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The thin layer of liquid at the surface of airway epithelium, the airway surface liquid (ASL), is important in normal airway physiology and in the pathophysiology of cystic fibrosis. At present, the best method to measure ASL depth involves scanning confocal microscopy after staining with an aqueous-phase fluorescent dye. We describe here a simple, noninvasive imaging method to measure ASL depth by reflectance imaging of an epithelial mucosa in which the surface is illuminated at a 45-degree angle by an elongated 13-[micro]m wide rectangular beam produced by a 670-nm micro-focus laser. The principle of the method is that air--liquid, liquid--liquid, and liquid--cell interfaces produce distinct specular or diffuse reflections that can be imaged to give a micron-resolution replica of the mucosal surface. The method was validated using fluid layers of specified thicknesses and applied to measure ASL depth in cell cultures and ex vivo fragments of pig trachea. In addition, the method was adapted to measure transepithelial fluid transport from the dynamics of fluid layer depth. Compared with confocal imaging, ASL depth measurement by surface laser reflectance microscopy does not require dye staining or costly instrumentation, and can potentially be adapted for in vivo measurements using fiberoptics. doi/10.1085/jgp.201010490

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated epithelial [Cl.sup.-] channel that, when defective, causes cystic fibrosis. Screening of a collection of 100,000 diverse small molecules revealed tour novel chemical classes of CFTR inhibitors with [K.sub.i] 10 [micro]M, 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 [C1.sup.-] conductance in KEY WORDS: cystic fibrosis * diarrhea * high-throughput screening * patch-clamp * drug discovery

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. 107:317-324.). 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.+]], [[C1.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 [+ or -] 2 mM, [[C1.sup.-]] was 123 [+ or -] 4 mM and pH was 7.28 [+ or -] 0.07, and not dependent on airway size (< 100- to >250-[micro]m diameter), ENaC inhibition by amiloride, or CFTR inhibition by the thiazolidinone CFT[R.sub.inh]-172. ASL depth was 8-35 [micro]m depending on airway size, substantially less than that in mouse trachea of ~55 [micro]m, 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. KEY WORDS: ASL * bronchioles * ratio imaging * lung * fluorescence microscopy

Aquaporin (AQP) water channels are expressed in the eye at sites of aqueous fluid production and outflow: AQP1 and AQP4 in nonpigmented ciliary epithelium, and AQP1 in trabecular meshwork endothelium. Novel methods were developed to compare aqueous fluid dynamics in wild-type mice versus mice lacking AQP1 and/or AQP4. Aqueous fluid production was measured by in vivo confocal microscopy after transcorneal iontophoretic introduction of fluorescein. Intraocular pressure (IOP), outflow, and anterior chamber compliance were determined from pressure measurements in response to fluid infusions using micropipettes. Aqueous fluid volume and [[Cl.sup.-]] were assayed in samples withdrawn by micropipettes. In wild-type mice (CD1 genetic background, age 4-6 wk), IOP was 16.0 [+ or -] 0.4 mmHg (SE), aqueous fluid volume 7.2 [+ or -] 0.3 [micro]l, fluid production 3.6 [+ or -] 0.2 [micro]l/h, fluid outflow 0.36 [+ or -] 0.06 [micro]l/h/mmHg, and compliance 0.036 [+ or -] 0.006 [micro]l/mmHg. IOP was significantly decreased by up to 1.8 mmHg (P < 0.002) and fluid production by up to 0.9 [micro]l/h in age/litter-matched mice lacking AQP1 and/or AQP4 (outbred CD1 and inbred C57/b16 genetic backgrounds). However, AQP deletion did not significantly affect outflow, [[Cl.sup.-]], volume, or compliance. These results provide evidence for the involvement of AQPs in intraocular pressure regulation by facilitating aqueous fluid secretion across the ciliary epithelium. AQP inhibition may thus provide a novel approach for the treatment of elevated IOP. KEY WORDS: water transport * AQP1 * AQP4 * anterior chamber * transgenic mice

The active absorption of fluid from the airspaces of the lung is important for the resolution of clinical pulmonary edema. Although ENaC channels provide a major route for [Na.sup.+] absorption, the route of [Cl.sup.-] transport has been unclear. We applied a series of complementary approaches to define the role of [Cl.sup.-] transport in fluid clearance in the distal airspaces of the intact mouse lung, using wild-type and cystic fibrosis [DELTA]F508 mice. Initial studies in wild-type mice showed marked inhibition of fluid clearance by [Cl.sup.-] channel inhibitors and [Cl.sup.-] ion substitution, providing evidence for a transcellular route for [Cl.sup.-] transport. In response to cAMP stimulation by isoproterenol, clearance was inhibited by the CFTR inhibitor glibenclamide in both wild-type mice and the normal human lung. Although isoproterenol markedly increased fluid absorption in wild-type mice, there was no effect in [DELTA]F508 mice. Radioisotopic clearance studies done at 23 [degrees] C (to block active fluid absorption) showed ~20% clearance of [sup.22]Na in 30 min both without and with isoproterenol. However, the clearance of [sup.36]Cl was increased by 47% by isoproterenol in wild-type mice but was not changed in [DELTA]F508 mice, providing independent evidence for involvement of CFTR in cAMP-stimulated [Cl.sup.-] transport. Further, CFTR played a major role in fluid clearance in a mouse model of acute volume-overload pulmonary edema. After infusion of saline (40% body weight), the lung wet-to-dry weight ratio increased by 28% in wild-type versus 64% in [DELTA]F508 mice. These results provide direct evidence for a functionally important role for CFTR in the distal airspaces of the lung. KEY WORDS: pulmonary edema * cystic fibrosis * lung epithelium * cAMP * lung fluid balance

A method to measure osmotic water permeabilty (Pf) of plasma membranes in cell layers was developed, and applied to cells and epithelia expressing molecular water channels. The integrated intensity f monochromatic light in phase contrast or dark field microscope was found to depend on relative cell voluem. The results confirm light microscopy with spatial filtering as a technically simple and quantitative way of measuring water permeability in cell layers.

A surface fluorescence method to quantify transalveolar transport of water, protons and solutes in lungs gives sensitive, time resolved biophysical data from intact organs. The lungs are removed and perfused via the pulmonary artery. The airspace is filled with a solution containing a membrane impermeant fluorescent indicator. Fluorescence signals from the lung surfaces originate from the indicator and provide data on intraalveolar fluid composition. The application of this method shows that the mouse lung has high water permeability which may involve water channels.

A study was conducted to validate methodology for measurement of osmotic water permeability in cultured cells grown on a glass coverslip. Light scattering methods were used and found to provide quantitative information about plasma membrane permeability with suitable calibration. Another aspect of the study evaluated the role of glucose in the water pathway. Results show that glucose transporters are not involved in the water transport pathway.

Several aquaporin-type water channels are expressed in mammalian airways and lung: AQP1 in microvascular endothelia, AQP3 in upper airway epithelia, AQP4 in upper and lower airway epithelia, and AQP5 in alveolar epithelia. Novel quantitative methods were developed to compare airway fluid transport-related functions in wild-type mice and knockout mice deficient in these aquaporins. Lower airway humidification, measured from the moisture content of expired air during mechanical ventilation with dry air through a tracheotomy, was 54-56% efficient in wild-type mice, and reduced by only 3-4% in AQP1/AQP5 or AQP3/AQP4 double knockout mice. Upper airway humidification, measured from the moisture gained by dry air passed through the upper airways in mice breathing through a tracheotomy, decreased from 91 to 50% with increasing ventilation from 20 to 220 ml/min, and reduced by 3-5% in AQP3/AQP4 knockout mice. The depth and salt concentration of the airway surface liquid in trachea was measured in vivo using fluorescent probes and confocal and ratio imaging microscopy. Airway surface liquid depth was 45 [+ or -] 5 [micro]m and [[Na.sup.+]] was 115 [+ or -] 4 mM in wild-type mice, and not significantly different in AQP3/AQP4 knockout mice. Osmotic water permeability in upper airways, measured by an in vivo instillation/sample method, was reduced by ~40% by AQP3/AQP4 deletion. In doing these measurements, we discovered a novel amiloride-sensitive isosmolar fluid absorption process in upper airways (13% in 5 min) that was not affected by aquaporin deletion. These results establish the fluid transporting properties of mouse airways, and indicate that aquaporins play at most a minor role in airway humidification, ASL hydration, and isosmolar fluid absorption. KEY WORDS: water permeability * trachea * airway surface liquid * ventilator * fluorescence microscopy

The airway surface liquid (ASL) is the thin layer of fluid coating the luminal surface of airway epithelial cells at an air interface. Its composition and osmolality are thought to be important in normal airway physiology and in airway diseases such as asthma and cystic fibrosis. The determinants of ASL osmolality include epithelial cell solute and water transport properties, evaporative water loss, and the composition of secreted fluids. We developed a noninvasive approach to measure ASL osmolality using osmotically sensitive 400-nm-diam liposomes composed of phosphatidylcholine/cholesterol/polyethylene glycol-phosphatidylcholine (1:0.3:0.08 molar ratio). Calcein was encapsulated in the liposomes at self-quenching concentrations (30 mM) as a volume-sensitive marker, together with sulforhodamine 101 (2 mM) as a volume-insensitive reference. Liposome calcein/sulforhodamine 101 fluorescence ratios responded rapidly ([is less than] 0.2 s) and stably to changes in solution osmolality. ASL osmolality was determined from calcein/sulforhodamine 101 fluorescence ratios after addition of microliter quantities of liposome suspensions to the ASL. In bovine airway epithelial cells cultured on porous supports at an air-liquid interface, ASL thickness (by confocal microscopy) was 22 [micro]m and osmolality was 325 [+ or -] 12 mOsm. In anesthetized mice in which a transparent window was created in the trachea, ASL thickness was 55 [micro]m and osmolality was 330 [+ or -] 36 mOsm. ASL osmolality was not affected by pharmacological inhibition of CFTR in airway cell cultures or by genetic deletion of CFTR in knockout mice. ASL osmolality could be increased substantially to [is greater than] 400 mOsm by exposure of the epithelium to dry air; the data were modeled mathematically using measured rates of osmosis and evaporative water loss. These results establish a ratio imaging method to map osmolality in biological compartments. ASL fluid is approximately isosmolar under normal physiological conditions, but can become hyperosmolar when exposed to dry air, which may induce cough and airway reactivity in some patients. KEY WORDS: water permeability * cystic fibrosis * fluorescence self-quenching * trachea * CFTR

The mammalian peripheral lung contains at least three aquaporin (AQP) water channels: AQP1 in microvascular endothelia, AQP4 in airway epithelia, and AQP5 in alveolar epithelia. In this study, we determined the role of AQP4 in airspace-to-capillary water transport by comparing water permeability in wild-type mice and transgenic null mice lacking AQP1, AQP4, or AQP1/AQP4 together. An apparatus was constructed to measure lung weight continuously during pulmonary artery perfusion of isolated mouse lungs. Osmotically induced water flux ([J.sub.v]) between the airspace and capillary compartments was measured from the kinetics of lung weight change in saline-filled lungs in response to changes in perfusate osmolality. [J.sub.v] in wild-type mice varied linearly with osmotic gradient size (4.4 x [10.sup.-5] [cm.sup.3] [s.sup.-1] [mOsm.sup.-1]) and was symmetric, independent of perfusate osmolyte size, weakly temperature dependent, and decreased 11-fold by AQP1 deletion. Transcapillary osmotic water permeability was greatly reduced by AQP1 deletion, as measured by the same method except that the airspace saline was replaced by an inert perfluorocarbon. Hydrostatically induced lung edema was characterized by lung weight changes in response to changes in pulmonary arterial inflow or pulmonary venous outflow pressure. At 5 cm H20 outflow pressure, the filtration coefficient was 4.7 [cm.sup.3] [s.sup.-1] [mOsm.sup.-1] and reduced 1.4-fold by AQP1 deletion. To study the role of AQP4 in lung water transport, AQP1/AQP4 double knockout mice were generated by crossbreeding of AQP1 and AQP4 null mice. [J.sub.v] were ([cm.sup.3] [s.sup.-1] [mOsm.sup.-1] x [10.sup.-5], SEM, n = 7-12 mice): 3.8 [+ or -] 0.4 (wild type), 0.35 [+ or -] 0.02 (AQP1 null), 3.7 [+ or -] 0.4 (AQP4 null), and 0.25 [+ or -] 0.01 (AQP1/AQP4 null). The significant reduction in [P.sub.f] in AQP1 vs. AQP1/AQP4 null mice was confirmed by an independent pleural surface fluorescence method showing a 1.6 [+ or -] 0.2-fold (SEM, five mice) reduced [P.sub.f] in the AQP1/AQP4 double knockout mice vs. AQP1 null mice. These results establish a simple gravimetric method to quantify osmosis and filtration in intact mouse lung and provide direct evidence for a contribution of the distal airways to airspace-to-capillary water transport. KEY WORDS: water permeability * airways * transgenic mouse * membrane transport * aquaporin-1

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]

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.

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.

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.