Objective: 

Chronic airway diseases, such as asthma or COPD, are characterized by excessive acetylcholine release and in some cases airway remodeling. The aim of this study was to investigate the effect of muscarinic agonists (acetylcholine or carbachol) on the proliferation and phenotype of rabbit tracheal airway smooth muscle cells (ASMC).

Methods: 

Serum starved cells for 3–15 days were treated with muscarinic agonists and proliferation was estimated using the Cell Titer 96® AQueous One Solution Assay (Promega) or the methyl-[3H]thymidine incorporation method. The involvement of signaling pathways was studied using Western blot analysis in total protein cell extracts and the inhibitors of the PI3K and the MAPK pathways LY294002 and PD89005 respectively. The cell phenotype was studied by indirect immunofluorescence, using antibodies against smooth muscle a- actin and Myosin Heavy Chain (MHC).

Results: 

In 3 days starved ASMC, both acetylcholine and carbachol increase the ratio of phospho-Akt and phospho-p42/44 to b-actin and DNA synthesis without an effect on cell number.

The percentage of cells expressing a-actin or MHC is increased correspondingly to the days of serum starvation. On the contrary in cells treated for 15 days with acetylcholine, carbachol or 10% FBS the percentage of cells expressing a-actin or MHC was significantly reduced compared to control cells.

In 7 days starved ASMC the treatment of cells for 24h with acetylcholine or carbachol induced an increase in cell number.

Conclusions: 

Muscarinic agonists affect ASMC phenotype and induce proliferation, via activation of the PI3K and the MAPK signaling pathways, in rabbit ASMC that have been exposed to prolonged serum starvation.