β₂-adrenoceptors and muscarinic receptors mediate opposing effects on endothelin-1 expression in human lung fibroblasts.

Human lung fibroblasts are a potential source of endothelin-1 (ET-1), a pro-fibrotic mediator. The present study explored possible muscarinic and β-adrenergic modulations of ET-1 expression in human lung fibroblasts. MRC-5 human lung fibroblasts were cultured. Expression of prepro-endothelin-1 (ppET-1) mRNA was determined by quantitative real time PCR. [(3)H]-Proline incorporation was determined as measure of collagen synthesis. The muscarinic agonist oxotremorine induced, in a tiotropium-sensitive manner, a three-fold increase in ppET-1 mRNA. The β(2)-adrenoceptor agonist olodaterol caused a reduction of ppET-1 mRNA by 45%. Olodaterol also opposed the stimulatory effect of oxotremorine. The effect of olodaterol was mimicked by the protein kinase A agonist 6-Bnz-cAMP, whereas the Epac (exchange protein activated by cAMP) agonist 8-CPT-2'-O-Me-cAMP was less effective. Transforming growth factor-β(1) (TGF-β, 0.3 and 1 ng/ml) induced a three- and eight-fold increase in pp-ET-1 mRNA, respectively. Olodaterol opposed the effect of 0.3, but not that of 1 ng/ml TGF-β. Likewise, 6-Bnz-cAMP opposed the effect of 0.3, but not that of 1 ng/ml TGF-β. TGF-β inhibited β(2)-adrenoceptor mRNA expression, maximally by 90%. Muscarinic agonist-induced stimulation of [(3)H]-proline incorporation was attenuated by the endothelin ET1 receptor antagonist bosentan. In conclusion, ET-1 expression in human lung fibroblasts is regulated by stimulatory muscarinic receptors and inhibitory β(2)-adrenoceptors. Since muscarinic up-regulation of ET-1 contributes to pro-fibrotic effects of muscarinic stimuli, inhibition of ET-1 expression could contribute to long-term beneficial effects of long-acting β(2)-adrenoceptor agonists and long-acting muscarinic antagonists. However, excessive exposure to TGF-β results in loss of β-adrenoceptor expression and function of its down-stream signaling.