ABSTRACT
Title
Inhibition of RhoA activation in human bronchial smooth muscle cells: novel insights into the molecular mechanism of salbutamol action
Authors
S. Fogli,1 L. Mattii,2 F. Stefanelli,1 B. Battolla2, C. Segnani2, M.C. Breschi1
1Dept. of Psychiatry, Neurobiology, Pharmacology and Biotechnology and 2Dept. of Human Morphology and Applied Biology, Section of Histology and General Embriology, University of Pisa
1Dept. of Psychiatry, Neurobiology, Pharmacology and Biotechnology and 2Dept. of Human Morphology and Applied Biology, Section of Histology and General Embriology, University of Pisa
Abstract
The monomeric G-protein RhoA has emerged to play a central role in several processes that contribute to asthma pathophysiology, including airway hyperresponsiveness, remodeling and inflammation (Schaafsma et al., 2008). The present study was aimed at investigating whether the β2-adrenoceptor (β2-AR) agonist, salbutamol, was able to modulate RhoA activation in human bronchial smooth muscle cells (BSMC). Serum starved BSMC were stimulated with the Rho activating compound calpeptin, in the presence or absence of salbutamol or the Epac activator 8-pCPT-2'-O-Me-cAMP. For RhoA immunocytochemical detection, cells were fixed with 1% formalin and sequentially exposed to anti-RhoA polyclonal immunoglobulins, biotinylated link antibodies, peroxidase-streptavidin complexes and diaminobenzadine. Activated RhoA expression was also assessed by RhoA G-LISATM quantitative assay. Stimulation with calpeptin caused a translocation of RhoA from the cytosol towards the membrane, as compared with untreated controls. Pre-treatment with salbutamol 10 µM was found to block calpeptin-induced activation of RhoA and such an effect was mitigated in homologously desensitized cells. These results were confirmed in ELISA experiments (approximately 80% and 50% reduction in activated RhoA expression after salbutamol pre-treatment in normal and desensitized cells, respectively). Finally, we also demonstrated that pre-treatment of calpeptin-stimulated BSMC with 8-pCPT-2'-O-Me-cAMP was able to reproduce the effect of salbutamol. In conclusion, our data suggest that (i) salbutamol inhibits RhoA activation in human BSMC through β2-AR/Epac pathway, and (ii) homologous β2-AR desensitization may blunt such an effect. Notwithstanding their preliminary nature, a potentially important pharmacological implication of these finding is the possible contribute of RhoA pathway to the molecular mechanism involved in airway smooth muscle relaxation/airway hyperresponsiveness caused by acute/chronic exposure to β2-AR agonists in the clinical setting.
Schaafsma et al. (2008). Eur J Pharmacol. 585, 398-406.
Schaafsma et al. (2008). Eur J Pharmacol. 585, 398-406.