Muscarinic M(3) receptor-dependent regulation of airway smooth muscle contractile phenotype

Abstract

1. Airway smooth muscle (ASM) cells are known to switch from a contractile to a proliferative and synthetic phenotype in culture in response to serum and growth factors. Phenotype switching in response to contractile agonists, however, is poorly characterised, despite the possible relationship between ASM phenotype and airway remodelling in asthma. 2. To investigate the effects of muscarinic receptor stimulation on ASM phenotype, we used organ-cultured bovine tracheal smooth muscle (BTSM) strips, in which contractile responsiveness, contractile protein expression and proliferation were measured after pretreatment with methacholine. 3. Long-term methacholine pretreatment (8 days) decreased maximal contraction and sensitivity to methacholine as well as to histamine and KCl. This decrease was dose-dependent (pEC(50)=5.2+/-0.1). Pretreatment with the highest concentration of methacholine applied (100 microm) could suppress maximal histamine-induced contraction to 8+/-1% of control. In addition, contractile protein expression (myosin, actin) was downregulated two-fold. No concomitant increase in proliferative capacity was observed. 4. The M(3)/M(2) muscarinic receptor antagonist DAU 5884 (0.1 microm) completely inhibited the observed decrease in contractility. In contrast, the M(2)/M(3) muscarinic receptor antagonist gallamine (10 microm) was ineffective, demonstrating that M(2) receptors were not involved. 5. Pretreatment (8 days) with 60 mm KCl could mimick the strong decreases in contractility. This was completely prevented by pretreatment with verapamil (1 microm). 6. Regulation of contractility was not affected by protein kinase C inhibition, whereas inhibitors of phosphatidyl inositol 3-kinase and p42/p44 mitogen activated protein kinase were partially effective. 7. These results show that long-term methacholine pretreatment (8 days) induces an M(3) receptor-dependent decrease in BTSM contractility without increased proliferative capacity.

Figure 1

Effect of 8-day methacholine pretreatment on BTSM contractility. BTSM strips were organ cultured for 8 days in the absence (open symbols) or presence (closed symbols) of 10 μM methacholine, after which contraction to (a) KCl, (b) histamine and (c) methacholine-induced was determined. Data represent means±s.e. mean from four (methacholine) to eight (KCl, histamine) experiments, each performed in duplicate.

Figure 2

Interaction of methacholine and PDGF pretreatment on histamine-induced BTSM contraction. BTSM strips were organ cultured for 8 days in the absence or presence of methacholine (10 μM), PDGF (10 ng ml⁻¹) and the combination hereof. Data represent means±s.e. mean from five experiments, each performed in duplicate.

Figure 3

Basal and PDGF-stimulated (10 ng ml⁻¹) [³H]thymidine incorporation in BTSM tissue slices, organ cultured for 8 days in the absence or presence of 10 μM methacholine. After the culture period, tissue slices were thoroughly washed and stimulated thereafter. Data represent means±s.e. mean from four experiments, each performed in duplicate. ^*P<0.05 compared to control.

Figure 4

Concentration dependency of the methacholine-induced pretreatment effects. (a) Histamine-induced contraction of BTSM strips, organ cultured for 8 days in the absence or presence of increasing concentrations of methacholine. (b) Maximal contraction (E_max) of histamine of BTSM strips, organ cultured with increasing concentrations of methacholine. Data in (b) represent maximal contractions measured under (a). Data represent means±s.e. mean from four experiments, each performed in duplicate.

Figure 5

Western analysis of contractile protein expression (sm-MHC, sm-α-actin) in BTSM strips, organ cultured for 8 days in the absence (C) or presence of increasing concentrations of methacholine. Blots shown are representative for four experiments. Graph shows the means±s.e. mean obtained after densitometry scans of the blots.

Figure 6

Role of M₂ and M₃ receptors in the methacholine-induced effects on BTSM contractility. BTSM strips were organ cultured for 8 days in the absence (open bars) or presence (closed bars) of 10 μM methacholine. During the entire culture period, BTSM strips were also incubated with DAU 5884 (0.1 μM, DAU) or gallamine (10 μM, Gall) where indicated. Untreated preparations served as controls (C). Data shown represent maximal contractile responses to histamine and are means±s.e. mean from four experiments, each performed in duplicate. ^*P<0.05.

Figure 7

Effects of prolonged increases in intracellular [Ca²⁺] on BTSM contractility. BTSM strips were organ cultured for 8 days in serum-free medium (open circles), in medium supplemented with CaCl₂ and KCl to produce final concentrations of 2.5 mM Ca²⁺ and 60 mM K⁺, respectively (closed circles), or in medium supplemented with verapamil (1 μM) and CaCl₂ and KCl (open triangles). Data shown represent contractile responses to histamine and are means± s.e. mean from three experiments, each performed in duplicate.

Figure 8

Role of PKC, p42/p44 MAP kinase and PI 3-kinase in the methacholine-induced effects on BTSM contractility. BTSM strips were organ cultured for 8 days in the absence (open bars) or presence (closed bars) of 10 μM methacholine. During the entire culture period, GF 109203X (10 μM, GF) PD 98059 (30 μM, PD) or LY 294002 (10 μM, LY) were also present where indicated. Vehicle treated (0.1% DMSO) preparations served as controls. Data shown represent maximal contractile responses to histamine and are means±s.e. mean from four experiments, each performed in duplicate. ^*P<0.05 compared to controls, # P<0.05 compared to methacholine (10 μM) and DMSO (0.1%)-pretreated.