Cholinergic Receptor and Cyclic Stretch-Mediated Inflammatory Gene Expression in Intact ASM

Abstract

We tested the hypothesis that cholinergic stimulation and cyclic stretch regulate inflammatory gene expression in intact airway smooth muscle by measuring mRNA expression in bovine tracheal smooth muscle using limited microarray analysis and RT-PCR. Carbachol (1 microM) induced significant increases in the expression of cyclooxygenase (COX)-1, COX-2, IL-8, and plasminogen activator, urokinase type (PLAU) to levels ranging from 1.3- to 3.1-fold of control. Sinusoidal length oscillation at an amplitude of 10% muscle length and a frequency of 1 Hz induced significant increases in the expression of CCL-2, COX-2, IL-1 beta, and IL-6 to levels ranging from 12- to 206-fold of control. Decreasing the oscillatory amplitude by 50% did not significantly change inflammatory gene expression. In contrast, decreasing the oscillatory frequency by 50% significantly attenuated inflammatory gene expression by 76-93%. Nifedipine (1 microM) had an insignificant effect on carbachol-induced gene expression, but significantly inhibited sinusoidal length oscillation-induced inflammatory gene expression by 40-78%. Correlation analysis revealed two groups of genes with differential responses to sinusoidal length oscillation. The highly responsive group included COX-2, IL-6, and IL-8, which exhibited 45- to 364-fold increases in gene expression in response to sinusoidal length oscillation. The moderately responsive group included CCL2 and PLAU, which exhibited 13- to 19-fold increases in gene expression in response to sinusoidal oscillation. These findings suggest that cyclic stretch regulates inflammatory gene expression in intact airway smooth muscle in an amplitude- and frequency-dependent manner by modulating the activity of L-type voltage-gated calcium channels.

Muscarinic M3 receptor stimulation increases cigarette smoke-induced IL-8 secretion by human airway smooth muscle cells

“…These results extend previous observations by others who have shown that muscarinic receptor stimulation can induce IL-8 production by bronchial epithelial cells, and leukotriene B 4 production by monocytes, macrophages and bronchial epithelial cells [6,[8][9][10]27]. Muscarinic receptor stimulation using carbachol was also previously shown to regulate pro-inflammatory gene transcription of IL-6 and -8 and COX-2 induced by mechanical stimulation of airway smooth muscle strips [19]. Our current findings confirm these studies and provide novel insights as we demonstrate a strong synergistic interaction with the response induced by cigarette smoke extract.…”

“…The regulatory role of acetylcholine in these synthetic airway smooth muscle cell responses is largely unknown. A study using bovine tracheal smooth muscle strips showed that stimulation with carbachol resulted in increased expression of IL-8, cyclooxygenase (COX)-1 and -2 at the mRNA level [19]. This study [19], together with the fact that airway smooth muscle expresses muscarinic M2 and M3 receptors in abundance, suggests a role for these receptors in release of mediators.…”

“…A study using bovine tracheal smooth muscle strips showed that stimulation with carbachol resulted in increased expression of IL-8, cyclooxygenase (COX)-1 and -2 at the mRNA level [19]. This study [19], together with the fact that airway smooth muscle expresses muscarinic M2 and M3 receptors in abundance, suggests a role for these receptors in release of mediators. Therefore airway smooth muscle may represent a primary target of acetylcholine released by nerve and other cell types in the airways.…”

Inhibition of allergen-induced airway remodelling by tiotropium and budesonide: a comparison

“…Furthermore, tiotropium bromide prevented airway eosinophilia in allergen-challenged animals. It is possible that acetylcholine promotes the release of chemokines and cytokines from airway structural cells, as reported for airway epithelial cells and airway smooth muscle [14][15][16], in order to attract inflammatory cells to the airways, and functionally interacts with the growth factors or cytokines that are released by these cells to induce direct remodelling effects on structural target cells, similar to what has been observed for airway smooth muscle remodelling [2,11]. Although transactivation of the epidermal growth factor receptor by muscarinic receptors has been reported in conjunctival goblet cells [33], no detailed molecular studies investigating the role of muscarinic receptors in the proliferation and hypertrophy of airway mucous glands exist.…”

“…In vitro studies have revealed that prolonged stimulation of muscarinic receptors enhances airway smooth muscle contractile protein expression, pro-mitogenic signalling and cell proliferation [1,11,12]. Moreover, muscarinic receptor stimulation induces cell proliferation of primary cultured pulmonary fibroblasts [13], and triggers the release of proinflammatory mediators, including leukotriene B 4 , from airway smooth muscle and airway epithelial and inflammatory cells [14][15][16][17]. These pro-inflammatory actions of acetylcholine may be enhanced in inflammatory airways diseases, since M 3 expression and function are increased on neutrophils from COPD patients [18].…”