β2-Agonist Induced cAMP Is Decreased in Asthmatic Airway Smooth Muscle Due to Increased PDE4D

Rory Edward Morty,Thomas Trian,Judith L Black,Patrick Berger,Qi Ge,Kyoko Niimi,Brian G Oliver,Stephen B Liggett,Lyn M Moir,Janette K Burgess

doi:10.1371/journal.pone.0020000

Abstract

Background and ObjectiveAsthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM) mass. In addition, some studies have suggested impaired β-agonist induced ASM relaxation in asthmatics, but the mechanism is not known.ObjectiveTo characterize the potential defect in β-agonist induced cAMP in ASM derived from asthmatic in comparison to non-asthmatic subjects and to investigate its mechanism.MethodsWe examined β2-adrenergic (β2AR) receptor expression and basal β-agonist and forskolin (direct activator of adenylyl cyclase) stimulated cAMP production in asthmatic cultured ASM (n = 15) and non-asthmatic ASM (n = 22). Based on these results, PDE activity, PDE4D expression and cell proliferation were determined.ResultsIn the presence of IBMX, a pan PDE inhibitor, asthmatic ASM had ∼50% lower cAMP production in response to isoproterenol, albuterol, formoterol, and forskolin compared to non-asthmatic ASM. However when PDE4 was specifically inhibited, cAMP production by the agonists and forskolin was normalized in asthmatic ASM. We then measured the amount and activity of PDE4, and found ∼2-fold greater expression and activity in asthmatic ASM compared to non-asthmatic ASM. Furthermore, inhibition of PDE4 reduced asthmatic ASM proliferation but not that of non-asthmatic ASM.ConclusionDecreased β-agonist induced cAMP in ASM from asthmatics results from enhanced degradation due to increased PDE4D expression. Clinical manifestations of this dysregulation would be suboptimal β-agonist-mediated bronchodilation and possibly reduced control over increasing ASM mass. These phenotypes appear to be “hard-wired” into ASM from asthmatics, as they do not require an inflammatory environment in culture to be observed.

Highlights

Asthma is characterized by variable degrees of obstruction due to active airway smooth muscle (ASM) contraction, increased ASM mass, and increased airway mucus
Since the measurement of Cyclic adenosine monophosphate (cAMP) in these cells is only possible in the presence of PDE inhibitors such as inhibitor 3Isobutyl-1-methylxanthine (IBMX) that prevent cAMP degradation, in these initial cAMP experiments, IBMX was used at the standard concentration of 0.5 mM (Fig. 1A)
These results revealed markedly lower cAMP levels in asthmatic ASM, which were statistically different at 1027 to 1025 M, but a trend was observed at all doses

Summary

Introduction

Asthma is characterized by variable degrees of obstruction due to active airway smooth muscle (ASM) contraction, increased ASM mass, and increased airway mucus. We and others have previously reported that calcium (Ca2+) homeostasis is altered in asthmatic ASM cells [11,12] The dysregulation of this second messenger is associated with an increase in asthmatic ASM cell proliferation that underlies the ASM remodelling which accompanies asthma [12,13], and accounts for the readily contracted ASM in asthmatics upon exposure to agents such as methacholine. B-agonists acting on b2AR expressed on ASM are responsible for cAMP production and the clinical effects. This pathway has been extensively dissected in cells which have been recombinantly manipulated, and endogenously expressing cells including human ASM. Some studies have suggested impaired b-agonist induced ASM relaxation in asthmatics, but the mechanism is not known

Methods

Results

Discussion

Conclusion