X-ray microanalysis of airway surface liquid in the mouse

Kozlova, Inna; Nilsson, Hans C.; Phillipson, Mia; Riederer, Brigitte; Seidler, Ursula; Colledge, William H; Roomans, Godfried M.

doi:10.1152/ajplung.00303.2004

Cited by 13 publications

(19 citation statements)

References 22 publications

(29 reference statements)

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“…The absolute osmolality of the airway surface liquid (ASL) has proven difficult to determine. Recent reports in mouse (42,43) and rat (44) airways indicate that ASL osmolality is hypotonic in basal conditions, whereas others suggest it is isotonic (32,45). More certainly, when ventilation is increased, for example with exercise, the ASL osmolality increases (31,32,46), with return to basal levels with cessation.…”

Section: Discussionmentioning

confidence: 97%

Transient receptor potential vanilloid 4 regulates aquaporin-5 abundance under hypotonic conditions

Sidhaye

Güler

Schweitzer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Aquaporin-5 (AQP5) is expressed in epithelia of lung, cornea, and various secretory glands, sites where extracellular osmolality is known to fluctuate. Hypertonic aquaporin (AQP) induction has been described, but little is known about the effects of a hypotonic environment on AQP abundance. We report that, when mouse lung epithelial cells were exposed to hypotonic medium, a doseresponsive decrease in AQP5 abundance was observed. Hypotonic reduction of AQP5 was blocked by ruthenium red, methanandamide, and miconazole, agents that inhibit the cation channel transient receptor potential vanilloid (TRPV) 4 present in lung epithelial cells. Several observations indicate that TRPV4 participates in hypotonic reduction of AQP5, including a requirement for extracellular calcium to achieve AQP5 reduction; an increase in intracellular calcium in mouse lung epithelial (MLE) cells after hypotonic stimulation; and reduction of AQP5 abundance after addition of the TRPV4 agonist 4␣-Phorbol-12,13-didecanoate (4␣-PDD). Similarly, addition of hypotonic PBS to mouse trachea in vivo decreased AQP5 within 1 h, an effect blocked by ruthenium red. To confirm a functional interaction, AQP5 was expressed in control or TRPV4-expressing human embryonic kidney (HEK) cells. Hypotonic reduction of AQP5 was observed only in the presence of TRPV4 and was blocked by ruthenium red. Combined with earlier studies, these observations indicate that AQP5 abundance is tightly regulated along a range of osmolalities and that AQP5 reduction by extracellular hypotonicity can be mediated by TRPV4. These findings have direct relevance to regulation of membrane water permeability and water homeostasis in epithelia of the lung and other organs.epithelium ͉ lung ͉ membrane permeability ͉ osmotic stress ͉ calcium channel A quaporin water channel proteins participate in a wide array of physiological processes and are the primary determinants of membrane osmotic water permeability (1). Aquaporin (AQP)-5 is on the apical membrane at several sites in mammals, including secretory cells in salivary, lacrimal, sweat, and airway submucosal glands, corneal epithelium, nasopharyngeal and bronchial epithelium, and type I pneumocytes of the lung (2-5). Studies of isolated salivary gland epithelial cells (6) and type I pneumocytes (7) demonstrate that AQP5 confers high level membrane water permeability to the cell and that cellular responses to an initial osmotic challenge as well as subsequent regulatory volume changes require AQP5 (6).AQP5 abundance is regulated by numerous stimuli, including TNF-␣ (8), adenoviral infection (9), cAMP (10, 11), and hypertonic stress (12, 13). The general paradigm of aquaporin induction by extracellular hypertonicity is well established because, in addition to AQP5, hypertonic induction of AQP1 (14-16), AQP3 (13,17,18), AQP4 (19,20), and AQP9 (20) has been reported. In contrast, the effects of reduced extracellular osmolality on aquaporin abundance have not been reported.Transient receptor potential-vanilloid (TRPV) 4 is a nonselective cati...

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Section: Discussionmentioning

confidence: 97%

Transient receptor potential vanilloid 4 regulates aquaporin-5 abundance under hypotonic conditions

Sidhaye

Güler

Schweitzer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…In some experiments, the anesthetized animals were injected intraperitoneally with pilocarpine (50 mg/kg body weight), or isoproterenol (10 mg/kg body weight), and the trachea was removed 15 min after the injection. In experiments where nasal fluid was collected, the animals received an intraperitoneal injection with pilocarpine, isoproterenol (dose as above) or phenylephrine (10 mg/kg body weight) [22].…”

Section: Micementioning

confidence: 99%

“…The CFTR (-/-) mice (female, age 15-18 months) [22,23] were transported by air to Uppsala and kept for one week before the start of the experiment, on a crude fiber deficient diet, and with addition of a laxating salt solution. These mice were compared with control NMRI mice of approximately the same age.…”

Section: Micementioning

confidence: 99%

“…The trachea or bronchi were removed from the anesthetized animals and dissected in a specially designed chamber, in which the humidity could be kept constant at close to 100% [19,20,22]. The chamber consisted of a Perspex box, with a retractable plastic sheath on one side, which could be opened for handling of the specimen.…”

Section: Analysis Of Animal Trachea In Situmentioning

confidence: 99%

“…We started with pig airways, which share many structural and physiological similarities with human airways [19,20]. We continued with rodent (mouse, rat) airways, which gave us the possibility to investigate CF mice [21][22][23]. We also carried out a study on human nasal fluid, where we investigated both normal humans, CF patients and patients with rhinitis (Vanthanouvong et al, submitted for publication), and we are investigating cell cultures of airway epithelial cells from CF patients and healthy controls (Kozlova et al, submitted for publication).. We have used two different techniques: X-ray microanalysis 1 of frozen-hydrated specimens [19], and X-ray microanalysis of ASL absorbed into small ion exchange beads [21].…”

Section: Introductionmentioning

confidence: 99%

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Composition of Airway Surface Liquid Determined by X-ray Microanalysis

Kozlova¹,

Vanthanouvong²,

Johannesson³

et al. 2006

Upsala Journal of Medical Sciences

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The composition of the airway surface liquid, a thin layer of fluid covering the airway wall, has been debated. Two new techniques to determine the ionic composition of the airway surface liquid are presented. In the first technique, pieces of the airway were shock-frozen and analyzed by X-ray microanalysis in the frozen state in the scanning electron microscope. In the second technique, the airway surface liquid was collected with the help of dextran beads that were allowed to absorb the fluid. The beads were collected in silicon oil, cleaned, dried, and analyzed. Airway surface liquid from pig airways was isotonic to lightly hypertonic, whereas airway surface liquid from mouse and rat airways was hypotonic. The ionic composition of airway surface liquid from rodent airways could be changed by pharmacological stimulation of fluid transport. Transgenic mice with cystic fibrosis (CF) had significantly higher Na and Cl concentrations in the airway surface liquid than normal mice. Nasal fluid was also collected from humans. In CF patients, CF heterozygotes, and rhinitis patients, the levels of Na and Cl in the nasal fluid were signifi- cantly higher than in healthy controls. In CF patients K levels were also significantly higher than in healthy controls. The ionic concentrations in fluid collected from patients with primary ciliary dyskinesia (PCD) were not different from normal. Females with CF had significantly higher concentrations of Na, Cl and K in their nasal fluid compared to male patients. The dextran bead technique was also used to determine the ionic composition of the apical fluid in cultures of respiratory epithelial cells from healthy controls and CF patients. In the healthy controls, the fluid was hypotonic. In the CF cell cultures, the apical fluid had a higher Na and Cl concentration than in the controls.

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Ionic composition of rat airway surface liquid determined by X-ray microanalysis

Vanthanouvong

Kozlova

Roomans

2005

Microsc. Res. Tech.

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The thin layer of liquid that lines the conducting airway epithelium, the airway surface liquid (ASL), is important for mucociliary clearance. Altered ionic composition and/ or volume of the ASL play a major role in the pathology of airway diseases such as cystic fibrosis. Since the ASL is a thin layer, it has been difficult to exactly determine its composition. The present paper describes two techniques that have been developed and used to study ASL composition: X-ray microanalysis of frozen hydrated rat trachea, and an ion-exchange (dextran) bead method, where dextran beads were placed on the airway epithelium to equilibrate with the ASL; the beads were then collected under silicone oil, dried and analyzed by X-ray microanalysis. The results from both frozen-hydrated specimens and from the dextran beads showed that ASL from rat trachea is hypotonic. Concentrations of Na, P, S, and K were higher in the frozen-hydrated ASL, in which mainly the mucus layer is analyzed, compared with the dextran bead method, in which mainly the periciliary liquid is sampled. Also the composition of rat nasal fluid was investigated by the dextran bead method. This fluid was somewhat hypertonic because of a high K concentration. The ionic composition of the nasal and tracheal fluid can be manipulated by cholinergic or alpha- or beta-adrenergic stimulation. Collecting ASL with dextran beads did not disturb the integrity of the airway epithelium. The ionic composition of the collected beads remained stable for several days during storage in silicone oil. It is concluded that X-ray microanalysis is a suitable method to determine the ionic composition of ASL.

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X-ray microanalysis of airway surface liquid in the mouse

Cited by 13 publications

References 22 publications

Transient receptor potential vanilloid 4 regulates aquaporin-5 abundance under hypotonic conditions

Transient receptor potential vanilloid 4 regulates aquaporin-5 abundance under hypotonic conditions

Composition of Airway Surface Liquid Determined by X-ray Microanalysis

Ionic composition of rat airway surface liquid determined by X-ray microanalysis

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