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Tracheal epithelium cell volume responses to hyperosmolar, isosmolar and hypoosmolar solutions: relation to epithelium-derived relaxing factor (EpDRF) effects.

Fedan JS, Thompson JA, Ismailoglu UB, Jing Y - Front Physiol (2013)

Bottom Line: Little is known of ASL hyperosmolarity effects on epithelial function.In previous studies amiloride and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) inhibited relaxation of IPT to hyperosmolar challenge, but had little effect on shrinkage of dispersed cells.Except for gadolinium and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), actin and microtubule inhibitors and membrane permeabilizing agents did not affect on ion transport by adherent epithelium or shrinkage responses of dispersed cells.

View Article: PubMed Central - PubMed

Affiliation: Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health Morgantown, WV, USA.

ABSTRACT
In asthmatic patients, inhalation of hyperosmolar saline or D-mannitol (D-M) elicits bronchoconstriction, but in healthy subjects exercise causes bronchodilation. Hyperventilation causes drying of airway surface liquid (ASL) and increases its osmolarity. Hyperosmolar challenge of airway epithelium releases epithelium-derived relaxing factor (EpDRF), which relaxes the airway smooth muscle. This pathway could be involved in exercise-induced bronchodilation. Little is known of ASL hyperosmolarity effects on epithelial function. We investigated the effects of osmolar challenge maneuvers on dispersed and adherent guinea-pig tracheal epithelial cells to examine the hypothesis that EpDRF-mediated relaxation is associated with epithelial cell shrinkage. Enzymatically-dispersed cells shrank when challenged with ≥10 mOsM added D-M, urea or NaCl with a concentration-dependence that mimics relaxation of the of isolated perfused tracheas (IPT). Cells shrank when incubated in isosmolar N-methyl-D-glucamine (NMDG) chloride, Na gluconate (Glu), NMDG-Glu, K-Glu and K2SO4, and swelled in isosmolar KBr and KCl. However, isosmolar challenge is not a strong stimulus of relaxation in IPTs. In previous studies amiloride and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) inhibited relaxation of IPT to hyperosmolar challenge, but had little effect on shrinkage of dispersed cells. Confocal microscopy in tracheal segments showed that adherent epithelium is refractory to low hyperosmolar concentrations that induce dispersed cell shrinkage and relaxation of IPT. Except for gadolinium and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), actin and microtubule inhibitors and membrane permeabilizing agents did not affect on ion transport by adherent epithelium or shrinkage responses of dispersed cells. Our studies dissociate relaxation of IPT from cell shrinkage after hyperosmolar challenge of airway epithelium.

No MeSH data available.


Related in: MedlinePlus

Characterization of dispersed tracheal epithelial cells. (A) Typical single, ciliated epithelial cell showing rounded appearance and polarized clustering of cilia. Bar = 50 μm. (B) Time-course of epithelial cell volume in un-stimulated and D-M (120 mOsM)-challenged cells after the 1 h equilibration period in MKH solution. n = 4. *Significantly different compared to t = 0 min. (C) Cell volume responses of epithelial cells following challenge with half-strength (hypotonic) MKH solution [0.5 (MKH); n = 5] and hyperosmolarity achieved with NaCl (240 mOsM; n = 4) added to the MKH solution. (D) Lack of effect of MCh on cell volume decrease initiated by challenge of epithelial cells with D-M (120 mOsM). n = 4. *Significantly different compared to t = 0 min.
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Figure 1: Characterization of dispersed tracheal epithelial cells. (A) Typical single, ciliated epithelial cell showing rounded appearance and polarized clustering of cilia. Bar = 50 μm. (B) Time-course of epithelial cell volume in un-stimulated and D-M (120 mOsM)-challenged cells after the 1 h equilibration period in MKH solution. n = 4. *Significantly different compared to t = 0 min. (C) Cell volume responses of epithelial cells following challenge with half-strength (hypotonic) MKH solution [0.5 (MKH); n = 5] and hyperosmolarity achieved with NaCl (240 mOsM; n = 4) added to the MKH solution. (D) Lack of effect of MCh on cell volume decrease initiated by challenge of epithelial cells with D-M (120 mOsM). n = 4. *Significantly different compared to t = 0 min.

Mentions: Guinea-pigs anesthetized with sodium pentobarbital (65 mg/kg, i.p.) were sacrificed by thoracotomy and bleeding and 4.2 cm long tracheal segments were removed. After cleaning in modified Krebs-Henseleit (MKH) solution (composition below) the tracheas were cut longitudinally through the smooth muscle band, and incubated with 2 ml 0.2% protease in EMEM at 37°C for 1 h. The digestion was stopped with 10% FBS/EMEM solution at 4°C. The epithelial cells were scraped off with a scalpel blade; clumps were rinsed and triturated in 10 ml of EMEM solution containing 0.1% DNase I. The digest was centrifuged (800 rpm) for 4–5 min at 10°C. Cells pooled from several animals, the number of which was determined by the particular experiment, were re-suspended in 5 ml of MKH solution, filtered (Falcon 40 mm nylon filter) and centrifuged. The cells were suspended in 1 ml of gassed MKH solution and incubated for 1 h at 37°C, to allow for re-establishment of ion gradients. Cell suspensions were divided into aliquots for the various experimental conditions. Cell integrity was assessed microscopically after adding 0.4% trypan blue solution. A typical ciliated cell in the suspension is shown in Figure 1A.


Tracheal epithelium cell volume responses to hyperosmolar, isosmolar and hypoosmolar solutions: relation to epithelium-derived relaxing factor (EpDRF) effects.

Fedan JS, Thompson JA, Ismailoglu UB, Jing Y - Front Physiol (2013)

Characterization of dispersed tracheal epithelial cells. (A) Typical single, ciliated epithelial cell showing rounded appearance and polarized clustering of cilia. Bar = 50 μm. (B) Time-course of epithelial cell volume in un-stimulated and D-M (120 mOsM)-challenged cells after the 1 h equilibration period in MKH solution. n = 4. *Significantly different compared to t = 0 min. (C) Cell volume responses of epithelial cells following challenge with half-strength (hypotonic) MKH solution [0.5 (MKH); n = 5] and hyperosmolarity achieved with NaCl (240 mOsM; n = 4) added to the MKH solution. (D) Lack of effect of MCh on cell volume decrease initiated by challenge of epithelial cells with D-M (120 mOsM). n = 4. *Significantly different compared to t = 0 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3795350&req=5

Figure 1: Characterization of dispersed tracheal epithelial cells. (A) Typical single, ciliated epithelial cell showing rounded appearance and polarized clustering of cilia. Bar = 50 μm. (B) Time-course of epithelial cell volume in un-stimulated and D-M (120 mOsM)-challenged cells after the 1 h equilibration period in MKH solution. n = 4. *Significantly different compared to t = 0 min. (C) Cell volume responses of epithelial cells following challenge with half-strength (hypotonic) MKH solution [0.5 (MKH); n = 5] and hyperosmolarity achieved with NaCl (240 mOsM; n = 4) added to the MKH solution. (D) Lack of effect of MCh on cell volume decrease initiated by challenge of epithelial cells with D-M (120 mOsM). n = 4. *Significantly different compared to t = 0 min.
Mentions: Guinea-pigs anesthetized with sodium pentobarbital (65 mg/kg, i.p.) were sacrificed by thoracotomy and bleeding and 4.2 cm long tracheal segments were removed. After cleaning in modified Krebs-Henseleit (MKH) solution (composition below) the tracheas were cut longitudinally through the smooth muscle band, and incubated with 2 ml 0.2% protease in EMEM at 37°C for 1 h. The digestion was stopped with 10% FBS/EMEM solution at 4°C. The epithelial cells were scraped off with a scalpel blade; clumps were rinsed and triturated in 10 ml of EMEM solution containing 0.1% DNase I. The digest was centrifuged (800 rpm) for 4–5 min at 10°C. Cells pooled from several animals, the number of which was determined by the particular experiment, were re-suspended in 5 ml of MKH solution, filtered (Falcon 40 mm nylon filter) and centrifuged. The cells were suspended in 1 ml of gassed MKH solution and incubated for 1 h at 37°C, to allow for re-establishment of ion gradients. Cell suspensions were divided into aliquots for the various experimental conditions. Cell integrity was assessed microscopically after adding 0.4% trypan blue solution. A typical ciliated cell in the suspension is shown in Figure 1A.

Bottom Line: Little is known of ASL hyperosmolarity effects on epithelial function.In previous studies amiloride and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) inhibited relaxation of IPT to hyperosmolar challenge, but had little effect on shrinkage of dispersed cells.Except for gadolinium and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), actin and microtubule inhibitors and membrane permeabilizing agents did not affect on ion transport by adherent epithelium or shrinkage responses of dispersed cells.

View Article: PubMed Central - PubMed

Affiliation: Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health Morgantown, WV, USA.

ABSTRACT
In asthmatic patients, inhalation of hyperosmolar saline or D-mannitol (D-M) elicits bronchoconstriction, but in healthy subjects exercise causes bronchodilation. Hyperventilation causes drying of airway surface liquid (ASL) and increases its osmolarity. Hyperosmolar challenge of airway epithelium releases epithelium-derived relaxing factor (EpDRF), which relaxes the airway smooth muscle. This pathway could be involved in exercise-induced bronchodilation. Little is known of ASL hyperosmolarity effects on epithelial function. We investigated the effects of osmolar challenge maneuvers on dispersed and adherent guinea-pig tracheal epithelial cells to examine the hypothesis that EpDRF-mediated relaxation is associated with epithelial cell shrinkage. Enzymatically-dispersed cells shrank when challenged with ≥10 mOsM added D-M, urea or NaCl with a concentration-dependence that mimics relaxation of the of isolated perfused tracheas (IPT). Cells shrank when incubated in isosmolar N-methyl-D-glucamine (NMDG) chloride, Na gluconate (Glu), NMDG-Glu, K-Glu and K2SO4, and swelled in isosmolar KBr and KCl. However, isosmolar challenge is not a strong stimulus of relaxation in IPTs. In previous studies amiloride and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) inhibited relaxation of IPT to hyperosmolar challenge, but had little effect on shrinkage of dispersed cells. Confocal microscopy in tracheal segments showed that adherent epithelium is refractory to low hyperosmolar concentrations that induce dispersed cell shrinkage and relaxation of IPT. Except for gadolinium and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), actin and microtubule inhibitors and membrane permeabilizing agents did not affect on ion transport by adherent epithelium or shrinkage responses of dispersed cells. Our studies dissociate relaxation of IPT from cell shrinkage after hyperosmolar challenge of airway epithelium.

No MeSH data available.


Related in: MedlinePlus