Muscarinic receptors mediate stimulation of human lung fibroblast proliferation

Airway remodeling is a structural alteration associated with chronic inflammatory and obstructive airway diseases, wherein fibroblasts are crucially involved. The present study investigates whether lung fibroblast proliferation is influenced by muscarinic mechanisms. For this purpose, expression of muscarinic receptors in MRC-5 human lung fibroblasts was characterized by semiquantitative RT-PCR, and the effects of muscarinic agonists and antagonists on (H-3)thymidine incorporation as a measure of proliferative activity were studied under different culture conditions. MRC-5 fibroblasts express mRNA encoding different subtypes of muscarinic receptors (M-2 > M-3 > M-4, traces for M, and no M-1). Expression Of M-2 and M-3 receptors was confirmed at the protein level by immunoblot analysis. Under different culture conditions, carbachol (up to 10 mu M) or oxotremorine (10 mu M) stimulated (H-3)-thymidine incorporation, with maximum increases between about 40 and 100%. The stimulatory effect of 10 mu M carbachol was prevented by pretreatment with pertussis toxin and antagonized in a concentration-dependent manner by the muscarinic receptor antagonists tiotropium, AQ-RA 741, AF-DX 384,4-diphenylacetoxy-N-methylpiperidine methoiodide, himbacine, p-fluorohexahydrosiladifenidol, and pirenzepine, with concentrations producing 50% inhibition of 14 pM, 24, 64,127,187, 452 nM, and 1.5 mu M, respectively. Primary human lung fibroblasts were also found to express mRNA for muscarinic receptors (M-2 > M-1 > M-3, traces for M-4 and no M-5), and showed a pertussis toxin-sensitive proliferative response to muscarinic receptor stimulation. In conclusion, proliferation of human lung fibroblasts can be stimulated by activation of muscarinic receptors with a pharmacologic profile correlating best to M-2 receptors.