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

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.

Keywords: cell volume; epithelium-derived relaxing factor; exercise asthma.

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.

Figure 2

Osmolar concentration-dependence of the effects of D-M (A), urea (B), or NaCl (C) on volume of dispersed epithelial cells. (A–C), n = 4, 5–9, and 5–9, respectively. ^*Significantly different compared to t = 0 min.

Figure 3

Effects of isosmolar solutions of ionic permeant and impermeant osmolytes on volume of dispersed epithelial cells. Cell volume was measured after the cells were placed into isosmolar solutions containing NaCl (A; n = 6), NMDG-Cl (C; n = 6), Na-Glu (E; n = 6), NMDG-Glu (G; n = 6), K-Glu (B; n = 6), K₂SO₄ (D; n = 6), KBr (F; n = 6), and KCl (H; n = 6). The order of increasing effectiveness at causing volume change was: K₂SO₄ = KGlu = NMDG-Cl (~15% decrease) < Na-Glu < NMDG-Glu (35% decrease). KCl and KBr caused increases in cell volume (~50 and 15%, respectively). D-M and urea could not be studied using this method. ^*Significantly different compared to t = 0 min.

Figure 4

Responses of IPT to perfusion with isosmolar (IO) K₂SO₄ (Top) or KBr (Bottom), followed by hyperosmolar (HO; 120 mOsM) challenge with the same osmolyte (hyperosmolar jump). These results are representative of n = 4 experiments for K₂SO₄ and n = 6 for KBr, in which contraction (shown) to isosmolar K₂SO₄ or KBr or no effect were observed (not shown). The discontinuities in the responses after the isosmolar additions occurred during perfusion solution changeover. Vertical bar, 5 cm H₂O; horizontal bar, 5 min.

Figure 5

Effects of isosmolar osmolyte solutions, and hyperosmolar osmolyte solutions added in the presence of isosmolar solution (osmolar jump), on volume of dispersed epithelial cells. n = 5, 5, and 4 for KCl, KBr, and NaCl, respectively. ^*Significantly different compared to t = 0 min.

Figure 6

Effects on cell volume of transport blockers alone (A panels), and the effects of the blockers on volume responses to MCh (B panels; 3 × 10⁻⁷ M) and D-M in the presence of the blockers and MCh (C panels; 30 mOsM) in dispersed cells. Cells were incubated with the blocker for 30 min (A panels), MCh plus blocker for 15 min (B panels), or MCh plus blocker plus D-M for 15 min (C panels). Control cells were not incubated with blockers. (A) ^*Significantly different compared to Control at t = 0 min. (B) ^*Significantly different from Control + bumetanide at t = 15 min. (C) ^*Significantly different from Control + MCh at t = 15 in; ^**significantly different from blocker + MCh at t = 15 min. n = 4 – 7.

Figure 7

Bioelectric responses of tracheal segments to MCh (basolateral; 3 × 10⁻⁷ M) and D-M (apical; 120 and 267 mOsM) obtained in the confocal imaging chamber. (A) Representative responses to MCh and D-M. Basal V_t average from all experiments was 6.7 ± 1.4 mV (n = 6). Bar = 5 min. (B) Summary of concentration-dependence of D-M-induced depolarization. Bioelectric responses to D-M in concentrations less than 120 mOsM were rarely produced. n = 2 and 2 for 120 and 267 mOsM D-M.

Figure 8

Representative confocal micrographs showing cell height of calcein-loaded control epithelium (left column) and after apical challenge with 120 mOsM D-M (middle column). (A) Three dimensional reconstruction of the image stack on the z-axis. Bar = 20 μM. (B) panel (A) in pseudocolor. Bar = 20 μM. (C) x-z plane (vertical red lines at arrows). (D) x-y plane of a single section in the middle of the image stack. (E) y-z plane (horizontal green lines at arrows). The bar graph in the right column depicts the concentration-dependence of volume responses to apically-applied D-M. Cell height was quantified from the image stack on the z-axis in confocal images and normalized with respect to control values. 120 mOsM D-M, n = 5; 267 mOsM D-M, n = 3.

Figure 9

Effects of apical isosmolar (IO) and hyperosmolar (HO) D-M on epithelial cell height in the confocal chamber. ^*Significantly less than Control and IO. n = 4.

Figure 10

Representative effects of apically-applied cytoskeleton/microtubule-interfering agents colchicine (2 × 10⁻⁴; n = 4), EHNA (5 × 10⁻⁴ M; n = 4), cytochalasins B (5 × 10⁻⁷ M; n = 6), and D (5 × 10⁻⁷ M; n = 6), latrunculin B (5 × 10⁻⁶ M; n = 4), jasplakinolide (5 × 10⁻⁶ M; n = 4), the pore-forming agent, nystatin (2.6 × 10⁻⁴ M; n = 6), and the HICC inhibitor, flufenamic acid (10⁻⁴ M), on V_t and R_t of tracheal segments in Ussing chambers. DMSO (0.1%), the solvent for these agents (except colchicine), was without effect on V_t and R_t. A summary of the results of all experiments is shown in Figure 11. Bar = 400 s. The baselines are provided in some cases for reference. The dots indicate the additions of the agents.

Figure 11

Effects of apically-applied cytoskeleton/microtubule- interfering agents, pore-forming agents and hypertonicity-induced cation channel blockers, on V_t and R_t (A), V_t and R_t responses to MCh (B), and V_t and R_t responses to 120 mOsM D-M (C). Top row: % change in V_t values above zero signify depolarization; negative values signify hyperpolarization. Bottom row: % change in R_t values above zero signify an increase in; negative values signify a decrease in R_t. DMSO (DMS, 0.1%) was the solvent for all agents except colchicine and gadolinium, which were dissolved in saline. Agent abbreviations and their concentrations are: Col, colchicine (2 × 10⁻⁴ M; n = 4); EHN, EHNA (5 × 10⁻⁴ M; n = 4); CyD and CyB, cytochalasins D and B, respectively (2 × 10⁻⁵ M; n = 6 and 6); Noc, nocodazole (2 × 10⁻⁵ M; n = 4); Lat, latrunculin B (5 × 10⁻⁶ M; n = 4); Jas, jasplakinalide (5 × 10⁻⁶ M; n = 4); Nys, nystatin (2.6 × 10⁻⁴ M; n = 6); α-H, α-hemolysin (100 units/ml; n = 4); Gad, gadolinium (10⁻⁴ M; n = 4); Flu, flufenamic acid (10⁻⁴ M; n = 5). A, summary of the responses elicited by the agents; B, responses to MCh obtained in the presence of the agents; C, responses to D-M in the presence of the agent and MCh (3 × 10⁻⁷ M). ^*Significantly different from DMSO (n = 4).

Figure 12

Effects EHNA (5 × 10⁻⁴ M) on cell volume and volume responses to D-M. Left panel (Basal response): % reduction in volume under basal conditions after addition of DMSO or EHNA dissolved in DMSO, during the 30 min incubation period. Right panel (D-M induced response): % reduction in volume after subsequent addition of 120 mOsM D-M dissolved in DMSO. In these experiments a control volume measurement was made before incubating the cells with vehicle or agents; the cells used in each replicate were from the same preparation. ^*Significantly different from D-M. Basal response, n = 4; DMSO-induced response.