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Eosinophils are abundant in inflammatory demyelinating lesions in neuromyelitis optica (NMO). We used cell culture, ex vivo spinal cord slices, and in vivo mouse models of NMO to investigate the [...]

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

Song Y, Namkung W, Nieison DW, Lee J, Finkbeiner WE, Verkman AS. Airway surface liquid depth measured in ex vivo fragments of pig and human trachea: dependence on [Na.sup.+] and [Cl.sup.-] channel function. Am J Physiol Lung Cell Mol Physiol 297: L1131-L1140, 2009. First published October 9, 2009; doi: 10.1152/ajplung.00085.2009.--The airway surface liquid (ASL) is the thin fluid layer lining the airways whose depth may be reduced in cystic fibrosis. Prior measurements of ASL depth have been made in airway epithelial cell cultures. Here, we established methodology to measure ASL depth to -1-[micro]m accuracy in ex vivo fragments of freshly obtained human and pig tracheas. Airway fragments were mounted in chambers designed for perfusion of the basal surface and observation of the apical, fluorescently stained ASL by scanning confocal microscopy using a high numerical aperture lens immersed in perfluorocarbon. Measurement accuracy was verified using standards of specified fluid thickness. ASL depth in well-differentiated primary cultures of human nasal respiratory epithelium was 8.0 [+ or -] 0.5 [micro]m (SE 10 cultures) under basal conditions, 8.4 [+ or -] 0.4 [micro]m following ENaC inhibition by amiloride, and 14.5 [+ or -] 1.2 [micro]m following CFTR stimulation by cAMP agonists. ASL depth in human trachea was 7.0 [+ or -] 0.7/[micro]m under basal conditions, 11.0 [+ or - ] 1.7 [mciro]m following amiloride, 17.0 [+ or -] 3.4 [micro]m following cAMP agonists, and 7.1 [+ or -] 0.5 [micro]m after CFTR inhibition. Similar results were found in pig trachea. This study provides the first direct measurements of ASL depth in intact human airways and indicates the involvement of ENaC sodium channels and CFTR chloride channels in determining ASL depth. We suggest that CF lung disease may be caused by the inability of CFTR-deficient airways to increase their ASL depth transiently following secretory stimuli that in non-CF airways produce transient increases in ASL depth. cystic fibrosis; fluorescence microscopy; cystic fibrosis transmembrahe conductance regulator doi: 10.1152/ajplung.00085.2009.

The cellular mechanisms by which loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel produce cystic fibrosis (CF) lung disease remain uncertain. Defective organellar function has been proposed as an important determinant in the pathogenesis of CF lung disease. According to one hypothesis, reduced CFTR chloride conductance in organelles in CF impairs their acidification by preventing chloride entry into the organelle lumen, which is needed to balance the positive charge produced by proton entry. According to a different hypothesis, CFTR mutation hyperacidifies organelles by an indirect mechanism involving unregulated sodium efflux through epithelial sodium channels. There are reports of defective Golgi, endosomal and lysosomal acidification in CF epithelial cells, defective phagolysosomal acidification in CF alveolar macrophages, and organellar hyperacidification in CF respiratory epithelial cells. The common theme relating too high or low organellar pH to cellular dysfunction and CF pathogenesis is impaired functioning of organellar enzymes, such as those involved in ceramide metabolism and protein processing in epithelial cells and antimicrobial activity in alveolar macrophages. We review here the evidence for defective organellar acidification in CF. Significant technical and conceptual concerns are discussed regarding the validity of data showing too high/low organellar pH in CF cells, and rigorous measurements of organellar pH in CF cells are reviewed that fail to support defective organellar acidification in CF. Indeed, there is an expanding body of evidence supporting the involvement of non-CFTR chloride channels in organellar acidification. We conclude that biologically significant involvement of CFTR in organellar acidification is unlikely. cystic fibrosis transmembrane conductance regulator; lysosome; endosome; Golgi; chloride channel

Water transport across gallbladder epithelium is driven by osmotic gradients generated from active salt absorption and secretion. Aquaporin (AQP) water channels have been proposed to facilitate transepithelial water transport in gallbladder and to modulate bile composition. We found strong AQP1 immunofluorescence at the apical membrane of mouse gallbladder epithelium. Transepithelial osmotic water permeability ([P.sub.f]) was measured in freshly isolated gallbladder sacs from the kinetics of luminal calcein self-quenching in response to an osmotic gradient. [P.sub.f] was very high (0.12 cm/s) in gallbladders from wild-type mice, cAMP independent, and independent of osmotic gradient size and direction. Although gallbladders from AQP1 knockout mice had similar size and morphology to those from wild-type mice, their [P.sub.f] was reduced by ~10-fold. Apical plasma membrane water permeability was greatly reduced in AQP1-deficient gallbladders, as measured by cytoplasmic calcein quenching in perfluorocarbon-filled, inverted gallbladder sacs. However, neither bile osmolality nor bile salt concentration differed in gallbladders from wild-type vs. AQP1 knockout mice. Our data indicate constitutively high water permeability in mouse gallbladder epithelium involving transcellular water transport through AQP1. The similar bile salt concentration in gallbladders from AQP1 knockout mice argues against a physiologically important role for AQP1 in mouse gallbladder. AQP1; transgenic mice; gastrointestinal system

The binding kinetics of [K.sup.} with TAC-Red and a new, longer-wavelength sensor, TAC-Crimson is quantified using fluorescence correlation spectroscopy. The rapid [K.sup.} binding kinetics with triazacryptand-based sensors support their utility for measuring changes in [K.sup.} concentrations during rapid neural signaling and ion channel gating.

Aquaporin (AQP)8-facilitated transport of NH3 has been suggested recently by increased NH3 permeability in Xenopus oocytes and yeast expressing human or rat AQP8. We tested the proposed roles of AQP8-facilitated N[H.sub.3] transport in mammalian physiology by comparative phenotype studies in wild-type vs. AQP8-null mice. AQP8-facilitated N[H.sub.3] transport was confirmed in mammalian cell cultures expressing rat or mouse AQP8, in which the fluorescence of a pH-sensing yellow fluorescent protein was measured in response to ammonia (N[H.sub.3]/N[H.sup.+.sub.4]) gradients. Relative AQP8 single-channel N[H.sub.3]-to-water permeability was ~0.03. AQPS-facilitated N[H.sub.3] and water permeability in a native tissue was confirmed in membrane vesicles isolated from testes of wild-type vs. AQPS-null mice, in which BCECF was used as an intravesicular pH indicator. A series of in vivo studies were done in mice, including 1) serum ammonia measurements before and after ammonia infusion, 2) renal ammonia clearance, 3) colonic ammonia absorption, and 4) liver ammonia accumulation and renal ammonia excretion after acute and chronic ammonia loading. Except for a small reduction in hepatic ammonia accumulation and increase in ammonia excretion in AQP8-null mice loaded with large amounts of ammonia, there were no significant differences in wild-type vs. AQPS-null mice. Our results support the conclusion that AQP8 can facilitate N[H.sub.3] transport but provide evidence against physiologically significant AQP8-facilitated N[H.sub.3] transport in mice. water transport; transgenic mouse; liver doi:10.1152/ajpcell.00057.2006.

Transgenic mouse models of defective urinary concentrating ability produced by deletion of various membrane transport or receptor proteins, including aquaporin-2 (AQP2), are associated with neonatal mortality from polyuria. Here, we report an inducible mouse model of AQP2 gene deletion with severe polyuria in adult mice. LoxP sequences were inserted into introns 1 and 2 in the mouse AQP2 gene by homologous recombination in embryonic stem cells. Mating of germ-line AQP2-loxP mice with tamoxifen-inducible Cre-expressing mice produced offspring with inducible homozygous Cre-AQP2-loxP, which had a normal phenotype. Tamoxifen injections over 10 days resulted in AQP2 gene excision, with undetectable full-length AQP2 transcript in kidney and a >95% reduction in immunoreactive AQP2 protein. Urine osmolality decreased from ~2,000 to water transport; water channel; transgenic mouse; NDI; polyuria doi:10.1152/ajprenal.00494.2005

Water accumulation in the heart is important in ischemia-reperfusion injury and operations performed by using cardiopulmonary bypass, with cardiac dysfunction associated with myocardial edema being the principal determinant of clinical outcome. As an initial step in determining the role of aquaporin (AQP) water channels in myocardial edema, we have assessed the myocardial expression of AQPs in humans, rats, and mice. RT-PCR revealed expression of AQP-1, -4, -6, -7, -8, and -11 transcripts in the mouse heart. AQP-1, -6, -7, and -11 mRNAs were found in the rat heart as well as low levels of AQP-4 and -9. Human hearts contained AQP-1, -3, -4, -5, -7, -9, -10, and -11 mRNAs. AQP-1 protein expression was confirmed by Western blot analysis in all three species. AQP-4 protein was detected in the mouse heart but not in the rat or human heart. To determine the potential functional consequences of myocardial AQP expression, water permeability was measured in plasma membrane vesicles from myocardial cells of wild-type versus various AQP knockout mice. Water permeability was reduced by AQP-1 knockout but not by AQP-4 or AQP-8 knockout. With the use of a model of isolated rat heart perfusion, it was found that osmotic and ischemic stresses are not associated with changes in AQP-1 or AQP-4 expression. These studies support a possible functional role of AQP-1 in myocardium but indicate that early adaptations to osmotic and ischemic stress do not involve transcriptional or posttranslational AQP-1 regulation. aquaporin knockout; cardiac myocyte; cardiopulmonary bypass doi:10.1152/ajpheart.00090.2006

Prior studies have shown that fluid secretions from airway submucosal glands in cystic fibrosis (CF) are reduced and hyperviscous, possibly contributing to the pathogenesis of CF airway disease. Because the CF transmembrane conductance regulator (CFTR) protein can transport both chloride and bicarbonate, we investigated whether gland fluid pH is abnormal in early CF, using nasal biopsies from pediatric subjects having minimal CF lung disease. Gland fluid pH, measured in freshly secreted droplets under oil stained with BCECF-dextran, was 6.57 [+ or -] 0.09 (mean [+ or -] SE) in biopsies from six CF subjects, significantly lower than 7.18 [+ or -] 0.06 in eight non-CF biopsies (P < 0.01). To rule out the possibility that the apparent gland fluid hyperacidity in CF results from modification of fluid pH by the airway surface, a microcannulation method was used to measure pH in fluid exiting gland orifices. In pig trachea and human bronchi, gland fluid pH was reduced by up to 0.45 units by CFTR inhibitors, but was not affected by amiloride. Acid base transport in the surface epithelium of pig trachea was studied from pH changes in 300-nl fluid droplets deposited onto the oil-covered airway surface. The droplets had specified ionic composition/pH and/or contained transporter activators/inhibitors. We found evidence for CFTR-dependent bicarbonate transport by the tracheal surface epithelium as well as ATP/bistamine-stimulated proton secretion, but not for sodium/proton or chloride/bicarbonate exchange. These results provide evidence for intrinsic hyperacidity in CF gland fluid secretions, which may contribute to CF airway pathology. cystic fibrosis transmembrane conductance regulator; airway; fluorescence microscopy; pH regulation

Fluorescence correlation spectroscopy (FCS) was applied to study diffusion and interactions of fluorescently labeled macromolecules in complex biological systems. The computational method is applicable for stimulating FCS measurement on system complicated by flourophore interactions or molecular crowding, and experimental protocols.

Previous studies in intact lung suggest that CFTR may play a role in cAMP-regulated fluid transport from the distal air spaces of the lung. However, the potential contribution of different epithelial cells (alveolar epithelial type I, type II, or bronchial epithelial cells) to CFTR-regulated fluid transport is unknown. In this study we determined whether the CFTR gene is expressed in human lung alveolar epithelial type II (AT II) cells and whether the CFTR chloride channel contributes to cAMP-regulated fluid transport in cultured human AT II cells. Human AT II ceils were isolated and cultured on collagen I-coated Transwell membranes for 120-144 h with an air-liquid interface. The cultured cells retained typical AT II-like features based on morphologic studies. Net basal fluid transport was 0.9 [+ or -] 0.1 [micro]*[cm.sup.-2]*[h.sup.-1] and increased to 1.35 [+ or -] 0.11 [micro]l*[cm.sup.-2]*[h.sup.-1] (mean [+ or -] SE, n = 18, P < 0.05) by stimulation with cAMP agonists. The CFTR inhibitor, [CFTR.sub.inh]-172, inhibited cAMP stimulated but not basal fluid transport. In short-circuit current ([I.sub.sc]) studies with an apical-to-basolateral transepithelial [Cl.sup.-] gradient, apical application of [CFTR.sub.inh]-172 reversed the forskolin-induced decrease in [I.sub.sc]. Real time RT-PCR demonstrated CFTR transcript expression in human AT II cells at a level similar to that in airway epithelial cells. We conclude that CFTR is expressed in cultured human AT II cells and may contribute to cAMP-regulated apical-basolateral fluid transport. lung epithelial cells; cystic fibrosis transmembrane conductance regulator; ion channel; cyclic adenosine monophosphate; pulmonary edema; alveolar fluid clearance

The aquaporin-7 (AQP7) water channel is known as a member of the aquaglyceroporins, which facilitate the transport of glycerol as well as water. Although AQP7 is abundantly expressed on the apical membrane of the proximal straight tubules in the kidney, the physiological role of AQP7 is still unknown. To investigate this, we generated AQP7 knockout mice. The water permeability of the proximal tubule brush-border membrane measured by the stopped-flow method was slightly but significantly reduced in the AQP7 knockout mice compared with that of wild-type mice (AQP7, 18.0 [+ or -] 0.4 x [10.sup.-3] cm/s vs. wild-type, 20.0 [+ or -] 0.3 x [10.sup.-3] cm/s). Although AQP7 solo-knockout mice did not exhibit a urinary concentrating defect, AQP1/AQP7 double-knockout mice had a reduction in urinary concentrating ability compared with AQP1 solok-nockout mice, suggesting that the amount of water reabsorbed through AQP7 in the proximal straight tubules is physiologically substantial. On the other hand, AQP7 knockout mice showed marked glyceroluria (AQP7, 1.7 [+ or -] 0.34 mg/ml vs. wild-type, 0.005 [+ or -] 0.002 mg/ml). This identified a novel glycerol reabsorption pathway in the proximal straight tubules. In two mouse models of proximal straight tubule injury, the cisplatin-induced acute renal failure (ARF) model and the ischemic ARF model, an increase in urine glycerol was observed (pretreatment, 0.007 [+ or -] 0.005 mg/ml; cisplatin, 0.063 [+ or -] 0.043 mg/ml; ischemia, 0.076 [+ or -] 0.02 mg/ml), suggesting that urine glycerol could be used as a new biomarker for detecting proximal straight tubule injury. aquaporin-7; kidney; urea; biomarker

Aquaporin-8 (AQP8) is a water-transporting protein expressed in organs of the mammalian gastrointestinal tract (salivary gland, liver, pancreas, small intestine, and colon) and in the testes, heart, kidney, and airways. We studied the phenotype of AQP8-null mice, and mice lacking AQP8, together with AQP1 or AQP5. AQP8-knockout mice lacked detectable AQP8 transcript and protein, and had reduced water permeability in plasma membranes from testes. Breeding of AQP8 heterozygous mice yielded AQP8-null mice, whose number, survival, and growth were not different from those of wild-type mice. Organ weight and serum/urine chemistries were similar in wild-type and AQP8-null mice, except for increased testicular weight in the null mice (4.8 [+ or -] 0.7 vs. 7.3 [+ or -] 0.3 mg/g body wt). Urinary concentrating ability in AQP8-null mice was unimpaired as assessed by urine osmolality (3,590 [+ or -] 360 mosmol/ kg[H.sub.2]O) and weight loss (22 [+ or -] 2%) after 36-h water deprivation; urinary concentrating ability was similarly impaired in AQP1-null mice vs. AQP8/AQP1 double-knockout mice. Agonist-driven fluid secretion in salivary gland was not different in AQP8 vs. wild-type mice (~1 [micro]lx[min.sup.-1]xg body [wt.sup.-1]) or in AQP5-null mice vs. AQP8/ AQP5 double-knockout mice. Closed intestinal loop measurements in vivo indicated unimpaired osmotically driven water transport, active fluid absorption, and cholera toxin-driven fluid secretion in AQP8-null mice. After 21 days on a 50% fat diet, wild-type and AQP8-null mice had similar weight gain (~15 g), with no evidence of steatorrhea or abnormalities in blood chemistries, except for mild hypertriglyceridemia in the null mice. The mild phenotype of AQP8-null mice was surprising in view of the multiple phenotype abnormalities found in mouse models of AQP1-5 deficiency. water channel; transgenic mice; testes; kidney; gastrointestinal tract

Intestinal infection with Vibrio cholerae results in secretory diarrhea with potentially massive fluid losses and volume depletion. Morbidity and mortality associated with cholera remain a major problem in the developing world despite the success of oral rehydration therapy. New research aiming to inhibit cholera toxin binding to receptors in the intestine provides an attractive strategy for cholera therapy. Together with anti-secretory agents, including inhibitors of enkephalinase and of the cystic fibrosis transmembrane conductance regulator, new treatment options for managing severe diarrhea in cholera could soon be available.

CFTR involvement in nasal potential differences in mice and pigs studied using a thiazolidinone CFTR inhibitor. Am J Physiol Lung Cell Mol Physiol 287: L936-L943, 2004. First published July 9, 2004; doi: 10.1152/ajplung.00354.2003.--Nasal potential difference (PD) measurements have been used to demonstrate defective CFTR function in cystic fibrosis (CF) and to evaluate potential CF therapies. We used the selective thiazolidinone CFTR inhibitor [CFTR.sub.inh]-172 to define the involvement of CFTR in nasal PD changes in mice and pigs. In normal mice infused intranasally with a physiological saline solution containing amiloride, nasal PD was -4.7 [+ or -] 0.7 mV, hyperpolarizing by 15 [+ or -] 1 mV after a low-[Cl.sup.-] solution, and a further 3.9 [+ or -] 0.5 mV after forskolin. [CFTR.sub.inh]-172 produced 1.1 [+ or -] 0.9- and 4.3 [+ or -] 0.7-mV depolarizations when added after low [Cl.sup.-] and forskolin, respectively. Systemically administered [CFTR.sub.inh]-172 reduced the forskolin-induced hyperpolarization from 4.7 [+ or -] 0.4 to 0.9 [+ or -] 0.1 mV but did not reduce the low [Cl.sup.-]-induced hyperpolarization. Nasal PD was -12 [+ or -] 1 mV in CF mice after amiloride, changing by cystic fibrosis; cystic fibrosis transmembrane conductance regulator; nasal potential difference; chloride channels

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

Drug discovery and development is generally done in the commercial rather than the academic realm. Drug discovery involves target discovery and validation, lead identification by high-throughput screening, and lead optimization by medicinal chemistry. Follow-up preclinical evaluation includes analysis in animal models of compound efficacy and pharmacology (ADME: administration, distribution, metabolism, elimination) and studies of toxicology, specificity, and drug interactions. Notwithstanding the high-cost, labor-intensive, and non-hypothesis-driven aspects of drug discovery, the academic setting has a unique and expanding niche in this important area of investigation. For example, academic drug discovery can focus on targets of limited commercial value, such as third-world and rare diseases, and on the development of research reagents such as high-affinity inhibitors for pharmacological 'gene knockout' in animal models ('chemical genetics'). This review describes the practical aspects of the preclinical drug discovery process for academic investigators. The discovery of small molecule inhibitors and activators of the cystic fibrosis transmembrane conductance regulator is presented as an example of an academic drug discovery program that has yielded new compounds for physiology research and clinical development. high-throughput screening; drug development; pharmacology; fluorescence; cystic fibrosis transmembrane conductance regulator

A calcein fluorescence quenching method was applied to measure osmotic water permeability in highly differentiated primary cultures of brain astrocytes from wild-type and aquaporin-4 (AQP-4)-deficient mice. Cells grown on coverglasses were loaded with calcein for measurement of volume changes after osmotic challenge. Hypotonic shock producing twofold cell swelling resulted in a reversible ~12% increase in calcein fluorescence, which was independent of cytosolic calcein concentration at levels well below where calcein self-quenching occurs. Calcein fluorescence was quenched in water transport; aquaporin-4; fluorescence quenching; brain swelling; cerebral edema

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

Phenotype analysis has demonstrated that aquaporin-1 (AQP1) null mice are polyuric and manifest a urinary concentrating defect because of an inability to create a hypertonic medullary interstitium. We report here that deletion of AQP1 is also associated with a decrease in urinary pH from 6.15 [+ or -] (SE) 0.1 to 5.63 [+ or -] 0.07. To explore the mechanism of the decrease in urinary pH, we examined the expression of [H.sup.+]-ATPase in kidneys of AQP1 null mice. There was strong labeling for [H.sup.+]-ATPase in intercalated cells and proximal tubule cells in both AQP1 null and wild-type mice. Strong [H.sup.+]-ATPase immunostaining was also present in the apical plasma membrane of inner medullary collecting duct (IMCD) cells in AQP1 null mice, whereas no [H.sup.+]-ATPase labeling was observed in IMCD cells in wild-type mice. In addition, there was an increase in the prevalence of type A intercalated cells in the IMCD of AQP1 null mice, suggesting that the deletion of intercalated cells from the IMCD, which normally occurs during postnatal kidney development, was impaired. Western blot analysis of [H.sup.+]-ATPase expression in the different regions of the kidney demonstrated a significant increase in [H.sup.+]-ATPase protein in the inner medulla of AQP1 null mice compared with wild-type mice. There were no changes in [H.sup.+]-ATPase expression in the cortex or outer medulla. These results represent the first demonstration of apical [H.sup.+]-ATPase immunoreactivity in IMCD cells in vivo and suggest that the decrease in urinary pH observed in AQP1 null mice is due to upregulation of [H.sup.+]-ATPase in the IMCD. The induction of [H.sup.+]-ATPase expression in IMCD cells of AQP1 null mice may be related to the chronically low interstitial osmolality in these animals. The challenge will be to identify the molecular signal(s) responsible for the de novo [H.sup.+]-ATPase expression. urine acidification; intercalated cells; immunohistochemistry

Activators of the CFTR [Cl.sup.-] channel may be useful for therapy of cystic fibrosis. Short-circuit current ([I.sub.sc]) measurements were done on human bronchial epithelial cells to characterize the best fiavone and benzimidazolone CFTR activators identified by lead-based combinatorial synthesis and high-throughput screening. The 7,8-benzoflavone UCCF-029 was a potent activator of [Cl.sup.-] transport, with activating potency (5 to airway epithelium; chloride secretion; drug discovery

Mice deficient in the epidermal water/glycerol transporter aquaporin-3 (AQP3) have reduced stratum corneum (SC) hydration and skin elasticity, and impaired barrier recovery after SC removal. SC glycerol content is reduced 3-fold in AQP3 null mice, whereas SC structure, protein/lipid composition, and ion/osmolyte content are not changed. We show here that glycerol replacement corrects each of the defects in AQP3 null mice. SC water content, measured by skin conductance and 3H20 accumulation, was 3-fold lower in AQP3 null vs. wild-type mice, but became similar after topical or systemic administration of glycerol in quantities that normalized SC glycerol content. SC water content was not corrected by glycerol-like osmolytes such as xylitol, erythritol, and propanediol. Orally administered glycerol fully corrected the reduced skin elasticity in AQP3 null mice as measured by the kinetics of skin displacement after suction, and the delayed barrier recovery as measured by transepidermal water loss after tape-stripping. Analysis of [[sup.14]C]glycerol kinetics indicated reduced blood-to-SC transport of glycerol in AQP3 null mice, resulting in slowed lipid biosynthesis. These data provide functional evidence for a physiological role of glycerol transport by an aquaglyceroporin, and indicate that glycerol is a major determinant of SC water retention, and mechanical and biosynthetic functions. Our findings establish a scientific basis for the >200-yr-old empirical practice of including glycerol in cosmetic and medicinal skin formulations. water transport | stratum corneum

Chloride concentration ([[Cl.sup.-]]) was measured in defined organellar compartments using fluorescently labeled transferrin, [[alpha].sub.2]-macroglobulin, and cholera toxin B-subunit conjugated with [Cl.sup.-]-sensitive and -insensitive dyes. In pulse-chase experiments, [[Cl.sup.-]] in Tf-labeled early/ recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over ~10 min in parallel to a drop in pH from 6.91 to 6.05. The low [[Cl.sup.-]] just after internalization (compared with 137 mM solution [[Cl.sup.-]]) was prevented by reducing the interior-negative Donnan potential. [[Cl.sup.-]] in [[alpha].sub.2]-macroglobulin-labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1-45 min after internalization, whereas pH decreased from 6.85 to 5.20. [Cl.sup.-] accumulation was prevented by bafilomycin but restored by valinomycin. A [Cl.sup.-] channel inhibitor slowed endosomal acidification and [Cl.sup.-] accumulation by ~2.5-fold. [[Cl.sup.-]] was 49 mM and pH was 6.42 in cholera toxin B subunit-labeled Golgi complex in Vero cells; Golgi compartment [Cl.sup.-] accumulation and acidification were reversed by bafilomycin. Our experiments provide evidence that [[Cl.sup.-] is the principal counter ion accompanying endosomal and Golgi compartment acidification, and that an interior-negative Donnan potential is responsible for low endosomal [[Cl.sup.-]] early after internalization. We propose that reduced [[Cl.sup.-]] and volume in early endosomes permits endosomal acidification and [[Cl.sup.-]] accumulation without lysis.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein, an epithelial chloride channel expressed in the airways, pancreas, testis, and other tissues. A central question is how defective CFTR function in CF leads to chronic lung infection and deterioration of lung function. Several mechanisms have been proposed to explain lung disease in CF, including abnormal airway surface liquid (ASL) properties, defective airway submucosal gland function, altered inflammatory response, defective organellar acidification, loss of CFTR regulation of plasma membrane ion transporters, and others. This review focuses on the physiology of the ASL and submucosal glands with regard to their proposed role in CF lung disease. Experimental evidence for defective ASL properties and gland function in CF is reviewed, and deficiencies in understanding ASL/gland physiology are identified as areas for further investigation. New model systems and measurement technologies are being developed to make progress in establishing lung disease mechanisms in CF, which should facilitate mechanism-based design of therapies for CF. cystic fibrosis transmembrane conductance regulator; epithelium; fluorescent indicators; Pseudomonas aeruginosa