Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA082

Poster Communications

The muscarinic receptor modulator gallamine induces proliferation of airway smooth muscle cells.

R. STAMATIOU1, E. Paraskeva1, A. Vasilaki1, A. Hatziefthimiou1


Abstract Background: Muscarinic receptor antagonists are a usual treatment for chronic airway diseases, characterised by increased bronchial contraction, like asthma and chronic obstructive pulmonary disease. Furthermore, these diseases are usually accompanied by airway remodelling, involving, among others airway smooth muscle cell (ASMC) proliferation. The purpose of this study was to examine the effect of the M2 muscarinic receptor modulator gallamine on rabbit tracheal ASMC proliferation and phenotype. Methods: Airway Smooth Muscle Cells were isolated from adult New Zealand rabbit tracheas and primary cultures were established. Cell proliferation was estimated by incorporation of radioactive thymidine, by the Cell Titer AQueus One Solution method, as well as by cell count after Trypan blue staining. The mechanism mediating the effect of gallamine on ASMC proliferation was elucidated by the use of the ΡΙ3Κ and ΜΑΡΚ inhibitors LY294002 (20 μΜ) and PD98059 (100 μΜ), respectively, muscarinic agonists and/or 10% FBS. The effect of gallamine on ASMC phenotype was studied by indirect immunofluorescence and Western blot analysis of α-actin and Myosin Heavy Chain (MHC) expression. All data are expressed as means ± standard error of the mean (SEM) and N represents the number of independent experiments. Statistical analysis was performed using GraphPad Image 5 (GraphPad Software, San Diego, CA, USA). Differences between means were analyzed using one-way ANOVA followed by Bonferonni's post-test. Results were considered significantly different when p<0.05. Results: Incubation with gallamine (1 nM-10 mM) for 24 h, increased methyl-[3H]thymidine incorporation and cell number of 24 h serum starved ASMCs, in a dose dependent way (LogEC50: -5.853, EC50: 1.4X10-6M, N= 4). The effect of gallamine involved the ΡΙ3Κ and ΜΑΡΚ pathways, since it was inhibited when cells were preincubated with LY294002 (20 μΜ) and PD98059 (100 μΜ), respectively. Specifically, LY294002 inhibited methyl-[3H]thymidine incorporation from 404.9±87.52 cpm (gallamine alone) to 201.1±39.96 cpm (p<0.05, N=6). A similar reduction was observed in the presence of PD98059 (p<0.05, N=6). Gallamine (10 mM), had a mitogenic effect (128.2±8.59 % of control, p<0.05, N=7) on ASMCs that acquired a contractile phenotype after 7 days of serum deprivation, as well. However, did not affect the proliferation induced by acetylocholine (10 μM), carbachol (300 nM) or 10% FBS. Finally, gallamine did not cause any change on the expression of the contractile phenotype markers, α-actin and MHC. Conclusions: Gallamine, in the absence of any agonist, induces proliferation of both synthetic and contractile ASMCs. The mitogenic effect of gallamine is dependent on the activation of the ΡΙ3Κ and ΜΑΡΚ signaling pathways and does not affect the expression of ASMC contractile protein markers.

Where applicable, experiments conform with Society ethical requirements