The present study aimed to establish an effective method for the
Asthma is one of the most common chronic diseases characterized by airway hyperresponsiveness (AHR) and airway remodeling. It has been well established that airway smooth muscle (ASM) cells are the main components of the respiratory tract. They are believed to have a role in the pathogenesis of asthma through their contractile properties (
Mabuterol hydrochloride (Mab) (
In the present study, a renewed and stable method of culturing guinea pig ASM cells was established. The suppression of increasing intracellular calcium by Mab was investigated with several detection methods and two agents Fura-2/AM, as well as Fluo-3/AM as a Ca2+ indicator.
Male or female Hartley guinea pigs, weighing 150–200 g, were provided by the Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, Liaoning, China). Animals were bred in a facility controlled by temperature (26±3°C), relative humidity (50±5%) and light (14 and 10 h of light and dark), with free access to food and water, with added vitamin C. All the experimental procedures in the present study were carried out in accordance with the Internationally Accepted Principles and the Guidelines for the Care and Use of Animal Center of Shenyang Pharmaceutical University.
Mab was supplied by the Pharmaceutical Engineering Department, Shenyang Pharmaceutical University (enantiomeric excess >99%). Ach was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) and Hanks' balanced salt solution (HBSS) were purchased from Gibco-BRL (Carlsbad, CA, USA) and type I collagenase from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Fetal bovine serum (FBS) was produced by Tianjin Hualida Biotechnology Co., Ltd. (Tianjin, China). Triton X-100 and 3-(4,5-dimethylthinazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Amresco LLC (Solon, OH, USA). Mouse anti-α-smooth muscle actin (α-SMA), 5% bovine serum albumin (BSA), streptavidin-biotin complex (SABC) immunohistochemical staining kit and 3,3′ diaminobenzidine (DAB) chromogenic reagent kit were all purchased from Wuhan Boster Biological Technology Co., Ltd. (Wuhan, China). Fura-2/AM, Fluo-3/AM and fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin G (IgG) were from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China).
One of the methods used collagenase to pretreat tracheal tissues (CPTT) and the other did not. Freshly dispersed tracheal smooth muscle strips of guinea pig were prepared as described previously (
When the cell density was ~80%, the ASM pieces were moved into another culture flask to be fully prepared as described above. Subsequently, the cells were detached with the mixed solution of 0.25% trypsin and 0.02% EDTA at 37°C for 3 min in preparation for cell generation. The MTT assay was used to determine cell viability (
To confirm that the cells were ASM cells and not epithelial cells or fibroblasts, homogeneity was confirmed with α-SMA according to a previously described method (
Intracellular Ca2+ was indicated with a fluorescent molecular probe, Fura-2/AM, as described previously (
A total of 2 µmol/l of the Ca2+-sensitive Fluo-3/AM was required according to the manufacturer's instruction when the intracellular Ca2+ level was determined with flow cytometry. Cells at the density of 3×106/ml were incubated at 37°C for 45 min in the dark after the treatment with Mab plus Ach, as described previously. The cells were gently rinsed with HBSS without Ca2+ 3 times. When Fluo-3/AM binds to cytoplasmic-free calcium, the complex emits green fluorescence under the stimulation of the 488 nm line of an argon ion laser. The fluorescent intensity at 525 nm was determined at 37°C with the Becton-Dickinson Immunocytometry system (FACSCalibur; BD Biosciences, San Jose, CA, USA) and the light signal was converted into an electric signal with linear amplification.
Results are expressed as mean ± standard error of the mean and statistical comparisons among groups were performed with one-way analysis of variance followed by least significant difference or independent samples t-test using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference in all the experiments. Figure plotting was conducted with the aid of software GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA).
As is shown in
Cells migrating from the ASM pieces treated with collagenase began to attach to the surface of the culture flask 6 h after they were generated and spread out gradually in the following 2 days. Their morphology was expressed in fusiform shown with arrowheads or an irregular triangle shown with arrows in
Cell viability was determined with the MTT assay at days 1 to 7, respectively, after they were generated. As was observed in the growth curve of
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
As shown in
Representative images of Ca2+ fluorescence obtained from the inverted fluorescent microscope are indicated in
The geometric mean (Geo Mean) of the M1 range in the diagram of flow cytometry (
ASM cells are involved in the pathophysiology of numerous airway diseases, such as airway remodeling and intracellular calcium overload (
Currently, two methods, i.e. with and without enzymes to digest ASM pieces, are commonly used to prepare primary ASM cells. It is known that the former may obtain the cells in a short period of time (
The function of ASM cells is clearly regulated by various signaling molecules. Activation of enzymes, protein phosphorylation and release of calcium pools are all involved in the transduction of the signaling molecules. Among them, Ca2+ may play a central role and free Ca2+ in the cytosol of ASM cells acts as a crucial secondary messenger in numerous biological processes, such as contraction, proliferation, gene transcription and secretion of signal mediators (
The drug is a selective long-acting β2-receptor agonist to be clinically used for asthma treatment (
Ach binds G-protein coupled receptors on the membrane of ASM cells to activate phospholipase C, and subsequently, inositoltrisphosphate (IP3) is produced under its catalysis (
In conclusion, the renewed method of ASM cell culture has successfully been proved. Additionally, it is clearly shown that Mab significantly suppresses the increased level of intracellular Ca2+ induced by Ach through three measurement methods with a specific fluorescent probe in the ASM cells. Due to the mechanism of calcium increase induced with Ach and the suppressive effect of Mab on the increased level of intracellular Ca2+, more studies should be performed to clarify the mechanism of the suppression in detail, in which RyR and/or the IP3R signaling pathway may provide innovative ideas with further research.
Molecular structure of mabuterol hydrochloride.
Comparison between the two different methods of culturing guinea pig airway smooth muscle (ASM) cells with or without collagenase to pretreat the ASM pieces. Number of days for the cells to (A) begin migrating out of the ASM pieces and the (B) cells having to generate due to their thick density. Data from five independent experiments were used and expressed as mean ± standard error of the mean. **P<0.01 by independent samples t-test with SPSS 16.0. The status of the cells on day 6 in the (C) with or (D) without collagenase groups, and that on days 2–4 of the ASM cells migrating from the second-hand pieces treated with the two methods is shown in (E) and (F), respectively (magnification, x100). Arrows in (C-F) indicate the ASM pieces.
(A-C) Morphology and viability of the third generation of guinea pig airway smooth muscle cells. (D) The growth curve recording cell growth condition over time. Data are expressed as mean ± standard error of the mean (n=8). Cells from days 4 to 8 proliferate fast and the density of the cells on days 4 or 6 was significantly increased compared with that on day 1 (*P<0.05). Images of the cells, i.e. (A-C), were respectively taken under an optical microscope (magnification, x100) on days 2, 4 and 6 after generation. The arrows in (A) indicate the cells in irregular triangle form and the arrowheads in fusiform, those in (B) expressed the cells in a good state, and those in (C) are the cells on ~day 6 that aligned so closely that their morphology became abnormal.
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.
Suppression of mabuterol hydrochloride (Mab) (10−3, 10−4, 10−5, 10−6 and 10−7 mmol/l) on the increased level of intracellular Ca2+ induced by acetylcholine (Ach) (10−4 M) in a concentration-dependent manner. Data are expressed as mean ± standard error of the mean obtained from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 compared to the group treated with Ach, and ###P<0.001 compared to the control by analysis of variance followed by least significant difference with SPSS 16.0. The percentage on the top of each column indicates the inhibitory rate of intracellular Ca2+.
Representative image of Ca2+ fluorescence obtained from the inverted system microscope (magnification, x200). The image in (A) comes from the sample treated with acetylcholine (Ach) (10−4 mmol/l) alone and (B-F) are from the samples pre-incubated with 10−3, 10−4, 10−5, 10−6 and 10−7 mmol/l of mabuterol hydrochloride, respectively, and subsequently with Ach (10−4 mmol/l). The scale bar represents 200 µm.
Geometric mean (Geo Mean) of range M1 in the column figure, calculated based on the diagrams of the immunocytometry systems. The cells loaded with Fluo-3/AM were respectively treated with 10−7, 10−6, 10−5, 10−4 and 10−3 mmol/l of mabuterol hydrochloride (Mab), and subsequently, their range of M1 in the flow cytometry diagram was decreased in a concentration-dependent manner as shown in (G), (F), (E), (D) and (C). The diagram in (B) illustrates the cells treated with 10−4 M acetylcholine (Ach) alone and (A) without treatment. Data are expressed as mean ± standard error of the mean obtained from three independent experiments. *P<0.05 and **P<0.01 compared to the group treated with Ach, and ###P<0.001 compared to the control by analysis of variance followed by least significant difference using SPSS 16.0.
Signaling pathways of Ca2+ mobilization in an airway smooth muscle (ASM) cell involving the inositoltrisphosphate receptor (IP3R) and ryanodine receptor (RyR), and the potential targets of mabuterol hydrochloride (Mab) that intervene in the increased level of intracellular Ca2+ induced with Ach. Binding with a G-protein coupled receptor, Ach activates phospholipase C (PLC) to generate IP3, which encourages the clusters of IP3R on SR to release Ca2+. This process may stimulate the adjacent RyR to increase Ca2+. RyR may also be activated or potentiated by cADP ribose (cADPR). It may be sequestered by the superficial sarcoplasmic reticulum (SR) through sarcoendoplasmic Ca2+ ATPase 2a (SERCA2a), although much of the calcium is released from stores and enters the cytoplasm. The increased level of intracellular Ca2+ leads to the contraction, proliferation and migration of the ASM.