β-Agonist-mediated Relaxation of Airway Smooth Muscle Is Protein Kinase A-dependent

Sarah J. Morgan,Deepak A. Deshpande,Brian C. Tiegs,Anna M. Misior,Huandong Yan,Alena V. Hershfeld,Thomas C. Rich,Reynold A. Panettieri,Steven S. An,Raymond B. Penn

doi:10.1074/jbc.m114.557652

Abstract

Inhaled β-agonists are effective at reversing bronchoconstriction in asthma, but the mechanism by which they exert this effect is unclear and controversial. PKA is the historically accepted effector, although this assumption is made on the basis of associative and not direct evidence. Recent studies have asserted that exchange protein activated by cAMP (Epac), not PKA, mediates the relaxation of airway smooth muscle (ASM) observed with β-agonist treatment. This study aims to clarify the role of PKA in the prorelaxant effects of β-agonists on ASM. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased β-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on histamine-induced intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Analyses of ASM cell and tissue contraction demonstrate that PKA inhibition eliminates most, if not all, β-agonist-mediated relaxation of contracted smooth muscle. Conversely, Epac knockdown had no effect on the regulation of contraction or procontractile signaling by isoproterenol. These findings suggest that PKA, not Epac, is the predominant and physiologically relevant effector through which β-agonists exert their relaxant effects.

Highlights

Mechanisms by which ␤-2-adrenoreceptor agonists effect bronchorelaxation remain unestablished
Recent studies have asserted that exchange protein activated by cAMP (Epac), not PKA, mediates the relaxation of airway smooth muscle (ASM) observed with ␤-agonist treatment
The effects of ␤-agonists have traditionally been attributed to activation of PKA, recent studies have demonstrated that ␤2AR signaling can initiate numerous other downstream events. cAMP-dependent but PKA-independent activation of Epac by the ␤2AR has been demonstrated in many cell types

Summary

Introduction

Mechanisms by which ␤-2-adrenoreceptor agonists effect bronchorelaxation remain unestablished. Results: Direct inhibition of PKA via molecular approaches reversed ␤-agonist-mediated antagonism of procontractile signaling and relaxation of contracted airway smooth muscle (ASM) despite augmenting intracellular cAMP. Recent studies have asserted that exchange protein activated by cAMP (Epac), not PKA, mediates the relaxation of airway smooth muscle (ASM) observed with ␤-agonist treatment. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased ␤-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on histamineinduced intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Epac knockdown had no effect on the regulation of contraction or procontractile signaling by isoproterenol. These findings suggest that PKA, not Epac, is the predominant and physiologically relevant effector through which ␤-agonists exert their relaxant effects

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Conclusion