Fig 1.
The chemical structure of STS.
Fig 2.
Effect of STS on Isc response in mouse tracheal epithelium.
(A) Apical application (ap) of 10 μM STS induced a fast and transient increase in Isc, as visible in the typical current trace. (B) Typical Isc response of basolateral application (bl) of 10 μM STS in normal K-H solution. (C) Comparison of ΔIsc induced by STS in apical (n = 9) and basolateral (n = 5) in mouse tracheal epithelium. Values are mean ± S.E.M. (Student’s t-test, *P < 0.05 compared with the apical group). (D) Dose-response curve of the apical application of STS-induced changes on Isc. The concentrations of STS are 0.1, 0.3, 1, 3, 6, 10, 30, 60, 100 and 300 μM. Each data point is mean ± S.E.M. (n = 3–6). EC50: effective concentration for half-maximal ΔIsc after apical STS application. The arrows mark the time at which the drugs were added.
Fig 3.
Cl− dependence of the Isc response to STS.
(A) Representative recording of the Isc activated by STS (10 μM, apical) in Cl− free K-H solution (n = 6). (B) Representative curve of the Isc activated by STS (10 μM, apical) in K-H solution pretreated with amiloride (100 μM, apical, n = 5). (C) Comparison of STS (10 μM, apical, n = 11) induced ΔIsc obtained in Cl− free K-H solution and normal K-H solution with or without amiloride (100 μM, apical). Values are mean ± S.E.M. (ANOVA, *P < 0.05 compared with the control). The arrows mark the time at which the drugs were added.
Fig 4.
Effect of different Cl− channel blockers on Isc induced by STS.
Representative recordings of Isc induced by STS (10 μM, apical) pretreated with (A) the non-specific Cl− channel blocker DPC (1 mM, apical, n = 6), (B) the Ca2+-activated Cl− channel (CaCC) blocker DIDS (100 μM, apical, n = 6), (C) tannic acid (100 μM, apical, n = 6), (D) the CFTR blocker CFTRinh172 (10 μM, apical, n = 6), (E) the adenylate cyclase inhibitor MDL-12330A (10 μM, apical, n = 5), for 15 min. (F) Comparison of the effects of different Cl− channel blockers and MDL-12330A on STS (10 μM, apical) induced ΔIsc. Values are mean ± S.E.M. (ANOVA, *P < 0.05 compared with the control). The arrows mark the time at which the drugs were added.
Fig 5.
Effect of atropine and Tetrodotoxin (TTX) on apical-applied STS-induced Isc.
(A) The effect of 2.8 μM atropine on apical applied STS-induced Isc. ATP was applied as a positive control to check the activity of mouse tracheal epithelium. (B) Apical application of 10 μM STS-induced Isc response in the presence of 1 μM TTX. (C) Atropine (2.8 μM, apical, n = 7) inhibited STS-induced Isc response significantly. In the presence of TTX (1 μM, basolateral, n = 8), ΔIsc induced by STS was not significantly altered compared to the control (10 μM, apical, n = 11). Values are mean ± S.E.M. (ANOVA, *P < 0.05 compared with the control). The arrows mark the time at which the drugs were added.
Fig 6.
Effect of Ca2+ signaling inhibitors on response to STS.
Representative current traces of Isc induced by STS (10 μM, apical) pretreated with (A) the IP3 receptor antagonist 2-APB (50 μM, apical, n = 6), (B) the intracellular Ca2+ chelator BAPTA-AM (100 μM, apical, n = 5). (C) Comparison of the effects of different Ca2+ signaling inhibitors on STS (10 μM, apical) induced ΔIsc. Values are mean ± S.E.M. (ANOVA, *P < 0.05 compared with the control). The arrows mark the time at which the drugs were added.
Fig 7.
Effects of STS on intracellular Ca2+ in mouse airway epithelial cells.
(A) Representative intracellular Ca2+ transients elicited by STS (10 μM) detected by fluo-3 fluorescence in the presence or absence of 2.8 μM atropine or in extracellular Ca2+ free solution. (B) Statistical analysis of intracellular Ca2+ in response to STS in N-PSS (control, 10 μM, n = 13) or in Ca2+ free PSS (n = 5) and the inhibitory effects of atropine (2.8 μM, n = 12), U73122 (20 μM, n = 3), 2-APB (50 μM, n = 6), Tg (10 μM, n = 7) and CPZ (10 μM, n = 3) on the Ca2+ transients elicited by STS (10 μM). Data is expressed as a percentage of the initial fluo-3 fluorescence. Values are mean ± S.E.M. (ANOVA, *P < 0.05 compared with the control).