Background: 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. Conclusion: PKA is the primary mechanism by which ␤-agonists relax ASM. Significance: PKA-dependent signaling and functions should guide the development of bronchodilator drugs.

Inhaled β-agonists are effective airway smooth muscle (ASM)-relaxing agents that help reverse bronchoconstriction in asthma, but their ability to affect the aberrant ASM growth that also occurs with asthma is poorly understood. β-Agonists exhibit PKA-dependent antimitogenic effects in several cell types. However, recent studies suggest that Epac, and not PKA, mediates the antimitogenic effect of cAMP in both ASM and fibroblasts. This study aims to clarify the role of PKA in mediating the effect of G(s)-coupled receptors on human ASM growth. Pretreatment of ASM cultures with β-agonists albuterol, isoproterenol, or salmeterol (100 nM to 10 μM) caused a significant (∼ 25-30%) inhibition of EGF-stimulated ASM thymidine incorporation and cell proliferation, whereas a much greater inhibition was observed from pretreatment with PGE(2) (75-80%). However, all agents were ineffective in cells expressing GFP chimeras of either PKI (a PKA inhibitor) or a mutant PKA regulatory subunit relative to the control cells expressing GFP. The antimitogenic efficacy of PGE(2) in inhibiting control cultures was associated with greater ability to stimulate sustained PKA activation and greater inhibition of late-phase promitogenic p42/p44 and PI3K activities. These findings suggest that therapeutic approaches enabling superior PKA activation in ASM will be most efficacious in deterring ASM growth.

Although G protein-coupled receptor (GPCR) kinases (GRKs) have been shown to mediate desensitization of numerous GPCRs in studies using cellular expression systems, their function under physiological conditions is less well understood. In the current study, we employed various strategies to assess the effect of inhibiting endogenous GRK2/3 on signaling and function of endogenously expressed Gs-coupled receptors in human airway smooth muscle (ASM) cells. GRK2/3 inhibition by expression of a Gβγ sequestrant, a GRK2/3 dominant-negative mutant, or siRNA-mediated knockdown increased intracellular cAMP accumulation mediated via β-agonist stimulation of the beta-2-adrenergic receptor (β2AR). Conversely, neither 5′-(N-ethylcarboxamido)-adenosine (NECA; activating the A2b adenosine receptor) nor prostaglandin E2 (PGE2; activating EP2 or EP4 receptors)-stimulated cAMP was significantly increased by GRK2/3 inhibition. Selective knockdown using siRNA suggested the majority of PGE2-stimulated cAMP in ASM was mediated by the EP2 receptor. Although a minor role for EP3 receptors in influencing PGE2-mediated cAMP was determined, the GRK2/3-resistant nature of EP2 receptor signaling in ASM was confirmed using the EP2-selective agonist butaprost. Somewhat surprisingly, GRK2/3 inhibition did not augment the inhibitory effect of the β-agonist on mitogen-stimulated increases in ASM growth. These findings demonstrate that with respect to Gs-coupled receptors in ASM, GRK2/3 selectively attenuates β2AR signaling, yet relief of GRK2/3-dependent β2AR desensitization does not influence at least one important physiological function of the receptor.

Excessive smooth muscle growth occurs within the context of inflammation associated with certain vascular and airway diseases. The inflammatory cytokines interleukin (IL)-1␤ and tumor necrosis factor-␣ (TNF-␣) have been shown previously to inhibit mitogen-stimulated smooth muscle growth through a mechanism presumed to be dependent on the induction of cyclooxygenase-2, prostaglandins, and activation of the cAMP-dependent protein kinase (PKA). Using both molecular and pharmacological strategies, we demonstrate that the mitogenic effects of IL-1␤ and TNF-␣ on cultured human airway smooth muscle (ASM) cells are tightly regulated by PKA activity. Suppression of induced PKA activity by either corticosteroids or cyclooxygenase inhibitors converts the cytokines from inhibitors to enhancers of mitogen-stimulated ASM growth, and biological variability in the capacity to activate PKA influences the modulatory effect of cytokines. Promitogenic effects of IL-1␤ are associated with delayed increases in p42/p44 and phosphoinositide-3 kinase activities, suggesting a role for induced autocrine factors. These findings suggest a mechanism by which mainstream therapies such as corticosteroids or cyclooxygenase inhibitors could fail to address or exacerbate the pathogenic smooth muscle growth that occurs in obstructive airway and cardiovascular diseases.

Glucocorticoids (GCs) and protein kinase A (PKA)-activating agents (beta-adrenergic receptor agonists) are mainstream asthma therapies based on their ability to prevent or reverse excessive airway smooth muscle (ASM) constriction. Their abilities to regulate another important feature of asthma--excessive ASM growth--are poorly understood. Recent studies have suggested that GCs render agents of inflammation such as IL-1 beta and TNF-alpha mitogenic to ASM, via suppression of (antimitogenic) induced cyclooxygenase-2-dependent PKA activity. To further explore the mechanistic basis of these observations, we assessed the effects of epidermal growth factor and IL-1 beta stimulation, and the modulatory effects of GC treatment and PKA inhibition, on the ASM transcriptome by microarray analysis. Results demonstrate that ASM stimulated with IL-1 beta, in a manner that is often cooperative with stimulation with epidermal growth factor, exhibit a profound capacity to function as immunomodulatory cells. Moreover, results implicate an important role for induced autocrine/paracrine factors (many whose regulation was minimally affected by GCs or PKA inhibition) as regulators of both airway inflammation and ASM growth. Induction of numerous chemokines, in conjunction with regulation of proteases and agents of extracellular matrix remodeling, is suggested as an important mechanism promoting upregulated G protein-coupled receptor signaling capable of stimulating ASM growth. Additional functional assays suggest that intracellular PKA plays a critical role in suppressing the promitogenic effects of induced autocrine factors in ASM. Finally, identification and comparison of GC- and PKA-sensitive genes in ASM provide insight into the complementary effects of beta-agonist/GC combination therapies, and suggest specific genes as important targets for guiding the development of new generations of GCs and adjunct asthma therapies.