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

1 Department of Pharmacology and Experimental Neuroscience and 2 Pulmonary, Critical Care, Sleep, and Allergy Section, University of Nebraska Medical Center, Omaha, Nebraska; and 3 Pulmonary, Allergy, and Critical Care Division, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania Submitted 12 September 2007 ; accepted in final form 3 November 2007 Mechanisms by which β-adrenergic receptor (βAR) 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 βAR agonists would inhibit mitogenesis in HASM cells via the β 2 AR, an increase in cAMP, and PKA activation. HASM cells were treated for 24 h with various agents and then analyzed for [ 3 H]thymidine incorporation as a measure of cell proliferation. EGF stimulated proliferation by 10-fold. The nonselective βAR agonist isoproterenol and the β 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 β 2 AR-selective antagonist inhibited the isoproterenol effect with 100-fold greater potency than a β 1 AR-selective antagonist, confirming β 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. β 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 µM), whereas a cAMP analog selective for the exchange protein directly activated by cAMP (EPAC), 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, maximally inhibited proliferation at a concentration that did not activate PKA (10 µM). These data show that β 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. exchange protein directly activated by cAMP; lysophosphatidic acid; epidermal growth factor; asthma Address for reprint requests and other correspondence: M. L. Toews, 985800 Nebraska Medical Center, Omaha, NE 68198-5800 (e-mail: mtoews{at}unmc.edu )