Formoterol-induced upregulation of the expression of M3R is associated with increased expression levels of PLCβ1 and IP3. (A) Rat ASMCs were randomly divided into seven groups and treated with formoterol with or without inhibitors and for different durations. Untreated cells were used as a control. The protein expression of rat ASMCs PLCβ1 was determined by western blot analysis. (B) Protein expression of PLCβ1 was normalized to the β-actin control. The data are expressed as the mean ± standard deviation from three independent experiments. (C) Expression of IP3 was determined by ELISA. (D and E) Correlations between the expression levels of PLCβ1 or IP3 and the expression of M3R were determined using linear regression. (F) Inhibitory rate of acetylcholine-induced IP3 accumulation (*P<0.01, compared with the 1 h incubation group; Δ P<0.05, compared with the 24 h formoterol only treatment group). F1h, 1 h formoterol treatment; F24h, 24 h formoterol treatment; FK, forskolin; ICI+F, formoterol+ICI118,551; SQ+F, formoterol+SQ22,536; H89+F, formoterol+H89; Con, control; IP3, inositol 1,4,5-trisphosphate; M3R, muscarinic M3 receptor, PLCβ, phospholipase C-β.