Pro-inflammatory mechanisms of muscarinic receptor stimulation in airway smooth muscle.
BACKGROUND
Acetylcholine, the primary parasympathetic neurotransmitter in the airways, plays an important role in bronchoconstriction and mucus production. Recently, it has been shown that acetylcholine, by acting on muscarinic receptors, is also involved in airway inflammation and remodelling. The mechanism(s) by which muscarinic receptors regulate inflammatory responses are, however, still unknown.


METHODS
The present study was aimed at characterizing the effect of muscarinic receptor stimulation on cytokine secretion by human airway smooth muscle cells (hASMc) and to dissect the intracellular signalling mechanisms involved. hASMc expressing functional muscarinic M2 and M3 receptors were stimulated with the muscarinic receptor agonist methacholine, alone, and in combination with cigarette smoke extract (CSE), TNF-α, PDGF-AB or IL-1β.


RESULTS
Muscarinic receptor stimulation induced modest IL-8 secretion by itself, yet augmented IL-8 secretion in combination with CSE, TNF-α or PDGF-AB, but not with IL-1β. Pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely normalized the effect of methacholine on CSE-induced IL-8 secretion, whereas PMA, a PKC activator, mimicked the effects of methacholine, inducing IL-8 secretion and augmenting the effects of CSE. Similar inhibition was observed using inhibitors of IκB-kinase-2 (SC514) and MEK1/2 (U0126), both downstream effectors of PKC. Accordingly, western blot analysis revealed that methacholine augmented the degradation of IκBα and the phosphorylation of ERK1/2 in combination with CSE, but not with IL-1β in hASMc.


CONCLUSIONS
We conclude that muscarinic receptors facilitate CSE-induced IL-8 secretion by hASMc via PKC dependent activation of IκBα and ERK1/2. This mechanism could be of importance for COPD patients using anticholinergics.
Oenema
Tjitske A
TA
Department of Molecular Pharmacology, University of Groningen, The Netherlands. t.a.oenema@rug.nl
Kolahian
Saeed
S
Nanninga
Janke E
JE
Rieks
Daniëlle
D
Hiemstra
Pieter S
PS
Zuyderduyn
Suzanne
S
Halayko
Andrew J
AJ
Meurs
Herman
H
Gosens
Reinoud
R
D000109:Acetylcholine / Q000494:pharmacology*; D001980:Bronchi / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism* / Q000502:physiology; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D043585:Receptor, Muscarinic M2 / Q000378:metabolism* / Q000502:physiology; D043587:Receptor, Muscarinic M3 / Q000378:metabolism* / Q000502:physiology; D011976:Receptors, Muscarinic / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction / Q000276:immunology; D012907:Smoking / Q000378:metabolism
D018836:Inflammation Mediators; D015850:Interleukin-6; D016209:Interleukin-8; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D000109:Acetylcholine