Nicotine stimulates ion transport via metabotropic β4 subunit containing nicotinic ACh receptors

Abstract

Background and purpose: Mucociliary clearance is an innate immune process of the airways, essential for removal of respiratory pathogens. It depends on ciliary beat and ion and fluid homeostasis of the epithelium. We have shown that nicotinic ACh receptors (nAChRs) activate ion transport in mouse tracheal epithelium. Yet the receptor subtypes and signalling pathways involved remained unknown.

Experimental approach: Transepithelial short circuit currents (I_SC ) of freshly isolated mouse tracheae were recorded using the Ussing chamber technique. Changes in [Ca²⁺ ]_i were studied on freshly dissociated mouse tracheal epithelial cells.

Key results: Apical application of the nAChR agonist nicotine transiently increased I_SC . The nicotine effect was abolished by the nAChR antagonist mecamylamine. α-Bungarotoxin (α7 antagonist) had no effect. The agonists epibatidine (α3β2, α4β2, α4β4 and α3β4) and A-85380 (α4β2 and α3β4) increased I_SC . The antagonists dihydro-β-erythroidine (α4β2, α3β2, α4β4 and α3β4), α-conotoxin MII (α3β2) and α-conotoxin PnIA (α3β2) reduced the nicotine effect. Nicotine- and epibatidine-induced currents were unaltered in β2^-/- mice, but in β4^-/- mice no increase was observed. In the presence of thapsigargin (endoplasmatic reticulum Ca²⁺ -ATPase inhibitor) or the ryanodine receptor antagonists JTV-519 and dantrolene there was a reduction in the nicotine-effect, indicating involvement of Ca²⁺ release from intracellular stores. Additionally, the PKA inhibitor H-89 and the TMEM16A (Ca²⁺ -activated chloride channel) inhibitor T16Ainh-A01 significantly reduced the nicotine-effect.

Conclusion and implications: α3β4 nAChRs are responsible for the nicotine-induced current changes via Ca²⁺ release from intracellular stores, PKA and ryanodine receptor activation. These nAChRs might be possible targets to stimulate chloride transport via TMEM16A.

Keywords: ACh receptors; Ussing chamber; epithelium; nicotine; non-neuronal cholinergic system; trachea.

FIGURE 1

Effect of nicotine on transepithelial ion transport of mouse tracheal epithelium. (a) Apical application of nicotine (100 μmol·L⁻¹) resulted in a transient increase in short circuit current (I_SC). Representative current trace. (b) The nicotine‐induced current increase was significant compared to baseline (n = 5, *P < 0.05). (c) The nicotine effect was dose dependent (n = 5 for each concentration). (d) Repeated apical nicotine application (100 μmol·L⁻¹) in the same trachea showed a reduced nicotine‐activated current (ΔI_SC) upon the second application (n = 5, *P < 0.05). (e) Mecamylamine (25 μmol·L⁻¹, apical) abolished the nicotine‐induced I_SC. Application of mecamylamine did not influence I_SC. Representative current trace. (f) In the presence of the non‐selective nAChR antagonist mecamylamine (MEC, 25 μmol·L⁻¹, apical), nicotine had no significant effect on I_SC (n = 5; ns, not significant)

FIGURE 2

Effect of nicotinic receptor agonists and antagonists on transepithelial ion transport of mouse tracheal epithelium. (a) The α7 nicotinic ACh receptor (nAChR) antagonist α‐bungarotoxin (αBTX, 100 nmol·L⁻¹, n = 5, apical) or the α7 nAChR antagonist α‐conotoxin ImI (C. ImI, 4 μmol·L⁻¹, apical) or the α9α10 nAChR antagonist ACV‐1 (100 nmol·L⁻¹, apical) did not influence the nicotine effect (100 μmol·L⁻¹, apical, ΔI_SC; ns, not significant). (b) The epibatidine‐induced (α4β2, α3β2, α4β4 and α3β4 nAChR agonist) current peak (1 μmol·L⁻¹) was significant compared to baseline current (n = 5, *P < 0.05). Application of the α4β2 and α3β4 nAChR agonist A‐85380 (100 μmol·L⁻¹, apical, n = 5) significantly increased I_SC (*P < 0.05). (c) The epibatidine effect was dose dependent (n = 5 for each concentration). (d) In the presence of 1 μmol·L⁻¹ of the α4β2, α3β2, α4β4 and α3β4 nAChR antagonist dihydro‐β‐erythroidine (DhβE), the nicotine effect (ΔI_SC) was similar to control conditions (n = 5; ns, not significant), but 10 μmol·L⁻¹ DhβE significantly reduced the nicotine‐induced current (ΔI_SC, n = 5, *P < 0.05). (e) The α3β2 nAChR antagonist α‐conotoxin MII (C. MII) did not influence the nicotine effect (ΔI_SC) in a concentration of 50 nmol·L⁻¹ (n = 5; ns, not significant) but significantly reduced the nicotine‐induced current (ΔI_SC, n = 5, *P < 0.05) in a concentration of 1 μmol·L⁻¹. (f) The α3β2 antagonist α‐conotoxin PnIA (C. PnIA) did not influence the nicotine effect (ΔI_SC) in a concentration of 100 nmol·L⁻¹ (n = 6; ns, not significant) but significantly reduced the nicotine‐induced current in a concentration of 500 mmol·L⁻¹ (ΔI_SC, n = 5, *P < 0.05)

FIGURE 3

Effect of nicotine or epibatidine on transepithelial ion transport in β2‐ or β4‐deficient mice. (a) Apical application of nicotine (100 μmol·L⁻¹) in β2^−/− mice resulted in a transient reversible increase in the short circuit current (I_SC). Representative current trace. (b) The nicotine‐induced peak (ΔI_SC) in β2^−/− mice was similar to wild‐type (WT) mice (n = 5; ns, not significant). (c) Apical application of epibatidine (1 μmol·L⁻¹) in β2^−/− mice resulted in a transient reversible increase in I_SC. Representative current trace. (d) The epibatidine‐induced peak (ΔI_SC) was similar in WT and β2^−/− mice (n = 5; ns, not significant). (e) Apical application of nicotine (100 μmol·L⁻¹) in β4^−/− mice did not influence I_SC. Representative current trace. (f) The nicotine‐induced peak (ΔI_SC) in β4^−/− mice was significantly reduced compared to WT mice (n = 5, *P < 0.05). (g) Apical application of epibatidine (1 μmol·L⁻¹) in β4^−/− mice had no effect on I_SC. Representative current trace. (h) The epibatidine‐induced peak (ΔI_SC) was significantly reduced in β4^−/− mice compared to WT mice (n = 5, *P < 0.05)

FIGURE 4

Downstream signalling involved in the nicotine‐induced activation of transepithelial ion transport in mouse trachea. (a) Application of 1‐μM thapsigargin (THA, apical, n = 5), an inhibitor of the Ca²⁺‐ATPase in the endoplasmatic reticulum, significantly reduced the nicotine effect (100 μM, apical, ΔI_SC, *P < 0.05). (b) In the presence of the ryanodine receptor antagonist JTV519 and dantrolene (each 10 μmol·L⁻¹, apical), nicotine (100 μmol·L⁻¹, apical) had no effect on I_SC (ns, not significant; n = 5). (c) The protein kinase C (PKC) inhibitor chelerythrine chloride (CC, 5 μmol·L⁻¹, apical and basolateral, n = 7) did not influence the nicotine effect (100 μmol·L⁻¹, apical, ΔI_SC; ns, not significant). (d) In the presence of the protein kinase A (PKA) inhibitor H‐89 (10 μmol·L⁻¹, apical and basolateral, n = 5), the current induced by nicotine (100 μmol·L⁻¹, apical, ΔI_SC, *P < 0.05) was significantly reduced. (e) Application of the JAK2 inhibitor AG 490 (50 μmol·L⁻¹, apical and basolateral, n = 5) did not lead to a significant change of the nicotine effect (100 μmol·L⁻¹, apical, ΔI_SC, *P < 0.05). (f) Application of the STAT3 inhibitor WP1066 (10 μmol·L⁻¹, apical, n = 6) did not influence the nicotine effect (100 μM, apical, ΔI_SC; ns, not significant)

FIGURE 5

Ca²⁺ imaging and immunohistochemistry experiments of tracheal epithelial cells of wild‐type mice. (a) Application of nicotine, ACh, or ATP significantly increased [Ca²⁺]_i compared to baseline in the presence of Ca²⁺‐containing external solution (normal Tyrode's). N, number of animals; n, number of cells. (b) Application of nicotine, ACh, or ATP increased [Ca²⁺]_i compared with baseline in the presence of Ca²⁺‐free external solution. N, number of animals; n, number of cells. (c) The nicotine‐induced [Ca²⁺]_i was significantly reduced in Ca²⁺‐free buffer compared to extracellular Ca²⁺‐containing solution (Tyrode's)

FIGURE 6

Role of chloride and potassium channels in the nicotine effect. (a) In the presence of the TMEM16A inhibitor T16Ainh‐A01, the nicotine effect was reduced. Representative current trace. (b) Apical application of T16Ainh‐A01 (10 μM) significantly reduced the nicotine effect (100 μmol·L⁻¹, apical, ΔI_SC, *P < 0.05, n = 5). (c) In the presence of the TMEM16A inhibitor Ani 9 (10 μmol·L⁻¹, apical), the nicotine effect (100 μmol·L⁻¹, apical, ΔI_SC, *P < 0.05, n = 5) was significantly reduced. (d) In the presence of the inhibitor of calcium‐activated chloride channels CaCCinh‐A01 (100 μmol·L⁻¹, apical), nicotine (100 μmol·L⁻¹, apical) had no significant effect on I_SC (n = 5; ns, not significant). (e) Basolateral application of the KCNQ1 inhibitor chromanol 293B (C. 293B, 100 μmol·L⁻¹) significantly reduced the nicotine effect (100 μmol·L⁻¹, apical, ΔI_SC, *P < 0.05, n = 5)

FIGURE 7

Schematic drawing of the signalling pathway delineated in our study. Activation of epithelial α3β4 nAChR by nicotine leads to a release of Ca²⁺ from the endoplasmatic reticulum (ER) via ryanodine receptors (RyR). This activates the Ca²⁺‐dependent chloride channel TMEM16A and the PKA via soluble ACs (sAC). PKA then activates the basolateral KCNQ1 potassium channel