Inhibition of human airway smooth muscle cell proliferation by beta(2)-adrenergic receptors and cAMP is PKA independent: evidence for EPAC involvement

Mechanisms by which beta-adrenergic receptor (beta AR) agonists inhibit proliferation of human airway smooth muscle (HASM) cells were investigated because of their potential relevance to smooth muscle hyperplasia in asthma. We hypothesized that beta AR agonists would inhibit mitogenesis in HASM cells via the beta(2)AR, an increase in cAMP, and PKA activation. HASM cells were treated for 24 h with various agents and then analyzed for [H-3] thymidine incorporation as a measure of cell proliferation. EGF stimulated proliferation by similar to 10-fold. The nonselective beta AR agonist isoproterenol and the beta(2)AR-selective agonists albuterol and salmeterol inhibited EGF-stimulated proliferation by more than 50%, with half-maximal effects at 4.8 nM, 110 nM, and 6.7 nM, respectively. A beta(2)AR-selective antagonist inhibited the isoproterenol effect with 100-fold greater potency than a beta(1)AR-selective antagonist, confirming beta(2)AR involvement in the inhibition of proliferation. The cAMP-elevating agents PGE(2) and forskolin decreased EGF-induced proliferation, suggesting cAMP as the mediator. beta(2)AR agonists and forskolin also inhibited proliferation stimulated by lysophosphatidic acid (LPA) as well as the synergistic proliferation stimulated by LPA + EGF. Importantly, PKA-selective cAMP analogs did not inhibit proliferation at concentrations that maximally activated PKA (10-100 mu M), whereas a cAMP analog selective for the exchange protein directly activated by cAMP (EPAC), 8-(4-chloro-phenylthio)2'-O-methyl-cAMP, maximally inhibited proliferation at a concentration that did not activate PKA (10 mu M). These data show that beta(2)AR agonists and other cAMP-elevating agents decrease proliferation in HASM cells via a PKA-independent mechanism, and they provide pharmacological evidence for involvement of EPAC or an EPAC-like cAMP effector protein instead.