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Muscarinic M₃-Receptors Mediate Cholinergic Synergism of Mitogenesis in Airway Smooth Muscle

Department of Molecular Pharmacology, University Centre for Pharmacy, Groningen, The Netherlands

Address correspondence to: Reinoud Gosens, Department of Molecular Pharmacology, University Centre for Pharmacy, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. E-mail: R.Gosens{at}farm.rug.nl

Muscarinic receptor agonists have been considered to act synergistically in combination with growth facors on airway smooth muscle growth. Characterization of the proliferative responses and of the receptor subtype(s) involved has not yet been studied. Therefore, we investigated mitogenesis induced by stimulation of muscarinic receptors, alone and in combination with stimulation by platelet-derived growth factor (PDGF). For this purpose, [³H]thymidine-incorporation was measured at different culture stages in bovine tracheal smooth muscle cells. Functional muscarinic M₃-receptors, as measured by formation of inositol phosphates, were present in unpassaged cells, but were lacking in passage 2 cells. Methacholine (10 µM) by itself was not able to induce a proliferative response in both cell culture stages. However, methacholine interacted synergistically with PDGF in a dose-dependent fashion (0.1–10 µM), but only in cells having functional muscarinic M₃-receptors. This synergism could be suppressed significantly by the selective M₃-receptor antagonists DAU 5884 (0.1 µM) and 4-DAMP (10 nM), but not at all by the M₂-subtype selective antagonist gallamine (10 µM). These results show that methacholine potentiates mitogenesis induced by PDGF solely through stimulation of muscarinic M₃-receptors in bovine tracheal smooth muscle cells.

Abbreviations: airway smooth muscle, ASM • bovine tracheal smooth muscle, BTSM • Chinese Hamster Ovary, CHO • Dulbecco's modified Eagle's medium, DMEM • extracellular signal regulated kinase, ERK • fetal bovine serum, FBS • mitogen-activated protein kinase, MAPK • phosphate-buffered saline, PBS • platelet-derived growth factor, PDGF • protein kinase C, PKC • pertussis toxin, PTX

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