Regulation of smooth muscle contractility by the epithelium in rat tracheas: role of prostaglandin E2 induced by the neurotransmitter acetylcholine

Abstract

Background: Previous studies have suggested the involvement of epithelium in modulating the contractility of neighboring smooth muscle cells. However, the mechanism underlying epithelium-derived relaxation in airways remains largely unclear. This study aimed to investigate the mechanism underlying epithelium-dependent smooth muscle relaxation mediated by neurotransmitters.

Methods: The contractile tension of Sprague-Dawley (SD) rat tracheal rings were measured using a mechanical recording system. Intracellular Ca²⁺ level was measured using a Ca²⁺ fluorescent probe Fluo-3 AM, and the fluorescence signal was recorded by a laser scanning confocal imaging system. The prostaglandin E₂ (PGE₂) content was measured using an enzyme-linked immunosorbent assay kit.

Results: We observed that the neurotransmitter acetylcholine (ACh) restrained the electric field stimulation (EFS)-induced contraction in the intact but not epithelium-denuded rat tracheal rings. After inhibiting the muscarinic ACh receptor (mAChR) or cyclooxygenase (COX), a critical enzyme in prostaglandin synthesis, the relaxant effect of ACh was attenuated. Exogenous PGE₂ showed a similar inhibitory effect on the EFS-evoked contraction of tracheal rings. Moreover, ACh triggered phospholipase C (PLC)-coupled Ca²⁺ release from intracellular Ca²⁺ stores and stimulated COX-dependent PGE₂ production in primary cultured rat tracheal epithelial cells.

Conclusions: Collectively, this study demonstrated that ACh induced rat tracheal smooth muscle relaxation by promoting PGE₂ release from tracheal epithelium, which might provide valuable insights into the cross-talk among neurons, epithelial cells and neighboring smooth muscle cells in airways.

Keywords: Cyclooxygenase (COX); neurotransmitter; prostaglandin E2 (PGE2); smooth muscle relaxation; tracheal epithelium.

Figure 1

Relaxant effect of acetylcholine (ACh) on electric field stimulation (EFS)-contracted rat tracheal rings. (A) Representative traces showing the relaxant effect of ACh (20 nM) on the contraction evoked by EFS in intact and epithelium-denuded rat tracheal rings with (B) the corresponding statistical analysis. Data were presented as a percentage of the initial contraction evoked by EFS. Each column and error bar indicated the mean ± SEM. n=9; ***, P<0.001 compared with the intact trachea group. The experiment was repeated at least three times.

Figure 2

The effect of atropine on acetylcholine (ACh)-induced relaxation in electric field stimulation (EFS)-contracted rat tracheal rings. (A) Representative traces showing the effect of atropine (2 µM) on the 20 nM ACh-induced relaxant effect on the contraction evoked by EFS in rat tracheal rings with (B) the corresponding statistical analysis. Data were presented as a percentage of the initial contraction evoked by EFS. Each column and error bar indicated the mean ± SEM. n=5; ***, P<0.001. The experiment was repeated three times.

Figure 3

The effect of L-NG-nitro-arginine (L-NNA) on acetylcholine (ACh)-induced relaxation in electric field stimulation (EFS)-contracted rat tracheal rings. (A) Representative traces showing the effect of L-NNA (300 µM) on the 20 nM ACh-induced relaxant effect on the contraction evoked by EFS in rat tracheal rings with (B) the corresponding statistical analysis. Data were presented as a percentage of the initial contraction evoked by EFS. Each column and error bar indicated the mean ± SEM. n=5; ns: P>0.05. The experiment was repeated at least three times.

Figure 4

The effect of indomethacin on acetylcholine (ACh)-induced relaxation and the relaxant effect of prostaglandin E₂ (PGE₂) on electric field stimulation (EFS)-contracted rat tracheal rings. (A) Representative traces showing the effect of indomethacin (10 µM) on the 20 nM ACh-induced relaxant effect on the contraction evoked by EFS in rat tracheal rings with (B) the corresponding statistical analysis. n=5; ***, P<0.001. (C) Representative traces showing the relaxant effect of PGE₂ (1 µM) on the contraction evoked by EFS in intact and epithelium-denuded rat tracheal rings with (D) the corresponding statistical analysis. Data were presented as a percentage of the initial contraction evoked by EFS. Each column and error bar indicated the mean ± SEM. n=5; ns: P>0.05. The experiment was repeated at least three times.

Figure 5

Effect of acetylcholine (ACh) on intracellular Ca²⁺ concentration {[Ca²⁺]_i} in primary cultured rat tracheal epithelial cells. (A) Average time-course trace of [Ca²⁺]_i after application of ACh (200 µM) in rat tracheal epithelial cells. (B) Average time-course trace of [Ca²⁺]_i after applying ACh in 2 µM Tg-pretreated rat tracheal epithelial cells. (C) Average time-course trace of [Ca²⁺]_i after applying ACh in 2 µM atropine-pretreated rat tracheal epithelial cells. Thapsigargin (Tg, 2 µM) was used as a positive control. (D) Average time-course trace of [Ca²⁺]_i after application of ACh in 10 µM U73122-pretreated rat tracheal epithelial cells. Tg (2 µM) was used as a positive control. Each symbol and error bar indicated the mean ± SEM. n=5. The experiment was repeated at least three times.

Figure 6

Effect of acetylcholine (ACh) on the release of prostaglandin E₂ (PGE₂) in primary cultured rat tracheal epithelial cells and schematic diagram of epithelium-derived PGE₂ in regulating smooth muscle contractility in rat tracheas. (A) Statistical analysis showing the effect of ACh (200 µM) on PGE₂ content in the presence or absence of indomethacin (10 µM) in the rat tracheal epithelial cells. Each column and error bar indicated the mean ± SEM. n=3–4; **, P<0.01. The experiment was repeated at least three times. (B) The neurotransmitter ACh released from parasympathetic nerves has a dual regulatory effect on rat tracheas. On the one hand, ACh directly evokes the contraction of the smooth muscles. On the other hand, ACh mediates epithelium-derived cyclooxygenase (COX)-dependent PGE₂ release from epithelial cells via muscarinic ACh receptors (mAChR)-phospholipase C (PLC)-Ca²⁺ signaling, thereby relaxing the smooth muscle cells.