salivary gland; however, pilocarpine stimulation studies showed no defect in the volume or composition of saliva in AQP1 and AQP4 knockout mice. These results implicate a key role for AQP5 in saliva fluid secretion and provide direct evidence that high epithelial cell membrane water permeability is required for active, near-isosmolar fluid transport.