Stereoselectivity of tradinterol's inhibition on proliferation of airway smooth muscle cells induced by acetylcholine through suppressing Ca(2+) signalling.

The objective of this study is to investigate whether the inhibition of tradinterol (SPFF) against acetylcholine (ACh)-induced proliferation is mediated by Ca(2+) signaling in airway smooth muscle cells (ASMCs), and whether stereoselectivity of the drug exists. Guinea pig ASMCs were primarily prepared with the method described and treated with ACh combined to SPFF isomers for 24 or 48 hours, respectively. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to determine the proliferation of the guinea pig ASMCs. Ca(2+) fluorescent intensity in the guinea pig ASMCs, expressed with percentage increase in fluorescence when the intensity was determined with varioskan flash or shown with percentage increase in Geo Mean (GM) measured with flow cytometry, was recorded. Images of the intensity were obtained with fluorescent microscope. 2-APB, an (inositol 1,4,5-trisphosphate receptor) IP3R blocker, and NiCl2, a store-operated channel (SOC) inhibitor, were used to investigate the mechanism of SPFF isomers regulating intracellular Ca(2+) via IP3R on sarcoplasmic reticulum (SR) and/or SOC on plasma membrane. (-)SPFF and (±)SPFF, treated for 48 hours, showed significant inhibition against ACh-induced proliferation. The Ca(2+) elevation induced by ACh was concentration-dependently suppressed by SPFF isomers. (-)SPFF is the most effective but the potency of (±)SPFF is less than that of the former and stronger than that of (+)SPFF based on the half maximal inhibitory concentration (IC50) value. No significant additive effect was observed when (-)SPFF/(±)SPFF was used alone and combined with NiCl2/2-APB. As far as (+)SPFF is concerned, no similar phenomenon was observed. (-)SPFF and (±)SPFF but (+)SPFF showed significant inhibition against the percentage increase in fluorescence induced by CaCl2. It is likely that the influence of IP2RSOC-mediated Ca(2+) signaling in ASMCs helps (-)SPFF and (±)SPFF contribute to the suppression of ASMCs proliferation. Stereoselectivity of SPFF isomers may lead to different levels of suppression of ACh-induced intracellular Ca(2+) and ASMCs proliferation. Moreover, cell cycle analysis with flow cytometry was applied to the evaluation of the action in human ASMCs in order to further confirm the anti-proliferative effect of the drugs. It was found that (-)SPFF, (±)SPFF but (+)SPFF suppressed the elevated rate of cell population in Phase S over all the cells stimulated with ACh, when SPFF and its isomers were individually exposed to the cells for 72 hours. These results that demonstrate the different stereoselective activities of SPFF are in consistent with those obtained from the guinea pig ASMCs.