Suppression of the increasing level of acetylcholine-stimulated intracellular Ca ²⁺ in guinea pig airway smooth muscle cells by mabuterol

Abstract

The present study aimed to establish an effective method for the in vitro culture of guinea pig airway smooth muscle (ASM) cells, and also investigate the suppressive effect of mabuterol hydrochloride (Mab) on the increased level of intracellular Ca ²⁺ in ASM cells induced with acetylcholine (Ach). Two different methods, i.e. with or without collagenase to pretreat tracheal tissues, were applied to the manufacture of ASM cells. Cell viability was determined with the 3-(4,5-dimethylthinazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Immunocytochemistry and immunofluorescence were used for the identification of ASM cells. Different concentration levels (10 ⁻³, 10 ⁻⁴, 10 ⁻⁵, 10 ⁻⁶ and 10 ⁻⁷ mmol/l) of Mab were administered 5 min before Ach (10 ⁻⁴ M) treatment, respectively. The Ca ²⁺ fluorescent probe, Fura-2/AM or Fluo-3/AM were applied to the inspection of Ca ²⁺ fluorescent intensity with Varioskan Flash, immunocytometry systems and an inverted system microscope, respectively. The results showed that the fresh method, in which isolated tracheal tissues were previously treated with collagenase for 20 min, was more advantageous for the preparation of guinea pig ASM cells compared to when the enzyme was not used. The time for the ASM cells to initially migrate out of the ‘tissue blocks’ and the culture having to be generated due to the thick cell density was significantly less. On identification with immunocytochemistry or immunofluorescent staining, >95% of the cells were ASM cells. Mab (10 ⁻³−10 ⁻⁷ mmol/l) significantly suppressed the elevation of intracellular Ca ²⁺ induced by Ach in a concentration-dependent manner. The inhibitory rates of intracellular Ca ²⁺ by different concentrations of Mab, from low to high, were 14.93, 24.73, 40.06, 48.54 and 57.13%, respectively, when Varioskan Flash was used for determination. In conclusion, this novel method has a shorter harvesting period for ASM cells. Mab can suppress the increasing level of intracellular Ca ²⁺ induced by Ach in guinea pig ASM cells. Further investigation into the precise mechanisms of action is required.