The Altered Calcium Release Function And MRNA Expression Of Ryanodine Receptors In Cultured Airway Smooth Muscle Cells Of Asthmatic Guinea Pig

PrefaceAsthma is a chronic inflammatory disease of airways with a multi-factorial pathogenesis. Airway hyperreactivity and airway remodeling are the important properties of asthma. Airway hyperreactivity is characterized by a hyperreactive response of airway smooth muscle cells(ASMCs) to spasmogens, also reflects a greater degree of ASMCs contraction caused by a bronchoconsricting agonist, these phenomenons are related to the rises of intracellular Ca²⁺ concentration ([Ca²⁺]i). Airway remodeling in the progress of asthma is mainly caused by hyperplasia and hypertrophy, whereas increases of [Ca²⁺]i would stimulate ASMCs growth and cause bronchial wall thickening. Airway inflammatory is another feature of asthma, ASMCs are also a rich source of biologically active cytokines, chemokines and growth factors, which may increase[Ca²⁺]i in ASMCs and modulate airway inflammation through chemotactic, autocrine or paracrine effect.Two cellular mechanisms contribute to increases in[Ca²⁺]i: Ca²⁺ release from intracellular stores and Ca²⁺ influx through cell membrane-associated Ca²⁺ channels. In clinical, calcium channel blockers by inhibiting transmembrane influx of Ca²⁺ act as membrane stabilizers, and it is effective in asthma. However, there are two intracellular Ca²⁺ channels in cells: inositol 1, 4, 5 trisphosphate receptors (IP₃Rs) and ryanodine receptors(RyRs). One study suggest ACH-induced Ca²⁺ oscillations in murine ASMCs are generated by Ca²⁺ release from the sarcoplasmic reticulum(SR) involving IP₃R and RyRs, the release of Ca²⁺ can partically inhibited by IP₃R inhibitor and completely inhibited by RyRs inhibitor. So we suppose that RyRs were more important than IP₃R for inducing the Ca²⁺ release from intracellular stores in some conditions. It maybe provides new insights into the complex calcium signaling in ASMCs and suggest that RyRs are potential therapeutic targets in bronchospastic disorders such as asthma. Therefore, we focus on the role of RyRs in the pathogenesis of asthma.Materiales and methods1. MaterialsCell culture reagents were obtained from Invitrogen Life Technologies-GIBCO. Ovalbumin, ryanodine, Flou-3/AM, EGTA and HEPES were obtained from Sigma chemical company. RT-PCR kit was obtained from TaKaRa company.Healthy male guinea pigs weighing 0.3～0.4kg. Animals is offered by experimental animals department of China Medical University.2. MethodsAnimal sensitization and allergen exposureThe method were performed as described previously. The guinea pigs were actively sensitized by intraperitoneal injections ovalbumin(OA) 1ml(10% W/V in 0.9%NaC1). Fourteen days after sensitization, they were challenged by exposure to aerosolized OA(1% W/V in 0.9% NaC1) for two minutes once every two days to induce asthma, by whole two weeks. The responsiveness of animals was observed when they were challenged. The animals gradually appearances accelerated breathing, at last appearances bucking. After challenging, the trachea was rapidly removed within 24h. Preparation of guinea pig ASMCs cultureThe trachea was isolated, the connective tissue and adventitia were carefully stripped off, and the epithelium was removed by gently scratching the intimal surface. The remaining smooth muscle was digested with 0.2%collagenase for 1 hour at 37℃. The cells were plated onto 12-well plates(for fluorescent experiments) or culture flask (for molecular biological experiments), and cultured in DMEM containing 20% fetal bovine serum(FBS) in a 37℃, 5% CO₂, humidified incubator. Upon reaching confluence, cells were passaged by blowing the cells with suctionpipes and reseeding them into two new 12-well plates. Primary cells and passage 2 to 3 were used.The verification of guinea pig ASMCsUnder microscope, the ASMCs exhibited the typical "hill and valley" aspect. Cell characterization was assessed by immunostaining using a monoclonal antibody againstα-smooth muscle actin. More than 95% of cells from each guinea pig used in the different experiments displayed positive immunohistochemical staining for antibodyMeasurement of[Ca²⁺]iIn single ASMC, [Ca²⁺]i was measured by using the Ca²⁺-sensitive fluorescent indicator, Flou-3/AM. It was added into 12-well plates(final concentration of Flou-3/AM was about 5×10^-6 mol·L^-1) in the dark at room temperature (35-37℃) under an atmosphere of 5% CO₂, 95% air for 30min. The Flou-3/AM-loaded cells on 12-well plates were then transferred to a recording cell chamber on the microscope stage and superfused with modified Hank' buffer for twice to remove extracellular Flou-3/AM. Flou-3 fluorescence (528-nm light emission excited by 488-nm illuminations) was collected from the cell. The fluorescence signals emitted from the cells were monitored continuously by using an intracellular imaging fluorescence microscopy system(MetaFlour 4.5 software) and recorded in a compatible computer for later analysis. F₀ was baseline fluorescence count, F was the fluorescence count after applicating agonist, AF/F₀(%)=100×(F-F₀) /F₀ represented the changes of [Ca²⁺]i, the changes in fluorescence provide only a relative, not an absolute, measurement of [Ca²⁺]i.RT-PCRTotal RNA was extracted and reverse transcribed from ASMCs of normal guinea pig and asthmatic guinea pig. Three sets of primers were used for PCR amplication of RyRs mRNA. The primer sequences were based on mouse and rat(GenBank2 accession numbers AF011788, U95157, AF011789 and AF130881 ) cDNAs for RyR types 1, 2 and 3, and all cross at least one intron in the genomic DNA.The consensus primer pairs,RyR1. Forward primer5'-GAAGGTTCTGGACAAACACGGG-3', Reverse primer5'-TCGCTCTTGTTGTAGAATTTGCGG-3', expected product size was 435 bp;RyR2: Forward primer5'-GAATCAGTGAGTTACTGGGCATGG-3', Reverse primer5'-CTGGTCTCTGAGTTCTCCAAAAGC-3', expected product size was 635 bp;RyR3. Forward primer5'-CCTTCGCTATCAACTTCATCCTGC-3', Reverse primer5'-TCTTCTACTGGGCTAAAGTCAAGG-3', expected product size was 505 bp.β-actin. Forward primer5'-AGAGCTACGAGCTGCCTGAC-3', Reverse primer5'-AGTACTTGCGCTCAGGAGGA-3', expected product size was 300 bp.The primers were synthesized by TaKaRa company. We used 50μl of PCR reaction solution. It contained 10μl 5×PCR buffer, 0.25μl Taq enzyme, 1μl upstream primer and 1μl downstream primer. Sterile water was added to make a final volume of 50μl and then mixed carefully. The amplication conditions for these primers were 94℃for 30.s, 58℃for 30.s and 72℃for 2 min, for 30 cycles. To verify their identity, PCR products were separated on 1.2% agarose gel stained with Gene finder. To quantify the PCR products, an invariant mRNA of humanβ-actin was used as an internal control. The optical density values for each band on the gel were measured by a gel documentation system. The results were expressed as the ratio of intensity of RyRs to that ofβ-actin in samples from control and asthmatic ASMCs.Results1. In extracellular Ca²⁺-free condition, different concentrations of ryanodine (5×10^-5 mol·L^-1, 10^-4 mol·L^-1, 2×10^-4 mol·L^-1)could induce[Ca²⁺]i increase in primary cultured ASMCs, but the increase of [Ca²⁺]i in primary cultured ASMCs of asthmatic guinea pig was much higher than those of control group(P＜0.01)2. In subcultured ASMCs of asthmatic guinea pig, although in the presence of 5×10^-5 mol·L^-1 ryanodine, the increase of [Ca²⁺]i was not so high as in primary cultured ASMCs (P＜0.01), in the presence of 10^-4, 2×10^-4 mol·L^-1 ryanodine, the increase of [Ca²⁺]i in both primary cultured ASMCs of asthmatic guinea pig and subcultured ASMCs of asthmatic guinea pig was at the similar level(P＞0.05). In extracellular Ca²⁺-free condition, histamine(10^-4 mol·L^-1), the increase of [Ca²⁺]i in primary cultured ASMCs of both asthmatic and control group was not significant different (P＞0.05).3. In ASMCs of normal guinea pig, RyR1 and RyR2 were expressioned, but RyR3 were not found.4. Similarly, in ASMCs of asthmatic guinea pig, only RyR1 and RyR2 were expressioned. Nevertheless, the expression of RyR1 increased obviously in asthmatic group compared with control group(P＜0.01).DiscussionAsthma is characterized by airway inflammatory, airway hyperreactivity and airway remodeling. In the present study, we know that abnormal calcium handling by ASMCs leading to an increase[Ca²⁺]i was considered to be responsible for the genesis of asthma. The increase of [Ca²⁺]i were by Ca²⁺ release from intracellular stores and Ca²⁺ influx through cell membrane-associated Ca²⁺ channels. We study on intracellular Ca²⁺ channels(RyRs), and we recognized that RyR1 has important roles in asthma.Ryanodine, a plant-derived insecticide, binds to RyRs and causes Ca²⁺ release from sarcoplasmic reticulum(SR), it is used to demonstrate the presence and study the behaviour of the RyRs in muscle and other cell types. In this study, we exposed ASMCs of guinea pig to ryanodine, the increase of [Ca²⁺]i in primary cultured ASMCs of asthmatic guinea pig is much higher than control group. The result indicates that the function of RyRs was increased in asthma. We suppose that the reason of hyperfunction of RyRs may be due to the increased sensitivity or up-expression of RyRs. From this experiment, we also consider that there are RyRs in ASMCs of guinea pig.Otherwise, we applied ryanodine to subcultured ASMCs of asthmatic guinea pig, in the presence of 10^-4 mol·L^-1, 2×10^-4 mol·L^-1 ryanodine, the increase level of[Ca²⁺]i in subcultured cells is similar to primary cultured cells, it demonstrates that thecharacteristics of RyRs in primary cultured ASMCs of asthmatic guinea pig still retained in subcultured ASMCs of asthmatic guinea pig. Moreover, in the presence of 5×10^-5 mol·L^-1 ryanodine, the responsiveness of subcultured cells is decreased comparing with primary cells, this phenomenon suggests that subcultured ASMCs of asthmatic guinea pig displays hyporesponsiveness to lower concentration of ryanodine.Recently, we believe that there are two known types of Ca²⁺ channel that regulate release from intracellular Ca²⁺ stores: RyRs and InsP₃ receptors(IP3R), there is only IP3R in smooth muscle for common idea. But Ryan E. Lesh found that RyRs are present in guinea pig aorta and vas deferens smooth muscle, RyRs are present in rat ASMCs, too. We suppose that RyRs are present in ASMCs of guinea pig from above results.We carry out RT-PCR for detecting the mRNA expression of RyR subtypes in primary cultured ASMCs of guinea pig. We found that RyR1 and RyR2 were expressed in ASMCs of both normal and asthmatic guinea pig, RyR3 were not found in both groups. The information is different from the expression of RyR subtypes in rat ASMCs, there are three subtypes coexpressioned. The matter of this result may be owing to the difference of genera. For the result, We also know that: the expression level of RyR2 was much higher than the expression level of RyR1 in both groups, but the expression of RyR1 increased obviously in asthmatic guinea pig, meanwhile, the expression of RyR2 are in the same level in both groups. At present, we know that RyR1 is found primarily in skeletal muscle. In our study, RyR1 is also found in ASMCs of guinea pig, furthermore, the overexpression of RyR1 is observed in asthmatic guinea pig. This discovery not only provide the theory for researching asthma, but also give an new therapeutic target site for treating asthma.RyR1 is one of the three subtypes RyRs, it can be activated by agonists, and induce Ca²⁺ release from SR. In asthmatic guinea pig, we speculate that the mRNA up-expression of RyR1 might be induce the protein up-expression of RyR1, it means that the density of RyR1 is increased, it makes the increase of [Ca²⁺]i, and this Could lead to airway hyperreactivity which appears in asthma. Therefore, we believe that RyR1 play an important role in asthma. In addition, it has been reported that RyR1 have important roles in Central core disease and Malignant hyperthermia, it shows that RyR1 may contribute to many other diseases, and it awaits research in the future. In contrast, the results suggest that RyR2 is not essential for asthma.In conclusion, our observations from this study indicate that RyRs shows hyperfunction in asthmatic guinea pig. We found that RyR1 and RyR2 were expressed in ASMCs of both normal and asthmatic guinea pig, and RyR1 increased obviously in asthmatic guinea pig. So we believe that the hyperfunction of RyRs is due to the overexpression of RyR1, and we think that RyR1 is involved in the pathogenesis of asthma. Meanwhile, we are interested in culturing the ASMCs of asthmatic guinea pig, we believe that the characteristics of "asthmatic properties" could still retains in subcultured ASMCs of asthmatic guinea pig under specified condition, but the methods have not been found yet. Conclusion1. Ryanodine receptors of asthmatic guinea pig showed hyperreactivity.2. Under specified condition, the characteristics of ryanodine receptor still retains in subcultured ASMCs of asthmatic guinea pig.3. RyR1 and RyR2 were expressed in ASMCs of normal and asthmatic guinea pig.4. Meanwhile, the up-expression of RyR1 were found in ASMCs of asthmatic guinea pig. It indicates that RyR1 play an important role in asthma.