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

Song X, Zhao C, Dai C, Ren Y, An N, Wen H, Pan LI, Cheng M, Zhang Y - Biomed Rep (2015)

Bottom Line: 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.Mab (10(-3)-10(-7) mmol/l) significantly suppressed the elevation of intracellular Ca(2+) induced by Ach in a concentration-dependent manner.Further investigation into the precise mechanisms of action is required.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P.R. China.

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.

No MeSH data available.


Related in: MedlinePlus

Data of (A and B) immunocytochemical identification of the cells preloaded with α-smooth muscle actin and (C and D) immunofluorescent identification of the cells preloaded with fluorescein isothiocyanate. The images in (A) and (B) were obtained under an inverted system microscope (magnification, x200 and x400, respectively) and those in (C) and (D) under an inverted fluorescent microscope (magnification, x200 and x400, respectively) in the condition of absorption peak at 492 nm and emission peak at 520 nm. The magnified cells in (B) are various shapes, including irregular triangle and fusiform, are illustrated with a thin arrow and common arrow, respectively. The arrow in (D) also indicates airway smooth muscle cells in fusiform identified with immunofluorescent staining.
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f4-br-0-0-502: Data of (A and B) immunocytochemical identification of the cells preloaded with α-smooth muscle actin and (C and D) immunofluorescent identification of the cells preloaded with fluorescein isothiocyanate. The images in (A) and (B) were obtained under an inverted system microscope (magnification, x200 and x400, respectively) and those in (C) and (D) under an inverted fluorescent microscope (magnification, x200 and x400, respectively) in the condition of absorption peak at 492 nm and emission peak at 520 nm. The magnified cells in (B) are various shapes, including irregular triangle and fusiform, are illustrated with a thin arrow and common arrow, respectively. The arrow in (D) also indicates airway smooth muscle cells in fusiform identified with immunofluorescent staining.

Mentions: Guinea pig ASM cells were identified with immunocytochemistry and immunofluorescent staining subsequently to being loaded with the specific α-SMA antibody. The results of immunocytochemistry were as stated in Fig. 4A and B. The magnified cells in Fig. 4 were in various shapes, including the irregular triangle form indicated with thin arrows and fusiform with common arrows. Green fluorescence could be observed at 492 nm under an inverted fluorescent microscope once the cells were loaded with FITC for identification with immunofluorescent staining, as expressed in Fig. 4C and D. The arrow in Fig. 4D indicates ASM cells in the fusiform. It was found that >95% of the cells were ASM cells in several randomly chosen perspectives.


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

Song X, Zhao C, Dai C, Ren Y, An N, Wen H, Pan LI, Cheng M, Zhang Y - Biomed Rep (2015)

Data of (A and B) immunocytochemical identification of the cells preloaded with α-smooth muscle actin and (C and D) immunofluorescent identification of the cells preloaded with fluorescein isothiocyanate. The images in (A) and (B) were obtained under an inverted system microscope (magnification, x200 and x400, respectively) and those in (C) and (D) under an inverted fluorescent microscope (magnification, x200 and x400, respectively) in the condition of absorption peak at 492 nm and emission peak at 520 nm. The magnified cells in (B) are various shapes, including irregular triangle and fusiform, are illustrated with a thin arrow and common arrow, respectively. The arrow in (D) also indicates airway smooth muscle cells in fusiform identified with immunofluorescent staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660599&req=5

f4-br-0-0-502: Data of (A and B) immunocytochemical identification of the cells preloaded with α-smooth muscle actin and (C and D) immunofluorescent identification of the cells preloaded with fluorescein isothiocyanate. The images in (A) and (B) were obtained under an inverted system microscope (magnification, x200 and x400, respectively) and those in (C) and (D) under an inverted fluorescent microscope (magnification, x200 and x400, respectively) in the condition of absorption peak at 492 nm and emission peak at 520 nm. The magnified cells in (B) are various shapes, including irregular triangle and fusiform, are illustrated with a thin arrow and common arrow, respectively. The arrow in (D) also indicates airway smooth muscle cells in fusiform identified with immunofluorescent staining.
Mentions: Guinea pig ASM cells were identified with immunocytochemistry and immunofluorescent staining subsequently to being loaded with the specific α-SMA antibody. The results of immunocytochemistry were as stated in Fig. 4A and B. The magnified cells in Fig. 4 were in various shapes, including the irregular triangle form indicated with thin arrows and fusiform with common arrows. Green fluorescence could be observed at 492 nm under an inverted fluorescent microscope once the cells were loaded with FITC for identification with immunofluorescent staining, as expressed in Fig. 4C and D. The arrow in Fig. 4D indicates ASM cells in the fusiform. It was found that >95% of the cells were ASM cells in several randomly chosen perspectives.

Bottom Line: 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.Mab (10(-3)-10(-7) mmol/l) significantly suppressed the elevation of intracellular Ca(2+) induced by Ach in a concentration-dependent manner.Further investigation into the precise mechanisms of action is required.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P.R. China.

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.

No MeSH data available.


Related in: MedlinePlus